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Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 239354 14 pageshttpdxdoiorg1011552013239354
Research ArticleSynthesis Characterisation and In Vitro AnticancerActivity of Curcumin Analogues Bearing PyrazolePyrimidineRing Targeting EGFR Tyrosine Kinase
1 Department of Pharmaceutical Chemistry Maharishi Arvind College of Pharmacy Ambabari Jaipur Rajasthan 302 023 India2Department of Pharmaceutical Chemistry Alwar Pharmacy College Alwar Rajasthan 301 030 India3 Department of Pharmaceutical Chemistry Birla Institute of Technology Mesra Ranchi Jharkhand 835 215 India
Correspondence should be addressed to Mohamed Jawed Ahsan jawedpharmagmailcom
Received 24 April 2013 Revised 20 July 2013 Accepted 23 July 2013
Academic Editor Vickram Ramkumar
Copyright copy 2013 Mohamed Jawed Ahsan et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
In search of potential therapeutics for cancer we described herein the synthesis characterization and in vitro anticancer activityof a novel series of curcumin analogues The anticancer effects were evaluated on a panel of 60 cell lines according to the NationalCancer Institute (NCI) screening protocol There were 10 tested compounds among 14 synthesized compounds which showedpotent anticancer activity in both one-dose and 5-dose assays The most active compound of the series was 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl(phenyl)methanone (10) which showed mean growth percent of minus2871 in one-dose assay and GI
50
values between 00079 and 186120583M in 5-dose assay
1 Introduction
Cancer is still continuing to be a major health problemworldwide The development of new anticancer therapeuticagents is one of the fundamental goals inmedicinal chemistryas cancer causes about 13 of all the death [1] Surpassingcardiovascular diseases it is taking the position numberone killer due to various factors [2] Also the treatment ofcancer is associated with various side effects which includebone marrow depression alopecia drug-induced cancerhepatotoxicity and many more Because of the need andvalue of anticancer drugs many laboratories are intensivelyinvestigating the chemistry and biology of novel anticanceragents Also the development of resistance against the exist-ing anticancer drugs and cytotoxicity and genotoxicity ofanticancer drugs to the normal cells are othermajor problemsin cancer therapy keeping research window open in searchfor newer anticancer molecules [3] But the window passagehas become narrower because it is rather hard to search
a molecule that can selectively inhibit the proliferation ofabnormal cells only with least or no affect on normal cells
In the last decade several pyrazole derivatives proved tohave anticancer activity [4ndash8] The other activities reportedfor pyrazole nucleus include antitubercular anticonvulsantanticancer antimicrobial anti-HIV antihepatotoxic anti-inflammatory and analgesic [9ndash16] Also curcumin a majoryellow pigment and active component of turmeric has beenshown to possess anti-inflammatory and anticancer activities[17] In some countries curcumin was consumed in thediet up to 4 g per adultday which appeared to lower theincidence rate of colorectal cancer In a study curcuminshowed autophagic and apoptotic death of K562 cell line(leukemia) [18] The cytotoxicity studies in different celllines indicated that the toxicity of curcumin was significantlyhigher in tumor cells if compared to the normal cells [19]Curcumin and its derivatives possess a wide variety ofpharmacological activities namely antibacterial anti-HIVanti-inflammatory antimalarial anticancer and many more
2 BioMed Research International
[20ndash24] Considering these recent discoveries curcumin canbe considered as an ideal lead compound for anticancer drugdevelopment Earlier we have reported the anticancer activityof pyrazoline and oxadiazole analogues [25 26] Receptortyrosine kinases (RTKs) have been shown to be key regulatorsof normal cellular processes and to additionally play a criticalrole in the development and progression of many types ofcancer by binding either with polypeptide growth factors orcytokines or hormones [27] Several transforming oncogeneslike src a gene obtained from Rous sarcoma virus abl a geneobtained from Abelson murine leukemia virus and so forthare known to possess tyrosine kinase activity Overexpressionof certain RTKs shows association with promotion andmaintenance of malignancies Thus inactivation of thespecific tyrosine kinase represents a potential approach fordesign of anticancer drugs Gefitinib and erlotinib usedin the treatment of certain types of cancer are epidermalgrowth factor receptor (EGFR) tyrosine kinase inhibitorsProtein tyrosine kinases occupy a central position in thecontrol of cellular proliferation and it is well recognizedthat the response of many cells to growth factors is initiatedby activation of tyrosine kinase [28] The involvement ofthe EGFR family of tyrosine kinases in cancer proliferationsuggests that an inhibitor which blocks the tyrosine kinaseactivity of the entire EGFR family could have significanttherapeutic potential [29] Hence enthused by all these factswe have synthesized a novel series of curcumin analoguesand evaluated their antitumor activity and selected EGFRfamily of tyrosine kinase as a biological target for carryingout the docking study of some of the active compounds
2 Materials and Methods
21 Chemistry All chemicals were supplied by E Merck(Germany) Konark Herbal (India) and S D Fine Chemicals(India) Melting points were determined by open tube capil-lary method and are uncorrected Purity of the compoundswas checked by elemental analysis and the progress ofreactions was monitored by TLC plates (silica gel G) usingmobile phase hexane ethylacetate (6 4) and the spots wereidentified by iodine vapours or UV light IR spectra wereobtained on a Shimadzu 8201 PC FT-IR spectrometer (KBrpellets) 1H NMR spectra were recorded on a Bruker AC300MHz spectrometer using TMS as internal standard inDMSO Mass spectra were recorded on a Bruker EsquireLCMS using ESI and elemental analyses were performed onPerkin-Elmer 2400 elemental analyzer
22 General Method for the Synthesis of 35-Bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carboxa-mide Analogues (1ndash9) 17-Bis(4-hydroxy-3-methoxyph-enyl)hepta-16-diene-35-dione (curcumin) (0005mol)and substituted phenyl semicarbazides (0005mol) wererefluxed in glacial acetic acid for 12 h The substitutedsemicarbazides were synthesized as per reported method[30] The excess of solvent was removed under reducedpressure and then the reaction mixture was poured intothe crushed ice The solid mass was filtered washed
dried and recrystallized with ethanol furnishing the title35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9)
(1H s CH=C) 673 (2H d J = 158Hz CH and CH) 678(2H d J = 158Hz CH and CH) 631ndash762 (10H m ArH)972 (2H s OH) 983 (1H s CONH) mz = 517 (M+) 519(M + 2)+
(1H s CH=C) 668 (2H d J = 162Hz CH and CH) 678(2H d J = 162Hz CH and CH) 692ndash760 (10H m ArH)962 (2H s OH) 997 (1H s CONH) 1297 (OH) 13C NMR(75MHzDMSO-d
CH) 676 (2H d J = 62Hz CH and CH) 689ndash792 (9H mArH) 997 (2H s OH) 1014 (1H s CONH)mz = 511 (M+)
23 General Method for the Synthesis of 35-Bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl (substituted phenyl)methanoneAnalogues (10-11) 17-Bis(4-hydroxy-3-methoxyphenyl) he-pta-16-diene-35-dione (curcumin) (0005mol) and substi-tuted phenyl hydrazides (0005mol) were refluxed in glacialacetic acid for 12 h The excess of solvent was removed underreduced pressure and then the reaction mixture was pouredinto the crushed ice The solid mass was filtered washeddried and recrystallized with ethanol furnishing the title 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl (substitutedphenyl)methanone analogues (10-11)
24 General Method for the Synthesis of DihydropyrimidineAnalogues (12ndash14) 17-Bis(4-hydroxy-3-methoxyphenyl)hepta-1 6-diene-35-dione (curcumin) (0005mol) and ureaguanidinethiourea (0005mol) were refluxed in glacialacetic acid for 12 h The excess of solvent was removedunder reduced pressure and then the reaction mixture waspoured into the crushed ice The solid mass was filteredwashed dried and recrystallized with ethanol furnishingthe title 35-bis(4-hydroxy-3-methylstyryl)-N-(substitutedphenyl)-1H-pyrazole-1-carboxamide analogues (12ndash14)
25 Anticancer Activity There were 10 compounds amongthe series selected and screened for their anticancer activityboth in one-dose and 5-dose assays by National CancerInstitute (NCI) on leukemia melanoma lung colon CNSovarian renal prostate and breast cancers cell lines nearly60 in number according to their screening protocol reportedelsewhere [31ndash34] All the curcumin analogues were synthe-sized and the structure of the compounds was submittedonline to the official site of NCI for anticancer screeningAmong 14 compounds only 10 compounds were selected foranticancer screening NCI has its own selection procedure ofthe compounds for anticancer screening based on the noveltyof heterocyclic ring system drug-like properties utilizing the
4 BioMed Research International
Table 1 Physical constants of the curcumin analogues (1ndash14)
concept of privileged scaffolds structure based on computer-aided drug design and so forth while the structures con-taining problematic linkage or functional groups (eg nitronitroso ndashNndashNndash ndashN=Nndash imine semicarbazone thioamidesand thioureas) for successful drug development are avoided[31]
The anticancer screening was carried out as per theNCI US protocol Using the seven absorbance measurements(time zero (119879
119894) control growth (119862) and test growth in
the presence of drug at the five concentration levels (119879119891))
the percentage growth was calculated at each of the drugconcentrations level as [(119879
119891minus 119879
119894)(119862 minus 119879
119894)] times 100 for
concentrations for which 119879119891ge 119879
119894and [(119879
119891minus 119879
119894)119879
119894] times 100
for concentrations for which 119879119891lt 119879
119894
Three-dose response parameters (GI50 TGI and LC
50)
were calculated for each of the experimental agents Growthinhibition of 50 (GI
50) was calculated from 100 times [(119879
119891minus
119879
119894)(119862 minus 119879
119894)] = 50 which was the drug concentration
resulting in a 50 reduction in the net protein increase (asmeasured by sulforhodamine B SRB staining) in controlcells during the drug incubation The total growth inhibi-tion (TGI) was calculated from 119879
119891= 119879
119894 which was the
drug concentration resulting in total growth inhibition andsignified the cytostatic effect The LC
50was calculated from
100 times [(119879
119891minus 119879
119894)119879
119894] = minus50 indicating a net loss of cells
following treatment which indicated the concentration ofdrug resulting in a 50 reduction in the measured proteinat the end of the drug treatment as compared to that at thebeginning Values were calculated for each of these threeparameters at the level of activity however if the effect didnot reach to the level of activity the value of parameter was
expressed as less than the minimum concentration testedor if the effect exceeded the level of activity the valueof parameter was expressed as greater than the maximumconcentration tested [31 35 36] LogGI
50 log TGI and
log LC50
are the logarithm molar concentrations producing50 growth inhibition (GI
50) a total growth inhibition
(TGI) and a 50 cellular death (LC50) respectively
26 Molecular Docking Studies
261 Protein Structure X-ray crystal structure EGFR kinase(PDB 2J5F) with resolution 300 A 119877-value 0194 (obs)was obtained from the protein data bank (Research Collab-oratory for Structural Bioinformatics (RCSB) (httpwwwrcsborgpdb))
262 Protein Preparation The protein (PDB 2J5F) wasprepared using the Protein PreparationWizard Preprocessedbond orders were assigned hydrogens were added metalswere treated and water molecules were deleted Heterostatefor cocrystallized ligand was generated using Epik proto-nation state and optimization of H-bonding of the proteinside chains were assigned using ProtAssign Energy wasminimized (Impref minimization) using RMSD 030 A andconverged by OPLS2005 force field utilities of Schrodingerrsquossuite 93
263 Receptor Grid Generation Receptor Grid has beengenerated with GLIDE module of Schrodinger with default
BioMed Research International 5
Table 2 Sixty human tumor cell lines anticancer screening data of curcumin analogues (1ndash14)
Panelcell line Growth percent in one-dose assay1 2 3 5 6 8 10 11 12 13
parameters and without any constraints Site has been speci-fied as centroid of the work space ligand (20 A) with van derWaals radius scaling factor 10 and partial charge cutoff 025
264 Molecular Docking Protocol The ligand docking wasperformed in GLIDE50 Ligands which show less than35 rotatable bonds and which are having less than 200atoms were selected The scaling factor will be 080 and thepotential charge cutoff is 015 All the conformers from theconfgen-ligprep output were docked in the EGFR TyrosineKinase active site All default parameters were used for extraprecision docking Glide extra precision mode was employedfor the current docking study Best poses were chosen forenergy minimization during docking a distance dependentdielectric constant of 20 and maximum number of min-imization step of 100 was used The docking simulations(Ligand receptor interactions) are scored using the Xtraprecision (XP) mode which is implemented in GLIDE50
27 Materials and Methods All computational analysis wascarried out on a Red Hat 50 Linux platform running on aDell Precision work station with Intel core 2 quad processorand 8GB of RAM
3 Results and Discussion
31 Chemistry The curcumin analogues (1ndash14) described inthe study are shown in Table 1 and the reaction sequencefor the synthesis is summarized in Scheme 1 In the first part
of the synthesis 17-bis(4-hydroxy-3-methoxyphenyl)hepta-16-diene-35-dione (curcumin) and substituted phenyl semi-carbazide were refluxed in glacial acetic acid to obtainthe 35-bis(4-hydroxy-3-methoxystyryl)-N-(substituted phe-nyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) Substi-tuted phenyl semicarbazides (ArNHCONHNH
2) were syn-
thesized as per reported method [30] In the second partof the synthesis 17-bis(4-hydroxy-3-methoxyphenyl)hepta-16-diene-35-dione (curcumin) and substituted phenylhy-drazide (ArCONHNH
2) were refluxed in glacial acetic acid to
obtain the 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl(substituted phenyl)methanone (10-11) In the third partof synthesis 17-bis(4-hydroxy-3-methoxyphenyl)hepta-16-diene-35-dione (curcumin) and ureaguanidinethioureawere refluxed in glacial acetic acid to obtain the pyrimidineanalogues (12ndash14) The plausible mechanism of reactions isgiven in Figure 1 The yields of the title compounds wereranging from 66 to 88 after recrystallization with absoluteethanol The completion of reaction was monitored by TLCusing mobile phase hexane ethylacetate (6 4) and purityof the compounds was checked by elemental analyses Boththe analytical and spectral data (IR NMR and MS) of thesynthesized compounds were in full accordance with theproposed structures In general IR spectra of the compoundsafforded C=N stretching at 1514ndash1583 cmminus1 and CndashN stretchat 1320ndash1335 cmminus1 and carbamoyl group NndashH stretching at3122ndash3210 cmminus1 andC=O stretching at 1680ndash1689 cmminus1 bandsfor carboxamide analogues (1ndash9) and at 1755ndash1757 cmminus1bands formethanone analogues (10-11)The 1HNMR spectra
BioMed Research International 7
Table 3 NCI in vitro testing results of compounds of the compounds 8 10 and 13 at five-dose level in 120583M
S no Panelcell line 8 (NSC 757926) 10 (NSC 757929) 13 (NSC 763375)GI50 TGI LC50 GI50 TGI LC50 GI50 TGI LC50
(1)
LeukemiaCCRF-CEM 0232 gt100 gt100 0127 gt100 gt100 0543 NT gt100HL-60(TB) 0237 0806 gt100 00884 NT gt100 218 617 gt100MOLT-4 0364 NT gt100 0347 gt100 gt100 101 519 gt100RPMI-8226 0336 NT gt100 0275 NT gt100 129 418 gt100SR 0004 gt100 gt100 00353 gt100 gt100 072 358 gt100
Colon cancerCOLO 205 205 362 638 186 334 597 18 324 585HCC-2998 064 58 gt100 0591 271 gt100 175 319 579HCT-116 0323 NT NT 0269 114 NT 11 239 518HCT-15 0335 166 NT 0167 152 NT 282 103 gt100HT29 0696 28 994 0463 319 gt100 314 0948 421KM12 0298 125 NT 00092 119 NT 163 332 679SW-620 0441 NT gt100 00079 NT gt100 165 368 NT
(4)
CNS cancerSF-268 0571 NT NT 0256 NT NT 211 156 gt100SF-295 0546 NT gt100 0538 NT NT 193 454 gt100SF-539 0354 15 NT 00604 153 NT 205 573 gt100SNB-19 0634 201 NT 0572 212 NT 181 795 gt100SNB-75 0416 182 NT 00099 187 NT 126 334 884U251 0471 159 NT 0428 170 NT 179 569 663
(5)
MelanomaLOX IMVI 0284 149 NT 00626 156 NT 127 259 NTMALME-3M 0563 198 NT 0525 217 NT 271 676 gt100M14 0372 141 NT 0155 148 NT 165 344 NTMDA-MB-435 00448 0383 NT 00175 000815 gt100 171 NT gt100SK-MEL-2 0366 182 NT 0266 203 gt100 198 41 NTSK-MEL-28 0509 247 861 0176 272 905 185 453 26SK-MEL-5 0317 129 367 0171 130 369 149 289 559UACC-257 154 547 319 114 429 533 153 35 8UACC-62 0413 165 489 0371 164 NT 149 323 NT
8 BioMed Research International
Table 3 Continued
S no Panelcell line 8 (NSC 757926) 10 (NSC 757929) 13 (NSC 763375)GI50 TGI LC50 GI50 TGI LC50 GI50 TGI LC50
showed singlet at 120575 119ndash128 ppm corresponding to CH3
a singlet at 120575 379ndash385 ppm corresponding to OCH3 a
singlet at 120575 652ndash662 ppm corresponding to CH=C proton(pyrazoledihydropyrimidine) a doublet at 120575 661ndash675 ppmcorresponding toCH=CHproton a doublet at 665ndash681 ppmcorresponding to CH=CH proton a multiplet at 120575 681ndash793 ppm corresponding to aromatic protons broad singlet at120575 993ndash1016 ppm corresponding to CONH
2 The compounds
(1ndash14) in 1HNMRspectra exhibited two doublets with 119869 valuebetween 146 and 166Hz confirming the trans coupling Themass spectra of the compounds revealed in each case a peakcorresponding to their molecular ion peaks The elementalanalysis results were within plusmn04 of the theoretical values
32 Anticancer Activity Ten compounds were evaluated fortheir anticancer activity in both one-dose and 5-dose assaysThe observed anticancer screening data of the compoundsare given in Table 2 The 5-dose assay screening data ofthree compounds are given in Table 3 Compound 1 wasfound to be highly active on COLO 205 (colon cancer)with cell promotion of minus7349 followed by RXF 393
BioMed Research International 9
HO
O
OH
O
HO
ONN
OH
ONH
OR
HO
ONN
OH
OO
R
HO
ONHN
OH
O
X
AcOHReflux
Curcumin
ArCONHNH2
ArNHCONHNH2
NH2CXNH2
1ndash9
12ndash14
10-11
OO
Scheme 1 Protocol for the synthesis of curcumin analogues
OO
HOO
OHO
OOH
HOO
OHO
Diketone (tautomer I) Keto-enol (tautomer II)
OOH
HOO
OHO O
NH NH
N N
OHOO
HO
O
NH
R
OHOH
HOO
OHO
NHNH
ONH
OH
HOO
OHO
NNH
X
NH
R
R
R
Curcumin
OOH
HOO
OHO
R
CurcuminO
NH
OHOH
HOO
OHO
NHNH
O
R
R
OH
HOO
OHO
NNH
OR
OOH
HOO
OHO X
OHOH
HOO
OHO
NH
O
OH
HOO
OHO
N
X
HN N
X
OHOO
HO
Curcumin
N N
OHOO
HO
O
H3C H3C
H3C
H3C
H3C
CH3
CH3
H3C CH3 H3C H3CCH3CH3
CH3
CH3
CH3
CH3CH3CH3
H2N
H2N
H2N
H3CH3CH3CCH3H3CH2N
H2N
minusH2O
minusH2O
minusH2O
minusH2O minusH2O
minusH2O
NH2
1ndash9
12ndash1410-11
X = S O
Figure 1 Plausible mechanism of reaction for the synthesis of pyrazole analogues (1ndash14)
(renal cancer) with cell promotion of minus5032 and HT29(colon cancer) with cell promotion of minus3495 while themaximum cell promotion was observed on NCIADR-RES(ovarian cancer) which showed 2083 growth promotion(7917 growth inhibition) at one-dose assay Compound2 was found to be highly active on RXF 393 (renal cancer)with cell promotion of minus5360 followed by SK-MEL-5(melanoma) with cell promotion of minus2966 and MDA-MB-468 (breast cancer) with cell promotion of minus2640 whilethe maximum cell promotion was observed on TK-10 (renalcancer) which showed 2310 growth promotion (7690growth inhibition) at one-dose assay The compound 3was found to be highly active on RXF 393 (renal cancer)
with cell promotion of minus4384 followed by HT29 (coloncancer) with cell promotion of minus4184 and SK-MEL-2(melanoma) with cell promotion of minus2707 while themaximum cell promotion was observed on TK-10 (renalcancer) which showed 3812 growth promotion (6188growth inhibition) at one-dose assay Compound 5 wasfound to be highly active on COLO 205 (colon cancer)with cell promotion of minus5943 followed by RXF 393 (renalcancer) with cell promotion of minus5761 and SF-295 (CNScancer) with cell promotion of minus5532 while the maximumcell promotion was observed on TK-10 (renal cancer)which showed 2404 growth promotion (7596 growthinhibition) at one-dose assay Compound 6 was found to
10 BioMed Research International
100
50
0
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
LeukemiaG
row
th (
)
log10 of sample concentration (molar)
CCRF-CEMMOLT-4HL-60(TB)
RPMI-8226K-562SR
(a)
100
50
0
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
Leukemia
Gro
wth
()
log10 of sample concentration (molar)
CCRF-CEMMOLT-4HL-60(TB)
RPMI-8226K-562SR
(b)
100
50
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
Gro
wth
()
log10 of sample concentration (molar)
0
COLO 205HCT-15SW-620HCC-2998
HT29HCT-116KM12
Colon cancer
(c)
Figure 2 (a) Five-dose anticancer screening of compound 8 on leukemia cell line (b) Five-dose anticancer screening of compound 10 onleukemia cell line (c) Five-dose anticancer screening of compound 13 on colon cancer cell line
Figure 3 Green colour stick model indicates the reference ligand and atom macromodel stick indicates our ligands
be highly active on RXF 393 (renal) with cell promotionof minus4746 followed by MDA-MB-468 (breast cancer)with cell promotion of minus2914 and SK-OV-3 (ovariancancer) with cell promotion of minus2909 while the maximumcell promotion was observed on TK-10 (renal cancer)
which showed 2855 growth promotion (7145 growthinhibition) at one-dose assay Compound 8 was foundto be highly active on SK-MEL-5 (melanoma) with cellpromotion of minus8344 followed by RXF 393 (renal cancer)with cell promotion of minus6729 and SK-OV-3 (ovarian
BioMed Research International 11
(a) (b)
Figure 4 (a) Compound 13 is represented with macromodel stick modelThe hydrogen bonds are shown with yellow colour dotted lines andhydrophobic interactions with orange dotted lines (b) Compound 13 at binding site on surface
(a) (b)
Figure 5 (a) Compound 3 is represented with macromodel stick modelThe hydrophobic cavity residues are represented with magenta stickmodelThe hydrogen bonds are shownwith yellow colour dotted lines and hydrophobic interactions with orange dotted lines (b) Compound3 at binding site on surface
cancer) with cell promotion of minus6358 while the maximumcell promotion was observed on TK-10 (renal cancer)which showed 1584 growth promotion (8416 growthinhibition) at one-dose assay The compound 10 was foundto be highly active on SK-MEL-5 (melanoma) with cellpromotion of minus8652 followed by UACC-62 (melanoma)with cell promotion of minus8439 and COLO 205 (coloncancer) with cell promotion of minus8405 while the maximumcell promotion was observed on UACC-257 (melanoma)which showed 1603 growth promotion (8397 growthinhibition) at one-dose assay Compound 11 was found to behighly active on CAKI-1 (renal cancer) with cell promotionof minus3477 followed by MDA-MB-435 (melanoma) with cellpromotion of minus3053 and SF-295 (CNS cancer) with cellpromotion of minus2968 while the maximum cell promotionwas observed on UACC-257 (melanoma) which showed8713 growth promotion (1287 growth inhibition) at one-dose assay Compound 12 was found to be highly active onHT29 (colon cancer) with cell promotion of minus641 followedby MDA-MB-468 (breast cancer) with cell promotion ofminus634 and CCRF-CEM (leukemia) with cell promotion ofminus1672 while the maximum cell promotion was observedon TK-10 (renal cancer) which showed 10274 growthpromotion at one-dose assay Compound 13 was found to behighly active on HT29 (colon cancer) with cell promotion ofminus7624 followed by 786-0 and A498 (renal cancer) with cellpromotion of minus5893 and minus5692 respectively while themaximum cell promotion was observed on NCIADR-RES(ovarian cancer) which showed 3932 growth promotion(1287 growth inhibition) at one-dose assay Among the35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) Compound
8 was found to be the most active compound of theseries with mean growth percent of minus1919 followed bycompound 5 which showed mean growth percent of minus1225and compound 1 which showed mean growth percent ofminus466 Among the 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl (substituted phenyl)methanone analogues(10-11) Compound 10 showed maximum activity with meangrowth percent of minus2871 while compound 13 was found tobe the most active compound among dihydropyrimidineanalogues (12ndash14) which showed mean growth percent ofminus546 Overall the most active compound of the serieswas compound 10 and most of the 35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carbox-amide analogues possessed potent anticancer activity Basedon our finding we can conclude that electronegative groupat position 2 (ie 2-chloro) on N-substituted phenyl ringamong 35-bis(4-hydroxy-3-methylstyryl)-N-(substitutedphenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) showedmore anticancer activity than at position 4 (ie 4-chloro and4-fluoro) Electron releasing group such as 24-dimethyl and4-methyl on N-substituted phenyl ring increased anticanceractivity and the activity was found to be more if thenumber of electron releasing groups was increased Amongthe 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl(substituted phenyl)methanone analogues (10 and 11) theactivity was found to bemore when there was no substitutionin the phenyl ring Among six member dihydropyrimidineanalogues (12ndash14) compound with X substitution as ldquoNHrdquoshowed more activity than as ldquoOrdquo substitution
The results of 5-dose assay of the most active compoundsamong their respective series are given in Table 3 Compound8 presented GI
50ranging between 0004 and 204 120583M The
12 BioMed Research International
best results were recorded on the leukemia cell lines withvalues ranging from 0004 to 0364 120583M (Figure 2(a)) Anumber of 3 tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 0383120583M Only in 23 cell linescompound 8 registered LC
50valuewithgt100120583MCompound
10 presented GI50ranging between 00079 and 186 120583M The
best results were recorded on the leukemia cell line withvalue ranging from 00353 to 0347 120583M (Figure 2(b)) Fourof the tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 000815120583M Only in 27 cell linescompound 10 registered LC
50value with gt100 120583M Com-
pound 13 presented GI50ranging between 0524 and 339 120583M
The best results were recorded on the colon cancer cell linewith values ranging from 0543 to 218 120583M(Figure 2(c)) Onlyone tested cancer cell line presented TGI value with gt100 120583Mthe best result value being noted on the HT29 (colon cancer)with value 0948 In 28 cell lines compound 13 registeredLC50value with gt100120583M
Some of the synthesized compounds were evaluated forEGFR tyrosine kinase inhibitory activity by recombinanttyrosine kinase assay using an ELISA-based assay withpoly(Glu Tyr 4 1) as a substrate [37] The results showedcompounds 8 10 and 13 shows inhibition of EGFR tyrosinekinase with IC
50values of 355 292 and 557120583Mrespectively
33 Molecular Docking The binding site of the referenceinhibitor is well defined by hydrophobic cavity The EGFRtyrosine kinase binding site contains the important residuesCys797 andThr790 re docking of reference ligand shown thatabove residues are important for hydrophobic interactionswith the same [38] The hydrophobic cavity with the residuesLeu788 Met766 Lys745 Glu762 Thr854 and Met793 makesthe binding site attractive for the design of new inhibitorsBased on our knowledge we designed some class of newpyrazole (1ndash11) dihydropyrimidine analogues (12ndash14) forEGFR tyrosine kinase inhibition But the binding sites ofour new molecules were found to be a little different withcomparison to reference inhibitor which is reported in thecrystal structure of EGFR tyrosine kinase (PDB 2J5F) Theseligandsrsquo interactions were shown in three different motifsThe first one is hydrophobic cavity in which the substitutedmethoxy hydroxy phenyl ring of the ligand is showinggood interactions as reference inhibitor The next bindingmotif contains the target residue Cys797 the N-substitutedpyrazole derivatives and six membered (dihydropyrimidine)are binding at this site Most of these interactions werehydrophobic The last binding motif contains the residuesLys875 and Asp831 which lie in side chain of the protein andshowed the side chain hydrogen bonding with the designedmolecules The docking score and Emodel score of some ofthe compounds selected for anticancer activity by NCI aregiven in Table 4 and binding modes are shown in Figure 3
Compound 13 showed the best hydrophobic interactionwith various residues such as Cys797 Met 793 Leu 844 Leu792 Ala 743 and Val 726 (Figures 4(a) and 4(b)) Negativelycharged Asp 837 interacted with methoxy hydroxy phenyl
ring (4-hydroxy-3-methoxyphenyl) Arg 841 showed 120587-120587cationic interaction The Met 793 present in the hydrophobiccavity showed H-bond backbone interaction and H-bond(side chain) interaction The positively charged Lys 845and Arg 841 destabilized interactions The interactions ofcompound 13 with the EGFR kinase receptor shown inribbon diagram the hydrophobic cavity is indicated withyellow colour loop motif-1 is indicated with green colourbeta sheet and the motif-2 is indicated with green colourloop The other ligands were also having good interactionswith the hydrophobic cavity and bindingmotifs the followingresidues leu718 Pro794 Met793 Ala743 Leu844 Val726and Cys797 were present around the ligand Thr790 Gln791having polar characteristics andMet 793 were responsible forbackbone H-bond interaction as well as H-bond (side chain)interaction Asp855 and Asp837 showed charged interactionslike destabilizing contacts Positively charged Lys875 andArg841 were found to have 120587-120587 cationic interaction and werepresent in bindingmotifmaking different binding patterns ofreported ligandsThe docking study for compound 3 is givenin Figures 5(a) and 5(b)
4 Conclusion
The novel series of curcumin analogues (pyrazoles) weresynthesized in satisfactory yields The anticancer activityshowed promising results The studies confirmed compound10 as potent lead compound for drug discovery and furtheroptimization The curcumin analogues discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer disease The pyrazole discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer
Acknowledgments
The authors are thankful to the staff members of the NationalCancer Institute (NCI) USA for in vitro anticancer screeningof the newly synthesized compounds They are especiallygrateful to Professor Dizon B Thelma Project ManagerNational Cancer Institute (NCI) USA
References
[1] WHO Cancer World Health Organization February 2006httpenwikipediaorgwikicancer
[2] M N Noolvi H M Patel V Bhardwaj and A ChauhanldquoSynthesis and in vitro antitumor activity of substituted quina-zoline and quinoxaline derivatives search for anticancer agentrdquoEuropean Journal of Medicinal Chemistry vol 46 no 6 pp2327ndash2346 2011
[3] N Aydemir and R Bilaloglu ldquoGenotoxicity of two anticancerdrugs gemcitabine and topotecan in mouse bone marrow invivordquoMutation Research vol 537 no 1 pp 43ndash51 2003
[4] I Bouabdallah L A MrsquoBarek A Zyad A Ramdani I Zidaneand A Melhaoui ldquoNew pyrazolic compounds as cytotoxicagentsrdquo Natural Product Research vol 21 no 4 pp 298ndash3022007
BioMed Research International 13
[5] D Havrylyuk B Zimenkovsky O Vasylenko L Zaprutko AGzella and R Lesyk ldquoSynthesis of novel thiazolone-based com-pounds containing pyrazoline moiety and evaluation of theiranticancer activityrdquo European Journal of Medicinal Chemistryvol 44 no 4 pp 1396ndash1404 2009
[6] M Shaharyar M M Abdullah M A Bakht and J MajeedldquoPyrazoline bearing benzimidazoles search for anticanceragentrdquo European Journal of Medicinal Chemistry vol 45 no 1pp 114ndash119 2010
[7] P-C Lv H-Q Li J Sun Y Zhou and H-L Zhu ldquoSynthesisand biological evaluation of pyrazole derivatives containingthiourea skeleton as anticancer agentsrdquo Bioorganic and Medici-nal Chemistry vol 18 no 13 pp 4606ndash4614 2010
[8] M S Christodoulou S Liekens K M Kasiotis and S AHaroutounian ldquoNovel pyrazole derivatives synthesis and eval-uation of anti-angiogenic activityrdquo Bioorganic and MedicinalChemistry vol 18 no 12 pp 4338ndash4350 2010
[9] M J Ahsan G J Samy H Khalillah R C Krit and SSoni ldquoMolecular properties prediction and synthesis of novelpyrazoline carboxamide analogs as antitubercular agentsrdquoAnti-Infective Agents vol 10 no 2 pp 117ndash123 2012
[10] M J Ahsan J G Samy H Khalilullah M A Bakht andM Z Hassan ldquoSynthesis and antimycobacterial evaluation of3a4-dihydro-3H-indeno [12-c] pyrazole-2-carboxamide ana-loguesrdquo European Journal of Medicinal Chemistry vol 46 no11 pp 5694ndash5697 2011
[11] M J Ahsan J G Samy K R Dutt et al ldquoDesign synthesis andantimycobacterial evaluation of novel 3-substituted-N-aryl-67-dimethoxy-3a4-dihydro-3H-indeno[12-c] pyrazole-2-carboxamide analoguesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 15 pp 4451ndash4453 2011
[12] M J Ahsan J G Samy S Soni et al ldquoDiscovery of novelantitubercular 3a4-dihydro-3H-indeno[12-c]pyrazole-2-carndashboxamidecarbothioamide analoguesrdquo Bioorganic and Medici-nal Chemistry Letters vol 21 no 18 pp 5259ndash5261 2011
[13] M J Ahsan H Khalilullah J P Stables and J GovindasamyldquoSynthesis and anticonvulsant activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide carbothioamide ana-loguesrdquo Journal of Enzyme Inhibition amp Medicinal Chemistryvol 28 no 3 pp 644ndash650 2013
[14] M J Ahsan J Govindasamy H Khalilullah et al ldquoPOMAanalyses as new efficient bioinformaticsrsquoplatform to predictand optimize bioactivity of synthesized 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamidecarbothioamide anal-oguesrdquo Bioorganic amp Medicinal Chemistry vol 22 no 24 pp7029ndash7035 2012
[15] H Khalilullah S Khan M J Ahsan and B AhmedldquoSynthesis and antihepatotoxic activity of 5-(23-dihydro-14-benzodioxane-6-yl)-3-substituted-phenyl-45-dihydro-1H-pyrazole derivativesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 24 pp 7251ndash7254 2011
[16] M J Alam M J Ahsan O Alam and S A KhanldquoSynthesis of 4-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)-6-(substituted phenyl)pyrimidin-2-ol analogues as 8 anti-inflammatory and analgesic agentsrdquo Letters in Drug Design ampDiscovery vol 10 no 8 pp 776ndash782 2013
[17] D P Chauhan ldquoChemotherapeutic potential of curcumin forcolorectal cancerrdquo Current Pharmaceutical Design vol 8 no 19pp 1695ndash1706 2002
[18] Y-L Jia J Li Z-H Qin and Z-Q Liang ldquoAutophagic andapoptotic mechanisms of curcumin-induced death in K562
cellsrdquo Journal of Asian Natural Products Research vol 11 no 11pp 918ndash928 2009
[19] A Kunwar A Barik BMishra K Rathinasamy R Pandey andK I Priyadarsini ldquoQuantitative cellular uptake localization andcytotoxicity of curcumin in normal and tumor cellsrdquoBiochimicaet Biophysica Acta vol 1780 no 4 pp 673ndash679 2008
[20] J Lal S K Gupta DThavaselvam and D D Agarwal ldquoDesignsynthesis synergistic antimicrobial activity and cytotoxicityof 4-aryl substituted 34-dihydropyrimidinones of curcuminrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 8 pp2872ndash2876 2012
[21] R K Singh D Rai D Yadav A Bhargava J Balzarini and EDe Clercq ldquoSynthesis antibacterial and antiviral properties ofcurcumin bioconjugates bearing dipeptide fatty acids and folicacidrdquo European Journal of Medicinal Chemistry vol 45 no 3pp 1078ndash1086 2010
[22] K Saja M S Babu D Karunagaran and P R SudhakaranldquoAnti-inflammatory effect of curcumin involves downregula-tion of MMP-9 in blood mononuclear cellsrdquo InternationalImmunopharmacology vol 7 no 13 pp 1659ndash1667 2007
[23] S Mishra K Karmodiya N Surolia and A Surolia ldquoSynthesisand exploration of novel curcumin analogues as anti-malarialagentsrdquo Bioorganic and Medicinal Chemistry vol 16 no 6 pp2894ndash2902 2008
[24] G Liang L Shao Y Wang et al ldquoExploration and synthesis ofcurcumin analogues with improved structural stability both invitro and in vivo as cytotoxic agentsrdquo Bioorganic and MedicinalChemistry vol 17 no 6 pp 2623ndash2631 2009
[25] M J Ahsan ldquoSynthesis and anticancer activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide analoguesrdquo Lettersin Drug Design amp Discovery vol 9 no 9 pp 823ndash827 2012
[26] Salahuddin M Shaharyar A Majumdar and M J AhsanldquoSynthesis characterization and anticancer evaluation of 2-(naphthalen-1-ylmethylnaphthalen-2-yloxymethyl)-1-[5-(sub-stituted phenyl)-[134]oxadiazol-2-ylmethyl]-1H-benzimida-zolerdquo Arabian Journal of Chemistry 2013
[27] E Zwick J Bange andAUllrich ldquoReceptor tyrosine kinase sig-nalling as a target for cancer intervention strategiesrdquo Endocrine-Related Cancer vol 8 no 3 pp 161ndash173 2001
[28] H A Bhuva and S G Kini ldquoSynthesis anticancer activity anddocking of some substituted benzothiazoles as tyrosine kinaseinhibitorsrdquo Journal ofMolecular Graphics andModelling vol 29no 1 pp 32ndash37 2010
[29] J Mendelsohn and J Baselga ldquoThe EGF receptor family astargets for cancer therapyrdquo Oncogene vol 19 no 56 pp 6550ndash6565 2000
[30] M Amir M J Ahsan and I Ali ldquoSynthesis of N1-(3-chloro-4-flouropheny)-N4-substituted semicarbazones as novel anticon-vulsant agentsrdquo Indian Journal of Chemistry B vol 49 no 11 pp1509ndash1514 2010
[31] httpdtpncinihgov[32] A Monks D Scudiero P Skehan et al ldquoFeasibility of a high-
flux anticancer drug screen using a diverse panel of culturedhuman tumor cell linesrdquo Journal of theNational Cancer Institutevol 83 no 11 pp 757ndash766 1991
[33] M R Body and K D Paull ldquoSome practical considerations andapplications of the national cancer institute in vitro anticancerdrug discovery screenrdquoDrug Development Research vol 34 no2 pp 91ndash109 1995
[34] R H Shoemaker ldquoThe NCI60 human tumour cell line anti-cancer drug screenrdquo Nature Reviews Cancer vol 6 no 10 pp813ndash823 2006
14 BioMed Research International
[35] M C Alley D A Scudiero A Monks et al ldquoFeasibility ofdrug screening with panels of human tumor cell lines using amicroculture tetrazolium assayrdquo Cancer Research vol 48 no 3pp 589ndash601 1988
[36] M R Grever S A Schepartz and B A Chabner ldquoTheNationalCancer Institute cancer drug discovery and development pro-gramrdquo Seminars in Oncology vol 19 no 6 pp 622ndash638 1992
[37] P Dubreuil S Letard M Ciufolini et al ldquoMasitinib (AB1010)a potent and selective tyrosine kinase inhibitor targeting KITrdquoPLoS ONE vol 4 no 9 Article ID e7258 2009
[38] J A Blair D Rauh C Kung et al ldquoStructure-guided devel-opment of affinity probes for tyrosine kinases using chemicalgeneticsrdquo Nature Chemical Biology vol 3 no 4 pp 229ndash2382007
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
[20ndash24] Considering these recent discoveries curcumin canbe considered as an ideal lead compound for anticancer drugdevelopment Earlier we have reported the anticancer activityof pyrazoline and oxadiazole analogues [25 26] Receptortyrosine kinases (RTKs) have been shown to be key regulatorsof normal cellular processes and to additionally play a criticalrole in the development and progression of many types ofcancer by binding either with polypeptide growth factors orcytokines or hormones [27] Several transforming oncogeneslike src a gene obtained from Rous sarcoma virus abl a geneobtained from Abelson murine leukemia virus and so forthare known to possess tyrosine kinase activity Overexpressionof certain RTKs shows association with promotion andmaintenance of malignancies Thus inactivation of thespecific tyrosine kinase represents a potential approach fordesign of anticancer drugs Gefitinib and erlotinib usedin the treatment of certain types of cancer are epidermalgrowth factor receptor (EGFR) tyrosine kinase inhibitorsProtein tyrosine kinases occupy a central position in thecontrol of cellular proliferation and it is well recognizedthat the response of many cells to growth factors is initiatedby activation of tyrosine kinase [28] The involvement ofthe EGFR family of tyrosine kinases in cancer proliferationsuggests that an inhibitor which blocks the tyrosine kinaseactivity of the entire EGFR family could have significanttherapeutic potential [29] Hence enthused by all these factswe have synthesized a novel series of curcumin analoguesand evaluated their antitumor activity and selected EGFRfamily of tyrosine kinase as a biological target for carryingout the docking study of some of the active compounds
2 Materials and Methods
21 Chemistry All chemicals were supplied by E Merck(Germany) Konark Herbal (India) and S D Fine Chemicals(India) Melting points were determined by open tube capil-lary method and are uncorrected Purity of the compoundswas checked by elemental analysis and the progress ofreactions was monitored by TLC plates (silica gel G) usingmobile phase hexane ethylacetate (6 4) and the spots wereidentified by iodine vapours or UV light IR spectra wereobtained on a Shimadzu 8201 PC FT-IR spectrometer (KBrpellets) 1H NMR spectra were recorded on a Bruker AC300MHz spectrometer using TMS as internal standard inDMSO Mass spectra were recorded on a Bruker EsquireLCMS using ESI and elemental analyses were performed onPerkin-Elmer 2400 elemental analyzer
22 General Method for the Synthesis of 35-Bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carboxa-mide Analogues (1ndash9) 17-Bis(4-hydroxy-3-methoxyph-enyl)hepta-16-diene-35-dione (curcumin) (0005mol)and substituted phenyl semicarbazides (0005mol) wererefluxed in glacial acetic acid for 12 h The substitutedsemicarbazides were synthesized as per reported method[30] The excess of solvent was removed under reducedpressure and then the reaction mixture was poured intothe crushed ice The solid mass was filtered washed
dried and recrystallized with ethanol furnishing the title35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9)
(1H s CH=C) 673 (2H d J = 158Hz CH and CH) 678(2H d J = 158Hz CH and CH) 631ndash762 (10H m ArH)972 (2H s OH) 983 (1H s CONH) mz = 517 (M+) 519(M + 2)+
(1H s CH=C) 668 (2H d J = 162Hz CH and CH) 678(2H d J = 162Hz CH and CH) 692ndash760 (10H m ArH)962 (2H s OH) 997 (1H s CONH) 1297 (OH) 13C NMR(75MHzDMSO-d
CH) 676 (2H d J = 62Hz CH and CH) 689ndash792 (9H mArH) 997 (2H s OH) 1014 (1H s CONH)mz = 511 (M+)
23 General Method for the Synthesis of 35-Bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl (substituted phenyl)methanoneAnalogues (10-11) 17-Bis(4-hydroxy-3-methoxyphenyl) he-pta-16-diene-35-dione (curcumin) (0005mol) and substi-tuted phenyl hydrazides (0005mol) were refluxed in glacialacetic acid for 12 h The excess of solvent was removed underreduced pressure and then the reaction mixture was pouredinto the crushed ice The solid mass was filtered washeddried and recrystallized with ethanol furnishing the title 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl (substitutedphenyl)methanone analogues (10-11)
24 General Method for the Synthesis of DihydropyrimidineAnalogues (12ndash14) 17-Bis(4-hydroxy-3-methoxyphenyl)hepta-1 6-diene-35-dione (curcumin) (0005mol) and ureaguanidinethiourea (0005mol) were refluxed in glacialacetic acid for 12 h The excess of solvent was removedunder reduced pressure and then the reaction mixture waspoured into the crushed ice The solid mass was filteredwashed dried and recrystallized with ethanol furnishingthe title 35-bis(4-hydroxy-3-methylstyryl)-N-(substitutedphenyl)-1H-pyrazole-1-carboxamide analogues (12ndash14)
25 Anticancer Activity There were 10 compounds amongthe series selected and screened for their anticancer activityboth in one-dose and 5-dose assays by National CancerInstitute (NCI) on leukemia melanoma lung colon CNSovarian renal prostate and breast cancers cell lines nearly60 in number according to their screening protocol reportedelsewhere [31ndash34] All the curcumin analogues were synthe-sized and the structure of the compounds was submittedonline to the official site of NCI for anticancer screeningAmong 14 compounds only 10 compounds were selected foranticancer screening NCI has its own selection procedure ofthe compounds for anticancer screening based on the noveltyof heterocyclic ring system drug-like properties utilizing the
4 BioMed Research International
Table 1 Physical constants of the curcumin analogues (1ndash14)
concept of privileged scaffolds structure based on computer-aided drug design and so forth while the structures con-taining problematic linkage or functional groups (eg nitronitroso ndashNndashNndash ndashN=Nndash imine semicarbazone thioamidesand thioureas) for successful drug development are avoided[31]
The anticancer screening was carried out as per theNCI US protocol Using the seven absorbance measurements(time zero (119879
119894) control growth (119862) and test growth in
the presence of drug at the five concentration levels (119879119891))
the percentage growth was calculated at each of the drugconcentrations level as [(119879
119891minus 119879
119894)(119862 minus 119879
119894)] times 100 for
concentrations for which 119879119891ge 119879
119894and [(119879
119891minus 119879
119894)119879
119894] times 100
for concentrations for which 119879119891lt 119879
119894
Three-dose response parameters (GI50 TGI and LC
50)
were calculated for each of the experimental agents Growthinhibition of 50 (GI
50) was calculated from 100 times [(119879
119891minus
119879
119894)(119862 minus 119879
119894)] = 50 which was the drug concentration
resulting in a 50 reduction in the net protein increase (asmeasured by sulforhodamine B SRB staining) in controlcells during the drug incubation The total growth inhibi-tion (TGI) was calculated from 119879
119891= 119879
119894 which was the
drug concentration resulting in total growth inhibition andsignified the cytostatic effect The LC
50was calculated from
100 times [(119879
119891minus 119879
119894)119879
119894] = minus50 indicating a net loss of cells
following treatment which indicated the concentration ofdrug resulting in a 50 reduction in the measured proteinat the end of the drug treatment as compared to that at thebeginning Values were calculated for each of these threeparameters at the level of activity however if the effect didnot reach to the level of activity the value of parameter was
expressed as less than the minimum concentration testedor if the effect exceeded the level of activity the valueof parameter was expressed as greater than the maximumconcentration tested [31 35 36] LogGI
50 log TGI and
log LC50
are the logarithm molar concentrations producing50 growth inhibition (GI
50) a total growth inhibition
(TGI) and a 50 cellular death (LC50) respectively
26 Molecular Docking Studies
261 Protein Structure X-ray crystal structure EGFR kinase(PDB 2J5F) with resolution 300 A 119877-value 0194 (obs)was obtained from the protein data bank (Research Collab-oratory for Structural Bioinformatics (RCSB) (httpwwwrcsborgpdb))
262 Protein Preparation The protein (PDB 2J5F) wasprepared using the Protein PreparationWizard Preprocessedbond orders were assigned hydrogens were added metalswere treated and water molecules were deleted Heterostatefor cocrystallized ligand was generated using Epik proto-nation state and optimization of H-bonding of the proteinside chains were assigned using ProtAssign Energy wasminimized (Impref minimization) using RMSD 030 A andconverged by OPLS2005 force field utilities of Schrodingerrsquossuite 93
263 Receptor Grid Generation Receptor Grid has beengenerated with GLIDE module of Schrodinger with default
BioMed Research International 5
Table 2 Sixty human tumor cell lines anticancer screening data of curcumin analogues (1ndash14)
Panelcell line Growth percent in one-dose assay1 2 3 5 6 8 10 11 12 13
parameters and without any constraints Site has been speci-fied as centroid of the work space ligand (20 A) with van derWaals radius scaling factor 10 and partial charge cutoff 025
264 Molecular Docking Protocol The ligand docking wasperformed in GLIDE50 Ligands which show less than35 rotatable bonds and which are having less than 200atoms were selected The scaling factor will be 080 and thepotential charge cutoff is 015 All the conformers from theconfgen-ligprep output were docked in the EGFR TyrosineKinase active site All default parameters were used for extraprecision docking Glide extra precision mode was employedfor the current docking study Best poses were chosen forenergy minimization during docking a distance dependentdielectric constant of 20 and maximum number of min-imization step of 100 was used The docking simulations(Ligand receptor interactions) are scored using the Xtraprecision (XP) mode which is implemented in GLIDE50
27 Materials and Methods All computational analysis wascarried out on a Red Hat 50 Linux platform running on aDell Precision work station with Intel core 2 quad processorand 8GB of RAM
3 Results and Discussion
31 Chemistry The curcumin analogues (1ndash14) described inthe study are shown in Table 1 and the reaction sequencefor the synthesis is summarized in Scheme 1 In the first part
of the synthesis 17-bis(4-hydroxy-3-methoxyphenyl)hepta-16-diene-35-dione (curcumin) and substituted phenyl semi-carbazide were refluxed in glacial acetic acid to obtainthe 35-bis(4-hydroxy-3-methoxystyryl)-N-(substituted phe-nyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) Substi-tuted phenyl semicarbazides (ArNHCONHNH
2) were syn-
thesized as per reported method [30] In the second partof the synthesis 17-bis(4-hydroxy-3-methoxyphenyl)hepta-16-diene-35-dione (curcumin) and substituted phenylhy-drazide (ArCONHNH
2) were refluxed in glacial acetic acid to
obtain the 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl(substituted phenyl)methanone (10-11) In the third partof synthesis 17-bis(4-hydroxy-3-methoxyphenyl)hepta-16-diene-35-dione (curcumin) and ureaguanidinethioureawere refluxed in glacial acetic acid to obtain the pyrimidineanalogues (12ndash14) The plausible mechanism of reactions isgiven in Figure 1 The yields of the title compounds wereranging from 66 to 88 after recrystallization with absoluteethanol The completion of reaction was monitored by TLCusing mobile phase hexane ethylacetate (6 4) and purityof the compounds was checked by elemental analyses Boththe analytical and spectral data (IR NMR and MS) of thesynthesized compounds were in full accordance with theproposed structures In general IR spectra of the compoundsafforded C=N stretching at 1514ndash1583 cmminus1 and CndashN stretchat 1320ndash1335 cmminus1 and carbamoyl group NndashH stretching at3122ndash3210 cmminus1 andC=O stretching at 1680ndash1689 cmminus1 bandsfor carboxamide analogues (1ndash9) and at 1755ndash1757 cmminus1bands formethanone analogues (10-11)The 1HNMR spectra
BioMed Research International 7
Table 3 NCI in vitro testing results of compounds of the compounds 8 10 and 13 at five-dose level in 120583M
S no Panelcell line 8 (NSC 757926) 10 (NSC 757929) 13 (NSC 763375)GI50 TGI LC50 GI50 TGI LC50 GI50 TGI LC50
(1)
LeukemiaCCRF-CEM 0232 gt100 gt100 0127 gt100 gt100 0543 NT gt100HL-60(TB) 0237 0806 gt100 00884 NT gt100 218 617 gt100MOLT-4 0364 NT gt100 0347 gt100 gt100 101 519 gt100RPMI-8226 0336 NT gt100 0275 NT gt100 129 418 gt100SR 0004 gt100 gt100 00353 gt100 gt100 072 358 gt100
Colon cancerCOLO 205 205 362 638 186 334 597 18 324 585HCC-2998 064 58 gt100 0591 271 gt100 175 319 579HCT-116 0323 NT NT 0269 114 NT 11 239 518HCT-15 0335 166 NT 0167 152 NT 282 103 gt100HT29 0696 28 994 0463 319 gt100 314 0948 421KM12 0298 125 NT 00092 119 NT 163 332 679SW-620 0441 NT gt100 00079 NT gt100 165 368 NT
(4)
CNS cancerSF-268 0571 NT NT 0256 NT NT 211 156 gt100SF-295 0546 NT gt100 0538 NT NT 193 454 gt100SF-539 0354 15 NT 00604 153 NT 205 573 gt100SNB-19 0634 201 NT 0572 212 NT 181 795 gt100SNB-75 0416 182 NT 00099 187 NT 126 334 884U251 0471 159 NT 0428 170 NT 179 569 663
(5)
MelanomaLOX IMVI 0284 149 NT 00626 156 NT 127 259 NTMALME-3M 0563 198 NT 0525 217 NT 271 676 gt100M14 0372 141 NT 0155 148 NT 165 344 NTMDA-MB-435 00448 0383 NT 00175 000815 gt100 171 NT gt100SK-MEL-2 0366 182 NT 0266 203 gt100 198 41 NTSK-MEL-28 0509 247 861 0176 272 905 185 453 26SK-MEL-5 0317 129 367 0171 130 369 149 289 559UACC-257 154 547 319 114 429 533 153 35 8UACC-62 0413 165 489 0371 164 NT 149 323 NT
8 BioMed Research International
Table 3 Continued
S no Panelcell line 8 (NSC 757926) 10 (NSC 757929) 13 (NSC 763375)GI50 TGI LC50 GI50 TGI LC50 GI50 TGI LC50
showed singlet at 120575 119ndash128 ppm corresponding to CH3
a singlet at 120575 379ndash385 ppm corresponding to OCH3 a
singlet at 120575 652ndash662 ppm corresponding to CH=C proton(pyrazoledihydropyrimidine) a doublet at 120575 661ndash675 ppmcorresponding toCH=CHproton a doublet at 665ndash681 ppmcorresponding to CH=CH proton a multiplet at 120575 681ndash793 ppm corresponding to aromatic protons broad singlet at120575 993ndash1016 ppm corresponding to CONH
2 The compounds
(1ndash14) in 1HNMRspectra exhibited two doublets with 119869 valuebetween 146 and 166Hz confirming the trans coupling Themass spectra of the compounds revealed in each case a peakcorresponding to their molecular ion peaks The elementalanalysis results were within plusmn04 of the theoretical values
32 Anticancer Activity Ten compounds were evaluated fortheir anticancer activity in both one-dose and 5-dose assaysThe observed anticancer screening data of the compoundsare given in Table 2 The 5-dose assay screening data ofthree compounds are given in Table 3 Compound 1 wasfound to be highly active on COLO 205 (colon cancer)with cell promotion of minus7349 followed by RXF 393
BioMed Research International 9
HO
O
OH
O
HO
ONN
OH
ONH
OR
HO
ONN
OH
OO
R
HO
ONHN
OH
O
X
AcOHReflux
Curcumin
ArCONHNH2
ArNHCONHNH2
NH2CXNH2
1ndash9
12ndash14
10-11
OO
Scheme 1 Protocol for the synthesis of curcumin analogues
OO
HOO
OHO
OOH
HOO
OHO
Diketone (tautomer I) Keto-enol (tautomer II)
OOH
HOO
OHO O
NH NH
N N
OHOO
HO
O
NH
R
OHOH
HOO
OHO
NHNH
ONH
OH
HOO
OHO
NNH
X
NH
R
R
R
Curcumin
OOH
HOO
OHO
R
CurcuminO
NH
OHOH
HOO
OHO
NHNH
O
R
R
OH
HOO
OHO
NNH
OR
OOH
HOO
OHO X
OHOH
HOO
OHO
NH
O
OH
HOO
OHO
N
X
HN N
X
OHOO
HO
Curcumin
N N
OHOO
HO
O
H3C H3C
H3C
H3C
H3C
CH3
CH3
H3C CH3 H3C H3CCH3CH3
CH3
CH3
CH3
CH3CH3CH3
H2N
H2N
H2N
H3CH3CH3CCH3H3CH2N
H2N
minusH2O
minusH2O
minusH2O
minusH2O minusH2O
minusH2O
NH2
1ndash9
12ndash1410-11
X = S O
Figure 1 Plausible mechanism of reaction for the synthesis of pyrazole analogues (1ndash14)
(renal cancer) with cell promotion of minus5032 and HT29(colon cancer) with cell promotion of minus3495 while themaximum cell promotion was observed on NCIADR-RES(ovarian cancer) which showed 2083 growth promotion(7917 growth inhibition) at one-dose assay Compound2 was found to be highly active on RXF 393 (renal cancer)with cell promotion of minus5360 followed by SK-MEL-5(melanoma) with cell promotion of minus2966 and MDA-MB-468 (breast cancer) with cell promotion of minus2640 whilethe maximum cell promotion was observed on TK-10 (renalcancer) which showed 2310 growth promotion (7690growth inhibition) at one-dose assay The compound 3was found to be highly active on RXF 393 (renal cancer)
with cell promotion of minus4384 followed by HT29 (coloncancer) with cell promotion of minus4184 and SK-MEL-2(melanoma) with cell promotion of minus2707 while themaximum cell promotion was observed on TK-10 (renalcancer) which showed 3812 growth promotion (6188growth inhibition) at one-dose assay Compound 5 wasfound to be highly active on COLO 205 (colon cancer)with cell promotion of minus5943 followed by RXF 393 (renalcancer) with cell promotion of minus5761 and SF-295 (CNScancer) with cell promotion of minus5532 while the maximumcell promotion was observed on TK-10 (renal cancer)which showed 2404 growth promotion (7596 growthinhibition) at one-dose assay Compound 6 was found to
10 BioMed Research International
100
50
0
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
LeukemiaG
row
th (
)
log10 of sample concentration (molar)
CCRF-CEMMOLT-4HL-60(TB)
RPMI-8226K-562SR
(a)
100
50
0
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
Leukemia
Gro
wth
()
log10 of sample concentration (molar)
CCRF-CEMMOLT-4HL-60(TB)
RPMI-8226K-562SR
(b)
100
50
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
Gro
wth
()
log10 of sample concentration (molar)
0
COLO 205HCT-15SW-620HCC-2998
HT29HCT-116KM12
Colon cancer
(c)
Figure 2 (a) Five-dose anticancer screening of compound 8 on leukemia cell line (b) Five-dose anticancer screening of compound 10 onleukemia cell line (c) Five-dose anticancer screening of compound 13 on colon cancer cell line
Figure 3 Green colour stick model indicates the reference ligand and atom macromodel stick indicates our ligands
be highly active on RXF 393 (renal) with cell promotionof minus4746 followed by MDA-MB-468 (breast cancer)with cell promotion of minus2914 and SK-OV-3 (ovariancancer) with cell promotion of minus2909 while the maximumcell promotion was observed on TK-10 (renal cancer)
which showed 2855 growth promotion (7145 growthinhibition) at one-dose assay Compound 8 was foundto be highly active on SK-MEL-5 (melanoma) with cellpromotion of minus8344 followed by RXF 393 (renal cancer)with cell promotion of minus6729 and SK-OV-3 (ovarian
BioMed Research International 11
(a) (b)
Figure 4 (a) Compound 13 is represented with macromodel stick modelThe hydrogen bonds are shown with yellow colour dotted lines andhydrophobic interactions with orange dotted lines (b) Compound 13 at binding site on surface
(a) (b)
Figure 5 (a) Compound 3 is represented with macromodel stick modelThe hydrophobic cavity residues are represented with magenta stickmodelThe hydrogen bonds are shownwith yellow colour dotted lines and hydrophobic interactions with orange dotted lines (b) Compound3 at binding site on surface
cancer) with cell promotion of minus6358 while the maximumcell promotion was observed on TK-10 (renal cancer)which showed 1584 growth promotion (8416 growthinhibition) at one-dose assay The compound 10 was foundto be highly active on SK-MEL-5 (melanoma) with cellpromotion of minus8652 followed by UACC-62 (melanoma)with cell promotion of minus8439 and COLO 205 (coloncancer) with cell promotion of minus8405 while the maximumcell promotion was observed on UACC-257 (melanoma)which showed 1603 growth promotion (8397 growthinhibition) at one-dose assay Compound 11 was found to behighly active on CAKI-1 (renal cancer) with cell promotionof minus3477 followed by MDA-MB-435 (melanoma) with cellpromotion of minus3053 and SF-295 (CNS cancer) with cellpromotion of minus2968 while the maximum cell promotionwas observed on UACC-257 (melanoma) which showed8713 growth promotion (1287 growth inhibition) at one-dose assay Compound 12 was found to be highly active onHT29 (colon cancer) with cell promotion of minus641 followedby MDA-MB-468 (breast cancer) with cell promotion ofminus634 and CCRF-CEM (leukemia) with cell promotion ofminus1672 while the maximum cell promotion was observedon TK-10 (renal cancer) which showed 10274 growthpromotion at one-dose assay Compound 13 was found to behighly active on HT29 (colon cancer) with cell promotion ofminus7624 followed by 786-0 and A498 (renal cancer) with cellpromotion of minus5893 and minus5692 respectively while themaximum cell promotion was observed on NCIADR-RES(ovarian cancer) which showed 3932 growth promotion(1287 growth inhibition) at one-dose assay Among the35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) Compound
8 was found to be the most active compound of theseries with mean growth percent of minus1919 followed bycompound 5 which showed mean growth percent of minus1225and compound 1 which showed mean growth percent ofminus466 Among the 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl (substituted phenyl)methanone analogues(10-11) Compound 10 showed maximum activity with meangrowth percent of minus2871 while compound 13 was found tobe the most active compound among dihydropyrimidineanalogues (12ndash14) which showed mean growth percent ofminus546 Overall the most active compound of the serieswas compound 10 and most of the 35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carbox-amide analogues possessed potent anticancer activity Basedon our finding we can conclude that electronegative groupat position 2 (ie 2-chloro) on N-substituted phenyl ringamong 35-bis(4-hydroxy-3-methylstyryl)-N-(substitutedphenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) showedmore anticancer activity than at position 4 (ie 4-chloro and4-fluoro) Electron releasing group such as 24-dimethyl and4-methyl on N-substituted phenyl ring increased anticanceractivity and the activity was found to be more if thenumber of electron releasing groups was increased Amongthe 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl(substituted phenyl)methanone analogues (10 and 11) theactivity was found to bemore when there was no substitutionin the phenyl ring Among six member dihydropyrimidineanalogues (12ndash14) compound with X substitution as ldquoNHrdquoshowed more activity than as ldquoOrdquo substitution
The results of 5-dose assay of the most active compoundsamong their respective series are given in Table 3 Compound8 presented GI
50ranging between 0004 and 204 120583M The
12 BioMed Research International
best results were recorded on the leukemia cell lines withvalues ranging from 0004 to 0364 120583M (Figure 2(a)) Anumber of 3 tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 0383120583M Only in 23 cell linescompound 8 registered LC
50valuewithgt100120583MCompound
10 presented GI50ranging between 00079 and 186 120583M The
best results were recorded on the leukemia cell line withvalue ranging from 00353 to 0347 120583M (Figure 2(b)) Fourof the tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 000815120583M Only in 27 cell linescompound 10 registered LC
50value with gt100 120583M Com-
pound 13 presented GI50ranging between 0524 and 339 120583M
The best results were recorded on the colon cancer cell linewith values ranging from 0543 to 218 120583M(Figure 2(c)) Onlyone tested cancer cell line presented TGI value with gt100 120583Mthe best result value being noted on the HT29 (colon cancer)with value 0948 In 28 cell lines compound 13 registeredLC50value with gt100120583M
Some of the synthesized compounds were evaluated forEGFR tyrosine kinase inhibitory activity by recombinanttyrosine kinase assay using an ELISA-based assay withpoly(Glu Tyr 4 1) as a substrate [37] The results showedcompounds 8 10 and 13 shows inhibition of EGFR tyrosinekinase with IC
50values of 355 292 and 557120583Mrespectively
33 Molecular Docking The binding site of the referenceinhibitor is well defined by hydrophobic cavity The EGFRtyrosine kinase binding site contains the important residuesCys797 andThr790 re docking of reference ligand shown thatabove residues are important for hydrophobic interactionswith the same [38] The hydrophobic cavity with the residuesLeu788 Met766 Lys745 Glu762 Thr854 and Met793 makesthe binding site attractive for the design of new inhibitorsBased on our knowledge we designed some class of newpyrazole (1ndash11) dihydropyrimidine analogues (12ndash14) forEGFR tyrosine kinase inhibition But the binding sites ofour new molecules were found to be a little different withcomparison to reference inhibitor which is reported in thecrystal structure of EGFR tyrosine kinase (PDB 2J5F) Theseligandsrsquo interactions were shown in three different motifsThe first one is hydrophobic cavity in which the substitutedmethoxy hydroxy phenyl ring of the ligand is showinggood interactions as reference inhibitor The next bindingmotif contains the target residue Cys797 the N-substitutedpyrazole derivatives and six membered (dihydropyrimidine)are binding at this site Most of these interactions werehydrophobic The last binding motif contains the residuesLys875 and Asp831 which lie in side chain of the protein andshowed the side chain hydrogen bonding with the designedmolecules The docking score and Emodel score of some ofthe compounds selected for anticancer activity by NCI aregiven in Table 4 and binding modes are shown in Figure 3
Compound 13 showed the best hydrophobic interactionwith various residues such as Cys797 Met 793 Leu 844 Leu792 Ala 743 and Val 726 (Figures 4(a) and 4(b)) Negativelycharged Asp 837 interacted with methoxy hydroxy phenyl
ring (4-hydroxy-3-methoxyphenyl) Arg 841 showed 120587-120587cationic interaction The Met 793 present in the hydrophobiccavity showed H-bond backbone interaction and H-bond(side chain) interaction The positively charged Lys 845and Arg 841 destabilized interactions The interactions ofcompound 13 with the EGFR kinase receptor shown inribbon diagram the hydrophobic cavity is indicated withyellow colour loop motif-1 is indicated with green colourbeta sheet and the motif-2 is indicated with green colourloop The other ligands were also having good interactionswith the hydrophobic cavity and bindingmotifs the followingresidues leu718 Pro794 Met793 Ala743 Leu844 Val726and Cys797 were present around the ligand Thr790 Gln791having polar characteristics andMet 793 were responsible forbackbone H-bond interaction as well as H-bond (side chain)interaction Asp855 and Asp837 showed charged interactionslike destabilizing contacts Positively charged Lys875 andArg841 were found to have 120587-120587 cationic interaction and werepresent in bindingmotifmaking different binding patterns ofreported ligandsThe docking study for compound 3 is givenin Figures 5(a) and 5(b)
4 Conclusion
The novel series of curcumin analogues (pyrazoles) weresynthesized in satisfactory yields The anticancer activityshowed promising results The studies confirmed compound10 as potent lead compound for drug discovery and furtheroptimization The curcumin analogues discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer disease The pyrazole discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer
Acknowledgments
The authors are thankful to the staff members of the NationalCancer Institute (NCI) USA for in vitro anticancer screeningof the newly synthesized compounds They are especiallygrateful to Professor Dizon B Thelma Project ManagerNational Cancer Institute (NCI) USA
References
[1] WHO Cancer World Health Organization February 2006httpenwikipediaorgwikicancer
[2] M N Noolvi H M Patel V Bhardwaj and A ChauhanldquoSynthesis and in vitro antitumor activity of substituted quina-zoline and quinoxaline derivatives search for anticancer agentrdquoEuropean Journal of Medicinal Chemistry vol 46 no 6 pp2327ndash2346 2011
[3] N Aydemir and R Bilaloglu ldquoGenotoxicity of two anticancerdrugs gemcitabine and topotecan in mouse bone marrow invivordquoMutation Research vol 537 no 1 pp 43ndash51 2003
[4] I Bouabdallah L A MrsquoBarek A Zyad A Ramdani I Zidaneand A Melhaoui ldquoNew pyrazolic compounds as cytotoxicagentsrdquo Natural Product Research vol 21 no 4 pp 298ndash3022007
BioMed Research International 13
[5] D Havrylyuk B Zimenkovsky O Vasylenko L Zaprutko AGzella and R Lesyk ldquoSynthesis of novel thiazolone-based com-pounds containing pyrazoline moiety and evaluation of theiranticancer activityrdquo European Journal of Medicinal Chemistryvol 44 no 4 pp 1396ndash1404 2009
[6] M Shaharyar M M Abdullah M A Bakht and J MajeedldquoPyrazoline bearing benzimidazoles search for anticanceragentrdquo European Journal of Medicinal Chemistry vol 45 no 1pp 114ndash119 2010
[7] P-C Lv H-Q Li J Sun Y Zhou and H-L Zhu ldquoSynthesisand biological evaluation of pyrazole derivatives containingthiourea skeleton as anticancer agentsrdquo Bioorganic and Medici-nal Chemistry vol 18 no 13 pp 4606ndash4614 2010
[8] M S Christodoulou S Liekens K M Kasiotis and S AHaroutounian ldquoNovel pyrazole derivatives synthesis and eval-uation of anti-angiogenic activityrdquo Bioorganic and MedicinalChemistry vol 18 no 12 pp 4338ndash4350 2010
[9] M J Ahsan G J Samy H Khalillah R C Krit and SSoni ldquoMolecular properties prediction and synthesis of novelpyrazoline carboxamide analogs as antitubercular agentsrdquoAnti-Infective Agents vol 10 no 2 pp 117ndash123 2012
[10] M J Ahsan J G Samy H Khalilullah M A Bakht andM Z Hassan ldquoSynthesis and antimycobacterial evaluation of3a4-dihydro-3H-indeno [12-c] pyrazole-2-carboxamide ana-loguesrdquo European Journal of Medicinal Chemistry vol 46 no11 pp 5694ndash5697 2011
[11] M J Ahsan J G Samy K R Dutt et al ldquoDesign synthesis andantimycobacterial evaluation of novel 3-substituted-N-aryl-67-dimethoxy-3a4-dihydro-3H-indeno[12-c] pyrazole-2-carboxamide analoguesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 15 pp 4451ndash4453 2011
[12] M J Ahsan J G Samy S Soni et al ldquoDiscovery of novelantitubercular 3a4-dihydro-3H-indeno[12-c]pyrazole-2-carndashboxamidecarbothioamide analoguesrdquo Bioorganic and Medici-nal Chemistry Letters vol 21 no 18 pp 5259ndash5261 2011
[13] M J Ahsan H Khalilullah J P Stables and J GovindasamyldquoSynthesis and anticonvulsant activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide carbothioamide ana-loguesrdquo Journal of Enzyme Inhibition amp Medicinal Chemistryvol 28 no 3 pp 644ndash650 2013
[14] M J Ahsan J Govindasamy H Khalilullah et al ldquoPOMAanalyses as new efficient bioinformaticsrsquoplatform to predictand optimize bioactivity of synthesized 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamidecarbothioamide anal-oguesrdquo Bioorganic amp Medicinal Chemistry vol 22 no 24 pp7029ndash7035 2012
[15] H Khalilullah S Khan M J Ahsan and B AhmedldquoSynthesis and antihepatotoxic activity of 5-(23-dihydro-14-benzodioxane-6-yl)-3-substituted-phenyl-45-dihydro-1H-pyrazole derivativesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 24 pp 7251ndash7254 2011
[16] M J Alam M J Ahsan O Alam and S A KhanldquoSynthesis of 4-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)-6-(substituted phenyl)pyrimidin-2-ol analogues as 8 anti-inflammatory and analgesic agentsrdquo Letters in Drug Design ampDiscovery vol 10 no 8 pp 776ndash782 2013
[17] D P Chauhan ldquoChemotherapeutic potential of curcumin forcolorectal cancerrdquo Current Pharmaceutical Design vol 8 no 19pp 1695ndash1706 2002
[18] Y-L Jia J Li Z-H Qin and Z-Q Liang ldquoAutophagic andapoptotic mechanisms of curcumin-induced death in K562
cellsrdquo Journal of Asian Natural Products Research vol 11 no 11pp 918ndash928 2009
[19] A Kunwar A Barik BMishra K Rathinasamy R Pandey andK I Priyadarsini ldquoQuantitative cellular uptake localization andcytotoxicity of curcumin in normal and tumor cellsrdquoBiochimicaet Biophysica Acta vol 1780 no 4 pp 673ndash679 2008
[20] J Lal S K Gupta DThavaselvam and D D Agarwal ldquoDesignsynthesis synergistic antimicrobial activity and cytotoxicityof 4-aryl substituted 34-dihydropyrimidinones of curcuminrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 8 pp2872ndash2876 2012
[21] R K Singh D Rai D Yadav A Bhargava J Balzarini and EDe Clercq ldquoSynthesis antibacterial and antiviral properties ofcurcumin bioconjugates bearing dipeptide fatty acids and folicacidrdquo European Journal of Medicinal Chemistry vol 45 no 3pp 1078ndash1086 2010
[22] K Saja M S Babu D Karunagaran and P R SudhakaranldquoAnti-inflammatory effect of curcumin involves downregula-tion of MMP-9 in blood mononuclear cellsrdquo InternationalImmunopharmacology vol 7 no 13 pp 1659ndash1667 2007
[23] S Mishra K Karmodiya N Surolia and A Surolia ldquoSynthesisand exploration of novel curcumin analogues as anti-malarialagentsrdquo Bioorganic and Medicinal Chemistry vol 16 no 6 pp2894ndash2902 2008
[24] G Liang L Shao Y Wang et al ldquoExploration and synthesis ofcurcumin analogues with improved structural stability both invitro and in vivo as cytotoxic agentsrdquo Bioorganic and MedicinalChemistry vol 17 no 6 pp 2623ndash2631 2009
[25] M J Ahsan ldquoSynthesis and anticancer activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide analoguesrdquo Lettersin Drug Design amp Discovery vol 9 no 9 pp 823ndash827 2012
[26] Salahuddin M Shaharyar A Majumdar and M J AhsanldquoSynthesis characterization and anticancer evaluation of 2-(naphthalen-1-ylmethylnaphthalen-2-yloxymethyl)-1-[5-(sub-stituted phenyl)-[134]oxadiazol-2-ylmethyl]-1H-benzimida-zolerdquo Arabian Journal of Chemistry 2013
[27] E Zwick J Bange andAUllrich ldquoReceptor tyrosine kinase sig-nalling as a target for cancer intervention strategiesrdquo Endocrine-Related Cancer vol 8 no 3 pp 161ndash173 2001
[28] H A Bhuva and S G Kini ldquoSynthesis anticancer activity anddocking of some substituted benzothiazoles as tyrosine kinaseinhibitorsrdquo Journal ofMolecular Graphics andModelling vol 29no 1 pp 32ndash37 2010
[29] J Mendelsohn and J Baselga ldquoThe EGF receptor family astargets for cancer therapyrdquo Oncogene vol 19 no 56 pp 6550ndash6565 2000
[30] M Amir M J Ahsan and I Ali ldquoSynthesis of N1-(3-chloro-4-flouropheny)-N4-substituted semicarbazones as novel anticon-vulsant agentsrdquo Indian Journal of Chemistry B vol 49 no 11 pp1509ndash1514 2010
[31] httpdtpncinihgov[32] A Monks D Scudiero P Skehan et al ldquoFeasibility of a high-
flux anticancer drug screen using a diverse panel of culturedhuman tumor cell linesrdquo Journal of theNational Cancer Institutevol 83 no 11 pp 757ndash766 1991
[33] M R Body and K D Paull ldquoSome practical considerations andapplications of the national cancer institute in vitro anticancerdrug discovery screenrdquoDrug Development Research vol 34 no2 pp 91ndash109 1995
[34] R H Shoemaker ldquoThe NCI60 human tumour cell line anti-cancer drug screenrdquo Nature Reviews Cancer vol 6 no 10 pp813ndash823 2006
14 BioMed Research International
[35] M C Alley D A Scudiero A Monks et al ldquoFeasibility ofdrug screening with panels of human tumor cell lines using amicroculture tetrazolium assayrdquo Cancer Research vol 48 no 3pp 589ndash601 1988
[36] M R Grever S A Schepartz and B A Chabner ldquoTheNationalCancer Institute cancer drug discovery and development pro-gramrdquo Seminars in Oncology vol 19 no 6 pp 622ndash638 1992
[37] P Dubreuil S Letard M Ciufolini et al ldquoMasitinib (AB1010)a potent and selective tyrosine kinase inhibitor targeting KITrdquoPLoS ONE vol 4 no 9 Article ID e7258 2009
[38] J A Blair D Rauh C Kung et al ldquoStructure-guided devel-opment of affinity probes for tyrosine kinases using chemicalgeneticsrdquo Nature Chemical Biology vol 3 no 4 pp 229ndash2382007
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
CH) 676 (2H d J = 62Hz CH and CH) 689ndash792 (9H mArH) 997 (2H s OH) 1014 (1H s CONH)mz = 511 (M+)
23 General Method for the Synthesis of 35-Bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl (substituted phenyl)methanoneAnalogues (10-11) 17-Bis(4-hydroxy-3-methoxyphenyl) he-pta-16-diene-35-dione (curcumin) (0005mol) and substi-tuted phenyl hydrazides (0005mol) were refluxed in glacialacetic acid for 12 h The excess of solvent was removed underreduced pressure and then the reaction mixture was pouredinto the crushed ice The solid mass was filtered washeddried and recrystallized with ethanol furnishing the title 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl (substitutedphenyl)methanone analogues (10-11)
24 General Method for the Synthesis of DihydropyrimidineAnalogues (12ndash14) 17-Bis(4-hydroxy-3-methoxyphenyl)hepta-1 6-diene-35-dione (curcumin) (0005mol) and ureaguanidinethiourea (0005mol) were refluxed in glacialacetic acid for 12 h The excess of solvent was removedunder reduced pressure and then the reaction mixture waspoured into the crushed ice The solid mass was filteredwashed dried and recrystallized with ethanol furnishingthe title 35-bis(4-hydroxy-3-methylstyryl)-N-(substitutedphenyl)-1H-pyrazole-1-carboxamide analogues (12ndash14)
25 Anticancer Activity There were 10 compounds amongthe series selected and screened for their anticancer activityboth in one-dose and 5-dose assays by National CancerInstitute (NCI) on leukemia melanoma lung colon CNSovarian renal prostate and breast cancers cell lines nearly60 in number according to their screening protocol reportedelsewhere [31ndash34] All the curcumin analogues were synthe-sized and the structure of the compounds was submittedonline to the official site of NCI for anticancer screeningAmong 14 compounds only 10 compounds were selected foranticancer screening NCI has its own selection procedure ofthe compounds for anticancer screening based on the noveltyof heterocyclic ring system drug-like properties utilizing the
4 BioMed Research International
Table 1 Physical constants of the curcumin analogues (1ndash14)
concept of privileged scaffolds structure based on computer-aided drug design and so forth while the structures con-taining problematic linkage or functional groups (eg nitronitroso ndashNndashNndash ndashN=Nndash imine semicarbazone thioamidesand thioureas) for successful drug development are avoided[31]
The anticancer screening was carried out as per theNCI US protocol Using the seven absorbance measurements(time zero (119879
119894) control growth (119862) and test growth in
the presence of drug at the five concentration levels (119879119891))
the percentage growth was calculated at each of the drugconcentrations level as [(119879
119891minus 119879
119894)(119862 minus 119879
119894)] times 100 for
concentrations for which 119879119891ge 119879
119894and [(119879
119891minus 119879
119894)119879
119894] times 100
for concentrations for which 119879119891lt 119879
119894
Three-dose response parameters (GI50 TGI and LC
50)
were calculated for each of the experimental agents Growthinhibition of 50 (GI
50) was calculated from 100 times [(119879
119891minus
119879
119894)(119862 minus 119879
119894)] = 50 which was the drug concentration
resulting in a 50 reduction in the net protein increase (asmeasured by sulforhodamine B SRB staining) in controlcells during the drug incubation The total growth inhibi-tion (TGI) was calculated from 119879
119891= 119879
119894 which was the
drug concentration resulting in total growth inhibition andsignified the cytostatic effect The LC
50was calculated from
100 times [(119879
119891minus 119879
119894)119879
119894] = minus50 indicating a net loss of cells
following treatment which indicated the concentration ofdrug resulting in a 50 reduction in the measured proteinat the end of the drug treatment as compared to that at thebeginning Values were calculated for each of these threeparameters at the level of activity however if the effect didnot reach to the level of activity the value of parameter was
expressed as less than the minimum concentration testedor if the effect exceeded the level of activity the valueof parameter was expressed as greater than the maximumconcentration tested [31 35 36] LogGI
50 log TGI and
log LC50
are the logarithm molar concentrations producing50 growth inhibition (GI
50) a total growth inhibition
(TGI) and a 50 cellular death (LC50) respectively
26 Molecular Docking Studies
261 Protein Structure X-ray crystal structure EGFR kinase(PDB 2J5F) with resolution 300 A 119877-value 0194 (obs)was obtained from the protein data bank (Research Collab-oratory for Structural Bioinformatics (RCSB) (httpwwwrcsborgpdb))
262 Protein Preparation The protein (PDB 2J5F) wasprepared using the Protein PreparationWizard Preprocessedbond orders were assigned hydrogens were added metalswere treated and water molecules were deleted Heterostatefor cocrystallized ligand was generated using Epik proto-nation state and optimization of H-bonding of the proteinside chains were assigned using ProtAssign Energy wasminimized (Impref minimization) using RMSD 030 A andconverged by OPLS2005 force field utilities of Schrodingerrsquossuite 93
263 Receptor Grid Generation Receptor Grid has beengenerated with GLIDE module of Schrodinger with default
BioMed Research International 5
Table 2 Sixty human tumor cell lines anticancer screening data of curcumin analogues (1ndash14)
Panelcell line Growth percent in one-dose assay1 2 3 5 6 8 10 11 12 13
parameters and without any constraints Site has been speci-fied as centroid of the work space ligand (20 A) with van derWaals radius scaling factor 10 and partial charge cutoff 025
264 Molecular Docking Protocol The ligand docking wasperformed in GLIDE50 Ligands which show less than35 rotatable bonds and which are having less than 200atoms were selected The scaling factor will be 080 and thepotential charge cutoff is 015 All the conformers from theconfgen-ligprep output were docked in the EGFR TyrosineKinase active site All default parameters were used for extraprecision docking Glide extra precision mode was employedfor the current docking study Best poses were chosen forenergy minimization during docking a distance dependentdielectric constant of 20 and maximum number of min-imization step of 100 was used The docking simulations(Ligand receptor interactions) are scored using the Xtraprecision (XP) mode which is implemented in GLIDE50
27 Materials and Methods All computational analysis wascarried out on a Red Hat 50 Linux platform running on aDell Precision work station with Intel core 2 quad processorand 8GB of RAM
3 Results and Discussion
31 Chemistry The curcumin analogues (1ndash14) described inthe study are shown in Table 1 and the reaction sequencefor the synthesis is summarized in Scheme 1 In the first part
of the synthesis 17-bis(4-hydroxy-3-methoxyphenyl)hepta-16-diene-35-dione (curcumin) and substituted phenyl semi-carbazide were refluxed in glacial acetic acid to obtainthe 35-bis(4-hydroxy-3-methoxystyryl)-N-(substituted phe-nyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) Substi-tuted phenyl semicarbazides (ArNHCONHNH
2) were syn-
thesized as per reported method [30] In the second partof the synthesis 17-bis(4-hydroxy-3-methoxyphenyl)hepta-16-diene-35-dione (curcumin) and substituted phenylhy-drazide (ArCONHNH
2) were refluxed in glacial acetic acid to
obtain the 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl(substituted phenyl)methanone (10-11) In the third partof synthesis 17-bis(4-hydroxy-3-methoxyphenyl)hepta-16-diene-35-dione (curcumin) and ureaguanidinethioureawere refluxed in glacial acetic acid to obtain the pyrimidineanalogues (12ndash14) The plausible mechanism of reactions isgiven in Figure 1 The yields of the title compounds wereranging from 66 to 88 after recrystallization with absoluteethanol The completion of reaction was monitored by TLCusing mobile phase hexane ethylacetate (6 4) and purityof the compounds was checked by elemental analyses Boththe analytical and spectral data (IR NMR and MS) of thesynthesized compounds were in full accordance with theproposed structures In general IR spectra of the compoundsafforded C=N stretching at 1514ndash1583 cmminus1 and CndashN stretchat 1320ndash1335 cmminus1 and carbamoyl group NndashH stretching at3122ndash3210 cmminus1 andC=O stretching at 1680ndash1689 cmminus1 bandsfor carboxamide analogues (1ndash9) and at 1755ndash1757 cmminus1bands formethanone analogues (10-11)The 1HNMR spectra
BioMed Research International 7
Table 3 NCI in vitro testing results of compounds of the compounds 8 10 and 13 at five-dose level in 120583M
S no Panelcell line 8 (NSC 757926) 10 (NSC 757929) 13 (NSC 763375)GI50 TGI LC50 GI50 TGI LC50 GI50 TGI LC50
(1)
LeukemiaCCRF-CEM 0232 gt100 gt100 0127 gt100 gt100 0543 NT gt100HL-60(TB) 0237 0806 gt100 00884 NT gt100 218 617 gt100MOLT-4 0364 NT gt100 0347 gt100 gt100 101 519 gt100RPMI-8226 0336 NT gt100 0275 NT gt100 129 418 gt100SR 0004 gt100 gt100 00353 gt100 gt100 072 358 gt100
Colon cancerCOLO 205 205 362 638 186 334 597 18 324 585HCC-2998 064 58 gt100 0591 271 gt100 175 319 579HCT-116 0323 NT NT 0269 114 NT 11 239 518HCT-15 0335 166 NT 0167 152 NT 282 103 gt100HT29 0696 28 994 0463 319 gt100 314 0948 421KM12 0298 125 NT 00092 119 NT 163 332 679SW-620 0441 NT gt100 00079 NT gt100 165 368 NT
(4)
CNS cancerSF-268 0571 NT NT 0256 NT NT 211 156 gt100SF-295 0546 NT gt100 0538 NT NT 193 454 gt100SF-539 0354 15 NT 00604 153 NT 205 573 gt100SNB-19 0634 201 NT 0572 212 NT 181 795 gt100SNB-75 0416 182 NT 00099 187 NT 126 334 884U251 0471 159 NT 0428 170 NT 179 569 663
(5)
MelanomaLOX IMVI 0284 149 NT 00626 156 NT 127 259 NTMALME-3M 0563 198 NT 0525 217 NT 271 676 gt100M14 0372 141 NT 0155 148 NT 165 344 NTMDA-MB-435 00448 0383 NT 00175 000815 gt100 171 NT gt100SK-MEL-2 0366 182 NT 0266 203 gt100 198 41 NTSK-MEL-28 0509 247 861 0176 272 905 185 453 26SK-MEL-5 0317 129 367 0171 130 369 149 289 559UACC-257 154 547 319 114 429 533 153 35 8UACC-62 0413 165 489 0371 164 NT 149 323 NT
8 BioMed Research International
Table 3 Continued
S no Panelcell line 8 (NSC 757926) 10 (NSC 757929) 13 (NSC 763375)GI50 TGI LC50 GI50 TGI LC50 GI50 TGI LC50
showed singlet at 120575 119ndash128 ppm corresponding to CH3
a singlet at 120575 379ndash385 ppm corresponding to OCH3 a
singlet at 120575 652ndash662 ppm corresponding to CH=C proton(pyrazoledihydropyrimidine) a doublet at 120575 661ndash675 ppmcorresponding toCH=CHproton a doublet at 665ndash681 ppmcorresponding to CH=CH proton a multiplet at 120575 681ndash793 ppm corresponding to aromatic protons broad singlet at120575 993ndash1016 ppm corresponding to CONH
2 The compounds
(1ndash14) in 1HNMRspectra exhibited two doublets with 119869 valuebetween 146 and 166Hz confirming the trans coupling Themass spectra of the compounds revealed in each case a peakcorresponding to their molecular ion peaks The elementalanalysis results were within plusmn04 of the theoretical values
32 Anticancer Activity Ten compounds were evaluated fortheir anticancer activity in both one-dose and 5-dose assaysThe observed anticancer screening data of the compoundsare given in Table 2 The 5-dose assay screening data ofthree compounds are given in Table 3 Compound 1 wasfound to be highly active on COLO 205 (colon cancer)with cell promotion of minus7349 followed by RXF 393
BioMed Research International 9
HO
O
OH
O
HO
ONN
OH
ONH
OR
HO
ONN
OH
OO
R
HO
ONHN
OH
O
X
AcOHReflux
Curcumin
ArCONHNH2
ArNHCONHNH2
NH2CXNH2
1ndash9
12ndash14
10-11
OO
Scheme 1 Protocol for the synthesis of curcumin analogues
OO
HOO
OHO
OOH
HOO
OHO
Diketone (tautomer I) Keto-enol (tautomer II)
OOH
HOO
OHO O
NH NH
N N
OHOO
HO
O
NH
R
OHOH
HOO
OHO
NHNH
ONH
OH
HOO
OHO
NNH
X
NH
R
R
R
Curcumin
OOH
HOO
OHO
R
CurcuminO
NH
OHOH
HOO
OHO
NHNH
O
R
R
OH
HOO
OHO
NNH
OR
OOH
HOO
OHO X
OHOH
HOO
OHO
NH
O
OH
HOO
OHO
N
X
HN N
X
OHOO
HO
Curcumin
N N
OHOO
HO
O
H3C H3C
H3C
H3C
H3C
CH3
CH3
H3C CH3 H3C H3CCH3CH3
CH3
CH3
CH3
CH3CH3CH3
H2N
H2N
H2N
H3CH3CH3CCH3H3CH2N
H2N
minusH2O
minusH2O
minusH2O
minusH2O minusH2O
minusH2O
NH2
1ndash9
12ndash1410-11
X = S O
Figure 1 Plausible mechanism of reaction for the synthesis of pyrazole analogues (1ndash14)
(renal cancer) with cell promotion of minus5032 and HT29(colon cancer) with cell promotion of minus3495 while themaximum cell promotion was observed on NCIADR-RES(ovarian cancer) which showed 2083 growth promotion(7917 growth inhibition) at one-dose assay Compound2 was found to be highly active on RXF 393 (renal cancer)with cell promotion of minus5360 followed by SK-MEL-5(melanoma) with cell promotion of minus2966 and MDA-MB-468 (breast cancer) with cell promotion of minus2640 whilethe maximum cell promotion was observed on TK-10 (renalcancer) which showed 2310 growth promotion (7690growth inhibition) at one-dose assay The compound 3was found to be highly active on RXF 393 (renal cancer)
with cell promotion of minus4384 followed by HT29 (coloncancer) with cell promotion of minus4184 and SK-MEL-2(melanoma) with cell promotion of minus2707 while themaximum cell promotion was observed on TK-10 (renalcancer) which showed 3812 growth promotion (6188growth inhibition) at one-dose assay Compound 5 wasfound to be highly active on COLO 205 (colon cancer)with cell promotion of minus5943 followed by RXF 393 (renalcancer) with cell promotion of minus5761 and SF-295 (CNScancer) with cell promotion of minus5532 while the maximumcell promotion was observed on TK-10 (renal cancer)which showed 2404 growth promotion (7596 growthinhibition) at one-dose assay Compound 6 was found to
10 BioMed Research International
100
50
0
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
LeukemiaG
row
th (
)
log10 of sample concentration (molar)
CCRF-CEMMOLT-4HL-60(TB)
RPMI-8226K-562SR
(a)
100
50
0
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
Leukemia
Gro
wth
()
log10 of sample concentration (molar)
CCRF-CEMMOLT-4HL-60(TB)
RPMI-8226K-562SR
(b)
100
50
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
Gro
wth
()
log10 of sample concentration (molar)
0
COLO 205HCT-15SW-620HCC-2998
HT29HCT-116KM12
Colon cancer
(c)
Figure 2 (a) Five-dose anticancer screening of compound 8 on leukemia cell line (b) Five-dose anticancer screening of compound 10 onleukemia cell line (c) Five-dose anticancer screening of compound 13 on colon cancer cell line
Figure 3 Green colour stick model indicates the reference ligand and atom macromodel stick indicates our ligands
be highly active on RXF 393 (renal) with cell promotionof minus4746 followed by MDA-MB-468 (breast cancer)with cell promotion of minus2914 and SK-OV-3 (ovariancancer) with cell promotion of minus2909 while the maximumcell promotion was observed on TK-10 (renal cancer)
which showed 2855 growth promotion (7145 growthinhibition) at one-dose assay Compound 8 was foundto be highly active on SK-MEL-5 (melanoma) with cellpromotion of minus8344 followed by RXF 393 (renal cancer)with cell promotion of minus6729 and SK-OV-3 (ovarian
BioMed Research International 11
(a) (b)
Figure 4 (a) Compound 13 is represented with macromodel stick modelThe hydrogen bonds are shown with yellow colour dotted lines andhydrophobic interactions with orange dotted lines (b) Compound 13 at binding site on surface
(a) (b)
Figure 5 (a) Compound 3 is represented with macromodel stick modelThe hydrophobic cavity residues are represented with magenta stickmodelThe hydrogen bonds are shownwith yellow colour dotted lines and hydrophobic interactions with orange dotted lines (b) Compound3 at binding site on surface
cancer) with cell promotion of minus6358 while the maximumcell promotion was observed on TK-10 (renal cancer)which showed 1584 growth promotion (8416 growthinhibition) at one-dose assay The compound 10 was foundto be highly active on SK-MEL-5 (melanoma) with cellpromotion of minus8652 followed by UACC-62 (melanoma)with cell promotion of minus8439 and COLO 205 (coloncancer) with cell promotion of minus8405 while the maximumcell promotion was observed on UACC-257 (melanoma)which showed 1603 growth promotion (8397 growthinhibition) at one-dose assay Compound 11 was found to behighly active on CAKI-1 (renal cancer) with cell promotionof minus3477 followed by MDA-MB-435 (melanoma) with cellpromotion of minus3053 and SF-295 (CNS cancer) with cellpromotion of minus2968 while the maximum cell promotionwas observed on UACC-257 (melanoma) which showed8713 growth promotion (1287 growth inhibition) at one-dose assay Compound 12 was found to be highly active onHT29 (colon cancer) with cell promotion of minus641 followedby MDA-MB-468 (breast cancer) with cell promotion ofminus634 and CCRF-CEM (leukemia) with cell promotion ofminus1672 while the maximum cell promotion was observedon TK-10 (renal cancer) which showed 10274 growthpromotion at one-dose assay Compound 13 was found to behighly active on HT29 (colon cancer) with cell promotion ofminus7624 followed by 786-0 and A498 (renal cancer) with cellpromotion of minus5893 and minus5692 respectively while themaximum cell promotion was observed on NCIADR-RES(ovarian cancer) which showed 3932 growth promotion(1287 growth inhibition) at one-dose assay Among the35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) Compound
8 was found to be the most active compound of theseries with mean growth percent of minus1919 followed bycompound 5 which showed mean growth percent of minus1225and compound 1 which showed mean growth percent ofminus466 Among the 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl (substituted phenyl)methanone analogues(10-11) Compound 10 showed maximum activity with meangrowth percent of minus2871 while compound 13 was found tobe the most active compound among dihydropyrimidineanalogues (12ndash14) which showed mean growth percent ofminus546 Overall the most active compound of the serieswas compound 10 and most of the 35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carbox-amide analogues possessed potent anticancer activity Basedon our finding we can conclude that electronegative groupat position 2 (ie 2-chloro) on N-substituted phenyl ringamong 35-bis(4-hydroxy-3-methylstyryl)-N-(substitutedphenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) showedmore anticancer activity than at position 4 (ie 4-chloro and4-fluoro) Electron releasing group such as 24-dimethyl and4-methyl on N-substituted phenyl ring increased anticanceractivity and the activity was found to be more if thenumber of electron releasing groups was increased Amongthe 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl(substituted phenyl)methanone analogues (10 and 11) theactivity was found to bemore when there was no substitutionin the phenyl ring Among six member dihydropyrimidineanalogues (12ndash14) compound with X substitution as ldquoNHrdquoshowed more activity than as ldquoOrdquo substitution
The results of 5-dose assay of the most active compoundsamong their respective series are given in Table 3 Compound8 presented GI
50ranging between 0004 and 204 120583M The
12 BioMed Research International
best results were recorded on the leukemia cell lines withvalues ranging from 0004 to 0364 120583M (Figure 2(a)) Anumber of 3 tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 0383120583M Only in 23 cell linescompound 8 registered LC
50valuewithgt100120583MCompound
10 presented GI50ranging between 00079 and 186 120583M The
best results were recorded on the leukemia cell line withvalue ranging from 00353 to 0347 120583M (Figure 2(b)) Fourof the tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 000815120583M Only in 27 cell linescompound 10 registered LC
50value with gt100 120583M Com-
pound 13 presented GI50ranging between 0524 and 339 120583M
The best results were recorded on the colon cancer cell linewith values ranging from 0543 to 218 120583M(Figure 2(c)) Onlyone tested cancer cell line presented TGI value with gt100 120583Mthe best result value being noted on the HT29 (colon cancer)with value 0948 In 28 cell lines compound 13 registeredLC50value with gt100120583M
Some of the synthesized compounds were evaluated forEGFR tyrosine kinase inhibitory activity by recombinanttyrosine kinase assay using an ELISA-based assay withpoly(Glu Tyr 4 1) as a substrate [37] The results showedcompounds 8 10 and 13 shows inhibition of EGFR tyrosinekinase with IC
50values of 355 292 and 557120583Mrespectively
33 Molecular Docking The binding site of the referenceinhibitor is well defined by hydrophobic cavity The EGFRtyrosine kinase binding site contains the important residuesCys797 andThr790 re docking of reference ligand shown thatabove residues are important for hydrophobic interactionswith the same [38] The hydrophobic cavity with the residuesLeu788 Met766 Lys745 Glu762 Thr854 and Met793 makesthe binding site attractive for the design of new inhibitorsBased on our knowledge we designed some class of newpyrazole (1ndash11) dihydropyrimidine analogues (12ndash14) forEGFR tyrosine kinase inhibition But the binding sites ofour new molecules were found to be a little different withcomparison to reference inhibitor which is reported in thecrystal structure of EGFR tyrosine kinase (PDB 2J5F) Theseligandsrsquo interactions were shown in three different motifsThe first one is hydrophobic cavity in which the substitutedmethoxy hydroxy phenyl ring of the ligand is showinggood interactions as reference inhibitor The next bindingmotif contains the target residue Cys797 the N-substitutedpyrazole derivatives and six membered (dihydropyrimidine)are binding at this site Most of these interactions werehydrophobic The last binding motif contains the residuesLys875 and Asp831 which lie in side chain of the protein andshowed the side chain hydrogen bonding with the designedmolecules The docking score and Emodel score of some ofthe compounds selected for anticancer activity by NCI aregiven in Table 4 and binding modes are shown in Figure 3
Compound 13 showed the best hydrophobic interactionwith various residues such as Cys797 Met 793 Leu 844 Leu792 Ala 743 and Val 726 (Figures 4(a) and 4(b)) Negativelycharged Asp 837 interacted with methoxy hydroxy phenyl
ring (4-hydroxy-3-methoxyphenyl) Arg 841 showed 120587-120587cationic interaction The Met 793 present in the hydrophobiccavity showed H-bond backbone interaction and H-bond(side chain) interaction The positively charged Lys 845and Arg 841 destabilized interactions The interactions ofcompound 13 with the EGFR kinase receptor shown inribbon diagram the hydrophobic cavity is indicated withyellow colour loop motif-1 is indicated with green colourbeta sheet and the motif-2 is indicated with green colourloop The other ligands were also having good interactionswith the hydrophobic cavity and bindingmotifs the followingresidues leu718 Pro794 Met793 Ala743 Leu844 Val726and Cys797 were present around the ligand Thr790 Gln791having polar characteristics andMet 793 were responsible forbackbone H-bond interaction as well as H-bond (side chain)interaction Asp855 and Asp837 showed charged interactionslike destabilizing contacts Positively charged Lys875 andArg841 were found to have 120587-120587 cationic interaction and werepresent in bindingmotifmaking different binding patterns ofreported ligandsThe docking study for compound 3 is givenin Figures 5(a) and 5(b)
4 Conclusion
The novel series of curcumin analogues (pyrazoles) weresynthesized in satisfactory yields The anticancer activityshowed promising results The studies confirmed compound10 as potent lead compound for drug discovery and furtheroptimization The curcumin analogues discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer disease The pyrazole discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer
Acknowledgments
The authors are thankful to the staff members of the NationalCancer Institute (NCI) USA for in vitro anticancer screeningof the newly synthesized compounds They are especiallygrateful to Professor Dizon B Thelma Project ManagerNational Cancer Institute (NCI) USA
References
[1] WHO Cancer World Health Organization February 2006httpenwikipediaorgwikicancer
[2] M N Noolvi H M Patel V Bhardwaj and A ChauhanldquoSynthesis and in vitro antitumor activity of substituted quina-zoline and quinoxaline derivatives search for anticancer agentrdquoEuropean Journal of Medicinal Chemistry vol 46 no 6 pp2327ndash2346 2011
[3] N Aydemir and R Bilaloglu ldquoGenotoxicity of two anticancerdrugs gemcitabine and topotecan in mouse bone marrow invivordquoMutation Research vol 537 no 1 pp 43ndash51 2003
[4] I Bouabdallah L A MrsquoBarek A Zyad A Ramdani I Zidaneand A Melhaoui ldquoNew pyrazolic compounds as cytotoxicagentsrdquo Natural Product Research vol 21 no 4 pp 298ndash3022007
BioMed Research International 13
[5] D Havrylyuk B Zimenkovsky O Vasylenko L Zaprutko AGzella and R Lesyk ldquoSynthesis of novel thiazolone-based com-pounds containing pyrazoline moiety and evaluation of theiranticancer activityrdquo European Journal of Medicinal Chemistryvol 44 no 4 pp 1396ndash1404 2009
[6] M Shaharyar M M Abdullah M A Bakht and J MajeedldquoPyrazoline bearing benzimidazoles search for anticanceragentrdquo European Journal of Medicinal Chemistry vol 45 no 1pp 114ndash119 2010
[7] P-C Lv H-Q Li J Sun Y Zhou and H-L Zhu ldquoSynthesisand biological evaluation of pyrazole derivatives containingthiourea skeleton as anticancer agentsrdquo Bioorganic and Medici-nal Chemistry vol 18 no 13 pp 4606ndash4614 2010
[8] M S Christodoulou S Liekens K M Kasiotis and S AHaroutounian ldquoNovel pyrazole derivatives synthesis and eval-uation of anti-angiogenic activityrdquo Bioorganic and MedicinalChemistry vol 18 no 12 pp 4338ndash4350 2010
[9] M J Ahsan G J Samy H Khalillah R C Krit and SSoni ldquoMolecular properties prediction and synthesis of novelpyrazoline carboxamide analogs as antitubercular agentsrdquoAnti-Infective Agents vol 10 no 2 pp 117ndash123 2012
[10] M J Ahsan J G Samy H Khalilullah M A Bakht andM Z Hassan ldquoSynthesis and antimycobacterial evaluation of3a4-dihydro-3H-indeno [12-c] pyrazole-2-carboxamide ana-loguesrdquo European Journal of Medicinal Chemistry vol 46 no11 pp 5694ndash5697 2011
[11] M J Ahsan J G Samy K R Dutt et al ldquoDesign synthesis andantimycobacterial evaluation of novel 3-substituted-N-aryl-67-dimethoxy-3a4-dihydro-3H-indeno[12-c] pyrazole-2-carboxamide analoguesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 15 pp 4451ndash4453 2011
[12] M J Ahsan J G Samy S Soni et al ldquoDiscovery of novelantitubercular 3a4-dihydro-3H-indeno[12-c]pyrazole-2-carndashboxamidecarbothioamide analoguesrdquo Bioorganic and Medici-nal Chemistry Letters vol 21 no 18 pp 5259ndash5261 2011
[13] M J Ahsan H Khalilullah J P Stables and J GovindasamyldquoSynthesis and anticonvulsant activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide carbothioamide ana-loguesrdquo Journal of Enzyme Inhibition amp Medicinal Chemistryvol 28 no 3 pp 644ndash650 2013
[14] M J Ahsan J Govindasamy H Khalilullah et al ldquoPOMAanalyses as new efficient bioinformaticsrsquoplatform to predictand optimize bioactivity of synthesized 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamidecarbothioamide anal-oguesrdquo Bioorganic amp Medicinal Chemistry vol 22 no 24 pp7029ndash7035 2012
[15] H Khalilullah S Khan M J Ahsan and B AhmedldquoSynthesis and antihepatotoxic activity of 5-(23-dihydro-14-benzodioxane-6-yl)-3-substituted-phenyl-45-dihydro-1H-pyrazole derivativesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 24 pp 7251ndash7254 2011
[16] M J Alam M J Ahsan O Alam and S A KhanldquoSynthesis of 4-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)-6-(substituted phenyl)pyrimidin-2-ol analogues as 8 anti-inflammatory and analgesic agentsrdquo Letters in Drug Design ampDiscovery vol 10 no 8 pp 776ndash782 2013
[17] D P Chauhan ldquoChemotherapeutic potential of curcumin forcolorectal cancerrdquo Current Pharmaceutical Design vol 8 no 19pp 1695ndash1706 2002
[18] Y-L Jia J Li Z-H Qin and Z-Q Liang ldquoAutophagic andapoptotic mechanisms of curcumin-induced death in K562
cellsrdquo Journal of Asian Natural Products Research vol 11 no 11pp 918ndash928 2009
[19] A Kunwar A Barik BMishra K Rathinasamy R Pandey andK I Priyadarsini ldquoQuantitative cellular uptake localization andcytotoxicity of curcumin in normal and tumor cellsrdquoBiochimicaet Biophysica Acta vol 1780 no 4 pp 673ndash679 2008
[20] J Lal S K Gupta DThavaselvam and D D Agarwal ldquoDesignsynthesis synergistic antimicrobial activity and cytotoxicityof 4-aryl substituted 34-dihydropyrimidinones of curcuminrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 8 pp2872ndash2876 2012
[21] R K Singh D Rai D Yadav A Bhargava J Balzarini and EDe Clercq ldquoSynthesis antibacterial and antiviral properties ofcurcumin bioconjugates bearing dipeptide fatty acids and folicacidrdquo European Journal of Medicinal Chemistry vol 45 no 3pp 1078ndash1086 2010
[22] K Saja M S Babu D Karunagaran and P R SudhakaranldquoAnti-inflammatory effect of curcumin involves downregula-tion of MMP-9 in blood mononuclear cellsrdquo InternationalImmunopharmacology vol 7 no 13 pp 1659ndash1667 2007
[23] S Mishra K Karmodiya N Surolia and A Surolia ldquoSynthesisand exploration of novel curcumin analogues as anti-malarialagentsrdquo Bioorganic and Medicinal Chemistry vol 16 no 6 pp2894ndash2902 2008
[24] G Liang L Shao Y Wang et al ldquoExploration and synthesis ofcurcumin analogues with improved structural stability both invitro and in vivo as cytotoxic agentsrdquo Bioorganic and MedicinalChemistry vol 17 no 6 pp 2623ndash2631 2009
[25] M J Ahsan ldquoSynthesis and anticancer activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide analoguesrdquo Lettersin Drug Design amp Discovery vol 9 no 9 pp 823ndash827 2012
[26] Salahuddin M Shaharyar A Majumdar and M J AhsanldquoSynthesis characterization and anticancer evaluation of 2-(naphthalen-1-ylmethylnaphthalen-2-yloxymethyl)-1-[5-(sub-stituted phenyl)-[134]oxadiazol-2-ylmethyl]-1H-benzimida-zolerdquo Arabian Journal of Chemistry 2013
[27] E Zwick J Bange andAUllrich ldquoReceptor tyrosine kinase sig-nalling as a target for cancer intervention strategiesrdquo Endocrine-Related Cancer vol 8 no 3 pp 161ndash173 2001
[28] H A Bhuva and S G Kini ldquoSynthesis anticancer activity anddocking of some substituted benzothiazoles as tyrosine kinaseinhibitorsrdquo Journal ofMolecular Graphics andModelling vol 29no 1 pp 32ndash37 2010
[29] J Mendelsohn and J Baselga ldquoThe EGF receptor family astargets for cancer therapyrdquo Oncogene vol 19 no 56 pp 6550ndash6565 2000
[30] M Amir M J Ahsan and I Ali ldquoSynthesis of N1-(3-chloro-4-flouropheny)-N4-substituted semicarbazones as novel anticon-vulsant agentsrdquo Indian Journal of Chemistry B vol 49 no 11 pp1509ndash1514 2010
[31] httpdtpncinihgov[32] A Monks D Scudiero P Skehan et al ldquoFeasibility of a high-
flux anticancer drug screen using a diverse panel of culturedhuman tumor cell linesrdquo Journal of theNational Cancer Institutevol 83 no 11 pp 757ndash766 1991
[33] M R Body and K D Paull ldquoSome practical considerations andapplications of the national cancer institute in vitro anticancerdrug discovery screenrdquoDrug Development Research vol 34 no2 pp 91ndash109 1995
[34] R H Shoemaker ldquoThe NCI60 human tumour cell line anti-cancer drug screenrdquo Nature Reviews Cancer vol 6 no 10 pp813ndash823 2006
14 BioMed Research International
[35] M C Alley D A Scudiero A Monks et al ldquoFeasibility ofdrug screening with panels of human tumor cell lines using amicroculture tetrazolium assayrdquo Cancer Research vol 48 no 3pp 589ndash601 1988
[36] M R Grever S A Schepartz and B A Chabner ldquoTheNationalCancer Institute cancer drug discovery and development pro-gramrdquo Seminars in Oncology vol 19 no 6 pp 622ndash638 1992
[37] P Dubreuil S Letard M Ciufolini et al ldquoMasitinib (AB1010)a potent and selective tyrosine kinase inhibitor targeting KITrdquoPLoS ONE vol 4 no 9 Article ID e7258 2009
[38] J A Blair D Rauh C Kung et al ldquoStructure-guided devel-opment of affinity probes for tyrosine kinases using chemicalgeneticsrdquo Nature Chemical Biology vol 3 no 4 pp 229ndash2382007
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
concept of privileged scaffolds structure based on computer-aided drug design and so forth while the structures con-taining problematic linkage or functional groups (eg nitronitroso ndashNndashNndash ndashN=Nndash imine semicarbazone thioamidesand thioureas) for successful drug development are avoided[31]
The anticancer screening was carried out as per theNCI US protocol Using the seven absorbance measurements(time zero (119879
119894) control growth (119862) and test growth in
the presence of drug at the five concentration levels (119879119891))
the percentage growth was calculated at each of the drugconcentrations level as [(119879
119891minus 119879
119894)(119862 minus 119879
119894)] times 100 for
concentrations for which 119879119891ge 119879
119894and [(119879
119891minus 119879
119894)119879
119894] times 100
for concentrations for which 119879119891lt 119879
119894
Three-dose response parameters (GI50 TGI and LC
50)
were calculated for each of the experimental agents Growthinhibition of 50 (GI
50) was calculated from 100 times [(119879
119891minus
119879
119894)(119862 minus 119879
119894)] = 50 which was the drug concentration
resulting in a 50 reduction in the net protein increase (asmeasured by sulforhodamine B SRB staining) in controlcells during the drug incubation The total growth inhibi-tion (TGI) was calculated from 119879
119891= 119879
119894 which was the
drug concentration resulting in total growth inhibition andsignified the cytostatic effect The LC
50was calculated from
100 times [(119879
119891minus 119879
119894)119879
119894] = minus50 indicating a net loss of cells
following treatment which indicated the concentration ofdrug resulting in a 50 reduction in the measured proteinat the end of the drug treatment as compared to that at thebeginning Values were calculated for each of these threeparameters at the level of activity however if the effect didnot reach to the level of activity the value of parameter was
expressed as less than the minimum concentration testedor if the effect exceeded the level of activity the valueof parameter was expressed as greater than the maximumconcentration tested [31 35 36] LogGI
50 log TGI and
log LC50
are the logarithm molar concentrations producing50 growth inhibition (GI
50) a total growth inhibition
(TGI) and a 50 cellular death (LC50) respectively
26 Molecular Docking Studies
261 Protein Structure X-ray crystal structure EGFR kinase(PDB 2J5F) with resolution 300 A 119877-value 0194 (obs)was obtained from the protein data bank (Research Collab-oratory for Structural Bioinformatics (RCSB) (httpwwwrcsborgpdb))
262 Protein Preparation The protein (PDB 2J5F) wasprepared using the Protein PreparationWizard Preprocessedbond orders were assigned hydrogens were added metalswere treated and water molecules were deleted Heterostatefor cocrystallized ligand was generated using Epik proto-nation state and optimization of H-bonding of the proteinside chains were assigned using ProtAssign Energy wasminimized (Impref minimization) using RMSD 030 A andconverged by OPLS2005 force field utilities of Schrodingerrsquossuite 93
263 Receptor Grid Generation Receptor Grid has beengenerated with GLIDE module of Schrodinger with default
BioMed Research International 5
Table 2 Sixty human tumor cell lines anticancer screening data of curcumin analogues (1ndash14)
Panelcell line Growth percent in one-dose assay1 2 3 5 6 8 10 11 12 13
parameters and without any constraints Site has been speci-fied as centroid of the work space ligand (20 A) with van derWaals radius scaling factor 10 and partial charge cutoff 025
264 Molecular Docking Protocol The ligand docking wasperformed in GLIDE50 Ligands which show less than35 rotatable bonds and which are having less than 200atoms were selected The scaling factor will be 080 and thepotential charge cutoff is 015 All the conformers from theconfgen-ligprep output were docked in the EGFR TyrosineKinase active site All default parameters were used for extraprecision docking Glide extra precision mode was employedfor the current docking study Best poses were chosen forenergy minimization during docking a distance dependentdielectric constant of 20 and maximum number of min-imization step of 100 was used The docking simulations(Ligand receptor interactions) are scored using the Xtraprecision (XP) mode which is implemented in GLIDE50
27 Materials and Methods All computational analysis wascarried out on a Red Hat 50 Linux platform running on aDell Precision work station with Intel core 2 quad processorand 8GB of RAM
3 Results and Discussion
31 Chemistry The curcumin analogues (1ndash14) described inthe study are shown in Table 1 and the reaction sequencefor the synthesis is summarized in Scheme 1 In the first part
of the synthesis 17-bis(4-hydroxy-3-methoxyphenyl)hepta-16-diene-35-dione (curcumin) and substituted phenyl semi-carbazide were refluxed in glacial acetic acid to obtainthe 35-bis(4-hydroxy-3-methoxystyryl)-N-(substituted phe-nyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) Substi-tuted phenyl semicarbazides (ArNHCONHNH
2) were syn-
thesized as per reported method [30] In the second partof the synthesis 17-bis(4-hydroxy-3-methoxyphenyl)hepta-16-diene-35-dione (curcumin) and substituted phenylhy-drazide (ArCONHNH
2) were refluxed in glacial acetic acid to
obtain the 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl(substituted phenyl)methanone (10-11) In the third partof synthesis 17-bis(4-hydroxy-3-methoxyphenyl)hepta-16-diene-35-dione (curcumin) and ureaguanidinethioureawere refluxed in glacial acetic acid to obtain the pyrimidineanalogues (12ndash14) The plausible mechanism of reactions isgiven in Figure 1 The yields of the title compounds wereranging from 66 to 88 after recrystallization with absoluteethanol The completion of reaction was monitored by TLCusing mobile phase hexane ethylacetate (6 4) and purityof the compounds was checked by elemental analyses Boththe analytical and spectral data (IR NMR and MS) of thesynthesized compounds were in full accordance with theproposed structures In general IR spectra of the compoundsafforded C=N stretching at 1514ndash1583 cmminus1 and CndashN stretchat 1320ndash1335 cmminus1 and carbamoyl group NndashH stretching at3122ndash3210 cmminus1 andC=O stretching at 1680ndash1689 cmminus1 bandsfor carboxamide analogues (1ndash9) and at 1755ndash1757 cmminus1bands formethanone analogues (10-11)The 1HNMR spectra
BioMed Research International 7
Table 3 NCI in vitro testing results of compounds of the compounds 8 10 and 13 at five-dose level in 120583M
S no Panelcell line 8 (NSC 757926) 10 (NSC 757929) 13 (NSC 763375)GI50 TGI LC50 GI50 TGI LC50 GI50 TGI LC50
(1)
LeukemiaCCRF-CEM 0232 gt100 gt100 0127 gt100 gt100 0543 NT gt100HL-60(TB) 0237 0806 gt100 00884 NT gt100 218 617 gt100MOLT-4 0364 NT gt100 0347 gt100 gt100 101 519 gt100RPMI-8226 0336 NT gt100 0275 NT gt100 129 418 gt100SR 0004 gt100 gt100 00353 gt100 gt100 072 358 gt100
Colon cancerCOLO 205 205 362 638 186 334 597 18 324 585HCC-2998 064 58 gt100 0591 271 gt100 175 319 579HCT-116 0323 NT NT 0269 114 NT 11 239 518HCT-15 0335 166 NT 0167 152 NT 282 103 gt100HT29 0696 28 994 0463 319 gt100 314 0948 421KM12 0298 125 NT 00092 119 NT 163 332 679SW-620 0441 NT gt100 00079 NT gt100 165 368 NT
(4)
CNS cancerSF-268 0571 NT NT 0256 NT NT 211 156 gt100SF-295 0546 NT gt100 0538 NT NT 193 454 gt100SF-539 0354 15 NT 00604 153 NT 205 573 gt100SNB-19 0634 201 NT 0572 212 NT 181 795 gt100SNB-75 0416 182 NT 00099 187 NT 126 334 884U251 0471 159 NT 0428 170 NT 179 569 663
(5)
MelanomaLOX IMVI 0284 149 NT 00626 156 NT 127 259 NTMALME-3M 0563 198 NT 0525 217 NT 271 676 gt100M14 0372 141 NT 0155 148 NT 165 344 NTMDA-MB-435 00448 0383 NT 00175 000815 gt100 171 NT gt100SK-MEL-2 0366 182 NT 0266 203 gt100 198 41 NTSK-MEL-28 0509 247 861 0176 272 905 185 453 26SK-MEL-5 0317 129 367 0171 130 369 149 289 559UACC-257 154 547 319 114 429 533 153 35 8UACC-62 0413 165 489 0371 164 NT 149 323 NT
8 BioMed Research International
Table 3 Continued
S no Panelcell line 8 (NSC 757926) 10 (NSC 757929) 13 (NSC 763375)GI50 TGI LC50 GI50 TGI LC50 GI50 TGI LC50
showed singlet at 120575 119ndash128 ppm corresponding to CH3
a singlet at 120575 379ndash385 ppm corresponding to OCH3 a
singlet at 120575 652ndash662 ppm corresponding to CH=C proton(pyrazoledihydropyrimidine) a doublet at 120575 661ndash675 ppmcorresponding toCH=CHproton a doublet at 665ndash681 ppmcorresponding to CH=CH proton a multiplet at 120575 681ndash793 ppm corresponding to aromatic protons broad singlet at120575 993ndash1016 ppm corresponding to CONH
2 The compounds
(1ndash14) in 1HNMRspectra exhibited two doublets with 119869 valuebetween 146 and 166Hz confirming the trans coupling Themass spectra of the compounds revealed in each case a peakcorresponding to their molecular ion peaks The elementalanalysis results were within plusmn04 of the theoretical values
32 Anticancer Activity Ten compounds were evaluated fortheir anticancer activity in both one-dose and 5-dose assaysThe observed anticancer screening data of the compoundsare given in Table 2 The 5-dose assay screening data ofthree compounds are given in Table 3 Compound 1 wasfound to be highly active on COLO 205 (colon cancer)with cell promotion of minus7349 followed by RXF 393
BioMed Research International 9
HO
O
OH
O
HO
ONN
OH
ONH
OR
HO
ONN
OH
OO
R
HO
ONHN
OH
O
X
AcOHReflux
Curcumin
ArCONHNH2
ArNHCONHNH2
NH2CXNH2
1ndash9
12ndash14
10-11
OO
Scheme 1 Protocol for the synthesis of curcumin analogues
OO
HOO
OHO
OOH
HOO
OHO
Diketone (tautomer I) Keto-enol (tautomer II)
OOH
HOO
OHO O
NH NH
N N
OHOO
HO
O
NH
R
OHOH
HOO
OHO
NHNH
ONH
OH
HOO
OHO
NNH
X
NH
R
R
R
Curcumin
OOH
HOO
OHO
R
CurcuminO
NH
OHOH
HOO
OHO
NHNH
O
R
R
OH
HOO
OHO
NNH
OR
OOH
HOO
OHO X
OHOH
HOO
OHO
NH
O
OH
HOO
OHO
N
X
HN N
X
OHOO
HO
Curcumin
N N
OHOO
HO
O
H3C H3C
H3C
H3C
H3C
CH3
CH3
H3C CH3 H3C H3CCH3CH3
CH3
CH3
CH3
CH3CH3CH3
H2N
H2N
H2N
H3CH3CH3CCH3H3CH2N
H2N
minusH2O
minusH2O
minusH2O
minusH2O minusH2O
minusH2O
NH2
1ndash9
12ndash1410-11
X = S O
Figure 1 Plausible mechanism of reaction for the synthesis of pyrazole analogues (1ndash14)
(renal cancer) with cell promotion of minus5032 and HT29(colon cancer) with cell promotion of minus3495 while themaximum cell promotion was observed on NCIADR-RES(ovarian cancer) which showed 2083 growth promotion(7917 growth inhibition) at one-dose assay Compound2 was found to be highly active on RXF 393 (renal cancer)with cell promotion of minus5360 followed by SK-MEL-5(melanoma) with cell promotion of minus2966 and MDA-MB-468 (breast cancer) with cell promotion of minus2640 whilethe maximum cell promotion was observed on TK-10 (renalcancer) which showed 2310 growth promotion (7690growth inhibition) at one-dose assay The compound 3was found to be highly active on RXF 393 (renal cancer)
with cell promotion of minus4384 followed by HT29 (coloncancer) with cell promotion of minus4184 and SK-MEL-2(melanoma) with cell promotion of minus2707 while themaximum cell promotion was observed on TK-10 (renalcancer) which showed 3812 growth promotion (6188growth inhibition) at one-dose assay Compound 5 wasfound to be highly active on COLO 205 (colon cancer)with cell promotion of minus5943 followed by RXF 393 (renalcancer) with cell promotion of minus5761 and SF-295 (CNScancer) with cell promotion of minus5532 while the maximumcell promotion was observed on TK-10 (renal cancer)which showed 2404 growth promotion (7596 growthinhibition) at one-dose assay Compound 6 was found to
10 BioMed Research International
100
50
0
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
LeukemiaG
row
th (
)
log10 of sample concentration (molar)
CCRF-CEMMOLT-4HL-60(TB)
RPMI-8226K-562SR
(a)
100
50
0
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
Leukemia
Gro
wth
()
log10 of sample concentration (molar)
CCRF-CEMMOLT-4HL-60(TB)
RPMI-8226K-562SR
(b)
100
50
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
Gro
wth
()
log10 of sample concentration (molar)
0
COLO 205HCT-15SW-620HCC-2998
HT29HCT-116KM12
Colon cancer
(c)
Figure 2 (a) Five-dose anticancer screening of compound 8 on leukemia cell line (b) Five-dose anticancer screening of compound 10 onleukemia cell line (c) Five-dose anticancer screening of compound 13 on colon cancer cell line
Figure 3 Green colour stick model indicates the reference ligand and atom macromodel stick indicates our ligands
be highly active on RXF 393 (renal) with cell promotionof minus4746 followed by MDA-MB-468 (breast cancer)with cell promotion of minus2914 and SK-OV-3 (ovariancancer) with cell promotion of minus2909 while the maximumcell promotion was observed on TK-10 (renal cancer)
which showed 2855 growth promotion (7145 growthinhibition) at one-dose assay Compound 8 was foundto be highly active on SK-MEL-5 (melanoma) with cellpromotion of minus8344 followed by RXF 393 (renal cancer)with cell promotion of minus6729 and SK-OV-3 (ovarian
BioMed Research International 11
(a) (b)
Figure 4 (a) Compound 13 is represented with macromodel stick modelThe hydrogen bonds are shown with yellow colour dotted lines andhydrophobic interactions with orange dotted lines (b) Compound 13 at binding site on surface
(a) (b)
Figure 5 (a) Compound 3 is represented with macromodel stick modelThe hydrophobic cavity residues are represented with magenta stickmodelThe hydrogen bonds are shownwith yellow colour dotted lines and hydrophobic interactions with orange dotted lines (b) Compound3 at binding site on surface
cancer) with cell promotion of minus6358 while the maximumcell promotion was observed on TK-10 (renal cancer)which showed 1584 growth promotion (8416 growthinhibition) at one-dose assay The compound 10 was foundto be highly active on SK-MEL-5 (melanoma) with cellpromotion of minus8652 followed by UACC-62 (melanoma)with cell promotion of minus8439 and COLO 205 (coloncancer) with cell promotion of minus8405 while the maximumcell promotion was observed on UACC-257 (melanoma)which showed 1603 growth promotion (8397 growthinhibition) at one-dose assay Compound 11 was found to behighly active on CAKI-1 (renal cancer) with cell promotionof minus3477 followed by MDA-MB-435 (melanoma) with cellpromotion of minus3053 and SF-295 (CNS cancer) with cellpromotion of minus2968 while the maximum cell promotionwas observed on UACC-257 (melanoma) which showed8713 growth promotion (1287 growth inhibition) at one-dose assay Compound 12 was found to be highly active onHT29 (colon cancer) with cell promotion of minus641 followedby MDA-MB-468 (breast cancer) with cell promotion ofminus634 and CCRF-CEM (leukemia) with cell promotion ofminus1672 while the maximum cell promotion was observedon TK-10 (renal cancer) which showed 10274 growthpromotion at one-dose assay Compound 13 was found to behighly active on HT29 (colon cancer) with cell promotion ofminus7624 followed by 786-0 and A498 (renal cancer) with cellpromotion of minus5893 and minus5692 respectively while themaximum cell promotion was observed on NCIADR-RES(ovarian cancer) which showed 3932 growth promotion(1287 growth inhibition) at one-dose assay Among the35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) Compound
8 was found to be the most active compound of theseries with mean growth percent of minus1919 followed bycompound 5 which showed mean growth percent of minus1225and compound 1 which showed mean growth percent ofminus466 Among the 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl (substituted phenyl)methanone analogues(10-11) Compound 10 showed maximum activity with meangrowth percent of minus2871 while compound 13 was found tobe the most active compound among dihydropyrimidineanalogues (12ndash14) which showed mean growth percent ofminus546 Overall the most active compound of the serieswas compound 10 and most of the 35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carbox-amide analogues possessed potent anticancer activity Basedon our finding we can conclude that electronegative groupat position 2 (ie 2-chloro) on N-substituted phenyl ringamong 35-bis(4-hydroxy-3-methylstyryl)-N-(substitutedphenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) showedmore anticancer activity than at position 4 (ie 4-chloro and4-fluoro) Electron releasing group such as 24-dimethyl and4-methyl on N-substituted phenyl ring increased anticanceractivity and the activity was found to be more if thenumber of electron releasing groups was increased Amongthe 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl(substituted phenyl)methanone analogues (10 and 11) theactivity was found to bemore when there was no substitutionin the phenyl ring Among six member dihydropyrimidineanalogues (12ndash14) compound with X substitution as ldquoNHrdquoshowed more activity than as ldquoOrdquo substitution
The results of 5-dose assay of the most active compoundsamong their respective series are given in Table 3 Compound8 presented GI
50ranging between 0004 and 204 120583M The
12 BioMed Research International
best results were recorded on the leukemia cell lines withvalues ranging from 0004 to 0364 120583M (Figure 2(a)) Anumber of 3 tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 0383120583M Only in 23 cell linescompound 8 registered LC
50valuewithgt100120583MCompound
10 presented GI50ranging between 00079 and 186 120583M The
best results were recorded on the leukemia cell line withvalue ranging from 00353 to 0347 120583M (Figure 2(b)) Fourof the tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 000815120583M Only in 27 cell linescompound 10 registered LC
50value with gt100 120583M Com-
pound 13 presented GI50ranging between 0524 and 339 120583M
The best results were recorded on the colon cancer cell linewith values ranging from 0543 to 218 120583M(Figure 2(c)) Onlyone tested cancer cell line presented TGI value with gt100 120583Mthe best result value being noted on the HT29 (colon cancer)with value 0948 In 28 cell lines compound 13 registeredLC50value with gt100120583M
Some of the synthesized compounds were evaluated forEGFR tyrosine kinase inhibitory activity by recombinanttyrosine kinase assay using an ELISA-based assay withpoly(Glu Tyr 4 1) as a substrate [37] The results showedcompounds 8 10 and 13 shows inhibition of EGFR tyrosinekinase with IC
50values of 355 292 and 557120583Mrespectively
33 Molecular Docking The binding site of the referenceinhibitor is well defined by hydrophobic cavity The EGFRtyrosine kinase binding site contains the important residuesCys797 andThr790 re docking of reference ligand shown thatabove residues are important for hydrophobic interactionswith the same [38] The hydrophobic cavity with the residuesLeu788 Met766 Lys745 Glu762 Thr854 and Met793 makesthe binding site attractive for the design of new inhibitorsBased on our knowledge we designed some class of newpyrazole (1ndash11) dihydropyrimidine analogues (12ndash14) forEGFR tyrosine kinase inhibition But the binding sites ofour new molecules were found to be a little different withcomparison to reference inhibitor which is reported in thecrystal structure of EGFR tyrosine kinase (PDB 2J5F) Theseligandsrsquo interactions were shown in three different motifsThe first one is hydrophobic cavity in which the substitutedmethoxy hydroxy phenyl ring of the ligand is showinggood interactions as reference inhibitor The next bindingmotif contains the target residue Cys797 the N-substitutedpyrazole derivatives and six membered (dihydropyrimidine)are binding at this site Most of these interactions werehydrophobic The last binding motif contains the residuesLys875 and Asp831 which lie in side chain of the protein andshowed the side chain hydrogen bonding with the designedmolecules The docking score and Emodel score of some ofthe compounds selected for anticancer activity by NCI aregiven in Table 4 and binding modes are shown in Figure 3
Compound 13 showed the best hydrophobic interactionwith various residues such as Cys797 Met 793 Leu 844 Leu792 Ala 743 and Val 726 (Figures 4(a) and 4(b)) Negativelycharged Asp 837 interacted with methoxy hydroxy phenyl
ring (4-hydroxy-3-methoxyphenyl) Arg 841 showed 120587-120587cationic interaction The Met 793 present in the hydrophobiccavity showed H-bond backbone interaction and H-bond(side chain) interaction The positively charged Lys 845and Arg 841 destabilized interactions The interactions ofcompound 13 with the EGFR kinase receptor shown inribbon diagram the hydrophobic cavity is indicated withyellow colour loop motif-1 is indicated with green colourbeta sheet and the motif-2 is indicated with green colourloop The other ligands were also having good interactionswith the hydrophobic cavity and bindingmotifs the followingresidues leu718 Pro794 Met793 Ala743 Leu844 Val726and Cys797 were present around the ligand Thr790 Gln791having polar characteristics andMet 793 were responsible forbackbone H-bond interaction as well as H-bond (side chain)interaction Asp855 and Asp837 showed charged interactionslike destabilizing contacts Positively charged Lys875 andArg841 were found to have 120587-120587 cationic interaction and werepresent in bindingmotifmaking different binding patterns ofreported ligandsThe docking study for compound 3 is givenin Figures 5(a) and 5(b)
4 Conclusion
The novel series of curcumin analogues (pyrazoles) weresynthesized in satisfactory yields The anticancer activityshowed promising results The studies confirmed compound10 as potent lead compound for drug discovery and furtheroptimization The curcumin analogues discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer disease The pyrazole discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer
Acknowledgments
The authors are thankful to the staff members of the NationalCancer Institute (NCI) USA for in vitro anticancer screeningof the newly synthesized compounds They are especiallygrateful to Professor Dizon B Thelma Project ManagerNational Cancer Institute (NCI) USA
References
[1] WHO Cancer World Health Organization February 2006httpenwikipediaorgwikicancer
[2] M N Noolvi H M Patel V Bhardwaj and A ChauhanldquoSynthesis and in vitro antitumor activity of substituted quina-zoline and quinoxaline derivatives search for anticancer agentrdquoEuropean Journal of Medicinal Chemistry vol 46 no 6 pp2327ndash2346 2011
[3] N Aydemir and R Bilaloglu ldquoGenotoxicity of two anticancerdrugs gemcitabine and topotecan in mouse bone marrow invivordquoMutation Research vol 537 no 1 pp 43ndash51 2003
[4] I Bouabdallah L A MrsquoBarek A Zyad A Ramdani I Zidaneand A Melhaoui ldquoNew pyrazolic compounds as cytotoxicagentsrdquo Natural Product Research vol 21 no 4 pp 298ndash3022007
BioMed Research International 13
[5] D Havrylyuk B Zimenkovsky O Vasylenko L Zaprutko AGzella and R Lesyk ldquoSynthesis of novel thiazolone-based com-pounds containing pyrazoline moiety and evaluation of theiranticancer activityrdquo European Journal of Medicinal Chemistryvol 44 no 4 pp 1396ndash1404 2009
[6] M Shaharyar M M Abdullah M A Bakht and J MajeedldquoPyrazoline bearing benzimidazoles search for anticanceragentrdquo European Journal of Medicinal Chemistry vol 45 no 1pp 114ndash119 2010
[7] P-C Lv H-Q Li J Sun Y Zhou and H-L Zhu ldquoSynthesisand biological evaluation of pyrazole derivatives containingthiourea skeleton as anticancer agentsrdquo Bioorganic and Medici-nal Chemistry vol 18 no 13 pp 4606ndash4614 2010
[8] M S Christodoulou S Liekens K M Kasiotis and S AHaroutounian ldquoNovel pyrazole derivatives synthesis and eval-uation of anti-angiogenic activityrdquo Bioorganic and MedicinalChemistry vol 18 no 12 pp 4338ndash4350 2010
[9] M J Ahsan G J Samy H Khalillah R C Krit and SSoni ldquoMolecular properties prediction and synthesis of novelpyrazoline carboxamide analogs as antitubercular agentsrdquoAnti-Infective Agents vol 10 no 2 pp 117ndash123 2012
[10] M J Ahsan J G Samy H Khalilullah M A Bakht andM Z Hassan ldquoSynthesis and antimycobacterial evaluation of3a4-dihydro-3H-indeno [12-c] pyrazole-2-carboxamide ana-loguesrdquo European Journal of Medicinal Chemistry vol 46 no11 pp 5694ndash5697 2011
[11] M J Ahsan J G Samy K R Dutt et al ldquoDesign synthesis andantimycobacterial evaluation of novel 3-substituted-N-aryl-67-dimethoxy-3a4-dihydro-3H-indeno[12-c] pyrazole-2-carboxamide analoguesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 15 pp 4451ndash4453 2011
[12] M J Ahsan J G Samy S Soni et al ldquoDiscovery of novelantitubercular 3a4-dihydro-3H-indeno[12-c]pyrazole-2-carndashboxamidecarbothioamide analoguesrdquo Bioorganic and Medici-nal Chemistry Letters vol 21 no 18 pp 5259ndash5261 2011
[13] M J Ahsan H Khalilullah J P Stables and J GovindasamyldquoSynthesis and anticonvulsant activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide carbothioamide ana-loguesrdquo Journal of Enzyme Inhibition amp Medicinal Chemistryvol 28 no 3 pp 644ndash650 2013
[14] M J Ahsan J Govindasamy H Khalilullah et al ldquoPOMAanalyses as new efficient bioinformaticsrsquoplatform to predictand optimize bioactivity of synthesized 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamidecarbothioamide anal-oguesrdquo Bioorganic amp Medicinal Chemistry vol 22 no 24 pp7029ndash7035 2012
[15] H Khalilullah S Khan M J Ahsan and B AhmedldquoSynthesis and antihepatotoxic activity of 5-(23-dihydro-14-benzodioxane-6-yl)-3-substituted-phenyl-45-dihydro-1H-pyrazole derivativesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 24 pp 7251ndash7254 2011
[16] M J Alam M J Ahsan O Alam and S A KhanldquoSynthesis of 4-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)-6-(substituted phenyl)pyrimidin-2-ol analogues as 8 anti-inflammatory and analgesic agentsrdquo Letters in Drug Design ampDiscovery vol 10 no 8 pp 776ndash782 2013
[17] D P Chauhan ldquoChemotherapeutic potential of curcumin forcolorectal cancerrdquo Current Pharmaceutical Design vol 8 no 19pp 1695ndash1706 2002
[18] Y-L Jia J Li Z-H Qin and Z-Q Liang ldquoAutophagic andapoptotic mechanisms of curcumin-induced death in K562
cellsrdquo Journal of Asian Natural Products Research vol 11 no 11pp 918ndash928 2009
[19] A Kunwar A Barik BMishra K Rathinasamy R Pandey andK I Priyadarsini ldquoQuantitative cellular uptake localization andcytotoxicity of curcumin in normal and tumor cellsrdquoBiochimicaet Biophysica Acta vol 1780 no 4 pp 673ndash679 2008
[20] J Lal S K Gupta DThavaselvam and D D Agarwal ldquoDesignsynthesis synergistic antimicrobial activity and cytotoxicityof 4-aryl substituted 34-dihydropyrimidinones of curcuminrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 8 pp2872ndash2876 2012
[21] R K Singh D Rai D Yadav A Bhargava J Balzarini and EDe Clercq ldquoSynthesis antibacterial and antiviral properties ofcurcumin bioconjugates bearing dipeptide fatty acids and folicacidrdquo European Journal of Medicinal Chemistry vol 45 no 3pp 1078ndash1086 2010
[22] K Saja M S Babu D Karunagaran and P R SudhakaranldquoAnti-inflammatory effect of curcumin involves downregula-tion of MMP-9 in blood mononuclear cellsrdquo InternationalImmunopharmacology vol 7 no 13 pp 1659ndash1667 2007
[23] S Mishra K Karmodiya N Surolia and A Surolia ldquoSynthesisand exploration of novel curcumin analogues as anti-malarialagentsrdquo Bioorganic and Medicinal Chemistry vol 16 no 6 pp2894ndash2902 2008
[24] G Liang L Shao Y Wang et al ldquoExploration and synthesis ofcurcumin analogues with improved structural stability both invitro and in vivo as cytotoxic agentsrdquo Bioorganic and MedicinalChemistry vol 17 no 6 pp 2623ndash2631 2009
[25] M J Ahsan ldquoSynthesis and anticancer activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide analoguesrdquo Lettersin Drug Design amp Discovery vol 9 no 9 pp 823ndash827 2012
[26] Salahuddin M Shaharyar A Majumdar and M J AhsanldquoSynthesis characterization and anticancer evaluation of 2-(naphthalen-1-ylmethylnaphthalen-2-yloxymethyl)-1-[5-(sub-stituted phenyl)-[134]oxadiazol-2-ylmethyl]-1H-benzimida-zolerdquo Arabian Journal of Chemistry 2013
[27] E Zwick J Bange andAUllrich ldquoReceptor tyrosine kinase sig-nalling as a target for cancer intervention strategiesrdquo Endocrine-Related Cancer vol 8 no 3 pp 161ndash173 2001
[28] H A Bhuva and S G Kini ldquoSynthesis anticancer activity anddocking of some substituted benzothiazoles as tyrosine kinaseinhibitorsrdquo Journal ofMolecular Graphics andModelling vol 29no 1 pp 32ndash37 2010
[29] J Mendelsohn and J Baselga ldquoThe EGF receptor family astargets for cancer therapyrdquo Oncogene vol 19 no 56 pp 6550ndash6565 2000
[30] M Amir M J Ahsan and I Ali ldquoSynthesis of N1-(3-chloro-4-flouropheny)-N4-substituted semicarbazones as novel anticon-vulsant agentsrdquo Indian Journal of Chemistry B vol 49 no 11 pp1509ndash1514 2010
[31] httpdtpncinihgov[32] A Monks D Scudiero P Skehan et al ldquoFeasibility of a high-
flux anticancer drug screen using a diverse panel of culturedhuman tumor cell linesrdquo Journal of theNational Cancer Institutevol 83 no 11 pp 757ndash766 1991
[33] M R Body and K D Paull ldquoSome practical considerations andapplications of the national cancer institute in vitro anticancerdrug discovery screenrdquoDrug Development Research vol 34 no2 pp 91ndash109 1995
[34] R H Shoemaker ldquoThe NCI60 human tumour cell line anti-cancer drug screenrdquo Nature Reviews Cancer vol 6 no 10 pp813ndash823 2006
14 BioMed Research International
[35] M C Alley D A Scudiero A Monks et al ldquoFeasibility ofdrug screening with panels of human tumor cell lines using amicroculture tetrazolium assayrdquo Cancer Research vol 48 no 3pp 589ndash601 1988
[36] M R Grever S A Schepartz and B A Chabner ldquoTheNationalCancer Institute cancer drug discovery and development pro-gramrdquo Seminars in Oncology vol 19 no 6 pp 622ndash638 1992
[37] P Dubreuil S Letard M Ciufolini et al ldquoMasitinib (AB1010)a potent and selective tyrosine kinase inhibitor targeting KITrdquoPLoS ONE vol 4 no 9 Article ID e7258 2009
[38] J A Blair D Rauh C Kung et al ldquoStructure-guided devel-opment of affinity probes for tyrosine kinases using chemicalgeneticsrdquo Nature Chemical Biology vol 3 no 4 pp 229ndash2382007
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
parameters and without any constraints Site has been speci-fied as centroid of the work space ligand (20 A) with van derWaals radius scaling factor 10 and partial charge cutoff 025
264 Molecular Docking Protocol The ligand docking wasperformed in GLIDE50 Ligands which show less than35 rotatable bonds and which are having less than 200atoms were selected The scaling factor will be 080 and thepotential charge cutoff is 015 All the conformers from theconfgen-ligprep output were docked in the EGFR TyrosineKinase active site All default parameters were used for extraprecision docking Glide extra precision mode was employedfor the current docking study Best poses were chosen forenergy minimization during docking a distance dependentdielectric constant of 20 and maximum number of min-imization step of 100 was used The docking simulations(Ligand receptor interactions) are scored using the Xtraprecision (XP) mode which is implemented in GLIDE50
27 Materials and Methods All computational analysis wascarried out on a Red Hat 50 Linux platform running on aDell Precision work station with Intel core 2 quad processorand 8GB of RAM
3 Results and Discussion
31 Chemistry The curcumin analogues (1ndash14) described inthe study are shown in Table 1 and the reaction sequencefor the synthesis is summarized in Scheme 1 In the first part
of the synthesis 17-bis(4-hydroxy-3-methoxyphenyl)hepta-16-diene-35-dione (curcumin) and substituted phenyl semi-carbazide were refluxed in glacial acetic acid to obtainthe 35-bis(4-hydroxy-3-methoxystyryl)-N-(substituted phe-nyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) Substi-tuted phenyl semicarbazides (ArNHCONHNH
2) were syn-
thesized as per reported method [30] In the second partof the synthesis 17-bis(4-hydroxy-3-methoxyphenyl)hepta-16-diene-35-dione (curcumin) and substituted phenylhy-drazide (ArCONHNH
2) were refluxed in glacial acetic acid to
obtain the 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl(substituted phenyl)methanone (10-11) In the third partof synthesis 17-bis(4-hydroxy-3-methoxyphenyl)hepta-16-diene-35-dione (curcumin) and ureaguanidinethioureawere refluxed in glacial acetic acid to obtain the pyrimidineanalogues (12ndash14) The plausible mechanism of reactions isgiven in Figure 1 The yields of the title compounds wereranging from 66 to 88 after recrystallization with absoluteethanol The completion of reaction was monitored by TLCusing mobile phase hexane ethylacetate (6 4) and purityof the compounds was checked by elemental analyses Boththe analytical and spectral data (IR NMR and MS) of thesynthesized compounds were in full accordance with theproposed structures In general IR spectra of the compoundsafforded C=N stretching at 1514ndash1583 cmminus1 and CndashN stretchat 1320ndash1335 cmminus1 and carbamoyl group NndashH stretching at3122ndash3210 cmminus1 andC=O stretching at 1680ndash1689 cmminus1 bandsfor carboxamide analogues (1ndash9) and at 1755ndash1757 cmminus1bands formethanone analogues (10-11)The 1HNMR spectra
BioMed Research International 7
Table 3 NCI in vitro testing results of compounds of the compounds 8 10 and 13 at five-dose level in 120583M
S no Panelcell line 8 (NSC 757926) 10 (NSC 757929) 13 (NSC 763375)GI50 TGI LC50 GI50 TGI LC50 GI50 TGI LC50
(1)
LeukemiaCCRF-CEM 0232 gt100 gt100 0127 gt100 gt100 0543 NT gt100HL-60(TB) 0237 0806 gt100 00884 NT gt100 218 617 gt100MOLT-4 0364 NT gt100 0347 gt100 gt100 101 519 gt100RPMI-8226 0336 NT gt100 0275 NT gt100 129 418 gt100SR 0004 gt100 gt100 00353 gt100 gt100 072 358 gt100
Colon cancerCOLO 205 205 362 638 186 334 597 18 324 585HCC-2998 064 58 gt100 0591 271 gt100 175 319 579HCT-116 0323 NT NT 0269 114 NT 11 239 518HCT-15 0335 166 NT 0167 152 NT 282 103 gt100HT29 0696 28 994 0463 319 gt100 314 0948 421KM12 0298 125 NT 00092 119 NT 163 332 679SW-620 0441 NT gt100 00079 NT gt100 165 368 NT
(4)
CNS cancerSF-268 0571 NT NT 0256 NT NT 211 156 gt100SF-295 0546 NT gt100 0538 NT NT 193 454 gt100SF-539 0354 15 NT 00604 153 NT 205 573 gt100SNB-19 0634 201 NT 0572 212 NT 181 795 gt100SNB-75 0416 182 NT 00099 187 NT 126 334 884U251 0471 159 NT 0428 170 NT 179 569 663
(5)
MelanomaLOX IMVI 0284 149 NT 00626 156 NT 127 259 NTMALME-3M 0563 198 NT 0525 217 NT 271 676 gt100M14 0372 141 NT 0155 148 NT 165 344 NTMDA-MB-435 00448 0383 NT 00175 000815 gt100 171 NT gt100SK-MEL-2 0366 182 NT 0266 203 gt100 198 41 NTSK-MEL-28 0509 247 861 0176 272 905 185 453 26SK-MEL-5 0317 129 367 0171 130 369 149 289 559UACC-257 154 547 319 114 429 533 153 35 8UACC-62 0413 165 489 0371 164 NT 149 323 NT
8 BioMed Research International
Table 3 Continued
S no Panelcell line 8 (NSC 757926) 10 (NSC 757929) 13 (NSC 763375)GI50 TGI LC50 GI50 TGI LC50 GI50 TGI LC50
showed singlet at 120575 119ndash128 ppm corresponding to CH3
a singlet at 120575 379ndash385 ppm corresponding to OCH3 a
singlet at 120575 652ndash662 ppm corresponding to CH=C proton(pyrazoledihydropyrimidine) a doublet at 120575 661ndash675 ppmcorresponding toCH=CHproton a doublet at 665ndash681 ppmcorresponding to CH=CH proton a multiplet at 120575 681ndash793 ppm corresponding to aromatic protons broad singlet at120575 993ndash1016 ppm corresponding to CONH
2 The compounds
(1ndash14) in 1HNMRspectra exhibited two doublets with 119869 valuebetween 146 and 166Hz confirming the trans coupling Themass spectra of the compounds revealed in each case a peakcorresponding to their molecular ion peaks The elementalanalysis results were within plusmn04 of the theoretical values
32 Anticancer Activity Ten compounds were evaluated fortheir anticancer activity in both one-dose and 5-dose assaysThe observed anticancer screening data of the compoundsare given in Table 2 The 5-dose assay screening data ofthree compounds are given in Table 3 Compound 1 wasfound to be highly active on COLO 205 (colon cancer)with cell promotion of minus7349 followed by RXF 393
BioMed Research International 9
HO
O
OH
O
HO
ONN
OH
ONH
OR
HO
ONN
OH
OO
R
HO
ONHN
OH
O
X
AcOHReflux
Curcumin
ArCONHNH2
ArNHCONHNH2
NH2CXNH2
1ndash9
12ndash14
10-11
OO
Scheme 1 Protocol for the synthesis of curcumin analogues
OO
HOO
OHO
OOH
HOO
OHO
Diketone (tautomer I) Keto-enol (tautomer II)
OOH
HOO
OHO O
NH NH
N N
OHOO
HO
O
NH
R
OHOH
HOO
OHO
NHNH
ONH
OH
HOO
OHO
NNH
X
NH
R
R
R
Curcumin
OOH
HOO
OHO
R
CurcuminO
NH
OHOH
HOO
OHO
NHNH
O
R
R
OH
HOO
OHO
NNH
OR
OOH
HOO
OHO X
OHOH
HOO
OHO
NH
O
OH
HOO
OHO
N
X
HN N
X
OHOO
HO
Curcumin
N N
OHOO
HO
O
H3C H3C
H3C
H3C
H3C
CH3
CH3
H3C CH3 H3C H3CCH3CH3
CH3
CH3
CH3
CH3CH3CH3
H2N
H2N
H2N
H3CH3CH3CCH3H3CH2N
H2N
minusH2O
minusH2O
minusH2O
minusH2O minusH2O
minusH2O
NH2
1ndash9
12ndash1410-11
X = S O
Figure 1 Plausible mechanism of reaction for the synthesis of pyrazole analogues (1ndash14)
(renal cancer) with cell promotion of minus5032 and HT29(colon cancer) with cell promotion of minus3495 while themaximum cell promotion was observed on NCIADR-RES(ovarian cancer) which showed 2083 growth promotion(7917 growth inhibition) at one-dose assay Compound2 was found to be highly active on RXF 393 (renal cancer)with cell promotion of minus5360 followed by SK-MEL-5(melanoma) with cell promotion of minus2966 and MDA-MB-468 (breast cancer) with cell promotion of minus2640 whilethe maximum cell promotion was observed on TK-10 (renalcancer) which showed 2310 growth promotion (7690growth inhibition) at one-dose assay The compound 3was found to be highly active on RXF 393 (renal cancer)
with cell promotion of minus4384 followed by HT29 (coloncancer) with cell promotion of minus4184 and SK-MEL-2(melanoma) with cell promotion of minus2707 while themaximum cell promotion was observed on TK-10 (renalcancer) which showed 3812 growth promotion (6188growth inhibition) at one-dose assay Compound 5 wasfound to be highly active on COLO 205 (colon cancer)with cell promotion of minus5943 followed by RXF 393 (renalcancer) with cell promotion of minus5761 and SF-295 (CNScancer) with cell promotion of minus5532 while the maximumcell promotion was observed on TK-10 (renal cancer)which showed 2404 growth promotion (7596 growthinhibition) at one-dose assay Compound 6 was found to
10 BioMed Research International
100
50
0
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
LeukemiaG
row
th (
)
log10 of sample concentration (molar)
CCRF-CEMMOLT-4HL-60(TB)
RPMI-8226K-562SR
(a)
100
50
0
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
Leukemia
Gro
wth
()
log10 of sample concentration (molar)
CCRF-CEMMOLT-4HL-60(TB)
RPMI-8226K-562SR
(b)
100
50
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
Gro
wth
()
log10 of sample concentration (molar)
0
COLO 205HCT-15SW-620HCC-2998
HT29HCT-116KM12
Colon cancer
(c)
Figure 2 (a) Five-dose anticancer screening of compound 8 on leukemia cell line (b) Five-dose anticancer screening of compound 10 onleukemia cell line (c) Five-dose anticancer screening of compound 13 on colon cancer cell line
Figure 3 Green colour stick model indicates the reference ligand and atom macromodel stick indicates our ligands
be highly active on RXF 393 (renal) with cell promotionof minus4746 followed by MDA-MB-468 (breast cancer)with cell promotion of minus2914 and SK-OV-3 (ovariancancer) with cell promotion of minus2909 while the maximumcell promotion was observed on TK-10 (renal cancer)
which showed 2855 growth promotion (7145 growthinhibition) at one-dose assay Compound 8 was foundto be highly active on SK-MEL-5 (melanoma) with cellpromotion of minus8344 followed by RXF 393 (renal cancer)with cell promotion of minus6729 and SK-OV-3 (ovarian
BioMed Research International 11
(a) (b)
Figure 4 (a) Compound 13 is represented with macromodel stick modelThe hydrogen bonds are shown with yellow colour dotted lines andhydrophobic interactions with orange dotted lines (b) Compound 13 at binding site on surface
(a) (b)
Figure 5 (a) Compound 3 is represented with macromodel stick modelThe hydrophobic cavity residues are represented with magenta stickmodelThe hydrogen bonds are shownwith yellow colour dotted lines and hydrophobic interactions with orange dotted lines (b) Compound3 at binding site on surface
cancer) with cell promotion of minus6358 while the maximumcell promotion was observed on TK-10 (renal cancer)which showed 1584 growth promotion (8416 growthinhibition) at one-dose assay The compound 10 was foundto be highly active on SK-MEL-5 (melanoma) with cellpromotion of minus8652 followed by UACC-62 (melanoma)with cell promotion of minus8439 and COLO 205 (coloncancer) with cell promotion of minus8405 while the maximumcell promotion was observed on UACC-257 (melanoma)which showed 1603 growth promotion (8397 growthinhibition) at one-dose assay Compound 11 was found to behighly active on CAKI-1 (renal cancer) with cell promotionof minus3477 followed by MDA-MB-435 (melanoma) with cellpromotion of minus3053 and SF-295 (CNS cancer) with cellpromotion of minus2968 while the maximum cell promotionwas observed on UACC-257 (melanoma) which showed8713 growth promotion (1287 growth inhibition) at one-dose assay Compound 12 was found to be highly active onHT29 (colon cancer) with cell promotion of minus641 followedby MDA-MB-468 (breast cancer) with cell promotion ofminus634 and CCRF-CEM (leukemia) with cell promotion ofminus1672 while the maximum cell promotion was observedon TK-10 (renal cancer) which showed 10274 growthpromotion at one-dose assay Compound 13 was found to behighly active on HT29 (colon cancer) with cell promotion ofminus7624 followed by 786-0 and A498 (renal cancer) with cellpromotion of minus5893 and minus5692 respectively while themaximum cell promotion was observed on NCIADR-RES(ovarian cancer) which showed 3932 growth promotion(1287 growth inhibition) at one-dose assay Among the35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) Compound
8 was found to be the most active compound of theseries with mean growth percent of minus1919 followed bycompound 5 which showed mean growth percent of minus1225and compound 1 which showed mean growth percent ofminus466 Among the 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl (substituted phenyl)methanone analogues(10-11) Compound 10 showed maximum activity with meangrowth percent of minus2871 while compound 13 was found tobe the most active compound among dihydropyrimidineanalogues (12ndash14) which showed mean growth percent ofminus546 Overall the most active compound of the serieswas compound 10 and most of the 35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carbox-amide analogues possessed potent anticancer activity Basedon our finding we can conclude that electronegative groupat position 2 (ie 2-chloro) on N-substituted phenyl ringamong 35-bis(4-hydroxy-3-methylstyryl)-N-(substitutedphenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) showedmore anticancer activity than at position 4 (ie 4-chloro and4-fluoro) Electron releasing group such as 24-dimethyl and4-methyl on N-substituted phenyl ring increased anticanceractivity and the activity was found to be more if thenumber of electron releasing groups was increased Amongthe 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl(substituted phenyl)methanone analogues (10 and 11) theactivity was found to bemore when there was no substitutionin the phenyl ring Among six member dihydropyrimidineanalogues (12ndash14) compound with X substitution as ldquoNHrdquoshowed more activity than as ldquoOrdquo substitution
The results of 5-dose assay of the most active compoundsamong their respective series are given in Table 3 Compound8 presented GI
50ranging between 0004 and 204 120583M The
12 BioMed Research International
best results were recorded on the leukemia cell lines withvalues ranging from 0004 to 0364 120583M (Figure 2(a)) Anumber of 3 tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 0383120583M Only in 23 cell linescompound 8 registered LC
50valuewithgt100120583MCompound
10 presented GI50ranging between 00079 and 186 120583M The
best results were recorded on the leukemia cell line withvalue ranging from 00353 to 0347 120583M (Figure 2(b)) Fourof the tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 000815120583M Only in 27 cell linescompound 10 registered LC
50value with gt100 120583M Com-
pound 13 presented GI50ranging between 0524 and 339 120583M
The best results were recorded on the colon cancer cell linewith values ranging from 0543 to 218 120583M(Figure 2(c)) Onlyone tested cancer cell line presented TGI value with gt100 120583Mthe best result value being noted on the HT29 (colon cancer)with value 0948 In 28 cell lines compound 13 registeredLC50value with gt100120583M
Some of the synthesized compounds were evaluated forEGFR tyrosine kinase inhibitory activity by recombinanttyrosine kinase assay using an ELISA-based assay withpoly(Glu Tyr 4 1) as a substrate [37] The results showedcompounds 8 10 and 13 shows inhibition of EGFR tyrosinekinase with IC
50values of 355 292 and 557120583Mrespectively
33 Molecular Docking The binding site of the referenceinhibitor is well defined by hydrophobic cavity The EGFRtyrosine kinase binding site contains the important residuesCys797 andThr790 re docking of reference ligand shown thatabove residues are important for hydrophobic interactionswith the same [38] The hydrophobic cavity with the residuesLeu788 Met766 Lys745 Glu762 Thr854 and Met793 makesthe binding site attractive for the design of new inhibitorsBased on our knowledge we designed some class of newpyrazole (1ndash11) dihydropyrimidine analogues (12ndash14) forEGFR tyrosine kinase inhibition But the binding sites ofour new molecules were found to be a little different withcomparison to reference inhibitor which is reported in thecrystal structure of EGFR tyrosine kinase (PDB 2J5F) Theseligandsrsquo interactions were shown in three different motifsThe first one is hydrophobic cavity in which the substitutedmethoxy hydroxy phenyl ring of the ligand is showinggood interactions as reference inhibitor The next bindingmotif contains the target residue Cys797 the N-substitutedpyrazole derivatives and six membered (dihydropyrimidine)are binding at this site Most of these interactions werehydrophobic The last binding motif contains the residuesLys875 and Asp831 which lie in side chain of the protein andshowed the side chain hydrogen bonding with the designedmolecules The docking score and Emodel score of some ofthe compounds selected for anticancer activity by NCI aregiven in Table 4 and binding modes are shown in Figure 3
Compound 13 showed the best hydrophobic interactionwith various residues such as Cys797 Met 793 Leu 844 Leu792 Ala 743 and Val 726 (Figures 4(a) and 4(b)) Negativelycharged Asp 837 interacted with methoxy hydroxy phenyl
ring (4-hydroxy-3-methoxyphenyl) Arg 841 showed 120587-120587cationic interaction The Met 793 present in the hydrophobiccavity showed H-bond backbone interaction and H-bond(side chain) interaction The positively charged Lys 845and Arg 841 destabilized interactions The interactions ofcompound 13 with the EGFR kinase receptor shown inribbon diagram the hydrophobic cavity is indicated withyellow colour loop motif-1 is indicated with green colourbeta sheet and the motif-2 is indicated with green colourloop The other ligands were also having good interactionswith the hydrophobic cavity and bindingmotifs the followingresidues leu718 Pro794 Met793 Ala743 Leu844 Val726and Cys797 were present around the ligand Thr790 Gln791having polar characteristics andMet 793 were responsible forbackbone H-bond interaction as well as H-bond (side chain)interaction Asp855 and Asp837 showed charged interactionslike destabilizing contacts Positively charged Lys875 andArg841 were found to have 120587-120587 cationic interaction and werepresent in bindingmotifmaking different binding patterns ofreported ligandsThe docking study for compound 3 is givenin Figures 5(a) and 5(b)
4 Conclusion
The novel series of curcumin analogues (pyrazoles) weresynthesized in satisfactory yields The anticancer activityshowed promising results The studies confirmed compound10 as potent lead compound for drug discovery and furtheroptimization The curcumin analogues discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer disease The pyrazole discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer
Acknowledgments
The authors are thankful to the staff members of the NationalCancer Institute (NCI) USA for in vitro anticancer screeningof the newly synthesized compounds They are especiallygrateful to Professor Dizon B Thelma Project ManagerNational Cancer Institute (NCI) USA
References
[1] WHO Cancer World Health Organization February 2006httpenwikipediaorgwikicancer
[2] M N Noolvi H M Patel V Bhardwaj and A ChauhanldquoSynthesis and in vitro antitumor activity of substituted quina-zoline and quinoxaline derivatives search for anticancer agentrdquoEuropean Journal of Medicinal Chemistry vol 46 no 6 pp2327ndash2346 2011
[3] N Aydemir and R Bilaloglu ldquoGenotoxicity of two anticancerdrugs gemcitabine and topotecan in mouse bone marrow invivordquoMutation Research vol 537 no 1 pp 43ndash51 2003
[4] I Bouabdallah L A MrsquoBarek A Zyad A Ramdani I Zidaneand A Melhaoui ldquoNew pyrazolic compounds as cytotoxicagentsrdquo Natural Product Research vol 21 no 4 pp 298ndash3022007
BioMed Research International 13
[5] D Havrylyuk B Zimenkovsky O Vasylenko L Zaprutko AGzella and R Lesyk ldquoSynthesis of novel thiazolone-based com-pounds containing pyrazoline moiety and evaluation of theiranticancer activityrdquo European Journal of Medicinal Chemistryvol 44 no 4 pp 1396ndash1404 2009
[6] M Shaharyar M M Abdullah M A Bakht and J MajeedldquoPyrazoline bearing benzimidazoles search for anticanceragentrdquo European Journal of Medicinal Chemistry vol 45 no 1pp 114ndash119 2010
[7] P-C Lv H-Q Li J Sun Y Zhou and H-L Zhu ldquoSynthesisand biological evaluation of pyrazole derivatives containingthiourea skeleton as anticancer agentsrdquo Bioorganic and Medici-nal Chemistry vol 18 no 13 pp 4606ndash4614 2010
[8] M S Christodoulou S Liekens K M Kasiotis and S AHaroutounian ldquoNovel pyrazole derivatives synthesis and eval-uation of anti-angiogenic activityrdquo Bioorganic and MedicinalChemistry vol 18 no 12 pp 4338ndash4350 2010
[9] M J Ahsan G J Samy H Khalillah R C Krit and SSoni ldquoMolecular properties prediction and synthesis of novelpyrazoline carboxamide analogs as antitubercular agentsrdquoAnti-Infective Agents vol 10 no 2 pp 117ndash123 2012
[10] M J Ahsan J G Samy H Khalilullah M A Bakht andM Z Hassan ldquoSynthesis and antimycobacterial evaluation of3a4-dihydro-3H-indeno [12-c] pyrazole-2-carboxamide ana-loguesrdquo European Journal of Medicinal Chemistry vol 46 no11 pp 5694ndash5697 2011
[11] M J Ahsan J G Samy K R Dutt et al ldquoDesign synthesis andantimycobacterial evaluation of novel 3-substituted-N-aryl-67-dimethoxy-3a4-dihydro-3H-indeno[12-c] pyrazole-2-carboxamide analoguesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 15 pp 4451ndash4453 2011
[12] M J Ahsan J G Samy S Soni et al ldquoDiscovery of novelantitubercular 3a4-dihydro-3H-indeno[12-c]pyrazole-2-carndashboxamidecarbothioamide analoguesrdquo Bioorganic and Medici-nal Chemistry Letters vol 21 no 18 pp 5259ndash5261 2011
[13] M J Ahsan H Khalilullah J P Stables and J GovindasamyldquoSynthesis and anticonvulsant activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide carbothioamide ana-loguesrdquo Journal of Enzyme Inhibition amp Medicinal Chemistryvol 28 no 3 pp 644ndash650 2013
[14] M J Ahsan J Govindasamy H Khalilullah et al ldquoPOMAanalyses as new efficient bioinformaticsrsquoplatform to predictand optimize bioactivity of synthesized 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamidecarbothioamide anal-oguesrdquo Bioorganic amp Medicinal Chemistry vol 22 no 24 pp7029ndash7035 2012
[15] H Khalilullah S Khan M J Ahsan and B AhmedldquoSynthesis and antihepatotoxic activity of 5-(23-dihydro-14-benzodioxane-6-yl)-3-substituted-phenyl-45-dihydro-1H-pyrazole derivativesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 24 pp 7251ndash7254 2011
[16] M J Alam M J Ahsan O Alam and S A KhanldquoSynthesis of 4-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)-6-(substituted phenyl)pyrimidin-2-ol analogues as 8 anti-inflammatory and analgesic agentsrdquo Letters in Drug Design ampDiscovery vol 10 no 8 pp 776ndash782 2013
[17] D P Chauhan ldquoChemotherapeutic potential of curcumin forcolorectal cancerrdquo Current Pharmaceutical Design vol 8 no 19pp 1695ndash1706 2002
[18] Y-L Jia J Li Z-H Qin and Z-Q Liang ldquoAutophagic andapoptotic mechanisms of curcumin-induced death in K562
cellsrdquo Journal of Asian Natural Products Research vol 11 no 11pp 918ndash928 2009
[19] A Kunwar A Barik BMishra K Rathinasamy R Pandey andK I Priyadarsini ldquoQuantitative cellular uptake localization andcytotoxicity of curcumin in normal and tumor cellsrdquoBiochimicaet Biophysica Acta vol 1780 no 4 pp 673ndash679 2008
[20] J Lal S K Gupta DThavaselvam and D D Agarwal ldquoDesignsynthesis synergistic antimicrobial activity and cytotoxicityof 4-aryl substituted 34-dihydropyrimidinones of curcuminrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 8 pp2872ndash2876 2012
[21] R K Singh D Rai D Yadav A Bhargava J Balzarini and EDe Clercq ldquoSynthesis antibacterial and antiviral properties ofcurcumin bioconjugates bearing dipeptide fatty acids and folicacidrdquo European Journal of Medicinal Chemistry vol 45 no 3pp 1078ndash1086 2010
[22] K Saja M S Babu D Karunagaran and P R SudhakaranldquoAnti-inflammatory effect of curcumin involves downregula-tion of MMP-9 in blood mononuclear cellsrdquo InternationalImmunopharmacology vol 7 no 13 pp 1659ndash1667 2007
[23] S Mishra K Karmodiya N Surolia and A Surolia ldquoSynthesisand exploration of novel curcumin analogues as anti-malarialagentsrdquo Bioorganic and Medicinal Chemistry vol 16 no 6 pp2894ndash2902 2008
[24] G Liang L Shao Y Wang et al ldquoExploration and synthesis ofcurcumin analogues with improved structural stability both invitro and in vivo as cytotoxic agentsrdquo Bioorganic and MedicinalChemistry vol 17 no 6 pp 2623ndash2631 2009
[25] M J Ahsan ldquoSynthesis and anticancer activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide analoguesrdquo Lettersin Drug Design amp Discovery vol 9 no 9 pp 823ndash827 2012
[26] Salahuddin M Shaharyar A Majumdar and M J AhsanldquoSynthesis characterization and anticancer evaluation of 2-(naphthalen-1-ylmethylnaphthalen-2-yloxymethyl)-1-[5-(sub-stituted phenyl)-[134]oxadiazol-2-ylmethyl]-1H-benzimida-zolerdquo Arabian Journal of Chemistry 2013
[27] E Zwick J Bange andAUllrich ldquoReceptor tyrosine kinase sig-nalling as a target for cancer intervention strategiesrdquo Endocrine-Related Cancer vol 8 no 3 pp 161ndash173 2001
[28] H A Bhuva and S G Kini ldquoSynthesis anticancer activity anddocking of some substituted benzothiazoles as tyrosine kinaseinhibitorsrdquo Journal ofMolecular Graphics andModelling vol 29no 1 pp 32ndash37 2010
[29] J Mendelsohn and J Baselga ldquoThe EGF receptor family astargets for cancer therapyrdquo Oncogene vol 19 no 56 pp 6550ndash6565 2000
[30] M Amir M J Ahsan and I Ali ldquoSynthesis of N1-(3-chloro-4-flouropheny)-N4-substituted semicarbazones as novel anticon-vulsant agentsrdquo Indian Journal of Chemistry B vol 49 no 11 pp1509ndash1514 2010
[31] httpdtpncinihgov[32] A Monks D Scudiero P Skehan et al ldquoFeasibility of a high-
flux anticancer drug screen using a diverse panel of culturedhuman tumor cell linesrdquo Journal of theNational Cancer Institutevol 83 no 11 pp 757ndash766 1991
[33] M R Body and K D Paull ldquoSome practical considerations andapplications of the national cancer institute in vitro anticancerdrug discovery screenrdquoDrug Development Research vol 34 no2 pp 91ndash109 1995
[34] R H Shoemaker ldquoThe NCI60 human tumour cell line anti-cancer drug screenrdquo Nature Reviews Cancer vol 6 no 10 pp813ndash823 2006
14 BioMed Research International
[35] M C Alley D A Scudiero A Monks et al ldquoFeasibility ofdrug screening with panels of human tumor cell lines using amicroculture tetrazolium assayrdquo Cancer Research vol 48 no 3pp 589ndash601 1988
[36] M R Grever S A Schepartz and B A Chabner ldquoTheNationalCancer Institute cancer drug discovery and development pro-gramrdquo Seminars in Oncology vol 19 no 6 pp 622ndash638 1992
[37] P Dubreuil S Letard M Ciufolini et al ldquoMasitinib (AB1010)a potent and selective tyrosine kinase inhibitor targeting KITrdquoPLoS ONE vol 4 no 9 Article ID e7258 2009
[38] J A Blair D Rauh C Kung et al ldquoStructure-guided devel-opment of affinity probes for tyrosine kinases using chemicalgeneticsrdquo Nature Chemical Biology vol 3 no 4 pp 229ndash2382007
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
parameters and without any constraints Site has been speci-fied as centroid of the work space ligand (20 A) with van derWaals radius scaling factor 10 and partial charge cutoff 025
264 Molecular Docking Protocol The ligand docking wasperformed in GLIDE50 Ligands which show less than35 rotatable bonds and which are having less than 200atoms were selected The scaling factor will be 080 and thepotential charge cutoff is 015 All the conformers from theconfgen-ligprep output were docked in the EGFR TyrosineKinase active site All default parameters were used for extraprecision docking Glide extra precision mode was employedfor the current docking study Best poses were chosen forenergy minimization during docking a distance dependentdielectric constant of 20 and maximum number of min-imization step of 100 was used The docking simulations(Ligand receptor interactions) are scored using the Xtraprecision (XP) mode which is implemented in GLIDE50
27 Materials and Methods All computational analysis wascarried out on a Red Hat 50 Linux platform running on aDell Precision work station with Intel core 2 quad processorand 8GB of RAM
3 Results and Discussion
31 Chemistry The curcumin analogues (1ndash14) described inthe study are shown in Table 1 and the reaction sequencefor the synthesis is summarized in Scheme 1 In the first part
of the synthesis 17-bis(4-hydroxy-3-methoxyphenyl)hepta-16-diene-35-dione (curcumin) and substituted phenyl semi-carbazide were refluxed in glacial acetic acid to obtainthe 35-bis(4-hydroxy-3-methoxystyryl)-N-(substituted phe-nyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) Substi-tuted phenyl semicarbazides (ArNHCONHNH
2) were syn-
thesized as per reported method [30] In the second partof the synthesis 17-bis(4-hydroxy-3-methoxyphenyl)hepta-16-diene-35-dione (curcumin) and substituted phenylhy-drazide (ArCONHNH
2) were refluxed in glacial acetic acid to
obtain the 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl(substituted phenyl)methanone (10-11) In the third partof synthesis 17-bis(4-hydroxy-3-methoxyphenyl)hepta-16-diene-35-dione (curcumin) and ureaguanidinethioureawere refluxed in glacial acetic acid to obtain the pyrimidineanalogues (12ndash14) The plausible mechanism of reactions isgiven in Figure 1 The yields of the title compounds wereranging from 66 to 88 after recrystallization with absoluteethanol The completion of reaction was monitored by TLCusing mobile phase hexane ethylacetate (6 4) and purityof the compounds was checked by elemental analyses Boththe analytical and spectral data (IR NMR and MS) of thesynthesized compounds were in full accordance with theproposed structures In general IR spectra of the compoundsafforded C=N stretching at 1514ndash1583 cmminus1 and CndashN stretchat 1320ndash1335 cmminus1 and carbamoyl group NndashH stretching at3122ndash3210 cmminus1 andC=O stretching at 1680ndash1689 cmminus1 bandsfor carboxamide analogues (1ndash9) and at 1755ndash1757 cmminus1bands formethanone analogues (10-11)The 1HNMR spectra
BioMed Research International 7
Table 3 NCI in vitro testing results of compounds of the compounds 8 10 and 13 at five-dose level in 120583M
S no Panelcell line 8 (NSC 757926) 10 (NSC 757929) 13 (NSC 763375)GI50 TGI LC50 GI50 TGI LC50 GI50 TGI LC50
(1)
LeukemiaCCRF-CEM 0232 gt100 gt100 0127 gt100 gt100 0543 NT gt100HL-60(TB) 0237 0806 gt100 00884 NT gt100 218 617 gt100MOLT-4 0364 NT gt100 0347 gt100 gt100 101 519 gt100RPMI-8226 0336 NT gt100 0275 NT gt100 129 418 gt100SR 0004 gt100 gt100 00353 gt100 gt100 072 358 gt100
Colon cancerCOLO 205 205 362 638 186 334 597 18 324 585HCC-2998 064 58 gt100 0591 271 gt100 175 319 579HCT-116 0323 NT NT 0269 114 NT 11 239 518HCT-15 0335 166 NT 0167 152 NT 282 103 gt100HT29 0696 28 994 0463 319 gt100 314 0948 421KM12 0298 125 NT 00092 119 NT 163 332 679SW-620 0441 NT gt100 00079 NT gt100 165 368 NT
(4)
CNS cancerSF-268 0571 NT NT 0256 NT NT 211 156 gt100SF-295 0546 NT gt100 0538 NT NT 193 454 gt100SF-539 0354 15 NT 00604 153 NT 205 573 gt100SNB-19 0634 201 NT 0572 212 NT 181 795 gt100SNB-75 0416 182 NT 00099 187 NT 126 334 884U251 0471 159 NT 0428 170 NT 179 569 663
(5)
MelanomaLOX IMVI 0284 149 NT 00626 156 NT 127 259 NTMALME-3M 0563 198 NT 0525 217 NT 271 676 gt100M14 0372 141 NT 0155 148 NT 165 344 NTMDA-MB-435 00448 0383 NT 00175 000815 gt100 171 NT gt100SK-MEL-2 0366 182 NT 0266 203 gt100 198 41 NTSK-MEL-28 0509 247 861 0176 272 905 185 453 26SK-MEL-5 0317 129 367 0171 130 369 149 289 559UACC-257 154 547 319 114 429 533 153 35 8UACC-62 0413 165 489 0371 164 NT 149 323 NT
8 BioMed Research International
Table 3 Continued
S no Panelcell line 8 (NSC 757926) 10 (NSC 757929) 13 (NSC 763375)GI50 TGI LC50 GI50 TGI LC50 GI50 TGI LC50
showed singlet at 120575 119ndash128 ppm corresponding to CH3
a singlet at 120575 379ndash385 ppm corresponding to OCH3 a
singlet at 120575 652ndash662 ppm corresponding to CH=C proton(pyrazoledihydropyrimidine) a doublet at 120575 661ndash675 ppmcorresponding toCH=CHproton a doublet at 665ndash681 ppmcorresponding to CH=CH proton a multiplet at 120575 681ndash793 ppm corresponding to aromatic protons broad singlet at120575 993ndash1016 ppm corresponding to CONH
2 The compounds
(1ndash14) in 1HNMRspectra exhibited two doublets with 119869 valuebetween 146 and 166Hz confirming the trans coupling Themass spectra of the compounds revealed in each case a peakcorresponding to their molecular ion peaks The elementalanalysis results were within plusmn04 of the theoretical values
32 Anticancer Activity Ten compounds were evaluated fortheir anticancer activity in both one-dose and 5-dose assaysThe observed anticancer screening data of the compoundsare given in Table 2 The 5-dose assay screening data ofthree compounds are given in Table 3 Compound 1 wasfound to be highly active on COLO 205 (colon cancer)with cell promotion of minus7349 followed by RXF 393
BioMed Research International 9
HO
O
OH
O
HO
ONN
OH
ONH
OR
HO
ONN
OH
OO
R
HO
ONHN
OH
O
X
AcOHReflux
Curcumin
ArCONHNH2
ArNHCONHNH2
NH2CXNH2
1ndash9
12ndash14
10-11
OO
Scheme 1 Protocol for the synthesis of curcumin analogues
OO
HOO
OHO
OOH
HOO
OHO
Diketone (tautomer I) Keto-enol (tautomer II)
OOH
HOO
OHO O
NH NH
N N
OHOO
HO
O
NH
R
OHOH
HOO
OHO
NHNH
ONH
OH
HOO
OHO
NNH
X
NH
R
R
R
Curcumin
OOH
HOO
OHO
R
CurcuminO
NH
OHOH
HOO
OHO
NHNH
O
R
R
OH
HOO
OHO
NNH
OR
OOH
HOO
OHO X
OHOH
HOO
OHO
NH
O
OH
HOO
OHO
N
X
HN N
X
OHOO
HO
Curcumin
N N
OHOO
HO
O
H3C H3C
H3C
H3C
H3C
CH3
CH3
H3C CH3 H3C H3CCH3CH3
CH3
CH3
CH3
CH3CH3CH3
H2N
H2N
H2N
H3CH3CH3CCH3H3CH2N
H2N
minusH2O
minusH2O
minusH2O
minusH2O minusH2O
minusH2O
NH2
1ndash9
12ndash1410-11
X = S O
Figure 1 Plausible mechanism of reaction for the synthesis of pyrazole analogues (1ndash14)
(renal cancer) with cell promotion of minus5032 and HT29(colon cancer) with cell promotion of minus3495 while themaximum cell promotion was observed on NCIADR-RES(ovarian cancer) which showed 2083 growth promotion(7917 growth inhibition) at one-dose assay Compound2 was found to be highly active on RXF 393 (renal cancer)with cell promotion of minus5360 followed by SK-MEL-5(melanoma) with cell promotion of minus2966 and MDA-MB-468 (breast cancer) with cell promotion of minus2640 whilethe maximum cell promotion was observed on TK-10 (renalcancer) which showed 2310 growth promotion (7690growth inhibition) at one-dose assay The compound 3was found to be highly active on RXF 393 (renal cancer)
with cell promotion of minus4384 followed by HT29 (coloncancer) with cell promotion of minus4184 and SK-MEL-2(melanoma) with cell promotion of minus2707 while themaximum cell promotion was observed on TK-10 (renalcancer) which showed 3812 growth promotion (6188growth inhibition) at one-dose assay Compound 5 wasfound to be highly active on COLO 205 (colon cancer)with cell promotion of minus5943 followed by RXF 393 (renalcancer) with cell promotion of minus5761 and SF-295 (CNScancer) with cell promotion of minus5532 while the maximumcell promotion was observed on TK-10 (renal cancer)which showed 2404 growth promotion (7596 growthinhibition) at one-dose assay Compound 6 was found to
10 BioMed Research International
100
50
0
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
LeukemiaG
row
th (
)
log10 of sample concentration (molar)
CCRF-CEMMOLT-4HL-60(TB)
RPMI-8226K-562SR
(a)
100
50
0
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
Leukemia
Gro
wth
()
log10 of sample concentration (molar)
CCRF-CEMMOLT-4HL-60(TB)
RPMI-8226K-562SR
(b)
100
50
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
Gro
wth
()
log10 of sample concentration (molar)
0
COLO 205HCT-15SW-620HCC-2998
HT29HCT-116KM12
Colon cancer
(c)
Figure 2 (a) Five-dose anticancer screening of compound 8 on leukemia cell line (b) Five-dose anticancer screening of compound 10 onleukemia cell line (c) Five-dose anticancer screening of compound 13 on colon cancer cell line
Figure 3 Green colour stick model indicates the reference ligand and atom macromodel stick indicates our ligands
be highly active on RXF 393 (renal) with cell promotionof minus4746 followed by MDA-MB-468 (breast cancer)with cell promotion of minus2914 and SK-OV-3 (ovariancancer) with cell promotion of minus2909 while the maximumcell promotion was observed on TK-10 (renal cancer)
which showed 2855 growth promotion (7145 growthinhibition) at one-dose assay Compound 8 was foundto be highly active on SK-MEL-5 (melanoma) with cellpromotion of minus8344 followed by RXF 393 (renal cancer)with cell promotion of minus6729 and SK-OV-3 (ovarian
BioMed Research International 11
(a) (b)
Figure 4 (a) Compound 13 is represented with macromodel stick modelThe hydrogen bonds are shown with yellow colour dotted lines andhydrophobic interactions with orange dotted lines (b) Compound 13 at binding site on surface
(a) (b)
Figure 5 (a) Compound 3 is represented with macromodel stick modelThe hydrophobic cavity residues are represented with magenta stickmodelThe hydrogen bonds are shownwith yellow colour dotted lines and hydrophobic interactions with orange dotted lines (b) Compound3 at binding site on surface
cancer) with cell promotion of minus6358 while the maximumcell promotion was observed on TK-10 (renal cancer)which showed 1584 growth promotion (8416 growthinhibition) at one-dose assay The compound 10 was foundto be highly active on SK-MEL-5 (melanoma) with cellpromotion of minus8652 followed by UACC-62 (melanoma)with cell promotion of minus8439 and COLO 205 (coloncancer) with cell promotion of minus8405 while the maximumcell promotion was observed on UACC-257 (melanoma)which showed 1603 growth promotion (8397 growthinhibition) at one-dose assay Compound 11 was found to behighly active on CAKI-1 (renal cancer) with cell promotionof minus3477 followed by MDA-MB-435 (melanoma) with cellpromotion of minus3053 and SF-295 (CNS cancer) with cellpromotion of minus2968 while the maximum cell promotionwas observed on UACC-257 (melanoma) which showed8713 growth promotion (1287 growth inhibition) at one-dose assay Compound 12 was found to be highly active onHT29 (colon cancer) with cell promotion of minus641 followedby MDA-MB-468 (breast cancer) with cell promotion ofminus634 and CCRF-CEM (leukemia) with cell promotion ofminus1672 while the maximum cell promotion was observedon TK-10 (renal cancer) which showed 10274 growthpromotion at one-dose assay Compound 13 was found to behighly active on HT29 (colon cancer) with cell promotion ofminus7624 followed by 786-0 and A498 (renal cancer) with cellpromotion of minus5893 and minus5692 respectively while themaximum cell promotion was observed on NCIADR-RES(ovarian cancer) which showed 3932 growth promotion(1287 growth inhibition) at one-dose assay Among the35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) Compound
8 was found to be the most active compound of theseries with mean growth percent of minus1919 followed bycompound 5 which showed mean growth percent of minus1225and compound 1 which showed mean growth percent ofminus466 Among the 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl (substituted phenyl)methanone analogues(10-11) Compound 10 showed maximum activity with meangrowth percent of minus2871 while compound 13 was found tobe the most active compound among dihydropyrimidineanalogues (12ndash14) which showed mean growth percent ofminus546 Overall the most active compound of the serieswas compound 10 and most of the 35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carbox-amide analogues possessed potent anticancer activity Basedon our finding we can conclude that electronegative groupat position 2 (ie 2-chloro) on N-substituted phenyl ringamong 35-bis(4-hydroxy-3-methylstyryl)-N-(substitutedphenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) showedmore anticancer activity than at position 4 (ie 4-chloro and4-fluoro) Electron releasing group such as 24-dimethyl and4-methyl on N-substituted phenyl ring increased anticanceractivity and the activity was found to be more if thenumber of electron releasing groups was increased Amongthe 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl(substituted phenyl)methanone analogues (10 and 11) theactivity was found to bemore when there was no substitutionin the phenyl ring Among six member dihydropyrimidineanalogues (12ndash14) compound with X substitution as ldquoNHrdquoshowed more activity than as ldquoOrdquo substitution
The results of 5-dose assay of the most active compoundsamong their respective series are given in Table 3 Compound8 presented GI
50ranging between 0004 and 204 120583M The
12 BioMed Research International
best results were recorded on the leukemia cell lines withvalues ranging from 0004 to 0364 120583M (Figure 2(a)) Anumber of 3 tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 0383120583M Only in 23 cell linescompound 8 registered LC
50valuewithgt100120583MCompound
10 presented GI50ranging between 00079 and 186 120583M The
best results were recorded on the leukemia cell line withvalue ranging from 00353 to 0347 120583M (Figure 2(b)) Fourof the tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 000815120583M Only in 27 cell linescompound 10 registered LC
50value with gt100 120583M Com-
pound 13 presented GI50ranging between 0524 and 339 120583M
The best results were recorded on the colon cancer cell linewith values ranging from 0543 to 218 120583M(Figure 2(c)) Onlyone tested cancer cell line presented TGI value with gt100 120583Mthe best result value being noted on the HT29 (colon cancer)with value 0948 In 28 cell lines compound 13 registeredLC50value with gt100120583M
Some of the synthesized compounds were evaluated forEGFR tyrosine kinase inhibitory activity by recombinanttyrosine kinase assay using an ELISA-based assay withpoly(Glu Tyr 4 1) as a substrate [37] The results showedcompounds 8 10 and 13 shows inhibition of EGFR tyrosinekinase with IC
50values of 355 292 and 557120583Mrespectively
33 Molecular Docking The binding site of the referenceinhibitor is well defined by hydrophobic cavity The EGFRtyrosine kinase binding site contains the important residuesCys797 andThr790 re docking of reference ligand shown thatabove residues are important for hydrophobic interactionswith the same [38] The hydrophobic cavity with the residuesLeu788 Met766 Lys745 Glu762 Thr854 and Met793 makesthe binding site attractive for the design of new inhibitorsBased on our knowledge we designed some class of newpyrazole (1ndash11) dihydropyrimidine analogues (12ndash14) forEGFR tyrosine kinase inhibition But the binding sites ofour new molecules were found to be a little different withcomparison to reference inhibitor which is reported in thecrystal structure of EGFR tyrosine kinase (PDB 2J5F) Theseligandsrsquo interactions were shown in three different motifsThe first one is hydrophobic cavity in which the substitutedmethoxy hydroxy phenyl ring of the ligand is showinggood interactions as reference inhibitor The next bindingmotif contains the target residue Cys797 the N-substitutedpyrazole derivatives and six membered (dihydropyrimidine)are binding at this site Most of these interactions werehydrophobic The last binding motif contains the residuesLys875 and Asp831 which lie in side chain of the protein andshowed the side chain hydrogen bonding with the designedmolecules The docking score and Emodel score of some ofthe compounds selected for anticancer activity by NCI aregiven in Table 4 and binding modes are shown in Figure 3
Compound 13 showed the best hydrophobic interactionwith various residues such as Cys797 Met 793 Leu 844 Leu792 Ala 743 and Val 726 (Figures 4(a) and 4(b)) Negativelycharged Asp 837 interacted with methoxy hydroxy phenyl
ring (4-hydroxy-3-methoxyphenyl) Arg 841 showed 120587-120587cationic interaction The Met 793 present in the hydrophobiccavity showed H-bond backbone interaction and H-bond(side chain) interaction The positively charged Lys 845and Arg 841 destabilized interactions The interactions ofcompound 13 with the EGFR kinase receptor shown inribbon diagram the hydrophobic cavity is indicated withyellow colour loop motif-1 is indicated with green colourbeta sheet and the motif-2 is indicated with green colourloop The other ligands were also having good interactionswith the hydrophobic cavity and bindingmotifs the followingresidues leu718 Pro794 Met793 Ala743 Leu844 Val726and Cys797 were present around the ligand Thr790 Gln791having polar characteristics andMet 793 were responsible forbackbone H-bond interaction as well as H-bond (side chain)interaction Asp855 and Asp837 showed charged interactionslike destabilizing contacts Positively charged Lys875 andArg841 were found to have 120587-120587 cationic interaction and werepresent in bindingmotifmaking different binding patterns ofreported ligandsThe docking study for compound 3 is givenin Figures 5(a) and 5(b)
4 Conclusion
The novel series of curcumin analogues (pyrazoles) weresynthesized in satisfactory yields The anticancer activityshowed promising results The studies confirmed compound10 as potent lead compound for drug discovery and furtheroptimization The curcumin analogues discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer disease The pyrazole discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer
Acknowledgments
The authors are thankful to the staff members of the NationalCancer Institute (NCI) USA for in vitro anticancer screeningof the newly synthesized compounds They are especiallygrateful to Professor Dizon B Thelma Project ManagerNational Cancer Institute (NCI) USA
References
[1] WHO Cancer World Health Organization February 2006httpenwikipediaorgwikicancer
[2] M N Noolvi H M Patel V Bhardwaj and A ChauhanldquoSynthesis and in vitro antitumor activity of substituted quina-zoline and quinoxaline derivatives search for anticancer agentrdquoEuropean Journal of Medicinal Chemistry vol 46 no 6 pp2327ndash2346 2011
[3] N Aydemir and R Bilaloglu ldquoGenotoxicity of two anticancerdrugs gemcitabine and topotecan in mouse bone marrow invivordquoMutation Research vol 537 no 1 pp 43ndash51 2003
[4] I Bouabdallah L A MrsquoBarek A Zyad A Ramdani I Zidaneand A Melhaoui ldquoNew pyrazolic compounds as cytotoxicagentsrdquo Natural Product Research vol 21 no 4 pp 298ndash3022007
BioMed Research International 13
[5] D Havrylyuk B Zimenkovsky O Vasylenko L Zaprutko AGzella and R Lesyk ldquoSynthesis of novel thiazolone-based com-pounds containing pyrazoline moiety and evaluation of theiranticancer activityrdquo European Journal of Medicinal Chemistryvol 44 no 4 pp 1396ndash1404 2009
[6] M Shaharyar M M Abdullah M A Bakht and J MajeedldquoPyrazoline bearing benzimidazoles search for anticanceragentrdquo European Journal of Medicinal Chemistry vol 45 no 1pp 114ndash119 2010
[7] P-C Lv H-Q Li J Sun Y Zhou and H-L Zhu ldquoSynthesisand biological evaluation of pyrazole derivatives containingthiourea skeleton as anticancer agentsrdquo Bioorganic and Medici-nal Chemistry vol 18 no 13 pp 4606ndash4614 2010
[8] M S Christodoulou S Liekens K M Kasiotis and S AHaroutounian ldquoNovel pyrazole derivatives synthesis and eval-uation of anti-angiogenic activityrdquo Bioorganic and MedicinalChemistry vol 18 no 12 pp 4338ndash4350 2010
[9] M J Ahsan G J Samy H Khalillah R C Krit and SSoni ldquoMolecular properties prediction and synthesis of novelpyrazoline carboxamide analogs as antitubercular agentsrdquoAnti-Infective Agents vol 10 no 2 pp 117ndash123 2012
[10] M J Ahsan J G Samy H Khalilullah M A Bakht andM Z Hassan ldquoSynthesis and antimycobacterial evaluation of3a4-dihydro-3H-indeno [12-c] pyrazole-2-carboxamide ana-loguesrdquo European Journal of Medicinal Chemistry vol 46 no11 pp 5694ndash5697 2011
[11] M J Ahsan J G Samy K R Dutt et al ldquoDesign synthesis andantimycobacterial evaluation of novel 3-substituted-N-aryl-67-dimethoxy-3a4-dihydro-3H-indeno[12-c] pyrazole-2-carboxamide analoguesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 15 pp 4451ndash4453 2011
[12] M J Ahsan J G Samy S Soni et al ldquoDiscovery of novelantitubercular 3a4-dihydro-3H-indeno[12-c]pyrazole-2-carndashboxamidecarbothioamide analoguesrdquo Bioorganic and Medici-nal Chemistry Letters vol 21 no 18 pp 5259ndash5261 2011
[13] M J Ahsan H Khalilullah J P Stables and J GovindasamyldquoSynthesis and anticonvulsant activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide carbothioamide ana-loguesrdquo Journal of Enzyme Inhibition amp Medicinal Chemistryvol 28 no 3 pp 644ndash650 2013
[14] M J Ahsan J Govindasamy H Khalilullah et al ldquoPOMAanalyses as new efficient bioinformaticsrsquoplatform to predictand optimize bioactivity of synthesized 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamidecarbothioamide anal-oguesrdquo Bioorganic amp Medicinal Chemistry vol 22 no 24 pp7029ndash7035 2012
[15] H Khalilullah S Khan M J Ahsan and B AhmedldquoSynthesis and antihepatotoxic activity of 5-(23-dihydro-14-benzodioxane-6-yl)-3-substituted-phenyl-45-dihydro-1H-pyrazole derivativesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 24 pp 7251ndash7254 2011
[16] M J Alam M J Ahsan O Alam and S A KhanldquoSynthesis of 4-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)-6-(substituted phenyl)pyrimidin-2-ol analogues as 8 anti-inflammatory and analgesic agentsrdquo Letters in Drug Design ampDiscovery vol 10 no 8 pp 776ndash782 2013
[17] D P Chauhan ldquoChemotherapeutic potential of curcumin forcolorectal cancerrdquo Current Pharmaceutical Design vol 8 no 19pp 1695ndash1706 2002
[18] Y-L Jia J Li Z-H Qin and Z-Q Liang ldquoAutophagic andapoptotic mechanisms of curcumin-induced death in K562
cellsrdquo Journal of Asian Natural Products Research vol 11 no 11pp 918ndash928 2009
[19] A Kunwar A Barik BMishra K Rathinasamy R Pandey andK I Priyadarsini ldquoQuantitative cellular uptake localization andcytotoxicity of curcumin in normal and tumor cellsrdquoBiochimicaet Biophysica Acta vol 1780 no 4 pp 673ndash679 2008
[20] J Lal S K Gupta DThavaselvam and D D Agarwal ldquoDesignsynthesis synergistic antimicrobial activity and cytotoxicityof 4-aryl substituted 34-dihydropyrimidinones of curcuminrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 8 pp2872ndash2876 2012
[21] R K Singh D Rai D Yadav A Bhargava J Balzarini and EDe Clercq ldquoSynthesis antibacterial and antiviral properties ofcurcumin bioconjugates bearing dipeptide fatty acids and folicacidrdquo European Journal of Medicinal Chemistry vol 45 no 3pp 1078ndash1086 2010
[22] K Saja M S Babu D Karunagaran and P R SudhakaranldquoAnti-inflammatory effect of curcumin involves downregula-tion of MMP-9 in blood mononuclear cellsrdquo InternationalImmunopharmacology vol 7 no 13 pp 1659ndash1667 2007
[23] S Mishra K Karmodiya N Surolia and A Surolia ldquoSynthesisand exploration of novel curcumin analogues as anti-malarialagentsrdquo Bioorganic and Medicinal Chemistry vol 16 no 6 pp2894ndash2902 2008
[24] G Liang L Shao Y Wang et al ldquoExploration and synthesis ofcurcumin analogues with improved structural stability both invitro and in vivo as cytotoxic agentsrdquo Bioorganic and MedicinalChemistry vol 17 no 6 pp 2623ndash2631 2009
[25] M J Ahsan ldquoSynthesis and anticancer activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide analoguesrdquo Lettersin Drug Design amp Discovery vol 9 no 9 pp 823ndash827 2012
[26] Salahuddin M Shaharyar A Majumdar and M J AhsanldquoSynthesis characterization and anticancer evaluation of 2-(naphthalen-1-ylmethylnaphthalen-2-yloxymethyl)-1-[5-(sub-stituted phenyl)-[134]oxadiazol-2-ylmethyl]-1H-benzimida-zolerdquo Arabian Journal of Chemistry 2013
[27] E Zwick J Bange andAUllrich ldquoReceptor tyrosine kinase sig-nalling as a target for cancer intervention strategiesrdquo Endocrine-Related Cancer vol 8 no 3 pp 161ndash173 2001
[28] H A Bhuva and S G Kini ldquoSynthesis anticancer activity anddocking of some substituted benzothiazoles as tyrosine kinaseinhibitorsrdquo Journal ofMolecular Graphics andModelling vol 29no 1 pp 32ndash37 2010
[29] J Mendelsohn and J Baselga ldquoThe EGF receptor family astargets for cancer therapyrdquo Oncogene vol 19 no 56 pp 6550ndash6565 2000
[30] M Amir M J Ahsan and I Ali ldquoSynthesis of N1-(3-chloro-4-flouropheny)-N4-substituted semicarbazones as novel anticon-vulsant agentsrdquo Indian Journal of Chemistry B vol 49 no 11 pp1509ndash1514 2010
[31] httpdtpncinihgov[32] A Monks D Scudiero P Skehan et al ldquoFeasibility of a high-
flux anticancer drug screen using a diverse panel of culturedhuman tumor cell linesrdquo Journal of theNational Cancer Institutevol 83 no 11 pp 757ndash766 1991
[33] M R Body and K D Paull ldquoSome practical considerations andapplications of the national cancer institute in vitro anticancerdrug discovery screenrdquoDrug Development Research vol 34 no2 pp 91ndash109 1995
[34] R H Shoemaker ldquoThe NCI60 human tumour cell line anti-cancer drug screenrdquo Nature Reviews Cancer vol 6 no 10 pp813ndash823 2006
14 BioMed Research International
[35] M C Alley D A Scudiero A Monks et al ldquoFeasibility ofdrug screening with panels of human tumor cell lines using amicroculture tetrazolium assayrdquo Cancer Research vol 48 no 3pp 589ndash601 1988
[36] M R Grever S A Schepartz and B A Chabner ldquoTheNationalCancer Institute cancer drug discovery and development pro-gramrdquo Seminars in Oncology vol 19 no 6 pp 622ndash638 1992
[37] P Dubreuil S Letard M Ciufolini et al ldquoMasitinib (AB1010)a potent and selective tyrosine kinase inhibitor targeting KITrdquoPLoS ONE vol 4 no 9 Article ID e7258 2009
[38] J A Blair D Rauh C Kung et al ldquoStructure-guided devel-opment of affinity probes for tyrosine kinases using chemicalgeneticsrdquo Nature Chemical Biology vol 3 no 4 pp 229ndash2382007
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Colon cancerCOLO 205 205 362 638 186 334 597 18 324 585HCC-2998 064 58 gt100 0591 271 gt100 175 319 579HCT-116 0323 NT NT 0269 114 NT 11 239 518HCT-15 0335 166 NT 0167 152 NT 282 103 gt100HT29 0696 28 994 0463 319 gt100 314 0948 421KM12 0298 125 NT 00092 119 NT 163 332 679SW-620 0441 NT gt100 00079 NT gt100 165 368 NT
(4)
CNS cancerSF-268 0571 NT NT 0256 NT NT 211 156 gt100SF-295 0546 NT gt100 0538 NT NT 193 454 gt100SF-539 0354 15 NT 00604 153 NT 205 573 gt100SNB-19 0634 201 NT 0572 212 NT 181 795 gt100SNB-75 0416 182 NT 00099 187 NT 126 334 884U251 0471 159 NT 0428 170 NT 179 569 663
(5)
MelanomaLOX IMVI 0284 149 NT 00626 156 NT 127 259 NTMALME-3M 0563 198 NT 0525 217 NT 271 676 gt100M14 0372 141 NT 0155 148 NT 165 344 NTMDA-MB-435 00448 0383 NT 00175 000815 gt100 171 NT gt100SK-MEL-2 0366 182 NT 0266 203 gt100 198 41 NTSK-MEL-28 0509 247 861 0176 272 905 185 453 26SK-MEL-5 0317 129 367 0171 130 369 149 289 559UACC-257 154 547 319 114 429 533 153 35 8UACC-62 0413 165 489 0371 164 NT 149 323 NT
8 BioMed Research International
Table 3 Continued
S no Panelcell line 8 (NSC 757926) 10 (NSC 757929) 13 (NSC 763375)GI50 TGI LC50 GI50 TGI LC50 GI50 TGI LC50
showed singlet at 120575 119ndash128 ppm corresponding to CH3
a singlet at 120575 379ndash385 ppm corresponding to OCH3 a
singlet at 120575 652ndash662 ppm corresponding to CH=C proton(pyrazoledihydropyrimidine) a doublet at 120575 661ndash675 ppmcorresponding toCH=CHproton a doublet at 665ndash681 ppmcorresponding to CH=CH proton a multiplet at 120575 681ndash793 ppm corresponding to aromatic protons broad singlet at120575 993ndash1016 ppm corresponding to CONH
2 The compounds
(1ndash14) in 1HNMRspectra exhibited two doublets with 119869 valuebetween 146 and 166Hz confirming the trans coupling Themass spectra of the compounds revealed in each case a peakcorresponding to their molecular ion peaks The elementalanalysis results were within plusmn04 of the theoretical values
32 Anticancer Activity Ten compounds were evaluated fortheir anticancer activity in both one-dose and 5-dose assaysThe observed anticancer screening data of the compoundsare given in Table 2 The 5-dose assay screening data ofthree compounds are given in Table 3 Compound 1 wasfound to be highly active on COLO 205 (colon cancer)with cell promotion of minus7349 followed by RXF 393
BioMed Research International 9
HO
O
OH
O
HO
ONN
OH
ONH
OR
HO
ONN
OH
OO
R
HO
ONHN
OH
O
X
AcOHReflux
Curcumin
ArCONHNH2
ArNHCONHNH2
NH2CXNH2
1ndash9
12ndash14
10-11
OO
Scheme 1 Protocol for the synthesis of curcumin analogues
OO
HOO
OHO
OOH
HOO
OHO
Diketone (tautomer I) Keto-enol (tautomer II)
OOH
HOO
OHO O
NH NH
N N
OHOO
HO
O
NH
R
OHOH
HOO
OHO
NHNH
ONH
OH
HOO
OHO
NNH
X
NH
R
R
R
Curcumin
OOH
HOO
OHO
R
CurcuminO
NH
OHOH
HOO
OHO
NHNH
O
R
R
OH
HOO
OHO
NNH
OR
OOH
HOO
OHO X
OHOH
HOO
OHO
NH
O
OH
HOO
OHO
N
X
HN N
X
OHOO
HO
Curcumin
N N
OHOO
HO
O
H3C H3C
H3C
H3C
H3C
CH3
CH3
H3C CH3 H3C H3CCH3CH3
CH3
CH3
CH3
CH3CH3CH3
H2N
H2N
H2N
H3CH3CH3CCH3H3CH2N
H2N
minusH2O
minusH2O
minusH2O
minusH2O minusH2O
minusH2O
NH2
1ndash9
12ndash1410-11
X = S O
Figure 1 Plausible mechanism of reaction for the synthesis of pyrazole analogues (1ndash14)
(renal cancer) with cell promotion of minus5032 and HT29(colon cancer) with cell promotion of minus3495 while themaximum cell promotion was observed on NCIADR-RES(ovarian cancer) which showed 2083 growth promotion(7917 growth inhibition) at one-dose assay Compound2 was found to be highly active on RXF 393 (renal cancer)with cell promotion of minus5360 followed by SK-MEL-5(melanoma) with cell promotion of minus2966 and MDA-MB-468 (breast cancer) with cell promotion of minus2640 whilethe maximum cell promotion was observed on TK-10 (renalcancer) which showed 2310 growth promotion (7690growth inhibition) at one-dose assay The compound 3was found to be highly active on RXF 393 (renal cancer)
with cell promotion of minus4384 followed by HT29 (coloncancer) with cell promotion of minus4184 and SK-MEL-2(melanoma) with cell promotion of minus2707 while themaximum cell promotion was observed on TK-10 (renalcancer) which showed 3812 growth promotion (6188growth inhibition) at one-dose assay Compound 5 wasfound to be highly active on COLO 205 (colon cancer)with cell promotion of minus5943 followed by RXF 393 (renalcancer) with cell promotion of minus5761 and SF-295 (CNScancer) with cell promotion of minus5532 while the maximumcell promotion was observed on TK-10 (renal cancer)which showed 2404 growth promotion (7596 growthinhibition) at one-dose assay Compound 6 was found to
10 BioMed Research International
100
50
0
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
LeukemiaG
row
th (
)
log10 of sample concentration (molar)
CCRF-CEMMOLT-4HL-60(TB)
RPMI-8226K-562SR
(a)
100
50
0
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
Leukemia
Gro
wth
()
log10 of sample concentration (molar)
CCRF-CEMMOLT-4HL-60(TB)
RPMI-8226K-562SR
(b)
100
50
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
Gro
wth
()
log10 of sample concentration (molar)
0
COLO 205HCT-15SW-620HCC-2998
HT29HCT-116KM12
Colon cancer
(c)
Figure 2 (a) Five-dose anticancer screening of compound 8 on leukemia cell line (b) Five-dose anticancer screening of compound 10 onleukemia cell line (c) Five-dose anticancer screening of compound 13 on colon cancer cell line
Figure 3 Green colour stick model indicates the reference ligand and atom macromodel stick indicates our ligands
be highly active on RXF 393 (renal) with cell promotionof minus4746 followed by MDA-MB-468 (breast cancer)with cell promotion of minus2914 and SK-OV-3 (ovariancancer) with cell promotion of minus2909 while the maximumcell promotion was observed on TK-10 (renal cancer)
which showed 2855 growth promotion (7145 growthinhibition) at one-dose assay Compound 8 was foundto be highly active on SK-MEL-5 (melanoma) with cellpromotion of minus8344 followed by RXF 393 (renal cancer)with cell promotion of minus6729 and SK-OV-3 (ovarian
BioMed Research International 11
(a) (b)
Figure 4 (a) Compound 13 is represented with macromodel stick modelThe hydrogen bonds are shown with yellow colour dotted lines andhydrophobic interactions with orange dotted lines (b) Compound 13 at binding site on surface
(a) (b)
Figure 5 (a) Compound 3 is represented with macromodel stick modelThe hydrophobic cavity residues are represented with magenta stickmodelThe hydrogen bonds are shownwith yellow colour dotted lines and hydrophobic interactions with orange dotted lines (b) Compound3 at binding site on surface
cancer) with cell promotion of minus6358 while the maximumcell promotion was observed on TK-10 (renal cancer)which showed 1584 growth promotion (8416 growthinhibition) at one-dose assay The compound 10 was foundto be highly active on SK-MEL-5 (melanoma) with cellpromotion of minus8652 followed by UACC-62 (melanoma)with cell promotion of minus8439 and COLO 205 (coloncancer) with cell promotion of minus8405 while the maximumcell promotion was observed on UACC-257 (melanoma)which showed 1603 growth promotion (8397 growthinhibition) at one-dose assay Compound 11 was found to behighly active on CAKI-1 (renal cancer) with cell promotionof minus3477 followed by MDA-MB-435 (melanoma) with cellpromotion of minus3053 and SF-295 (CNS cancer) with cellpromotion of minus2968 while the maximum cell promotionwas observed on UACC-257 (melanoma) which showed8713 growth promotion (1287 growth inhibition) at one-dose assay Compound 12 was found to be highly active onHT29 (colon cancer) with cell promotion of minus641 followedby MDA-MB-468 (breast cancer) with cell promotion ofminus634 and CCRF-CEM (leukemia) with cell promotion ofminus1672 while the maximum cell promotion was observedon TK-10 (renal cancer) which showed 10274 growthpromotion at one-dose assay Compound 13 was found to behighly active on HT29 (colon cancer) with cell promotion ofminus7624 followed by 786-0 and A498 (renal cancer) with cellpromotion of minus5893 and minus5692 respectively while themaximum cell promotion was observed on NCIADR-RES(ovarian cancer) which showed 3932 growth promotion(1287 growth inhibition) at one-dose assay Among the35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) Compound
8 was found to be the most active compound of theseries with mean growth percent of minus1919 followed bycompound 5 which showed mean growth percent of minus1225and compound 1 which showed mean growth percent ofminus466 Among the 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl (substituted phenyl)methanone analogues(10-11) Compound 10 showed maximum activity with meangrowth percent of minus2871 while compound 13 was found tobe the most active compound among dihydropyrimidineanalogues (12ndash14) which showed mean growth percent ofminus546 Overall the most active compound of the serieswas compound 10 and most of the 35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carbox-amide analogues possessed potent anticancer activity Basedon our finding we can conclude that electronegative groupat position 2 (ie 2-chloro) on N-substituted phenyl ringamong 35-bis(4-hydroxy-3-methylstyryl)-N-(substitutedphenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) showedmore anticancer activity than at position 4 (ie 4-chloro and4-fluoro) Electron releasing group such as 24-dimethyl and4-methyl on N-substituted phenyl ring increased anticanceractivity and the activity was found to be more if thenumber of electron releasing groups was increased Amongthe 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl(substituted phenyl)methanone analogues (10 and 11) theactivity was found to bemore when there was no substitutionin the phenyl ring Among six member dihydropyrimidineanalogues (12ndash14) compound with X substitution as ldquoNHrdquoshowed more activity than as ldquoOrdquo substitution
The results of 5-dose assay of the most active compoundsamong their respective series are given in Table 3 Compound8 presented GI
50ranging between 0004 and 204 120583M The
12 BioMed Research International
best results were recorded on the leukemia cell lines withvalues ranging from 0004 to 0364 120583M (Figure 2(a)) Anumber of 3 tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 0383120583M Only in 23 cell linescompound 8 registered LC
50valuewithgt100120583MCompound
10 presented GI50ranging between 00079 and 186 120583M The
best results were recorded on the leukemia cell line withvalue ranging from 00353 to 0347 120583M (Figure 2(b)) Fourof the tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 000815120583M Only in 27 cell linescompound 10 registered LC
50value with gt100 120583M Com-
pound 13 presented GI50ranging between 0524 and 339 120583M
The best results were recorded on the colon cancer cell linewith values ranging from 0543 to 218 120583M(Figure 2(c)) Onlyone tested cancer cell line presented TGI value with gt100 120583Mthe best result value being noted on the HT29 (colon cancer)with value 0948 In 28 cell lines compound 13 registeredLC50value with gt100120583M
Some of the synthesized compounds were evaluated forEGFR tyrosine kinase inhibitory activity by recombinanttyrosine kinase assay using an ELISA-based assay withpoly(Glu Tyr 4 1) as a substrate [37] The results showedcompounds 8 10 and 13 shows inhibition of EGFR tyrosinekinase with IC
50values of 355 292 and 557120583Mrespectively
33 Molecular Docking The binding site of the referenceinhibitor is well defined by hydrophobic cavity The EGFRtyrosine kinase binding site contains the important residuesCys797 andThr790 re docking of reference ligand shown thatabove residues are important for hydrophobic interactionswith the same [38] The hydrophobic cavity with the residuesLeu788 Met766 Lys745 Glu762 Thr854 and Met793 makesthe binding site attractive for the design of new inhibitorsBased on our knowledge we designed some class of newpyrazole (1ndash11) dihydropyrimidine analogues (12ndash14) forEGFR tyrosine kinase inhibition But the binding sites ofour new molecules were found to be a little different withcomparison to reference inhibitor which is reported in thecrystal structure of EGFR tyrosine kinase (PDB 2J5F) Theseligandsrsquo interactions were shown in three different motifsThe first one is hydrophobic cavity in which the substitutedmethoxy hydroxy phenyl ring of the ligand is showinggood interactions as reference inhibitor The next bindingmotif contains the target residue Cys797 the N-substitutedpyrazole derivatives and six membered (dihydropyrimidine)are binding at this site Most of these interactions werehydrophobic The last binding motif contains the residuesLys875 and Asp831 which lie in side chain of the protein andshowed the side chain hydrogen bonding with the designedmolecules The docking score and Emodel score of some ofthe compounds selected for anticancer activity by NCI aregiven in Table 4 and binding modes are shown in Figure 3
Compound 13 showed the best hydrophobic interactionwith various residues such as Cys797 Met 793 Leu 844 Leu792 Ala 743 and Val 726 (Figures 4(a) and 4(b)) Negativelycharged Asp 837 interacted with methoxy hydroxy phenyl
ring (4-hydroxy-3-methoxyphenyl) Arg 841 showed 120587-120587cationic interaction The Met 793 present in the hydrophobiccavity showed H-bond backbone interaction and H-bond(side chain) interaction The positively charged Lys 845and Arg 841 destabilized interactions The interactions ofcompound 13 with the EGFR kinase receptor shown inribbon diagram the hydrophobic cavity is indicated withyellow colour loop motif-1 is indicated with green colourbeta sheet and the motif-2 is indicated with green colourloop The other ligands were also having good interactionswith the hydrophobic cavity and bindingmotifs the followingresidues leu718 Pro794 Met793 Ala743 Leu844 Val726and Cys797 were present around the ligand Thr790 Gln791having polar characteristics andMet 793 were responsible forbackbone H-bond interaction as well as H-bond (side chain)interaction Asp855 and Asp837 showed charged interactionslike destabilizing contacts Positively charged Lys875 andArg841 were found to have 120587-120587 cationic interaction and werepresent in bindingmotifmaking different binding patterns ofreported ligandsThe docking study for compound 3 is givenin Figures 5(a) and 5(b)
4 Conclusion
The novel series of curcumin analogues (pyrazoles) weresynthesized in satisfactory yields The anticancer activityshowed promising results The studies confirmed compound10 as potent lead compound for drug discovery and furtheroptimization The curcumin analogues discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer disease The pyrazole discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer
Acknowledgments
The authors are thankful to the staff members of the NationalCancer Institute (NCI) USA for in vitro anticancer screeningof the newly synthesized compounds They are especiallygrateful to Professor Dizon B Thelma Project ManagerNational Cancer Institute (NCI) USA
References
[1] WHO Cancer World Health Organization February 2006httpenwikipediaorgwikicancer
[2] M N Noolvi H M Patel V Bhardwaj and A ChauhanldquoSynthesis and in vitro antitumor activity of substituted quina-zoline and quinoxaline derivatives search for anticancer agentrdquoEuropean Journal of Medicinal Chemistry vol 46 no 6 pp2327ndash2346 2011
[3] N Aydemir and R Bilaloglu ldquoGenotoxicity of two anticancerdrugs gemcitabine and topotecan in mouse bone marrow invivordquoMutation Research vol 537 no 1 pp 43ndash51 2003
[4] I Bouabdallah L A MrsquoBarek A Zyad A Ramdani I Zidaneand A Melhaoui ldquoNew pyrazolic compounds as cytotoxicagentsrdquo Natural Product Research vol 21 no 4 pp 298ndash3022007
BioMed Research International 13
[5] D Havrylyuk B Zimenkovsky O Vasylenko L Zaprutko AGzella and R Lesyk ldquoSynthesis of novel thiazolone-based com-pounds containing pyrazoline moiety and evaluation of theiranticancer activityrdquo European Journal of Medicinal Chemistryvol 44 no 4 pp 1396ndash1404 2009
[6] M Shaharyar M M Abdullah M A Bakht and J MajeedldquoPyrazoline bearing benzimidazoles search for anticanceragentrdquo European Journal of Medicinal Chemistry vol 45 no 1pp 114ndash119 2010
[7] P-C Lv H-Q Li J Sun Y Zhou and H-L Zhu ldquoSynthesisand biological evaluation of pyrazole derivatives containingthiourea skeleton as anticancer agentsrdquo Bioorganic and Medici-nal Chemistry vol 18 no 13 pp 4606ndash4614 2010
[8] M S Christodoulou S Liekens K M Kasiotis and S AHaroutounian ldquoNovel pyrazole derivatives synthesis and eval-uation of anti-angiogenic activityrdquo Bioorganic and MedicinalChemistry vol 18 no 12 pp 4338ndash4350 2010
[9] M J Ahsan G J Samy H Khalillah R C Krit and SSoni ldquoMolecular properties prediction and synthesis of novelpyrazoline carboxamide analogs as antitubercular agentsrdquoAnti-Infective Agents vol 10 no 2 pp 117ndash123 2012
[10] M J Ahsan J G Samy H Khalilullah M A Bakht andM Z Hassan ldquoSynthesis and antimycobacterial evaluation of3a4-dihydro-3H-indeno [12-c] pyrazole-2-carboxamide ana-loguesrdquo European Journal of Medicinal Chemistry vol 46 no11 pp 5694ndash5697 2011
[11] M J Ahsan J G Samy K R Dutt et al ldquoDesign synthesis andantimycobacterial evaluation of novel 3-substituted-N-aryl-67-dimethoxy-3a4-dihydro-3H-indeno[12-c] pyrazole-2-carboxamide analoguesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 15 pp 4451ndash4453 2011
[12] M J Ahsan J G Samy S Soni et al ldquoDiscovery of novelantitubercular 3a4-dihydro-3H-indeno[12-c]pyrazole-2-carndashboxamidecarbothioamide analoguesrdquo Bioorganic and Medici-nal Chemistry Letters vol 21 no 18 pp 5259ndash5261 2011
[13] M J Ahsan H Khalilullah J P Stables and J GovindasamyldquoSynthesis and anticonvulsant activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide carbothioamide ana-loguesrdquo Journal of Enzyme Inhibition amp Medicinal Chemistryvol 28 no 3 pp 644ndash650 2013
[14] M J Ahsan J Govindasamy H Khalilullah et al ldquoPOMAanalyses as new efficient bioinformaticsrsquoplatform to predictand optimize bioactivity of synthesized 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamidecarbothioamide anal-oguesrdquo Bioorganic amp Medicinal Chemistry vol 22 no 24 pp7029ndash7035 2012
[15] H Khalilullah S Khan M J Ahsan and B AhmedldquoSynthesis and antihepatotoxic activity of 5-(23-dihydro-14-benzodioxane-6-yl)-3-substituted-phenyl-45-dihydro-1H-pyrazole derivativesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 24 pp 7251ndash7254 2011
[16] M J Alam M J Ahsan O Alam and S A KhanldquoSynthesis of 4-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)-6-(substituted phenyl)pyrimidin-2-ol analogues as 8 anti-inflammatory and analgesic agentsrdquo Letters in Drug Design ampDiscovery vol 10 no 8 pp 776ndash782 2013
[17] D P Chauhan ldquoChemotherapeutic potential of curcumin forcolorectal cancerrdquo Current Pharmaceutical Design vol 8 no 19pp 1695ndash1706 2002
[18] Y-L Jia J Li Z-H Qin and Z-Q Liang ldquoAutophagic andapoptotic mechanisms of curcumin-induced death in K562
cellsrdquo Journal of Asian Natural Products Research vol 11 no 11pp 918ndash928 2009
[19] A Kunwar A Barik BMishra K Rathinasamy R Pandey andK I Priyadarsini ldquoQuantitative cellular uptake localization andcytotoxicity of curcumin in normal and tumor cellsrdquoBiochimicaet Biophysica Acta vol 1780 no 4 pp 673ndash679 2008
[20] J Lal S K Gupta DThavaselvam and D D Agarwal ldquoDesignsynthesis synergistic antimicrobial activity and cytotoxicityof 4-aryl substituted 34-dihydropyrimidinones of curcuminrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 8 pp2872ndash2876 2012
[21] R K Singh D Rai D Yadav A Bhargava J Balzarini and EDe Clercq ldquoSynthesis antibacterial and antiviral properties ofcurcumin bioconjugates bearing dipeptide fatty acids and folicacidrdquo European Journal of Medicinal Chemistry vol 45 no 3pp 1078ndash1086 2010
[22] K Saja M S Babu D Karunagaran and P R SudhakaranldquoAnti-inflammatory effect of curcumin involves downregula-tion of MMP-9 in blood mononuclear cellsrdquo InternationalImmunopharmacology vol 7 no 13 pp 1659ndash1667 2007
[23] S Mishra K Karmodiya N Surolia and A Surolia ldquoSynthesisand exploration of novel curcumin analogues as anti-malarialagentsrdquo Bioorganic and Medicinal Chemistry vol 16 no 6 pp2894ndash2902 2008
[24] G Liang L Shao Y Wang et al ldquoExploration and synthesis ofcurcumin analogues with improved structural stability both invitro and in vivo as cytotoxic agentsrdquo Bioorganic and MedicinalChemistry vol 17 no 6 pp 2623ndash2631 2009
[25] M J Ahsan ldquoSynthesis and anticancer activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide analoguesrdquo Lettersin Drug Design amp Discovery vol 9 no 9 pp 823ndash827 2012
[26] Salahuddin M Shaharyar A Majumdar and M J AhsanldquoSynthesis characterization and anticancer evaluation of 2-(naphthalen-1-ylmethylnaphthalen-2-yloxymethyl)-1-[5-(sub-stituted phenyl)-[134]oxadiazol-2-ylmethyl]-1H-benzimida-zolerdquo Arabian Journal of Chemistry 2013
[27] E Zwick J Bange andAUllrich ldquoReceptor tyrosine kinase sig-nalling as a target for cancer intervention strategiesrdquo Endocrine-Related Cancer vol 8 no 3 pp 161ndash173 2001
[28] H A Bhuva and S G Kini ldquoSynthesis anticancer activity anddocking of some substituted benzothiazoles as tyrosine kinaseinhibitorsrdquo Journal ofMolecular Graphics andModelling vol 29no 1 pp 32ndash37 2010
[29] J Mendelsohn and J Baselga ldquoThe EGF receptor family astargets for cancer therapyrdquo Oncogene vol 19 no 56 pp 6550ndash6565 2000
[30] M Amir M J Ahsan and I Ali ldquoSynthesis of N1-(3-chloro-4-flouropheny)-N4-substituted semicarbazones as novel anticon-vulsant agentsrdquo Indian Journal of Chemistry B vol 49 no 11 pp1509ndash1514 2010
[31] httpdtpncinihgov[32] A Monks D Scudiero P Skehan et al ldquoFeasibility of a high-
flux anticancer drug screen using a diverse panel of culturedhuman tumor cell linesrdquo Journal of theNational Cancer Institutevol 83 no 11 pp 757ndash766 1991
[33] M R Body and K D Paull ldquoSome practical considerations andapplications of the national cancer institute in vitro anticancerdrug discovery screenrdquoDrug Development Research vol 34 no2 pp 91ndash109 1995
[34] R H Shoemaker ldquoThe NCI60 human tumour cell line anti-cancer drug screenrdquo Nature Reviews Cancer vol 6 no 10 pp813ndash823 2006
14 BioMed Research International
[35] M C Alley D A Scudiero A Monks et al ldquoFeasibility ofdrug screening with panels of human tumor cell lines using amicroculture tetrazolium assayrdquo Cancer Research vol 48 no 3pp 589ndash601 1988
[36] M R Grever S A Schepartz and B A Chabner ldquoTheNationalCancer Institute cancer drug discovery and development pro-gramrdquo Seminars in Oncology vol 19 no 6 pp 622ndash638 1992
[37] P Dubreuil S Letard M Ciufolini et al ldquoMasitinib (AB1010)a potent and selective tyrosine kinase inhibitor targeting KITrdquoPLoS ONE vol 4 no 9 Article ID e7258 2009
[38] J A Blair D Rauh C Kung et al ldquoStructure-guided devel-opment of affinity probes for tyrosine kinases using chemicalgeneticsrdquo Nature Chemical Biology vol 3 no 4 pp 229ndash2382007
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
showed singlet at 120575 119ndash128 ppm corresponding to CH3
a singlet at 120575 379ndash385 ppm corresponding to OCH3 a
singlet at 120575 652ndash662 ppm corresponding to CH=C proton(pyrazoledihydropyrimidine) a doublet at 120575 661ndash675 ppmcorresponding toCH=CHproton a doublet at 665ndash681 ppmcorresponding to CH=CH proton a multiplet at 120575 681ndash793 ppm corresponding to aromatic protons broad singlet at120575 993ndash1016 ppm corresponding to CONH
2 The compounds
(1ndash14) in 1HNMRspectra exhibited two doublets with 119869 valuebetween 146 and 166Hz confirming the trans coupling Themass spectra of the compounds revealed in each case a peakcorresponding to their molecular ion peaks The elementalanalysis results were within plusmn04 of the theoretical values
32 Anticancer Activity Ten compounds were evaluated fortheir anticancer activity in both one-dose and 5-dose assaysThe observed anticancer screening data of the compoundsare given in Table 2 The 5-dose assay screening data ofthree compounds are given in Table 3 Compound 1 wasfound to be highly active on COLO 205 (colon cancer)with cell promotion of minus7349 followed by RXF 393
BioMed Research International 9
HO
O
OH
O
HO
ONN
OH
ONH
OR
HO
ONN
OH
OO
R
HO
ONHN
OH
O
X
AcOHReflux
Curcumin
ArCONHNH2
ArNHCONHNH2
NH2CXNH2
1ndash9
12ndash14
10-11
OO
Scheme 1 Protocol for the synthesis of curcumin analogues
OO
HOO
OHO
OOH
HOO
OHO
Diketone (tautomer I) Keto-enol (tautomer II)
OOH
HOO
OHO O
NH NH
N N
OHOO
HO
O
NH
R
OHOH
HOO
OHO
NHNH
ONH
OH
HOO
OHO
NNH
X
NH
R
R
R
Curcumin
OOH
HOO
OHO
R
CurcuminO
NH
OHOH
HOO
OHO
NHNH
O
R
R
OH
HOO
OHO
NNH
OR
OOH
HOO
OHO X
OHOH
HOO
OHO
NH
O
OH
HOO
OHO
N
X
HN N
X
OHOO
HO
Curcumin
N N
OHOO
HO
O
H3C H3C
H3C
H3C
H3C
CH3
CH3
H3C CH3 H3C H3CCH3CH3
CH3
CH3
CH3
CH3CH3CH3
H2N
H2N
H2N
H3CH3CH3CCH3H3CH2N
H2N
minusH2O
minusH2O
minusH2O
minusH2O minusH2O
minusH2O
NH2
1ndash9
12ndash1410-11
X = S O
Figure 1 Plausible mechanism of reaction for the synthesis of pyrazole analogues (1ndash14)
(renal cancer) with cell promotion of minus5032 and HT29(colon cancer) with cell promotion of minus3495 while themaximum cell promotion was observed on NCIADR-RES(ovarian cancer) which showed 2083 growth promotion(7917 growth inhibition) at one-dose assay Compound2 was found to be highly active on RXF 393 (renal cancer)with cell promotion of minus5360 followed by SK-MEL-5(melanoma) with cell promotion of minus2966 and MDA-MB-468 (breast cancer) with cell promotion of minus2640 whilethe maximum cell promotion was observed on TK-10 (renalcancer) which showed 2310 growth promotion (7690growth inhibition) at one-dose assay The compound 3was found to be highly active on RXF 393 (renal cancer)
with cell promotion of minus4384 followed by HT29 (coloncancer) with cell promotion of minus4184 and SK-MEL-2(melanoma) with cell promotion of minus2707 while themaximum cell promotion was observed on TK-10 (renalcancer) which showed 3812 growth promotion (6188growth inhibition) at one-dose assay Compound 5 wasfound to be highly active on COLO 205 (colon cancer)with cell promotion of minus5943 followed by RXF 393 (renalcancer) with cell promotion of minus5761 and SF-295 (CNScancer) with cell promotion of minus5532 while the maximumcell promotion was observed on TK-10 (renal cancer)which showed 2404 growth promotion (7596 growthinhibition) at one-dose assay Compound 6 was found to
10 BioMed Research International
100
50
0
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
LeukemiaG
row
th (
)
log10 of sample concentration (molar)
CCRF-CEMMOLT-4HL-60(TB)
RPMI-8226K-562SR
(a)
100
50
0
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
Leukemia
Gro
wth
()
log10 of sample concentration (molar)
CCRF-CEMMOLT-4HL-60(TB)
RPMI-8226K-562SR
(b)
100
50
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
Gro
wth
()
log10 of sample concentration (molar)
0
COLO 205HCT-15SW-620HCC-2998
HT29HCT-116KM12
Colon cancer
(c)
Figure 2 (a) Five-dose anticancer screening of compound 8 on leukemia cell line (b) Five-dose anticancer screening of compound 10 onleukemia cell line (c) Five-dose anticancer screening of compound 13 on colon cancer cell line
Figure 3 Green colour stick model indicates the reference ligand and atom macromodel stick indicates our ligands
be highly active on RXF 393 (renal) with cell promotionof minus4746 followed by MDA-MB-468 (breast cancer)with cell promotion of minus2914 and SK-OV-3 (ovariancancer) with cell promotion of minus2909 while the maximumcell promotion was observed on TK-10 (renal cancer)
which showed 2855 growth promotion (7145 growthinhibition) at one-dose assay Compound 8 was foundto be highly active on SK-MEL-5 (melanoma) with cellpromotion of minus8344 followed by RXF 393 (renal cancer)with cell promotion of minus6729 and SK-OV-3 (ovarian
BioMed Research International 11
(a) (b)
Figure 4 (a) Compound 13 is represented with macromodel stick modelThe hydrogen bonds are shown with yellow colour dotted lines andhydrophobic interactions with orange dotted lines (b) Compound 13 at binding site on surface
(a) (b)
Figure 5 (a) Compound 3 is represented with macromodel stick modelThe hydrophobic cavity residues are represented with magenta stickmodelThe hydrogen bonds are shownwith yellow colour dotted lines and hydrophobic interactions with orange dotted lines (b) Compound3 at binding site on surface
cancer) with cell promotion of minus6358 while the maximumcell promotion was observed on TK-10 (renal cancer)which showed 1584 growth promotion (8416 growthinhibition) at one-dose assay The compound 10 was foundto be highly active on SK-MEL-5 (melanoma) with cellpromotion of minus8652 followed by UACC-62 (melanoma)with cell promotion of minus8439 and COLO 205 (coloncancer) with cell promotion of minus8405 while the maximumcell promotion was observed on UACC-257 (melanoma)which showed 1603 growth promotion (8397 growthinhibition) at one-dose assay Compound 11 was found to behighly active on CAKI-1 (renal cancer) with cell promotionof minus3477 followed by MDA-MB-435 (melanoma) with cellpromotion of minus3053 and SF-295 (CNS cancer) with cellpromotion of minus2968 while the maximum cell promotionwas observed on UACC-257 (melanoma) which showed8713 growth promotion (1287 growth inhibition) at one-dose assay Compound 12 was found to be highly active onHT29 (colon cancer) with cell promotion of minus641 followedby MDA-MB-468 (breast cancer) with cell promotion ofminus634 and CCRF-CEM (leukemia) with cell promotion ofminus1672 while the maximum cell promotion was observedon TK-10 (renal cancer) which showed 10274 growthpromotion at one-dose assay Compound 13 was found to behighly active on HT29 (colon cancer) with cell promotion ofminus7624 followed by 786-0 and A498 (renal cancer) with cellpromotion of minus5893 and minus5692 respectively while themaximum cell promotion was observed on NCIADR-RES(ovarian cancer) which showed 3932 growth promotion(1287 growth inhibition) at one-dose assay Among the35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) Compound
8 was found to be the most active compound of theseries with mean growth percent of minus1919 followed bycompound 5 which showed mean growth percent of minus1225and compound 1 which showed mean growth percent ofminus466 Among the 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl (substituted phenyl)methanone analogues(10-11) Compound 10 showed maximum activity with meangrowth percent of minus2871 while compound 13 was found tobe the most active compound among dihydropyrimidineanalogues (12ndash14) which showed mean growth percent ofminus546 Overall the most active compound of the serieswas compound 10 and most of the 35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carbox-amide analogues possessed potent anticancer activity Basedon our finding we can conclude that electronegative groupat position 2 (ie 2-chloro) on N-substituted phenyl ringamong 35-bis(4-hydroxy-3-methylstyryl)-N-(substitutedphenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) showedmore anticancer activity than at position 4 (ie 4-chloro and4-fluoro) Electron releasing group such as 24-dimethyl and4-methyl on N-substituted phenyl ring increased anticanceractivity and the activity was found to be more if thenumber of electron releasing groups was increased Amongthe 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl(substituted phenyl)methanone analogues (10 and 11) theactivity was found to bemore when there was no substitutionin the phenyl ring Among six member dihydropyrimidineanalogues (12ndash14) compound with X substitution as ldquoNHrdquoshowed more activity than as ldquoOrdquo substitution
The results of 5-dose assay of the most active compoundsamong their respective series are given in Table 3 Compound8 presented GI
50ranging between 0004 and 204 120583M The
12 BioMed Research International
best results were recorded on the leukemia cell lines withvalues ranging from 0004 to 0364 120583M (Figure 2(a)) Anumber of 3 tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 0383120583M Only in 23 cell linescompound 8 registered LC
50valuewithgt100120583MCompound
10 presented GI50ranging between 00079 and 186 120583M The
best results were recorded on the leukemia cell line withvalue ranging from 00353 to 0347 120583M (Figure 2(b)) Fourof the tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 000815120583M Only in 27 cell linescompound 10 registered LC
50value with gt100 120583M Com-
pound 13 presented GI50ranging between 0524 and 339 120583M
The best results were recorded on the colon cancer cell linewith values ranging from 0543 to 218 120583M(Figure 2(c)) Onlyone tested cancer cell line presented TGI value with gt100 120583Mthe best result value being noted on the HT29 (colon cancer)with value 0948 In 28 cell lines compound 13 registeredLC50value with gt100120583M
Some of the synthesized compounds were evaluated forEGFR tyrosine kinase inhibitory activity by recombinanttyrosine kinase assay using an ELISA-based assay withpoly(Glu Tyr 4 1) as a substrate [37] The results showedcompounds 8 10 and 13 shows inhibition of EGFR tyrosinekinase with IC
50values of 355 292 and 557120583Mrespectively
33 Molecular Docking The binding site of the referenceinhibitor is well defined by hydrophobic cavity The EGFRtyrosine kinase binding site contains the important residuesCys797 andThr790 re docking of reference ligand shown thatabove residues are important for hydrophobic interactionswith the same [38] The hydrophobic cavity with the residuesLeu788 Met766 Lys745 Glu762 Thr854 and Met793 makesthe binding site attractive for the design of new inhibitorsBased on our knowledge we designed some class of newpyrazole (1ndash11) dihydropyrimidine analogues (12ndash14) forEGFR tyrosine kinase inhibition But the binding sites ofour new molecules were found to be a little different withcomparison to reference inhibitor which is reported in thecrystal structure of EGFR tyrosine kinase (PDB 2J5F) Theseligandsrsquo interactions were shown in three different motifsThe first one is hydrophobic cavity in which the substitutedmethoxy hydroxy phenyl ring of the ligand is showinggood interactions as reference inhibitor The next bindingmotif contains the target residue Cys797 the N-substitutedpyrazole derivatives and six membered (dihydropyrimidine)are binding at this site Most of these interactions werehydrophobic The last binding motif contains the residuesLys875 and Asp831 which lie in side chain of the protein andshowed the side chain hydrogen bonding with the designedmolecules The docking score and Emodel score of some ofthe compounds selected for anticancer activity by NCI aregiven in Table 4 and binding modes are shown in Figure 3
Compound 13 showed the best hydrophobic interactionwith various residues such as Cys797 Met 793 Leu 844 Leu792 Ala 743 and Val 726 (Figures 4(a) and 4(b)) Negativelycharged Asp 837 interacted with methoxy hydroxy phenyl
ring (4-hydroxy-3-methoxyphenyl) Arg 841 showed 120587-120587cationic interaction The Met 793 present in the hydrophobiccavity showed H-bond backbone interaction and H-bond(side chain) interaction The positively charged Lys 845and Arg 841 destabilized interactions The interactions ofcompound 13 with the EGFR kinase receptor shown inribbon diagram the hydrophobic cavity is indicated withyellow colour loop motif-1 is indicated with green colourbeta sheet and the motif-2 is indicated with green colourloop The other ligands were also having good interactionswith the hydrophobic cavity and bindingmotifs the followingresidues leu718 Pro794 Met793 Ala743 Leu844 Val726and Cys797 were present around the ligand Thr790 Gln791having polar characteristics andMet 793 were responsible forbackbone H-bond interaction as well as H-bond (side chain)interaction Asp855 and Asp837 showed charged interactionslike destabilizing contacts Positively charged Lys875 andArg841 were found to have 120587-120587 cationic interaction and werepresent in bindingmotifmaking different binding patterns ofreported ligandsThe docking study for compound 3 is givenin Figures 5(a) and 5(b)
4 Conclusion
The novel series of curcumin analogues (pyrazoles) weresynthesized in satisfactory yields The anticancer activityshowed promising results The studies confirmed compound10 as potent lead compound for drug discovery and furtheroptimization The curcumin analogues discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer disease The pyrazole discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer
Acknowledgments
The authors are thankful to the staff members of the NationalCancer Institute (NCI) USA for in vitro anticancer screeningof the newly synthesized compounds They are especiallygrateful to Professor Dizon B Thelma Project ManagerNational Cancer Institute (NCI) USA
References
[1] WHO Cancer World Health Organization February 2006httpenwikipediaorgwikicancer
[2] M N Noolvi H M Patel V Bhardwaj and A ChauhanldquoSynthesis and in vitro antitumor activity of substituted quina-zoline and quinoxaline derivatives search for anticancer agentrdquoEuropean Journal of Medicinal Chemistry vol 46 no 6 pp2327ndash2346 2011
[3] N Aydemir and R Bilaloglu ldquoGenotoxicity of two anticancerdrugs gemcitabine and topotecan in mouse bone marrow invivordquoMutation Research vol 537 no 1 pp 43ndash51 2003
[4] I Bouabdallah L A MrsquoBarek A Zyad A Ramdani I Zidaneand A Melhaoui ldquoNew pyrazolic compounds as cytotoxicagentsrdquo Natural Product Research vol 21 no 4 pp 298ndash3022007
BioMed Research International 13
[5] D Havrylyuk B Zimenkovsky O Vasylenko L Zaprutko AGzella and R Lesyk ldquoSynthesis of novel thiazolone-based com-pounds containing pyrazoline moiety and evaluation of theiranticancer activityrdquo European Journal of Medicinal Chemistryvol 44 no 4 pp 1396ndash1404 2009
[6] M Shaharyar M M Abdullah M A Bakht and J MajeedldquoPyrazoline bearing benzimidazoles search for anticanceragentrdquo European Journal of Medicinal Chemistry vol 45 no 1pp 114ndash119 2010
[7] P-C Lv H-Q Li J Sun Y Zhou and H-L Zhu ldquoSynthesisand biological evaluation of pyrazole derivatives containingthiourea skeleton as anticancer agentsrdquo Bioorganic and Medici-nal Chemistry vol 18 no 13 pp 4606ndash4614 2010
[8] M S Christodoulou S Liekens K M Kasiotis and S AHaroutounian ldquoNovel pyrazole derivatives synthesis and eval-uation of anti-angiogenic activityrdquo Bioorganic and MedicinalChemistry vol 18 no 12 pp 4338ndash4350 2010
[9] M J Ahsan G J Samy H Khalillah R C Krit and SSoni ldquoMolecular properties prediction and synthesis of novelpyrazoline carboxamide analogs as antitubercular agentsrdquoAnti-Infective Agents vol 10 no 2 pp 117ndash123 2012
[10] M J Ahsan J G Samy H Khalilullah M A Bakht andM Z Hassan ldquoSynthesis and antimycobacterial evaluation of3a4-dihydro-3H-indeno [12-c] pyrazole-2-carboxamide ana-loguesrdquo European Journal of Medicinal Chemistry vol 46 no11 pp 5694ndash5697 2011
[11] M J Ahsan J G Samy K R Dutt et al ldquoDesign synthesis andantimycobacterial evaluation of novel 3-substituted-N-aryl-67-dimethoxy-3a4-dihydro-3H-indeno[12-c] pyrazole-2-carboxamide analoguesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 15 pp 4451ndash4453 2011
[12] M J Ahsan J G Samy S Soni et al ldquoDiscovery of novelantitubercular 3a4-dihydro-3H-indeno[12-c]pyrazole-2-carndashboxamidecarbothioamide analoguesrdquo Bioorganic and Medici-nal Chemistry Letters vol 21 no 18 pp 5259ndash5261 2011
[13] M J Ahsan H Khalilullah J P Stables and J GovindasamyldquoSynthesis and anticonvulsant activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide carbothioamide ana-loguesrdquo Journal of Enzyme Inhibition amp Medicinal Chemistryvol 28 no 3 pp 644ndash650 2013
[14] M J Ahsan J Govindasamy H Khalilullah et al ldquoPOMAanalyses as new efficient bioinformaticsrsquoplatform to predictand optimize bioactivity of synthesized 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamidecarbothioamide anal-oguesrdquo Bioorganic amp Medicinal Chemistry vol 22 no 24 pp7029ndash7035 2012
[15] H Khalilullah S Khan M J Ahsan and B AhmedldquoSynthesis and antihepatotoxic activity of 5-(23-dihydro-14-benzodioxane-6-yl)-3-substituted-phenyl-45-dihydro-1H-pyrazole derivativesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 24 pp 7251ndash7254 2011
[16] M J Alam M J Ahsan O Alam and S A KhanldquoSynthesis of 4-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)-6-(substituted phenyl)pyrimidin-2-ol analogues as 8 anti-inflammatory and analgesic agentsrdquo Letters in Drug Design ampDiscovery vol 10 no 8 pp 776ndash782 2013
[17] D P Chauhan ldquoChemotherapeutic potential of curcumin forcolorectal cancerrdquo Current Pharmaceutical Design vol 8 no 19pp 1695ndash1706 2002
[18] Y-L Jia J Li Z-H Qin and Z-Q Liang ldquoAutophagic andapoptotic mechanisms of curcumin-induced death in K562
cellsrdquo Journal of Asian Natural Products Research vol 11 no 11pp 918ndash928 2009
[19] A Kunwar A Barik BMishra K Rathinasamy R Pandey andK I Priyadarsini ldquoQuantitative cellular uptake localization andcytotoxicity of curcumin in normal and tumor cellsrdquoBiochimicaet Biophysica Acta vol 1780 no 4 pp 673ndash679 2008
[20] J Lal S K Gupta DThavaselvam and D D Agarwal ldquoDesignsynthesis synergistic antimicrobial activity and cytotoxicityof 4-aryl substituted 34-dihydropyrimidinones of curcuminrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 8 pp2872ndash2876 2012
[21] R K Singh D Rai D Yadav A Bhargava J Balzarini and EDe Clercq ldquoSynthesis antibacterial and antiviral properties ofcurcumin bioconjugates bearing dipeptide fatty acids and folicacidrdquo European Journal of Medicinal Chemistry vol 45 no 3pp 1078ndash1086 2010
[22] K Saja M S Babu D Karunagaran and P R SudhakaranldquoAnti-inflammatory effect of curcumin involves downregula-tion of MMP-9 in blood mononuclear cellsrdquo InternationalImmunopharmacology vol 7 no 13 pp 1659ndash1667 2007
[23] S Mishra K Karmodiya N Surolia and A Surolia ldquoSynthesisand exploration of novel curcumin analogues as anti-malarialagentsrdquo Bioorganic and Medicinal Chemistry vol 16 no 6 pp2894ndash2902 2008
[24] G Liang L Shao Y Wang et al ldquoExploration and synthesis ofcurcumin analogues with improved structural stability both invitro and in vivo as cytotoxic agentsrdquo Bioorganic and MedicinalChemistry vol 17 no 6 pp 2623ndash2631 2009
[25] M J Ahsan ldquoSynthesis and anticancer activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide analoguesrdquo Lettersin Drug Design amp Discovery vol 9 no 9 pp 823ndash827 2012
[26] Salahuddin M Shaharyar A Majumdar and M J AhsanldquoSynthesis characterization and anticancer evaluation of 2-(naphthalen-1-ylmethylnaphthalen-2-yloxymethyl)-1-[5-(sub-stituted phenyl)-[134]oxadiazol-2-ylmethyl]-1H-benzimida-zolerdquo Arabian Journal of Chemistry 2013
[27] E Zwick J Bange andAUllrich ldquoReceptor tyrosine kinase sig-nalling as a target for cancer intervention strategiesrdquo Endocrine-Related Cancer vol 8 no 3 pp 161ndash173 2001
[28] H A Bhuva and S G Kini ldquoSynthesis anticancer activity anddocking of some substituted benzothiazoles as tyrosine kinaseinhibitorsrdquo Journal ofMolecular Graphics andModelling vol 29no 1 pp 32ndash37 2010
[29] J Mendelsohn and J Baselga ldquoThe EGF receptor family astargets for cancer therapyrdquo Oncogene vol 19 no 56 pp 6550ndash6565 2000
[30] M Amir M J Ahsan and I Ali ldquoSynthesis of N1-(3-chloro-4-flouropheny)-N4-substituted semicarbazones as novel anticon-vulsant agentsrdquo Indian Journal of Chemistry B vol 49 no 11 pp1509ndash1514 2010
[31] httpdtpncinihgov[32] A Monks D Scudiero P Skehan et al ldquoFeasibility of a high-
flux anticancer drug screen using a diverse panel of culturedhuman tumor cell linesrdquo Journal of theNational Cancer Institutevol 83 no 11 pp 757ndash766 1991
[33] M R Body and K D Paull ldquoSome practical considerations andapplications of the national cancer institute in vitro anticancerdrug discovery screenrdquoDrug Development Research vol 34 no2 pp 91ndash109 1995
[34] R H Shoemaker ldquoThe NCI60 human tumour cell line anti-cancer drug screenrdquo Nature Reviews Cancer vol 6 no 10 pp813ndash823 2006
14 BioMed Research International
[35] M C Alley D A Scudiero A Monks et al ldquoFeasibility ofdrug screening with panels of human tumor cell lines using amicroculture tetrazolium assayrdquo Cancer Research vol 48 no 3pp 589ndash601 1988
[36] M R Grever S A Schepartz and B A Chabner ldquoTheNationalCancer Institute cancer drug discovery and development pro-gramrdquo Seminars in Oncology vol 19 no 6 pp 622ndash638 1992
[37] P Dubreuil S Letard M Ciufolini et al ldquoMasitinib (AB1010)a potent and selective tyrosine kinase inhibitor targeting KITrdquoPLoS ONE vol 4 no 9 Article ID e7258 2009
[38] J A Blair D Rauh C Kung et al ldquoStructure-guided devel-opment of affinity probes for tyrosine kinases using chemicalgeneticsrdquo Nature Chemical Biology vol 3 no 4 pp 229ndash2382007
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Scheme 1 Protocol for the synthesis of curcumin analogues
OO
HOO
OHO
OOH
HOO
OHO
Diketone (tautomer I) Keto-enol (tautomer II)
OOH
HOO
OHO O
NH NH
N N
OHOO
HO
O
NH
R
OHOH
HOO
OHO
NHNH
ONH
OH
HOO
OHO
NNH
X
NH
R
R
R
Curcumin
OOH
HOO
OHO
R
CurcuminO
NH
OHOH
HOO
OHO
NHNH
O
R
R
OH
HOO
OHO
NNH
OR
OOH
HOO
OHO X
OHOH
HOO
OHO
NH
O
OH
HOO
OHO
N
X
HN N
X
OHOO
HO
Curcumin
N N
OHOO
HO
O
H3C H3C
H3C
H3C
H3C
CH3
CH3
H3C CH3 H3C H3CCH3CH3
CH3
CH3
CH3
CH3CH3CH3
H2N
H2N
H2N
H3CH3CH3CCH3H3CH2N
H2N
minusH2O
minusH2O
minusH2O
minusH2O minusH2O
minusH2O
NH2
1ndash9
12ndash1410-11
X = S O
Figure 1 Plausible mechanism of reaction for the synthesis of pyrazole analogues (1ndash14)
(renal cancer) with cell promotion of minus5032 and HT29(colon cancer) with cell promotion of minus3495 while themaximum cell promotion was observed on NCIADR-RES(ovarian cancer) which showed 2083 growth promotion(7917 growth inhibition) at one-dose assay Compound2 was found to be highly active on RXF 393 (renal cancer)with cell promotion of minus5360 followed by SK-MEL-5(melanoma) with cell promotion of minus2966 and MDA-MB-468 (breast cancer) with cell promotion of minus2640 whilethe maximum cell promotion was observed on TK-10 (renalcancer) which showed 2310 growth promotion (7690growth inhibition) at one-dose assay The compound 3was found to be highly active on RXF 393 (renal cancer)
with cell promotion of minus4384 followed by HT29 (coloncancer) with cell promotion of minus4184 and SK-MEL-2(melanoma) with cell promotion of minus2707 while themaximum cell promotion was observed on TK-10 (renalcancer) which showed 3812 growth promotion (6188growth inhibition) at one-dose assay Compound 5 wasfound to be highly active on COLO 205 (colon cancer)with cell promotion of minus5943 followed by RXF 393 (renalcancer) with cell promotion of minus5761 and SF-295 (CNScancer) with cell promotion of minus5532 while the maximumcell promotion was observed on TK-10 (renal cancer)which showed 2404 growth promotion (7596 growthinhibition) at one-dose assay Compound 6 was found to
10 BioMed Research International
100
50
0
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
LeukemiaG
row
th (
)
log10 of sample concentration (molar)
CCRF-CEMMOLT-4HL-60(TB)
RPMI-8226K-562SR
(a)
100
50
0
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
Leukemia
Gro
wth
()
log10 of sample concentration (molar)
CCRF-CEMMOLT-4HL-60(TB)
RPMI-8226K-562SR
(b)
100
50
minus50
minus100
minus9 minus8 minus7 minus6 minus5 minus4
Gro
wth
()
log10 of sample concentration (molar)
0
COLO 205HCT-15SW-620HCC-2998
HT29HCT-116KM12
Colon cancer
(c)
Figure 2 (a) Five-dose anticancer screening of compound 8 on leukemia cell line (b) Five-dose anticancer screening of compound 10 onleukemia cell line (c) Five-dose anticancer screening of compound 13 on colon cancer cell line
Figure 3 Green colour stick model indicates the reference ligand and atom macromodel stick indicates our ligands
be highly active on RXF 393 (renal) with cell promotionof minus4746 followed by MDA-MB-468 (breast cancer)with cell promotion of minus2914 and SK-OV-3 (ovariancancer) with cell promotion of minus2909 while the maximumcell promotion was observed on TK-10 (renal cancer)
which showed 2855 growth promotion (7145 growthinhibition) at one-dose assay Compound 8 was foundto be highly active on SK-MEL-5 (melanoma) with cellpromotion of minus8344 followed by RXF 393 (renal cancer)with cell promotion of minus6729 and SK-OV-3 (ovarian
BioMed Research International 11
(a) (b)
Figure 4 (a) Compound 13 is represented with macromodel stick modelThe hydrogen bonds are shown with yellow colour dotted lines andhydrophobic interactions with orange dotted lines (b) Compound 13 at binding site on surface
(a) (b)
Figure 5 (a) Compound 3 is represented with macromodel stick modelThe hydrophobic cavity residues are represented with magenta stickmodelThe hydrogen bonds are shownwith yellow colour dotted lines and hydrophobic interactions with orange dotted lines (b) Compound3 at binding site on surface
cancer) with cell promotion of minus6358 while the maximumcell promotion was observed on TK-10 (renal cancer)which showed 1584 growth promotion (8416 growthinhibition) at one-dose assay The compound 10 was foundto be highly active on SK-MEL-5 (melanoma) with cellpromotion of minus8652 followed by UACC-62 (melanoma)with cell promotion of minus8439 and COLO 205 (coloncancer) with cell promotion of minus8405 while the maximumcell promotion was observed on UACC-257 (melanoma)which showed 1603 growth promotion (8397 growthinhibition) at one-dose assay Compound 11 was found to behighly active on CAKI-1 (renal cancer) with cell promotionof minus3477 followed by MDA-MB-435 (melanoma) with cellpromotion of minus3053 and SF-295 (CNS cancer) with cellpromotion of minus2968 while the maximum cell promotionwas observed on UACC-257 (melanoma) which showed8713 growth promotion (1287 growth inhibition) at one-dose assay Compound 12 was found to be highly active onHT29 (colon cancer) with cell promotion of minus641 followedby MDA-MB-468 (breast cancer) with cell promotion ofminus634 and CCRF-CEM (leukemia) with cell promotion ofminus1672 while the maximum cell promotion was observedon TK-10 (renal cancer) which showed 10274 growthpromotion at one-dose assay Compound 13 was found to behighly active on HT29 (colon cancer) with cell promotion ofminus7624 followed by 786-0 and A498 (renal cancer) with cellpromotion of minus5893 and minus5692 respectively while themaximum cell promotion was observed on NCIADR-RES(ovarian cancer) which showed 3932 growth promotion(1287 growth inhibition) at one-dose assay Among the35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) Compound
8 was found to be the most active compound of theseries with mean growth percent of minus1919 followed bycompound 5 which showed mean growth percent of minus1225and compound 1 which showed mean growth percent ofminus466 Among the 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl (substituted phenyl)methanone analogues(10-11) Compound 10 showed maximum activity with meangrowth percent of minus2871 while compound 13 was found tobe the most active compound among dihydropyrimidineanalogues (12ndash14) which showed mean growth percent ofminus546 Overall the most active compound of the serieswas compound 10 and most of the 35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carbox-amide analogues possessed potent anticancer activity Basedon our finding we can conclude that electronegative groupat position 2 (ie 2-chloro) on N-substituted phenyl ringamong 35-bis(4-hydroxy-3-methylstyryl)-N-(substitutedphenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) showedmore anticancer activity than at position 4 (ie 4-chloro and4-fluoro) Electron releasing group such as 24-dimethyl and4-methyl on N-substituted phenyl ring increased anticanceractivity and the activity was found to be more if thenumber of electron releasing groups was increased Amongthe 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl(substituted phenyl)methanone analogues (10 and 11) theactivity was found to bemore when there was no substitutionin the phenyl ring Among six member dihydropyrimidineanalogues (12ndash14) compound with X substitution as ldquoNHrdquoshowed more activity than as ldquoOrdquo substitution
The results of 5-dose assay of the most active compoundsamong their respective series are given in Table 3 Compound8 presented GI
50ranging between 0004 and 204 120583M The
12 BioMed Research International
best results were recorded on the leukemia cell lines withvalues ranging from 0004 to 0364 120583M (Figure 2(a)) Anumber of 3 tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 0383120583M Only in 23 cell linescompound 8 registered LC
50valuewithgt100120583MCompound
10 presented GI50ranging between 00079 and 186 120583M The
best results were recorded on the leukemia cell line withvalue ranging from 00353 to 0347 120583M (Figure 2(b)) Fourof the tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 000815120583M Only in 27 cell linescompound 10 registered LC
50value with gt100 120583M Com-
pound 13 presented GI50ranging between 0524 and 339 120583M
The best results were recorded on the colon cancer cell linewith values ranging from 0543 to 218 120583M(Figure 2(c)) Onlyone tested cancer cell line presented TGI value with gt100 120583Mthe best result value being noted on the HT29 (colon cancer)with value 0948 In 28 cell lines compound 13 registeredLC50value with gt100120583M
Some of the synthesized compounds were evaluated forEGFR tyrosine kinase inhibitory activity by recombinanttyrosine kinase assay using an ELISA-based assay withpoly(Glu Tyr 4 1) as a substrate [37] The results showedcompounds 8 10 and 13 shows inhibition of EGFR tyrosinekinase with IC
50values of 355 292 and 557120583Mrespectively
33 Molecular Docking The binding site of the referenceinhibitor is well defined by hydrophobic cavity The EGFRtyrosine kinase binding site contains the important residuesCys797 andThr790 re docking of reference ligand shown thatabove residues are important for hydrophobic interactionswith the same [38] The hydrophobic cavity with the residuesLeu788 Met766 Lys745 Glu762 Thr854 and Met793 makesthe binding site attractive for the design of new inhibitorsBased on our knowledge we designed some class of newpyrazole (1ndash11) dihydropyrimidine analogues (12ndash14) forEGFR tyrosine kinase inhibition But the binding sites ofour new molecules were found to be a little different withcomparison to reference inhibitor which is reported in thecrystal structure of EGFR tyrosine kinase (PDB 2J5F) Theseligandsrsquo interactions were shown in three different motifsThe first one is hydrophobic cavity in which the substitutedmethoxy hydroxy phenyl ring of the ligand is showinggood interactions as reference inhibitor The next bindingmotif contains the target residue Cys797 the N-substitutedpyrazole derivatives and six membered (dihydropyrimidine)are binding at this site Most of these interactions werehydrophobic The last binding motif contains the residuesLys875 and Asp831 which lie in side chain of the protein andshowed the side chain hydrogen bonding with the designedmolecules The docking score and Emodel score of some ofthe compounds selected for anticancer activity by NCI aregiven in Table 4 and binding modes are shown in Figure 3
Compound 13 showed the best hydrophobic interactionwith various residues such as Cys797 Met 793 Leu 844 Leu792 Ala 743 and Val 726 (Figures 4(a) and 4(b)) Negativelycharged Asp 837 interacted with methoxy hydroxy phenyl
ring (4-hydroxy-3-methoxyphenyl) Arg 841 showed 120587-120587cationic interaction The Met 793 present in the hydrophobiccavity showed H-bond backbone interaction and H-bond(side chain) interaction The positively charged Lys 845and Arg 841 destabilized interactions The interactions ofcompound 13 with the EGFR kinase receptor shown inribbon diagram the hydrophobic cavity is indicated withyellow colour loop motif-1 is indicated with green colourbeta sheet and the motif-2 is indicated with green colourloop The other ligands were also having good interactionswith the hydrophobic cavity and bindingmotifs the followingresidues leu718 Pro794 Met793 Ala743 Leu844 Val726and Cys797 were present around the ligand Thr790 Gln791having polar characteristics andMet 793 were responsible forbackbone H-bond interaction as well as H-bond (side chain)interaction Asp855 and Asp837 showed charged interactionslike destabilizing contacts Positively charged Lys875 andArg841 were found to have 120587-120587 cationic interaction and werepresent in bindingmotifmaking different binding patterns ofreported ligandsThe docking study for compound 3 is givenin Figures 5(a) and 5(b)
4 Conclusion
The novel series of curcumin analogues (pyrazoles) weresynthesized in satisfactory yields The anticancer activityshowed promising results The studies confirmed compound10 as potent lead compound for drug discovery and furtheroptimization The curcumin analogues discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer disease The pyrazole discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer
Acknowledgments
The authors are thankful to the staff members of the NationalCancer Institute (NCI) USA for in vitro anticancer screeningof the newly synthesized compounds They are especiallygrateful to Professor Dizon B Thelma Project ManagerNational Cancer Institute (NCI) USA
References
[1] WHO Cancer World Health Organization February 2006httpenwikipediaorgwikicancer
[2] M N Noolvi H M Patel V Bhardwaj and A ChauhanldquoSynthesis and in vitro antitumor activity of substituted quina-zoline and quinoxaline derivatives search for anticancer agentrdquoEuropean Journal of Medicinal Chemistry vol 46 no 6 pp2327ndash2346 2011
[3] N Aydemir and R Bilaloglu ldquoGenotoxicity of two anticancerdrugs gemcitabine and topotecan in mouse bone marrow invivordquoMutation Research vol 537 no 1 pp 43ndash51 2003
[4] I Bouabdallah L A MrsquoBarek A Zyad A Ramdani I Zidaneand A Melhaoui ldquoNew pyrazolic compounds as cytotoxicagentsrdquo Natural Product Research vol 21 no 4 pp 298ndash3022007
BioMed Research International 13
[5] D Havrylyuk B Zimenkovsky O Vasylenko L Zaprutko AGzella and R Lesyk ldquoSynthesis of novel thiazolone-based com-pounds containing pyrazoline moiety and evaluation of theiranticancer activityrdquo European Journal of Medicinal Chemistryvol 44 no 4 pp 1396ndash1404 2009
[6] M Shaharyar M M Abdullah M A Bakht and J MajeedldquoPyrazoline bearing benzimidazoles search for anticanceragentrdquo European Journal of Medicinal Chemistry vol 45 no 1pp 114ndash119 2010
[7] P-C Lv H-Q Li J Sun Y Zhou and H-L Zhu ldquoSynthesisand biological evaluation of pyrazole derivatives containingthiourea skeleton as anticancer agentsrdquo Bioorganic and Medici-nal Chemistry vol 18 no 13 pp 4606ndash4614 2010
[8] M S Christodoulou S Liekens K M Kasiotis and S AHaroutounian ldquoNovel pyrazole derivatives synthesis and eval-uation of anti-angiogenic activityrdquo Bioorganic and MedicinalChemistry vol 18 no 12 pp 4338ndash4350 2010
[9] M J Ahsan G J Samy H Khalillah R C Krit and SSoni ldquoMolecular properties prediction and synthesis of novelpyrazoline carboxamide analogs as antitubercular agentsrdquoAnti-Infective Agents vol 10 no 2 pp 117ndash123 2012
[10] M J Ahsan J G Samy H Khalilullah M A Bakht andM Z Hassan ldquoSynthesis and antimycobacterial evaluation of3a4-dihydro-3H-indeno [12-c] pyrazole-2-carboxamide ana-loguesrdquo European Journal of Medicinal Chemistry vol 46 no11 pp 5694ndash5697 2011
[11] M J Ahsan J G Samy K R Dutt et al ldquoDesign synthesis andantimycobacterial evaluation of novel 3-substituted-N-aryl-67-dimethoxy-3a4-dihydro-3H-indeno[12-c] pyrazole-2-carboxamide analoguesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 15 pp 4451ndash4453 2011
[12] M J Ahsan J G Samy S Soni et al ldquoDiscovery of novelantitubercular 3a4-dihydro-3H-indeno[12-c]pyrazole-2-carndashboxamidecarbothioamide analoguesrdquo Bioorganic and Medici-nal Chemistry Letters vol 21 no 18 pp 5259ndash5261 2011
[13] M J Ahsan H Khalilullah J P Stables and J GovindasamyldquoSynthesis and anticonvulsant activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide carbothioamide ana-loguesrdquo Journal of Enzyme Inhibition amp Medicinal Chemistryvol 28 no 3 pp 644ndash650 2013
[14] M J Ahsan J Govindasamy H Khalilullah et al ldquoPOMAanalyses as new efficient bioinformaticsrsquoplatform to predictand optimize bioactivity of synthesized 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamidecarbothioamide anal-oguesrdquo Bioorganic amp Medicinal Chemistry vol 22 no 24 pp7029ndash7035 2012
[15] H Khalilullah S Khan M J Ahsan and B AhmedldquoSynthesis and antihepatotoxic activity of 5-(23-dihydro-14-benzodioxane-6-yl)-3-substituted-phenyl-45-dihydro-1H-pyrazole derivativesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 24 pp 7251ndash7254 2011
[16] M J Alam M J Ahsan O Alam and S A KhanldquoSynthesis of 4-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)-6-(substituted phenyl)pyrimidin-2-ol analogues as 8 anti-inflammatory and analgesic agentsrdquo Letters in Drug Design ampDiscovery vol 10 no 8 pp 776ndash782 2013
[17] D P Chauhan ldquoChemotherapeutic potential of curcumin forcolorectal cancerrdquo Current Pharmaceutical Design vol 8 no 19pp 1695ndash1706 2002
[18] Y-L Jia J Li Z-H Qin and Z-Q Liang ldquoAutophagic andapoptotic mechanisms of curcumin-induced death in K562
cellsrdquo Journal of Asian Natural Products Research vol 11 no 11pp 918ndash928 2009
[19] A Kunwar A Barik BMishra K Rathinasamy R Pandey andK I Priyadarsini ldquoQuantitative cellular uptake localization andcytotoxicity of curcumin in normal and tumor cellsrdquoBiochimicaet Biophysica Acta vol 1780 no 4 pp 673ndash679 2008
[20] J Lal S K Gupta DThavaselvam and D D Agarwal ldquoDesignsynthesis synergistic antimicrobial activity and cytotoxicityof 4-aryl substituted 34-dihydropyrimidinones of curcuminrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 8 pp2872ndash2876 2012
[21] R K Singh D Rai D Yadav A Bhargava J Balzarini and EDe Clercq ldquoSynthesis antibacterial and antiviral properties ofcurcumin bioconjugates bearing dipeptide fatty acids and folicacidrdquo European Journal of Medicinal Chemistry vol 45 no 3pp 1078ndash1086 2010
[22] K Saja M S Babu D Karunagaran and P R SudhakaranldquoAnti-inflammatory effect of curcumin involves downregula-tion of MMP-9 in blood mononuclear cellsrdquo InternationalImmunopharmacology vol 7 no 13 pp 1659ndash1667 2007
[23] S Mishra K Karmodiya N Surolia and A Surolia ldquoSynthesisand exploration of novel curcumin analogues as anti-malarialagentsrdquo Bioorganic and Medicinal Chemistry vol 16 no 6 pp2894ndash2902 2008
[24] G Liang L Shao Y Wang et al ldquoExploration and synthesis ofcurcumin analogues with improved structural stability both invitro and in vivo as cytotoxic agentsrdquo Bioorganic and MedicinalChemistry vol 17 no 6 pp 2623ndash2631 2009
[25] M J Ahsan ldquoSynthesis and anticancer activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide analoguesrdquo Lettersin Drug Design amp Discovery vol 9 no 9 pp 823ndash827 2012
[26] Salahuddin M Shaharyar A Majumdar and M J AhsanldquoSynthesis characterization and anticancer evaluation of 2-(naphthalen-1-ylmethylnaphthalen-2-yloxymethyl)-1-[5-(sub-stituted phenyl)-[134]oxadiazol-2-ylmethyl]-1H-benzimida-zolerdquo Arabian Journal of Chemistry 2013
[27] E Zwick J Bange andAUllrich ldquoReceptor tyrosine kinase sig-nalling as a target for cancer intervention strategiesrdquo Endocrine-Related Cancer vol 8 no 3 pp 161ndash173 2001
[28] H A Bhuva and S G Kini ldquoSynthesis anticancer activity anddocking of some substituted benzothiazoles as tyrosine kinaseinhibitorsrdquo Journal ofMolecular Graphics andModelling vol 29no 1 pp 32ndash37 2010
[29] J Mendelsohn and J Baselga ldquoThe EGF receptor family astargets for cancer therapyrdquo Oncogene vol 19 no 56 pp 6550ndash6565 2000
[30] M Amir M J Ahsan and I Ali ldquoSynthesis of N1-(3-chloro-4-flouropheny)-N4-substituted semicarbazones as novel anticon-vulsant agentsrdquo Indian Journal of Chemistry B vol 49 no 11 pp1509ndash1514 2010
[31] httpdtpncinihgov[32] A Monks D Scudiero P Skehan et al ldquoFeasibility of a high-
flux anticancer drug screen using a diverse panel of culturedhuman tumor cell linesrdquo Journal of theNational Cancer Institutevol 83 no 11 pp 757ndash766 1991
[33] M R Body and K D Paull ldquoSome practical considerations andapplications of the national cancer institute in vitro anticancerdrug discovery screenrdquoDrug Development Research vol 34 no2 pp 91ndash109 1995
[34] R H Shoemaker ldquoThe NCI60 human tumour cell line anti-cancer drug screenrdquo Nature Reviews Cancer vol 6 no 10 pp813ndash823 2006
14 BioMed Research International
[35] M C Alley D A Scudiero A Monks et al ldquoFeasibility ofdrug screening with panels of human tumor cell lines using amicroculture tetrazolium assayrdquo Cancer Research vol 48 no 3pp 589ndash601 1988
[36] M R Grever S A Schepartz and B A Chabner ldquoTheNationalCancer Institute cancer drug discovery and development pro-gramrdquo Seminars in Oncology vol 19 no 6 pp 622ndash638 1992
[37] P Dubreuil S Letard M Ciufolini et al ldquoMasitinib (AB1010)a potent and selective tyrosine kinase inhibitor targeting KITrdquoPLoS ONE vol 4 no 9 Article ID e7258 2009
[38] J A Blair D Rauh C Kung et al ldquoStructure-guided devel-opment of affinity probes for tyrosine kinases using chemicalgeneticsrdquo Nature Chemical Biology vol 3 no 4 pp 229ndash2382007
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Figure 2 (a) Five-dose anticancer screening of compound 8 on leukemia cell line (b) Five-dose anticancer screening of compound 10 onleukemia cell line (c) Five-dose anticancer screening of compound 13 on colon cancer cell line
Figure 3 Green colour stick model indicates the reference ligand and atom macromodel stick indicates our ligands
be highly active on RXF 393 (renal) with cell promotionof minus4746 followed by MDA-MB-468 (breast cancer)with cell promotion of minus2914 and SK-OV-3 (ovariancancer) with cell promotion of minus2909 while the maximumcell promotion was observed on TK-10 (renal cancer)
which showed 2855 growth promotion (7145 growthinhibition) at one-dose assay Compound 8 was foundto be highly active on SK-MEL-5 (melanoma) with cellpromotion of minus8344 followed by RXF 393 (renal cancer)with cell promotion of minus6729 and SK-OV-3 (ovarian
BioMed Research International 11
(a) (b)
Figure 4 (a) Compound 13 is represented with macromodel stick modelThe hydrogen bonds are shown with yellow colour dotted lines andhydrophobic interactions with orange dotted lines (b) Compound 13 at binding site on surface
(a) (b)
Figure 5 (a) Compound 3 is represented with macromodel stick modelThe hydrophobic cavity residues are represented with magenta stickmodelThe hydrogen bonds are shownwith yellow colour dotted lines and hydrophobic interactions with orange dotted lines (b) Compound3 at binding site on surface
cancer) with cell promotion of minus6358 while the maximumcell promotion was observed on TK-10 (renal cancer)which showed 1584 growth promotion (8416 growthinhibition) at one-dose assay The compound 10 was foundto be highly active on SK-MEL-5 (melanoma) with cellpromotion of minus8652 followed by UACC-62 (melanoma)with cell promotion of minus8439 and COLO 205 (coloncancer) with cell promotion of minus8405 while the maximumcell promotion was observed on UACC-257 (melanoma)which showed 1603 growth promotion (8397 growthinhibition) at one-dose assay Compound 11 was found to behighly active on CAKI-1 (renal cancer) with cell promotionof minus3477 followed by MDA-MB-435 (melanoma) with cellpromotion of minus3053 and SF-295 (CNS cancer) with cellpromotion of minus2968 while the maximum cell promotionwas observed on UACC-257 (melanoma) which showed8713 growth promotion (1287 growth inhibition) at one-dose assay Compound 12 was found to be highly active onHT29 (colon cancer) with cell promotion of minus641 followedby MDA-MB-468 (breast cancer) with cell promotion ofminus634 and CCRF-CEM (leukemia) with cell promotion ofminus1672 while the maximum cell promotion was observedon TK-10 (renal cancer) which showed 10274 growthpromotion at one-dose assay Compound 13 was found to behighly active on HT29 (colon cancer) with cell promotion ofminus7624 followed by 786-0 and A498 (renal cancer) with cellpromotion of minus5893 and minus5692 respectively while themaximum cell promotion was observed on NCIADR-RES(ovarian cancer) which showed 3932 growth promotion(1287 growth inhibition) at one-dose assay Among the35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) Compound
8 was found to be the most active compound of theseries with mean growth percent of minus1919 followed bycompound 5 which showed mean growth percent of minus1225and compound 1 which showed mean growth percent ofminus466 Among the 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl (substituted phenyl)methanone analogues(10-11) Compound 10 showed maximum activity with meangrowth percent of minus2871 while compound 13 was found tobe the most active compound among dihydropyrimidineanalogues (12ndash14) which showed mean growth percent ofminus546 Overall the most active compound of the serieswas compound 10 and most of the 35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carbox-amide analogues possessed potent anticancer activity Basedon our finding we can conclude that electronegative groupat position 2 (ie 2-chloro) on N-substituted phenyl ringamong 35-bis(4-hydroxy-3-methylstyryl)-N-(substitutedphenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) showedmore anticancer activity than at position 4 (ie 4-chloro and4-fluoro) Electron releasing group such as 24-dimethyl and4-methyl on N-substituted phenyl ring increased anticanceractivity and the activity was found to be more if thenumber of electron releasing groups was increased Amongthe 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl(substituted phenyl)methanone analogues (10 and 11) theactivity was found to bemore when there was no substitutionin the phenyl ring Among six member dihydropyrimidineanalogues (12ndash14) compound with X substitution as ldquoNHrdquoshowed more activity than as ldquoOrdquo substitution
The results of 5-dose assay of the most active compoundsamong their respective series are given in Table 3 Compound8 presented GI
50ranging between 0004 and 204 120583M The
12 BioMed Research International
best results were recorded on the leukemia cell lines withvalues ranging from 0004 to 0364 120583M (Figure 2(a)) Anumber of 3 tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 0383120583M Only in 23 cell linescompound 8 registered LC
50valuewithgt100120583MCompound
10 presented GI50ranging between 00079 and 186 120583M The
best results were recorded on the leukemia cell line withvalue ranging from 00353 to 0347 120583M (Figure 2(b)) Fourof the tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 000815120583M Only in 27 cell linescompound 10 registered LC
50value with gt100 120583M Com-
pound 13 presented GI50ranging between 0524 and 339 120583M
The best results were recorded on the colon cancer cell linewith values ranging from 0543 to 218 120583M(Figure 2(c)) Onlyone tested cancer cell line presented TGI value with gt100 120583Mthe best result value being noted on the HT29 (colon cancer)with value 0948 In 28 cell lines compound 13 registeredLC50value with gt100120583M
Some of the synthesized compounds were evaluated forEGFR tyrosine kinase inhibitory activity by recombinanttyrosine kinase assay using an ELISA-based assay withpoly(Glu Tyr 4 1) as a substrate [37] The results showedcompounds 8 10 and 13 shows inhibition of EGFR tyrosinekinase with IC
50values of 355 292 and 557120583Mrespectively
33 Molecular Docking The binding site of the referenceinhibitor is well defined by hydrophobic cavity The EGFRtyrosine kinase binding site contains the important residuesCys797 andThr790 re docking of reference ligand shown thatabove residues are important for hydrophobic interactionswith the same [38] The hydrophobic cavity with the residuesLeu788 Met766 Lys745 Glu762 Thr854 and Met793 makesthe binding site attractive for the design of new inhibitorsBased on our knowledge we designed some class of newpyrazole (1ndash11) dihydropyrimidine analogues (12ndash14) forEGFR tyrosine kinase inhibition But the binding sites ofour new molecules were found to be a little different withcomparison to reference inhibitor which is reported in thecrystal structure of EGFR tyrosine kinase (PDB 2J5F) Theseligandsrsquo interactions were shown in three different motifsThe first one is hydrophobic cavity in which the substitutedmethoxy hydroxy phenyl ring of the ligand is showinggood interactions as reference inhibitor The next bindingmotif contains the target residue Cys797 the N-substitutedpyrazole derivatives and six membered (dihydropyrimidine)are binding at this site Most of these interactions werehydrophobic The last binding motif contains the residuesLys875 and Asp831 which lie in side chain of the protein andshowed the side chain hydrogen bonding with the designedmolecules The docking score and Emodel score of some ofthe compounds selected for anticancer activity by NCI aregiven in Table 4 and binding modes are shown in Figure 3
Compound 13 showed the best hydrophobic interactionwith various residues such as Cys797 Met 793 Leu 844 Leu792 Ala 743 and Val 726 (Figures 4(a) and 4(b)) Negativelycharged Asp 837 interacted with methoxy hydroxy phenyl
ring (4-hydroxy-3-methoxyphenyl) Arg 841 showed 120587-120587cationic interaction The Met 793 present in the hydrophobiccavity showed H-bond backbone interaction and H-bond(side chain) interaction The positively charged Lys 845and Arg 841 destabilized interactions The interactions ofcompound 13 with the EGFR kinase receptor shown inribbon diagram the hydrophobic cavity is indicated withyellow colour loop motif-1 is indicated with green colourbeta sheet and the motif-2 is indicated with green colourloop The other ligands were also having good interactionswith the hydrophobic cavity and bindingmotifs the followingresidues leu718 Pro794 Met793 Ala743 Leu844 Val726and Cys797 were present around the ligand Thr790 Gln791having polar characteristics andMet 793 were responsible forbackbone H-bond interaction as well as H-bond (side chain)interaction Asp855 and Asp837 showed charged interactionslike destabilizing contacts Positively charged Lys875 andArg841 were found to have 120587-120587 cationic interaction and werepresent in bindingmotifmaking different binding patterns ofreported ligandsThe docking study for compound 3 is givenin Figures 5(a) and 5(b)
4 Conclusion
The novel series of curcumin analogues (pyrazoles) weresynthesized in satisfactory yields The anticancer activityshowed promising results The studies confirmed compound10 as potent lead compound for drug discovery and furtheroptimization The curcumin analogues discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer disease The pyrazole discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer
Acknowledgments
The authors are thankful to the staff members of the NationalCancer Institute (NCI) USA for in vitro anticancer screeningof the newly synthesized compounds They are especiallygrateful to Professor Dizon B Thelma Project ManagerNational Cancer Institute (NCI) USA
References
[1] WHO Cancer World Health Organization February 2006httpenwikipediaorgwikicancer
[2] M N Noolvi H M Patel V Bhardwaj and A ChauhanldquoSynthesis and in vitro antitumor activity of substituted quina-zoline and quinoxaline derivatives search for anticancer agentrdquoEuropean Journal of Medicinal Chemistry vol 46 no 6 pp2327ndash2346 2011
[3] N Aydemir and R Bilaloglu ldquoGenotoxicity of two anticancerdrugs gemcitabine and topotecan in mouse bone marrow invivordquoMutation Research vol 537 no 1 pp 43ndash51 2003
[4] I Bouabdallah L A MrsquoBarek A Zyad A Ramdani I Zidaneand A Melhaoui ldquoNew pyrazolic compounds as cytotoxicagentsrdquo Natural Product Research vol 21 no 4 pp 298ndash3022007
BioMed Research International 13
[5] D Havrylyuk B Zimenkovsky O Vasylenko L Zaprutko AGzella and R Lesyk ldquoSynthesis of novel thiazolone-based com-pounds containing pyrazoline moiety and evaluation of theiranticancer activityrdquo European Journal of Medicinal Chemistryvol 44 no 4 pp 1396ndash1404 2009
[6] M Shaharyar M M Abdullah M A Bakht and J MajeedldquoPyrazoline bearing benzimidazoles search for anticanceragentrdquo European Journal of Medicinal Chemistry vol 45 no 1pp 114ndash119 2010
[7] P-C Lv H-Q Li J Sun Y Zhou and H-L Zhu ldquoSynthesisand biological evaluation of pyrazole derivatives containingthiourea skeleton as anticancer agentsrdquo Bioorganic and Medici-nal Chemistry vol 18 no 13 pp 4606ndash4614 2010
[8] M S Christodoulou S Liekens K M Kasiotis and S AHaroutounian ldquoNovel pyrazole derivatives synthesis and eval-uation of anti-angiogenic activityrdquo Bioorganic and MedicinalChemistry vol 18 no 12 pp 4338ndash4350 2010
[9] M J Ahsan G J Samy H Khalillah R C Krit and SSoni ldquoMolecular properties prediction and synthesis of novelpyrazoline carboxamide analogs as antitubercular agentsrdquoAnti-Infective Agents vol 10 no 2 pp 117ndash123 2012
[10] M J Ahsan J G Samy H Khalilullah M A Bakht andM Z Hassan ldquoSynthesis and antimycobacterial evaluation of3a4-dihydro-3H-indeno [12-c] pyrazole-2-carboxamide ana-loguesrdquo European Journal of Medicinal Chemistry vol 46 no11 pp 5694ndash5697 2011
[11] M J Ahsan J G Samy K R Dutt et al ldquoDesign synthesis andantimycobacterial evaluation of novel 3-substituted-N-aryl-67-dimethoxy-3a4-dihydro-3H-indeno[12-c] pyrazole-2-carboxamide analoguesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 15 pp 4451ndash4453 2011
[12] M J Ahsan J G Samy S Soni et al ldquoDiscovery of novelantitubercular 3a4-dihydro-3H-indeno[12-c]pyrazole-2-carndashboxamidecarbothioamide analoguesrdquo Bioorganic and Medici-nal Chemistry Letters vol 21 no 18 pp 5259ndash5261 2011
[13] M J Ahsan H Khalilullah J P Stables and J GovindasamyldquoSynthesis and anticonvulsant activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide carbothioamide ana-loguesrdquo Journal of Enzyme Inhibition amp Medicinal Chemistryvol 28 no 3 pp 644ndash650 2013
[14] M J Ahsan J Govindasamy H Khalilullah et al ldquoPOMAanalyses as new efficient bioinformaticsrsquoplatform to predictand optimize bioactivity of synthesized 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamidecarbothioamide anal-oguesrdquo Bioorganic amp Medicinal Chemistry vol 22 no 24 pp7029ndash7035 2012
[15] H Khalilullah S Khan M J Ahsan and B AhmedldquoSynthesis and antihepatotoxic activity of 5-(23-dihydro-14-benzodioxane-6-yl)-3-substituted-phenyl-45-dihydro-1H-pyrazole derivativesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 24 pp 7251ndash7254 2011
[16] M J Alam M J Ahsan O Alam and S A KhanldquoSynthesis of 4-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)-6-(substituted phenyl)pyrimidin-2-ol analogues as 8 anti-inflammatory and analgesic agentsrdquo Letters in Drug Design ampDiscovery vol 10 no 8 pp 776ndash782 2013
[17] D P Chauhan ldquoChemotherapeutic potential of curcumin forcolorectal cancerrdquo Current Pharmaceutical Design vol 8 no 19pp 1695ndash1706 2002
[18] Y-L Jia J Li Z-H Qin and Z-Q Liang ldquoAutophagic andapoptotic mechanisms of curcumin-induced death in K562
cellsrdquo Journal of Asian Natural Products Research vol 11 no 11pp 918ndash928 2009
[19] A Kunwar A Barik BMishra K Rathinasamy R Pandey andK I Priyadarsini ldquoQuantitative cellular uptake localization andcytotoxicity of curcumin in normal and tumor cellsrdquoBiochimicaet Biophysica Acta vol 1780 no 4 pp 673ndash679 2008
[20] J Lal S K Gupta DThavaselvam and D D Agarwal ldquoDesignsynthesis synergistic antimicrobial activity and cytotoxicityof 4-aryl substituted 34-dihydropyrimidinones of curcuminrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 8 pp2872ndash2876 2012
[21] R K Singh D Rai D Yadav A Bhargava J Balzarini and EDe Clercq ldquoSynthesis antibacterial and antiviral properties ofcurcumin bioconjugates bearing dipeptide fatty acids and folicacidrdquo European Journal of Medicinal Chemistry vol 45 no 3pp 1078ndash1086 2010
[22] K Saja M S Babu D Karunagaran and P R SudhakaranldquoAnti-inflammatory effect of curcumin involves downregula-tion of MMP-9 in blood mononuclear cellsrdquo InternationalImmunopharmacology vol 7 no 13 pp 1659ndash1667 2007
[23] S Mishra K Karmodiya N Surolia and A Surolia ldquoSynthesisand exploration of novel curcumin analogues as anti-malarialagentsrdquo Bioorganic and Medicinal Chemistry vol 16 no 6 pp2894ndash2902 2008
[24] G Liang L Shao Y Wang et al ldquoExploration and synthesis ofcurcumin analogues with improved structural stability both invitro and in vivo as cytotoxic agentsrdquo Bioorganic and MedicinalChemistry vol 17 no 6 pp 2623ndash2631 2009
[25] M J Ahsan ldquoSynthesis and anticancer activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide analoguesrdquo Lettersin Drug Design amp Discovery vol 9 no 9 pp 823ndash827 2012
[26] Salahuddin M Shaharyar A Majumdar and M J AhsanldquoSynthesis characterization and anticancer evaluation of 2-(naphthalen-1-ylmethylnaphthalen-2-yloxymethyl)-1-[5-(sub-stituted phenyl)-[134]oxadiazol-2-ylmethyl]-1H-benzimida-zolerdquo Arabian Journal of Chemistry 2013
[27] E Zwick J Bange andAUllrich ldquoReceptor tyrosine kinase sig-nalling as a target for cancer intervention strategiesrdquo Endocrine-Related Cancer vol 8 no 3 pp 161ndash173 2001
[28] H A Bhuva and S G Kini ldquoSynthesis anticancer activity anddocking of some substituted benzothiazoles as tyrosine kinaseinhibitorsrdquo Journal ofMolecular Graphics andModelling vol 29no 1 pp 32ndash37 2010
[29] J Mendelsohn and J Baselga ldquoThe EGF receptor family astargets for cancer therapyrdquo Oncogene vol 19 no 56 pp 6550ndash6565 2000
[30] M Amir M J Ahsan and I Ali ldquoSynthesis of N1-(3-chloro-4-flouropheny)-N4-substituted semicarbazones as novel anticon-vulsant agentsrdquo Indian Journal of Chemistry B vol 49 no 11 pp1509ndash1514 2010
[31] httpdtpncinihgov[32] A Monks D Scudiero P Skehan et al ldquoFeasibility of a high-
flux anticancer drug screen using a diverse panel of culturedhuman tumor cell linesrdquo Journal of theNational Cancer Institutevol 83 no 11 pp 757ndash766 1991
[33] M R Body and K D Paull ldquoSome practical considerations andapplications of the national cancer institute in vitro anticancerdrug discovery screenrdquoDrug Development Research vol 34 no2 pp 91ndash109 1995
[34] R H Shoemaker ldquoThe NCI60 human tumour cell line anti-cancer drug screenrdquo Nature Reviews Cancer vol 6 no 10 pp813ndash823 2006
14 BioMed Research International
[35] M C Alley D A Scudiero A Monks et al ldquoFeasibility ofdrug screening with panels of human tumor cell lines using amicroculture tetrazolium assayrdquo Cancer Research vol 48 no 3pp 589ndash601 1988
[36] M R Grever S A Schepartz and B A Chabner ldquoTheNationalCancer Institute cancer drug discovery and development pro-gramrdquo Seminars in Oncology vol 19 no 6 pp 622ndash638 1992
[37] P Dubreuil S Letard M Ciufolini et al ldquoMasitinib (AB1010)a potent and selective tyrosine kinase inhibitor targeting KITrdquoPLoS ONE vol 4 no 9 Article ID e7258 2009
[38] J A Blair D Rauh C Kung et al ldquoStructure-guided devel-opment of affinity probes for tyrosine kinases using chemicalgeneticsrdquo Nature Chemical Biology vol 3 no 4 pp 229ndash2382007
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Figure 4 (a) Compound 13 is represented with macromodel stick modelThe hydrogen bonds are shown with yellow colour dotted lines andhydrophobic interactions with orange dotted lines (b) Compound 13 at binding site on surface
(a) (b)
Figure 5 (a) Compound 3 is represented with macromodel stick modelThe hydrophobic cavity residues are represented with magenta stickmodelThe hydrogen bonds are shownwith yellow colour dotted lines and hydrophobic interactions with orange dotted lines (b) Compound3 at binding site on surface
cancer) with cell promotion of minus6358 while the maximumcell promotion was observed on TK-10 (renal cancer)which showed 1584 growth promotion (8416 growthinhibition) at one-dose assay The compound 10 was foundto be highly active on SK-MEL-5 (melanoma) with cellpromotion of minus8652 followed by UACC-62 (melanoma)with cell promotion of minus8439 and COLO 205 (coloncancer) with cell promotion of minus8405 while the maximumcell promotion was observed on UACC-257 (melanoma)which showed 1603 growth promotion (8397 growthinhibition) at one-dose assay Compound 11 was found to behighly active on CAKI-1 (renal cancer) with cell promotionof minus3477 followed by MDA-MB-435 (melanoma) with cellpromotion of minus3053 and SF-295 (CNS cancer) with cellpromotion of minus2968 while the maximum cell promotionwas observed on UACC-257 (melanoma) which showed8713 growth promotion (1287 growth inhibition) at one-dose assay Compound 12 was found to be highly active onHT29 (colon cancer) with cell promotion of minus641 followedby MDA-MB-468 (breast cancer) with cell promotion ofminus634 and CCRF-CEM (leukemia) with cell promotion ofminus1672 while the maximum cell promotion was observedon TK-10 (renal cancer) which showed 10274 growthpromotion at one-dose assay Compound 13 was found to behighly active on HT29 (colon cancer) with cell promotion ofminus7624 followed by 786-0 and A498 (renal cancer) with cellpromotion of minus5893 and minus5692 respectively while themaximum cell promotion was observed on NCIADR-RES(ovarian cancer) which showed 3932 growth promotion(1287 growth inhibition) at one-dose assay Among the35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) Compound
8 was found to be the most active compound of theseries with mean growth percent of minus1919 followed bycompound 5 which showed mean growth percent of minus1225and compound 1 which showed mean growth percent ofminus466 Among the 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl (substituted phenyl)methanone analogues(10-11) Compound 10 showed maximum activity with meangrowth percent of minus2871 while compound 13 was found tobe the most active compound among dihydropyrimidineanalogues (12ndash14) which showed mean growth percent ofminus546 Overall the most active compound of the serieswas compound 10 and most of the 35-bis(4-hydroxy-3-methylstyryl)-N-(substituted phenyl)-1H-pyrazole-1-carbox-amide analogues possessed potent anticancer activity Basedon our finding we can conclude that electronegative groupat position 2 (ie 2-chloro) on N-substituted phenyl ringamong 35-bis(4-hydroxy-3-methylstyryl)-N-(substitutedphenyl)-1H-pyrazole-1-carboxamide analogues (1ndash9) showedmore anticancer activity than at position 4 (ie 4-chloro and4-fluoro) Electron releasing group such as 24-dimethyl and4-methyl on N-substituted phenyl ring increased anticanceractivity and the activity was found to be more if thenumber of electron releasing groups was increased Amongthe 35-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-yl(substituted phenyl)methanone analogues (10 and 11) theactivity was found to bemore when there was no substitutionin the phenyl ring Among six member dihydropyrimidineanalogues (12ndash14) compound with X substitution as ldquoNHrdquoshowed more activity than as ldquoOrdquo substitution
The results of 5-dose assay of the most active compoundsamong their respective series are given in Table 3 Compound8 presented GI
50ranging between 0004 and 204 120583M The
12 BioMed Research International
best results were recorded on the leukemia cell lines withvalues ranging from 0004 to 0364 120583M (Figure 2(a)) Anumber of 3 tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 0383120583M Only in 23 cell linescompound 8 registered LC
50valuewithgt100120583MCompound
10 presented GI50ranging between 00079 and 186 120583M The
best results were recorded on the leukemia cell line withvalue ranging from 00353 to 0347 120583M (Figure 2(b)) Fourof the tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 000815120583M Only in 27 cell linescompound 10 registered LC
50value with gt100 120583M Com-
pound 13 presented GI50ranging between 0524 and 339 120583M
The best results were recorded on the colon cancer cell linewith values ranging from 0543 to 218 120583M(Figure 2(c)) Onlyone tested cancer cell line presented TGI value with gt100 120583Mthe best result value being noted on the HT29 (colon cancer)with value 0948 In 28 cell lines compound 13 registeredLC50value with gt100120583M
Some of the synthesized compounds were evaluated forEGFR tyrosine kinase inhibitory activity by recombinanttyrosine kinase assay using an ELISA-based assay withpoly(Glu Tyr 4 1) as a substrate [37] The results showedcompounds 8 10 and 13 shows inhibition of EGFR tyrosinekinase with IC
50values of 355 292 and 557120583Mrespectively
33 Molecular Docking The binding site of the referenceinhibitor is well defined by hydrophobic cavity The EGFRtyrosine kinase binding site contains the important residuesCys797 andThr790 re docking of reference ligand shown thatabove residues are important for hydrophobic interactionswith the same [38] The hydrophobic cavity with the residuesLeu788 Met766 Lys745 Glu762 Thr854 and Met793 makesthe binding site attractive for the design of new inhibitorsBased on our knowledge we designed some class of newpyrazole (1ndash11) dihydropyrimidine analogues (12ndash14) forEGFR tyrosine kinase inhibition But the binding sites ofour new molecules were found to be a little different withcomparison to reference inhibitor which is reported in thecrystal structure of EGFR tyrosine kinase (PDB 2J5F) Theseligandsrsquo interactions were shown in three different motifsThe first one is hydrophobic cavity in which the substitutedmethoxy hydroxy phenyl ring of the ligand is showinggood interactions as reference inhibitor The next bindingmotif contains the target residue Cys797 the N-substitutedpyrazole derivatives and six membered (dihydropyrimidine)are binding at this site Most of these interactions werehydrophobic The last binding motif contains the residuesLys875 and Asp831 which lie in side chain of the protein andshowed the side chain hydrogen bonding with the designedmolecules The docking score and Emodel score of some ofthe compounds selected for anticancer activity by NCI aregiven in Table 4 and binding modes are shown in Figure 3
Compound 13 showed the best hydrophobic interactionwith various residues such as Cys797 Met 793 Leu 844 Leu792 Ala 743 and Val 726 (Figures 4(a) and 4(b)) Negativelycharged Asp 837 interacted with methoxy hydroxy phenyl
ring (4-hydroxy-3-methoxyphenyl) Arg 841 showed 120587-120587cationic interaction The Met 793 present in the hydrophobiccavity showed H-bond backbone interaction and H-bond(side chain) interaction The positively charged Lys 845and Arg 841 destabilized interactions The interactions ofcompound 13 with the EGFR kinase receptor shown inribbon diagram the hydrophobic cavity is indicated withyellow colour loop motif-1 is indicated with green colourbeta sheet and the motif-2 is indicated with green colourloop The other ligands were also having good interactionswith the hydrophobic cavity and bindingmotifs the followingresidues leu718 Pro794 Met793 Ala743 Leu844 Val726and Cys797 were present around the ligand Thr790 Gln791having polar characteristics andMet 793 were responsible forbackbone H-bond interaction as well as H-bond (side chain)interaction Asp855 and Asp837 showed charged interactionslike destabilizing contacts Positively charged Lys875 andArg841 were found to have 120587-120587 cationic interaction and werepresent in bindingmotifmaking different binding patterns ofreported ligandsThe docking study for compound 3 is givenin Figures 5(a) and 5(b)
4 Conclusion
The novel series of curcumin analogues (pyrazoles) weresynthesized in satisfactory yields The anticancer activityshowed promising results The studies confirmed compound10 as potent lead compound for drug discovery and furtheroptimization The curcumin analogues discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer disease The pyrazole discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer
Acknowledgments
The authors are thankful to the staff members of the NationalCancer Institute (NCI) USA for in vitro anticancer screeningof the newly synthesized compounds They are especiallygrateful to Professor Dizon B Thelma Project ManagerNational Cancer Institute (NCI) USA
References
[1] WHO Cancer World Health Organization February 2006httpenwikipediaorgwikicancer
[2] M N Noolvi H M Patel V Bhardwaj and A ChauhanldquoSynthesis and in vitro antitumor activity of substituted quina-zoline and quinoxaline derivatives search for anticancer agentrdquoEuropean Journal of Medicinal Chemistry vol 46 no 6 pp2327ndash2346 2011
[3] N Aydemir and R Bilaloglu ldquoGenotoxicity of two anticancerdrugs gemcitabine and topotecan in mouse bone marrow invivordquoMutation Research vol 537 no 1 pp 43ndash51 2003
[4] I Bouabdallah L A MrsquoBarek A Zyad A Ramdani I Zidaneand A Melhaoui ldquoNew pyrazolic compounds as cytotoxicagentsrdquo Natural Product Research vol 21 no 4 pp 298ndash3022007
BioMed Research International 13
[5] D Havrylyuk B Zimenkovsky O Vasylenko L Zaprutko AGzella and R Lesyk ldquoSynthesis of novel thiazolone-based com-pounds containing pyrazoline moiety and evaluation of theiranticancer activityrdquo European Journal of Medicinal Chemistryvol 44 no 4 pp 1396ndash1404 2009
[6] M Shaharyar M M Abdullah M A Bakht and J MajeedldquoPyrazoline bearing benzimidazoles search for anticanceragentrdquo European Journal of Medicinal Chemistry vol 45 no 1pp 114ndash119 2010
[7] P-C Lv H-Q Li J Sun Y Zhou and H-L Zhu ldquoSynthesisand biological evaluation of pyrazole derivatives containingthiourea skeleton as anticancer agentsrdquo Bioorganic and Medici-nal Chemistry vol 18 no 13 pp 4606ndash4614 2010
[8] M S Christodoulou S Liekens K M Kasiotis and S AHaroutounian ldquoNovel pyrazole derivatives synthesis and eval-uation of anti-angiogenic activityrdquo Bioorganic and MedicinalChemistry vol 18 no 12 pp 4338ndash4350 2010
[9] M J Ahsan G J Samy H Khalillah R C Krit and SSoni ldquoMolecular properties prediction and synthesis of novelpyrazoline carboxamide analogs as antitubercular agentsrdquoAnti-Infective Agents vol 10 no 2 pp 117ndash123 2012
[10] M J Ahsan J G Samy H Khalilullah M A Bakht andM Z Hassan ldquoSynthesis and antimycobacterial evaluation of3a4-dihydro-3H-indeno [12-c] pyrazole-2-carboxamide ana-loguesrdquo European Journal of Medicinal Chemistry vol 46 no11 pp 5694ndash5697 2011
[11] M J Ahsan J G Samy K R Dutt et al ldquoDesign synthesis andantimycobacterial evaluation of novel 3-substituted-N-aryl-67-dimethoxy-3a4-dihydro-3H-indeno[12-c] pyrazole-2-carboxamide analoguesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 15 pp 4451ndash4453 2011
[12] M J Ahsan J G Samy S Soni et al ldquoDiscovery of novelantitubercular 3a4-dihydro-3H-indeno[12-c]pyrazole-2-carndashboxamidecarbothioamide analoguesrdquo Bioorganic and Medici-nal Chemistry Letters vol 21 no 18 pp 5259ndash5261 2011
[13] M J Ahsan H Khalilullah J P Stables and J GovindasamyldquoSynthesis and anticonvulsant activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide carbothioamide ana-loguesrdquo Journal of Enzyme Inhibition amp Medicinal Chemistryvol 28 no 3 pp 644ndash650 2013
[14] M J Ahsan J Govindasamy H Khalilullah et al ldquoPOMAanalyses as new efficient bioinformaticsrsquoplatform to predictand optimize bioactivity of synthesized 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamidecarbothioamide anal-oguesrdquo Bioorganic amp Medicinal Chemistry vol 22 no 24 pp7029ndash7035 2012
[15] H Khalilullah S Khan M J Ahsan and B AhmedldquoSynthesis and antihepatotoxic activity of 5-(23-dihydro-14-benzodioxane-6-yl)-3-substituted-phenyl-45-dihydro-1H-pyrazole derivativesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 24 pp 7251ndash7254 2011
[16] M J Alam M J Ahsan O Alam and S A KhanldquoSynthesis of 4-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)-6-(substituted phenyl)pyrimidin-2-ol analogues as 8 anti-inflammatory and analgesic agentsrdquo Letters in Drug Design ampDiscovery vol 10 no 8 pp 776ndash782 2013
[17] D P Chauhan ldquoChemotherapeutic potential of curcumin forcolorectal cancerrdquo Current Pharmaceutical Design vol 8 no 19pp 1695ndash1706 2002
[18] Y-L Jia J Li Z-H Qin and Z-Q Liang ldquoAutophagic andapoptotic mechanisms of curcumin-induced death in K562
cellsrdquo Journal of Asian Natural Products Research vol 11 no 11pp 918ndash928 2009
[19] A Kunwar A Barik BMishra K Rathinasamy R Pandey andK I Priyadarsini ldquoQuantitative cellular uptake localization andcytotoxicity of curcumin in normal and tumor cellsrdquoBiochimicaet Biophysica Acta vol 1780 no 4 pp 673ndash679 2008
[20] J Lal S K Gupta DThavaselvam and D D Agarwal ldquoDesignsynthesis synergistic antimicrobial activity and cytotoxicityof 4-aryl substituted 34-dihydropyrimidinones of curcuminrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 8 pp2872ndash2876 2012
[21] R K Singh D Rai D Yadav A Bhargava J Balzarini and EDe Clercq ldquoSynthesis antibacterial and antiviral properties ofcurcumin bioconjugates bearing dipeptide fatty acids and folicacidrdquo European Journal of Medicinal Chemistry vol 45 no 3pp 1078ndash1086 2010
[22] K Saja M S Babu D Karunagaran and P R SudhakaranldquoAnti-inflammatory effect of curcumin involves downregula-tion of MMP-9 in blood mononuclear cellsrdquo InternationalImmunopharmacology vol 7 no 13 pp 1659ndash1667 2007
[23] S Mishra K Karmodiya N Surolia and A Surolia ldquoSynthesisand exploration of novel curcumin analogues as anti-malarialagentsrdquo Bioorganic and Medicinal Chemistry vol 16 no 6 pp2894ndash2902 2008
[24] G Liang L Shao Y Wang et al ldquoExploration and synthesis ofcurcumin analogues with improved structural stability both invitro and in vivo as cytotoxic agentsrdquo Bioorganic and MedicinalChemistry vol 17 no 6 pp 2623ndash2631 2009
[25] M J Ahsan ldquoSynthesis and anticancer activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide analoguesrdquo Lettersin Drug Design amp Discovery vol 9 no 9 pp 823ndash827 2012
[26] Salahuddin M Shaharyar A Majumdar and M J AhsanldquoSynthesis characterization and anticancer evaluation of 2-(naphthalen-1-ylmethylnaphthalen-2-yloxymethyl)-1-[5-(sub-stituted phenyl)-[134]oxadiazol-2-ylmethyl]-1H-benzimida-zolerdquo Arabian Journal of Chemistry 2013
[27] E Zwick J Bange andAUllrich ldquoReceptor tyrosine kinase sig-nalling as a target for cancer intervention strategiesrdquo Endocrine-Related Cancer vol 8 no 3 pp 161ndash173 2001
[28] H A Bhuva and S G Kini ldquoSynthesis anticancer activity anddocking of some substituted benzothiazoles as tyrosine kinaseinhibitorsrdquo Journal ofMolecular Graphics andModelling vol 29no 1 pp 32ndash37 2010
[29] J Mendelsohn and J Baselga ldquoThe EGF receptor family astargets for cancer therapyrdquo Oncogene vol 19 no 56 pp 6550ndash6565 2000
[30] M Amir M J Ahsan and I Ali ldquoSynthesis of N1-(3-chloro-4-flouropheny)-N4-substituted semicarbazones as novel anticon-vulsant agentsrdquo Indian Journal of Chemistry B vol 49 no 11 pp1509ndash1514 2010
[31] httpdtpncinihgov[32] A Monks D Scudiero P Skehan et al ldquoFeasibility of a high-
flux anticancer drug screen using a diverse panel of culturedhuman tumor cell linesrdquo Journal of theNational Cancer Institutevol 83 no 11 pp 757ndash766 1991
[33] M R Body and K D Paull ldquoSome practical considerations andapplications of the national cancer institute in vitro anticancerdrug discovery screenrdquoDrug Development Research vol 34 no2 pp 91ndash109 1995
[34] R H Shoemaker ldquoThe NCI60 human tumour cell line anti-cancer drug screenrdquo Nature Reviews Cancer vol 6 no 10 pp813ndash823 2006
14 BioMed Research International
[35] M C Alley D A Scudiero A Monks et al ldquoFeasibility ofdrug screening with panels of human tumor cell lines using amicroculture tetrazolium assayrdquo Cancer Research vol 48 no 3pp 589ndash601 1988
[36] M R Grever S A Schepartz and B A Chabner ldquoTheNationalCancer Institute cancer drug discovery and development pro-gramrdquo Seminars in Oncology vol 19 no 6 pp 622ndash638 1992
[37] P Dubreuil S Letard M Ciufolini et al ldquoMasitinib (AB1010)a potent and selective tyrosine kinase inhibitor targeting KITrdquoPLoS ONE vol 4 no 9 Article ID e7258 2009
[38] J A Blair D Rauh C Kung et al ldquoStructure-guided devel-opment of affinity probes for tyrosine kinases using chemicalgeneticsrdquo Nature Chemical Biology vol 3 no 4 pp 229ndash2382007
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
best results were recorded on the leukemia cell lines withvalues ranging from 0004 to 0364 120583M (Figure 2(a)) Anumber of 3 tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 0383120583M Only in 23 cell linescompound 8 registered LC
50valuewithgt100120583MCompound
10 presented GI50ranging between 00079 and 186 120583M The
best results were recorded on the leukemia cell line withvalue ranging from 00353 to 0347 120583M (Figure 2(b)) Fourof the tested cancer cell lines presented TGI value withgt100120583M the best result value being noted on the MDA-MB-435 (melanoma) with value 000815120583M Only in 27 cell linescompound 10 registered LC
50value with gt100 120583M Com-
pound 13 presented GI50ranging between 0524 and 339 120583M
The best results were recorded on the colon cancer cell linewith values ranging from 0543 to 218 120583M(Figure 2(c)) Onlyone tested cancer cell line presented TGI value with gt100 120583Mthe best result value being noted on the HT29 (colon cancer)with value 0948 In 28 cell lines compound 13 registeredLC50value with gt100120583M
Some of the synthesized compounds were evaluated forEGFR tyrosine kinase inhibitory activity by recombinanttyrosine kinase assay using an ELISA-based assay withpoly(Glu Tyr 4 1) as a substrate [37] The results showedcompounds 8 10 and 13 shows inhibition of EGFR tyrosinekinase with IC
50values of 355 292 and 557120583Mrespectively
33 Molecular Docking The binding site of the referenceinhibitor is well defined by hydrophobic cavity The EGFRtyrosine kinase binding site contains the important residuesCys797 andThr790 re docking of reference ligand shown thatabove residues are important for hydrophobic interactionswith the same [38] The hydrophobic cavity with the residuesLeu788 Met766 Lys745 Glu762 Thr854 and Met793 makesthe binding site attractive for the design of new inhibitorsBased on our knowledge we designed some class of newpyrazole (1ndash11) dihydropyrimidine analogues (12ndash14) forEGFR tyrosine kinase inhibition But the binding sites ofour new molecules were found to be a little different withcomparison to reference inhibitor which is reported in thecrystal structure of EGFR tyrosine kinase (PDB 2J5F) Theseligandsrsquo interactions were shown in three different motifsThe first one is hydrophobic cavity in which the substitutedmethoxy hydroxy phenyl ring of the ligand is showinggood interactions as reference inhibitor The next bindingmotif contains the target residue Cys797 the N-substitutedpyrazole derivatives and six membered (dihydropyrimidine)are binding at this site Most of these interactions werehydrophobic The last binding motif contains the residuesLys875 and Asp831 which lie in side chain of the protein andshowed the side chain hydrogen bonding with the designedmolecules The docking score and Emodel score of some ofthe compounds selected for anticancer activity by NCI aregiven in Table 4 and binding modes are shown in Figure 3
Compound 13 showed the best hydrophobic interactionwith various residues such as Cys797 Met 793 Leu 844 Leu792 Ala 743 and Val 726 (Figures 4(a) and 4(b)) Negativelycharged Asp 837 interacted with methoxy hydroxy phenyl
ring (4-hydroxy-3-methoxyphenyl) Arg 841 showed 120587-120587cationic interaction The Met 793 present in the hydrophobiccavity showed H-bond backbone interaction and H-bond(side chain) interaction The positively charged Lys 845and Arg 841 destabilized interactions The interactions ofcompound 13 with the EGFR kinase receptor shown inribbon diagram the hydrophobic cavity is indicated withyellow colour loop motif-1 is indicated with green colourbeta sheet and the motif-2 is indicated with green colourloop The other ligands were also having good interactionswith the hydrophobic cavity and bindingmotifs the followingresidues leu718 Pro794 Met793 Ala743 Leu844 Val726and Cys797 were present around the ligand Thr790 Gln791having polar characteristics andMet 793 were responsible forbackbone H-bond interaction as well as H-bond (side chain)interaction Asp855 and Asp837 showed charged interactionslike destabilizing contacts Positively charged Lys875 andArg841 were found to have 120587-120587 cationic interaction and werepresent in bindingmotifmaking different binding patterns ofreported ligandsThe docking study for compound 3 is givenin Figures 5(a) and 5(b)
4 Conclusion
The novel series of curcumin analogues (pyrazoles) weresynthesized in satisfactory yields The anticancer activityshowed promising results The studies confirmed compound10 as potent lead compound for drug discovery and furtheroptimization The curcumin analogues discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer disease The pyrazole discovered in thisstudy may provide valuable therapeutic intervention for thetreatment of cancer
Acknowledgments
The authors are thankful to the staff members of the NationalCancer Institute (NCI) USA for in vitro anticancer screeningof the newly synthesized compounds They are especiallygrateful to Professor Dizon B Thelma Project ManagerNational Cancer Institute (NCI) USA
References
[1] WHO Cancer World Health Organization February 2006httpenwikipediaorgwikicancer
[2] M N Noolvi H M Patel V Bhardwaj and A ChauhanldquoSynthesis and in vitro antitumor activity of substituted quina-zoline and quinoxaline derivatives search for anticancer agentrdquoEuropean Journal of Medicinal Chemistry vol 46 no 6 pp2327ndash2346 2011
[3] N Aydemir and R Bilaloglu ldquoGenotoxicity of two anticancerdrugs gemcitabine and topotecan in mouse bone marrow invivordquoMutation Research vol 537 no 1 pp 43ndash51 2003
[4] I Bouabdallah L A MrsquoBarek A Zyad A Ramdani I Zidaneand A Melhaoui ldquoNew pyrazolic compounds as cytotoxicagentsrdquo Natural Product Research vol 21 no 4 pp 298ndash3022007
BioMed Research International 13
[5] D Havrylyuk B Zimenkovsky O Vasylenko L Zaprutko AGzella and R Lesyk ldquoSynthesis of novel thiazolone-based com-pounds containing pyrazoline moiety and evaluation of theiranticancer activityrdquo European Journal of Medicinal Chemistryvol 44 no 4 pp 1396ndash1404 2009
[6] M Shaharyar M M Abdullah M A Bakht and J MajeedldquoPyrazoline bearing benzimidazoles search for anticanceragentrdquo European Journal of Medicinal Chemistry vol 45 no 1pp 114ndash119 2010
[7] P-C Lv H-Q Li J Sun Y Zhou and H-L Zhu ldquoSynthesisand biological evaluation of pyrazole derivatives containingthiourea skeleton as anticancer agentsrdquo Bioorganic and Medici-nal Chemistry vol 18 no 13 pp 4606ndash4614 2010
[8] M S Christodoulou S Liekens K M Kasiotis and S AHaroutounian ldquoNovel pyrazole derivatives synthesis and eval-uation of anti-angiogenic activityrdquo Bioorganic and MedicinalChemistry vol 18 no 12 pp 4338ndash4350 2010
[9] M J Ahsan G J Samy H Khalillah R C Krit and SSoni ldquoMolecular properties prediction and synthesis of novelpyrazoline carboxamide analogs as antitubercular agentsrdquoAnti-Infective Agents vol 10 no 2 pp 117ndash123 2012
[10] M J Ahsan J G Samy H Khalilullah M A Bakht andM Z Hassan ldquoSynthesis and antimycobacterial evaluation of3a4-dihydro-3H-indeno [12-c] pyrazole-2-carboxamide ana-loguesrdquo European Journal of Medicinal Chemistry vol 46 no11 pp 5694ndash5697 2011
[11] M J Ahsan J G Samy K R Dutt et al ldquoDesign synthesis andantimycobacterial evaluation of novel 3-substituted-N-aryl-67-dimethoxy-3a4-dihydro-3H-indeno[12-c] pyrazole-2-carboxamide analoguesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 15 pp 4451ndash4453 2011
[12] M J Ahsan J G Samy S Soni et al ldquoDiscovery of novelantitubercular 3a4-dihydro-3H-indeno[12-c]pyrazole-2-carndashboxamidecarbothioamide analoguesrdquo Bioorganic and Medici-nal Chemistry Letters vol 21 no 18 pp 5259ndash5261 2011
[13] M J Ahsan H Khalilullah J P Stables and J GovindasamyldquoSynthesis and anticonvulsant activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide carbothioamide ana-loguesrdquo Journal of Enzyme Inhibition amp Medicinal Chemistryvol 28 no 3 pp 644ndash650 2013
[14] M J Ahsan J Govindasamy H Khalilullah et al ldquoPOMAanalyses as new efficient bioinformaticsrsquoplatform to predictand optimize bioactivity of synthesized 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamidecarbothioamide anal-oguesrdquo Bioorganic amp Medicinal Chemistry vol 22 no 24 pp7029ndash7035 2012
[15] H Khalilullah S Khan M J Ahsan and B AhmedldquoSynthesis and antihepatotoxic activity of 5-(23-dihydro-14-benzodioxane-6-yl)-3-substituted-phenyl-45-dihydro-1H-pyrazole derivativesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 24 pp 7251ndash7254 2011
[16] M J Alam M J Ahsan O Alam and S A KhanldquoSynthesis of 4-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)-6-(substituted phenyl)pyrimidin-2-ol analogues as 8 anti-inflammatory and analgesic agentsrdquo Letters in Drug Design ampDiscovery vol 10 no 8 pp 776ndash782 2013
[17] D P Chauhan ldquoChemotherapeutic potential of curcumin forcolorectal cancerrdquo Current Pharmaceutical Design vol 8 no 19pp 1695ndash1706 2002
[18] Y-L Jia J Li Z-H Qin and Z-Q Liang ldquoAutophagic andapoptotic mechanisms of curcumin-induced death in K562
cellsrdquo Journal of Asian Natural Products Research vol 11 no 11pp 918ndash928 2009
[19] A Kunwar A Barik BMishra K Rathinasamy R Pandey andK I Priyadarsini ldquoQuantitative cellular uptake localization andcytotoxicity of curcumin in normal and tumor cellsrdquoBiochimicaet Biophysica Acta vol 1780 no 4 pp 673ndash679 2008
[20] J Lal S K Gupta DThavaselvam and D D Agarwal ldquoDesignsynthesis synergistic antimicrobial activity and cytotoxicityof 4-aryl substituted 34-dihydropyrimidinones of curcuminrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 8 pp2872ndash2876 2012
[21] R K Singh D Rai D Yadav A Bhargava J Balzarini and EDe Clercq ldquoSynthesis antibacterial and antiviral properties ofcurcumin bioconjugates bearing dipeptide fatty acids and folicacidrdquo European Journal of Medicinal Chemistry vol 45 no 3pp 1078ndash1086 2010
[22] K Saja M S Babu D Karunagaran and P R SudhakaranldquoAnti-inflammatory effect of curcumin involves downregula-tion of MMP-9 in blood mononuclear cellsrdquo InternationalImmunopharmacology vol 7 no 13 pp 1659ndash1667 2007
[23] S Mishra K Karmodiya N Surolia and A Surolia ldquoSynthesisand exploration of novel curcumin analogues as anti-malarialagentsrdquo Bioorganic and Medicinal Chemistry vol 16 no 6 pp2894ndash2902 2008
[24] G Liang L Shao Y Wang et al ldquoExploration and synthesis ofcurcumin analogues with improved structural stability both invitro and in vivo as cytotoxic agentsrdquo Bioorganic and MedicinalChemistry vol 17 no 6 pp 2623ndash2631 2009
[25] M J Ahsan ldquoSynthesis and anticancer activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide analoguesrdquo Lettersin Drug Design amp Discovery vol 9 no 9 pp 823ndash827 2012
[26] Salahuddin M Shaharyar A Majumdar and M J AhsanldquoSynthesis characterization and anticancer evaluation of 2-(naphthalen-1-ylmethylnaphthalen-2-yloxymethyl)-1-[5-(sub-stituted phenyl)-[134]oxadiazol-2-ylmethyl]-1H-benzimida-zolerdquo Arabian Journal of Chemistry 2013
[27] E Zwick J Bange andAUllrich ldquoReceptor tyrosine kinase sig-nalling as a target for cancer intervention strategiesrdquo Endocrine-Related Cancer vol 8 no 3 pp 161ndash173 2001
[28] H A Bhuva and S G Kini ldquoSynthesis anticancer activity anddocking of some substituted benzothiazoles as tyrosine kinaseinhibitorsrdquo Journal ofMolecular Graphics andModelling vol 29no 1 pp 32ndash37 2010
[29] J Mendelsohn and J Baselga ldquoThe EGF receptor family astargets for cancer therapyrdquo Oncogene vol 19 no 56 pp 6550ndash6565 2000
[30] M Amir M J Ahsan and I Ali ldquoSynthesis of N1-(3-chloro-4-flouropheny)-N4-substituted semicarbazones as novel anticon-vulsant agentsrdquo Indian Journal of Chemistry B vol 49 no 11 pp1509ndash1514 2010
[31] httpdtpncinihgov[32] A Monks D Scudiero P Skehan et al ldquoFeasibility of a high-
flux anticancer drug screen using a diverse panel of culturedhuman tumor cell linesrdquo Journal of theNational Cancer Institutevol 83 no 11 pp 757ndash766 1991
[33] M R Body and K D Paull ldquoSome practical considerations andapplications of the national cancer institute in vitro anticancerdrug discovery screenrdquoDrug Development Research vol 34 no2 pp 91ndash109 1995
[34] R H Shoemaker ldquoThe NCI60 human tumour cell line anti-cancer drug screenrdquo Nature Reviews Cancer vol 6 no 10 pp813ndash823 2006
14 BioMed Research International
[35] M C Alley D A Scudiero A Monks et al ldquoFeasibility ofdrug screening with panels of human tumor cell lines using amicroculture tetrazolium assayrdquo Cancer Research vol 48 no 3pp 589ndash601 1988
[36] M R Grever S A Schepartz and B A Chabner ldquoTheNationalCancer Institute cancer drug discovery and development pro-gramrdquo Seminars in Oncology vol 19 no 6 pp 622ndash638 1992
[37] P Dubreuil S Letard M Ciufolini et al ldquoMasitinib (AB1010)a potent and selective tyrosine kinase inhibitor targeting KITrdquoPLoS ONE vol 4 no 9 Article ID e7258 2009
[38] J A Blair D Rauh C Kung et al ldquoStructure-guided devel-opment of affinity probes for tyrosine kinases using chemicalgeneticsrdquo Nature Chemical Biology vol 3 no 4 pp 229ndash2382007
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
[5] D Havrylyuk B Zimenkovsky O Vasylenko L Zaprutko AGzella and R Lesyk ldquoSynthesis of novel thiazolone-based com-pounds containing pyrazoline moiety and evaluation of theiranticancer activityrdquo European Journal of Medicinal Chemistryvol 44 no 4 pp 1396ndash1404 2009
[6] M Shaharyar M M Abdullah M A Bakht and J MajeedldquoPyrazoline bearing benzimidazoles search for anticanceragentrdquo European Journal of Medicinal Chemistry vol 45 no 1pp 114ndash119 2010
[7] P-C Lv H-Q Li J Sun Y Zhou and H-L Zhu ldquoSynthesisand biological evaluation of pyrazole derivatives containingthiourea skeleton as anticancer agentsrdquo Bioorganic and Medici-nal Chemistry vol 18 no 13 pp 4606ndash4614 2010
[8] M S Christodoulou S Liekens K M Kasiotis and S AHaroutounian ldquoNovel pyrazole derivatives synthesis and eval-uation of anti-angiogenic activityrdquo Bioorganic and MedicinalChemistry vol 18 no 12 pp 4338ndash4350 2010
[9] M J Ahsan G J Samy H Khalillah R C Krit and SSoni ldquoMolecular properties prediction and synthesis of novelpyrazoline carboxamide analogs as antitubercular agentsrdquoAnti-Infective Agents vol 10 no 2 pp 117ndash123 2012
[10] M J Ahsan J G Samy H Khalilullah M A Bakht andM Z Hassan ldquoSynthesis and antimycobacterial evaluation of3a4-dihydro-3H-indeno [12-c] pyrazole-2-carboxamide ana-loguesrdquo European Journal of Medicinal Chemistry vol 46 no11 pp 5694ndash5697 2011
[11] M J Ahsan J G Samy K R Dutt et al ldquoDesign synthesis andantimycobacterial evaluation of novel 3-substituted-N-aryl-67-dimethoxy-3a4-dihydro-3H-indeno[12-c] pyrazole-2-carboxamide analoguesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 15 pp 4451ndash4453 2011
[12] M J Ahsan J G Samy S Soni et al ldquoDiscovery of novelantitubercular 3a4-dihydro-3H-indeno[12-c]pyrazole-2-carndashboxamidecarbothioamide analoguesrdquo Bioorganic and Medici-nal Chemistry Letters vol 21 no 18 pp 5259ndash5261 2011
[13] M J Ahsan H Khalilullah J P Stables and J GovindasamyldquoSynthesis and anticonvulsant activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide carbothioamide ana-loguesrdquo Journal of Enzyme Inhibition amp Medicinal Chemistryvol 28 no 3 pp 644ndash650 2013
[14] M J Ahsan J Govindasamy H Khalilullah et al ldquoPOMAanalyses as new efficient bioinformaticsrsquoplatform to predictand optimize bioactivity of synthesized 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamidecarbothioamide anal-oguesrdquo Bioorganic amp Medicinal Chemistry vol 22 no 24 pp7029ndash7035 2012
[15] H Khalilullah S Khan M J Ahsan and B AhmedldquoSynthesis and antihepatotoxic activity of 5-(23-dihydro-14-benzodioxane-6-yl)-3-substituted-phenyl-45-dihydro-1H-pyrazole derivativesrdquo Bioorganic and Medicinal ChemistryLetters vol 21 no 24 pp 7251ndash7254 2011
[16] M J Alam M J Ahsan O Alam and S A KhanldquoSynthesis of 4-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)-6-(substituted phenyl)pyrimidin-2-ol analogues as 8 anti-inflammatory and analgesic agentsrdquo Letters in Drug Design ampDiscovery vol 10 no 8 pp 776ndash782 2013
[17] D P Chauhan ldquoChemotherapeutic potential of curcumin forcolorectal cancerrdquo Current Pharmaceutical Design vol 8 no 19pp 1695ndash1706 2002
[18] Y-L Jia J Li Z-H Qin and Z-Q Liang ldquoAutophagic andapoptotic mechanisms of curcumin-induced death in K562
cellsrdquo Journal of Asian Natural Products Research vol 11 no 11pp 918ndash928 2009
[19] A Kunwar A Barik BMishra K Rathinasamy R Pandey andK I Priyadarsini ldquoQuantitative cellular uptake localization andcytotoxicity of curcumin in normal and tumor cellsrdquoBiochimicaet Biophysica Acta vol 1780 no 4 pp 673ndash679 2008
[20] J Lal S K Gupta DThavaselvam and D D Agarwal ldquoDesignsynthesis synergistic antimicrobial activity and cytotoxicityof 4-aryl substituted 34-dihydropyrimidinones of curcuminrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 8 pp2872ndash2876 2012
[21] R K Singh D Rai D Yadav A Bhargava J Balzarini and EDe Clercq ldquoSynthesis antibacterial and antiviral properties ofcurcumin bioconjugates bearing dipeptide fatty acids and folicacidrdquo European Journal of Medicinal Chemistry vol 45 no 3pp 1078ndash1086 2010
[22] K Saja M S Babu D Karunagaran and P R SudhakaranldquoAnti-inflammatory effect of curcumin involves downregula-tion of MMP-9 in blood mononuclear cellsrdquo InternationalImmunopharmacology vol 7 no 13 pp 1659ndash1667 2007
[23] S Mishra K Karmodiya N Surolia and A Surolia ldquoSynthesisand exploration of novel curcumin analogues as anti-malarialagentsrdquo Bioorganic and Medicinal Chemistry vol 16 no 6 pp2894ndash2902 2008
[24] G Liang L Shao Y Wang et al ldquoExploration and synthesis ofcurcumin analogues with improved structural stability both invitro and in vivo as cytotoxic agentsrdquo Bioorganic and MedicinalChemistry vol 17 no 6 pp 2623ndash2631 2009
[25] M J Ahsan ldquoSynthesis and anticancer activity of 3a 4-dihydro-3H-indeno[1 2-c]pyrazole-2-carboxamide analoguesrdquo Lettersin Drug Design amp Discovery vol 9 no 9 pp 823ndash827 2012
[26] Salahuddin M Shaharyar A Majumdar and M J AhsanldquoSynthesis characterization and anticancer evaluation of 2-(naphthalen-1-ylmethylnaphthalen-2-yloxymethyl)-1-[5-(sub-stituted phenyl)-[134]oxadiazol-2-ylmethyl]-1H-benzimida-zolerdquo Arabian Journal of Chemistry 2013
[27] E Zwick J Bange andAUllrich ldquoReceptor tyrosine kinase sig-nalling as a target for cancer intervention strategiesrdquo Endocrine-Related Cancer vol 8 no 3 pp 161ndash173 2001
[28] H A Bhuva and S G Kini ldquoSynthesis anticancer activity anddocking of some substituted benzothiazoles as tyrosine kinaseinhibitorsrdquo Journal ofMolecular Graphics andModelling vol 29no 1 pp 32ndash37 2010
[29] J Mendelsohn and J Baselga ldquoThe EGF receptor family astargets for cancer therapyrdquo Oncogene vol 19 no 56 pp 6550ndash6565 2000
[30] M Amir M J Ahsan and I Ali ldquoSynthesis of N1-(3-chloro-4-flouropheny)-N4-substituted semicarbazones as novel anticon-vulsant agentsrdquo Indian Journal of Chemistry B vol 49 no 11 pp1509ndash1514 2010
[31] httpdtpncinihgov[32] A Monks D Scudiero P Skehan et al ldquoFeasibility of a high-
flux anticancer drug screen using a diverse panel of culturedhuman tumor cell linesrdquo Journal of theNational Cancer Institutevol 83 no 11 pp 757ndash766 1991
[33] M R Body and K D Paull ldquoSome practical considerations andapplications of the national cancer institute in vitro anticancerdrug discovery screenrdquoDrug Development Research vol 34 no2 pp 91ndash109 1995
[34] R H Shoemaker ldquoThe NCI60 human tumour cell line anti-cancer drug screenrdquo Nature Reviews Cancer vol 6 no 10 pp813ndash823 2006
14 BioMed Research International
[35] M C Alley D A Scudiero A Monks et al ldquoFeasibility ofdrug screening with panels of human tumor cell lines using amicroculture tetrazolium assayrdquo Cancer Research vol 48 no 3pp 589ndash601 1988
[36] M R Grever S A Schepartz and B A Chabner ldquoTheNationalCancer Institute cancer drug discovery and development pro-gramrdquo Seminars in Oncology vol 19 no 6 pp 622ndash638 1992
[37] P Dubreuil S Letard M Ciufolini et al ldquoMasitinib (AB1010)a potent and selective tyrosine kinase inhibitor targeting KITrdquoPLoS ONE vol 4 no 9 Article ID e7258 2009
[38] J A Blair D Rauh C Kung et al ldquoStructure-guided devel-opment of affinity probes for tyrosine kinases using chemicalgeneticsrdquo Nature Chemical Biology vol 3 no 4 pp 229ndash2382007
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
[35] M C Alley D A Scudiero A Monks et al ldquoFeasibility ofdrug screening with panels of human tumor cell lines using amicroculture tetrazolium assayrdquo Cancer Research vol 48 no 3pp 589ndash601 1988
[36] M R Grever S A Schepartz and B A Chabner ldquoTheNationalCancer Institute cancer drug discovery and development pro-gramrdquo Seminars in Oncology vol 19 no 6 pp 622ndash638 1992
[37] P Dubreuil S Letard M Ciufolini et al ldquoMasitinib (AB1010)a potent and selective tyrosine kinase inhibitor targeting KITrdquoPLoS ONE vol 4 no 9 Article ID e7258 2009
[38] J A Blair D Rauh C Kung et al ldquoStructure-guided devel-opment of affinity probes for tyrosine kinases using chemicalgeneticsrdquo Nature Chemical Biology vol 3 no 4 pp 229ndash2382007
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014