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Research ArticleDesign and Microwave Assisted Synthesis of CoumarinDerivatives as PDE Inhibitors
Mahadev N Kumbar1 Ravindra R Kamble1 Atulkumar A Kamble1
1Department of Chemistry Karnatak University Pavate Nagar Dharwad 580003 India2Centre of Excellence in Clinical Embryology Level 2 Central Research Lab Kasturba Medical College Manipal UniversityManipal Karnataka 576104 India3Department of Studies in Chemistry Mangalore University Konaje Karnataka 574199 India
Correspondence should be addressed to Ravindra R Kamble kamchem9gmailcom
Received 30 October 2015 Revised 26 December 2015 Accepted 27 December 2015
Academic Editor Hussein El-Subbagh
Copyright copy 2016 Mahadev N Kumbar et al This 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
Coumarins appended to benzimidazole through pyrazole are designed and synthesized using microwave irradiation Thesecompounds were analyzed for phosphodiesterase (PDE) inhibition indirectly by motility pattern in human spermatozoa Someof the synthesized compounds namely 5d 5e 5f 5g 5h and 5k have exhibited potent inhibitory activity on PDE
1 Introduction
The development of simple mild practicable cheap eco-benign method for the synthesis of heterocycles has grabbedthe attention of researchers In particular microwave assistedorganic synthesis has become a rapidly growing field inorganic chemistry as this technique makes reaction timeshorter and tolerates wide range reactions which are bestsuited to the increased demands of industry [1] and some ofthe synthesized compounds were screened by PDE
PDE inhibitors are therapeutic agents which target PDEisoenzymes and inhibit the catabolism of the secondary mes-sengers such as cyclic adenosine monophosphate (cAMP)and cyclic guanosine monophosphate (cGMP) thus pro-longing the biological effect determined by the type ofcell involved Cyclic nucleotide phosphodiesterases (PDEs)catalyze the hydrolysis of cAMP andor cGMPThey functionin conjunction with adenylyl and guanylyl cyclases to regulatethe amplitude and duration of cell signaling mechanismsmediated via cAMP and cGMP They therefore serve to reg-ulate a range of biological responses to first messengers suchas light vascular resistance cardiac output visceral motility
immune response [2] inflammation [3] neuroplasticityvision [4] and reproduction [5] Phosphodiesterases (PDEs)modulate the activity of cyclic nucleotides by regulating theirdegradation PDEs are critical determinants for modulationof cellular levels of cAMP andor cGMP by many stimuli[6] Thus the ubiquitously present PDEs play a pivotal rolein regulating cell signaling the breakdown of cAMP andcGMP
The fold selectivity for PDE5 over PDE11A4 for Sildenafil(1000-fold selectivity) and Vardenafil (9000-fold selectivity)reveals that these drugs are very unlikely to cross-react withPDE11A4 in patients treated with these medications On thecontrary it was suggested to use the newest PDE5 inhibitorTadalafil with caution In fact the 40-fold selectivity ratio ofTadalafil for PDE5 over PDE11A4 is significantly lower thanthose reported with the other two drugs and is nearly thesame as that reported for Sildenafil over PDE1 (41-fold) Inthis case it has been suggested that the 41-fold selectivity ofSildenafil for PDE5 over PDE1 may induce vasodilatationflushing and tachycardia Within human PDE11A familythe alternative splicing leads to generation of proteins whichdisplay unique properties [7 8]
Hindawi Publishing CorporationInternational Journal of Medicinal ChemistryVolume 2016 Article ID 9890630 16 pageshttpdxdoiorg10115520169890630
2 International Journal of Medicinal Chemistry
X
R
N
N
NN
N
O
R = 5-MeO 5-Me 4-MeO X = CH N
OO
N
N
NH
N NHN
F
S
O
O
R = methyl phenyl
V VI VII
NH
NNH
SO
N
HN
NH
OO
Ph
NH
N
NS
S
O
N
NH
I II III IV
OCH3OCH3O(CH2)3 3OCH
R998400
H3C
Figure 1 Some of the benzimidazole drugs and known PDE inhibitors
NO
NN
Ph NO N
N
Ph
RN
ON
O
Ph
VIII IX X
Figure 2 Representative drug molecules as PDE inhibitors VIIIndashX
Benzimidazole nucleus is the key building block fornumerous drugs that play beneficial roles in the functioningof biologically important molecules Specifically this nucleusis a constituent of Vit B
12and many currently existing
medications Almost all benzimidazoles with different het-erocyclic substituents led to essential modification in theirphysicochemical metabolic and pharmacokinetic properties[9] Benzimidazole is a core structural moiety found in someof the important drugs like albendazole (I)mebendazole (II)thiabendazole (III) rabeprazole (IV) and so forth Literaturesurvey revealed that coumarin (V) scaffolds were proved toincrease the cAMP levels through the specific inhibition ofPDE3 in accordance with their common structural features[10] and pyrazole (VI) derivatives have been explored for theidentification of phosphodiesterase (PDE4) inhibitors as isexemplified by the discovery and development of tofimilast[11] also most of the benzimidazole (VII) derivatives haveshown very prominent PDE (10A) activity [12] Coumarinpyrazole and benzimidazole core structuralmoiety are foundin some of the important PDE inhibition compounds by Yanget al [13] (Figure 1)
Also the scaffolds containing coumarin pyrazole andbenzimidazole are the key moieties in heterocyclic chemistryand are important structural units of various natural andsynthetic biologically active molecules They are known
to possess a wide range of pharmacological activities thatinclude antimicrobial and anti-inflammatory activitiesManycoumarin derivatives have shown anticancer anticoagulantanti-inflammatory antimicrobial antioxidant antiviral andcardiovascular activities [14ndash22]
During drug development of PDEs it was believed thatSAR of pyrazole and pyrazole replacements that remove thehydrogen bond donor were very promising The structuralanalogs viz compounds VIII and X contain benzoxazoleand benzimidazole attached to quinoline through oxygen aslinker groupThese have shown possess promising PDE inhi-bition as reported by Hamaguchi et al [23] Also substitutionof the pyrazole with simple alkyl groups retained potencywhile adding minimal molecular weight The methyl sub-stituted pyrazole IX (Figure 2) in an in vitro P-glycoprotein(PgP) overexpressing cell line had improved the efflux ratiosThese above drug development results and the structures ofvarious classes of clinically established PDEs conform to abroadly accepted pharmacophore (Figure 3) This suggeststhat three important structural requirements are to be presentto show PDE inhibition Those three important parametersare viz (i) fused heterocyclic ring for good oral bioavail-ability (ring A)and (ii) a five-membered heterocycle or alkylchain (B)which is connected to another fused five-memberedheterocycle (ring C) [24]
International Journal of Medicinal Chemistry 3
O O
NN
Ph
NH
N A B
C
Figure 3 Designed 3-(4-(1H-benzo[d]imidazol-2-yl)-1-phenyl-1H-pyrazol-3-yl)-2H-chromen-2-ones derivatives 5andashx
Reflux or MW
YX
1andashd 2andashd
3andashd
4andashf
5andashx
O O
NNPh
NH
N Y
O
O
O
R EtOHAcOH
RT stir
O
N
O
R
NH
Ph
O O
R
NN
CHO
Ph
R
X
DMFPOCl3
PhNHNH2
50ndash60∘C
4h
NH2
NH2
Scheme 1 Synthetic route for title compounds 5andashx where 5a R = -H X = -H Y = H 5b R = -H X = 6-Cl Y = H 5c R = -H X = 6-CH3
Y = H 5d R = -H X = 6-NO2 Y = H 5e R = -H X = 6-Br Y = N 5f R = -H X = 57-dimethyl Y = H 5g R = -6Cl X = -H Y = H 5h
R = -6Cl X = -4Cl Y = H 5i R = -6Cl X = -CH3 Y = H 5j R = -6Cl X = 6-NO
2 Y = H 5k R = -6Cl X = 6-Br Y = N 5l R = -6Cl X =
57-dimethyl Y = H 5m R = -6Br X = -H Y = H 5n R = -6Br X = -4Cl Y = H 5o R = -6Br X = -CH3 Y = H 5p R = -6Br X = 6-NO
2 Y =
H 5q R = -6Br X = 5-Br Y = N 5r R = -6Br X = 57-dimethyl Y = H 5s R = 8-OCH3 X = -H Y = H 5t R = 8-OCH
3 X = 4-Cl Y = H 5u
R = 8-OCH3 X = 6-CH
3 Y = H 5v R = 8-OCH
3 X = 6-NO
2 Y = H 5w R = 8-OCH
3 X = 5-Br Y = N 5x R = 8-OCH
3 X = 57-dimethyl
Y = H
In view of the above and in search of biologically potentnovel heterocyclic scaffolds herein we report facile and inex-pensive method for the synthesis of coumarin with pyrazolenucleus and functionalized benzimidazoles in a singlemoietywith an expectation to obtain potent PDE inhibitor
2 Results and Discussion
21 Chemistry The starting materials 3-acetyl-2H-chromen-2-one 1andashd and 3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde 3andashdwere obtained using literaturemethods [25]The synthetic protocols for the title compounds3-(4-(1H-benzo[d]imidazol-2-yl)-1-phenyl-1H-pyrazol-3-yl)-2H-chromen-2-ones 5andashx are outlined in Scheme 1 Alsoan optimum condition was established under microwaveirradiation for the synthesis of the title compounds 5andashxby condensation of o-arylenediamines 4andashf and 3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-carbaldehydes
3andashd in ethanol in 5ndash8min with excellent yields Comparedto conventional method (4ndash6 hrs) microwave irradiationgreatly reduced the reaction time from 3-4 h to 6ndash8min Theyield of the product was also increased up to 94 (Table 1)
Structures of all the synthesized compounds 5andashx wereconfirmed by various spectroscopic techniques viz IR 1H13CNMR MS and elemental analyses The compounds haveshown strong adsorption band for carbonyl of coumarin andN-H of benzimidazole ring at 1711ndash1737 and 3345ndash3368 cmminus1respectively In case of 1HNMR spectra all the compoundsexhibited a singlet in the range 1042ndash1336 ppm for benzim-idazole N-H ring and coumarin C
4H at 769ndash792 ppm The
aromatic protons of all pyrazole rings appeared as multipletsin the range 732ndash798 ppm In case of 13CNMR spectralstudy the numbers of signals are consistent with number ofmagnetically nonequivalent carbon atoms in the moleculeand in mass spectra all the synthesized title compounds haveshown the molecular ion peaks at their respectivemz values
4 International Journal of Medicinal Chemistry
Table1Com
paris
onof
conventio
naland
microwaves
ynthesisof
titlecompo
unds
5andashx
from
3andashd
and4andashf
inabsoluteethano
lund
erop
timized
cond
ition
s
3andashd
4andashf
5andashx
Con
ventional
Microwave
Time(hr)
Yield(
)Time(min)
Yield
NN
Ph
CHO
OO 3a
4a
H2N
H2N
NN
Ph
N H
NO
O 5a
439
594
NN
Ph
CHO
OO
3a
Cl4b
H2N
H2N
NN
Ph
N H
NCl
OO
5b
435
590
NN
Ph
CHO
OO
3a4c
H2N
H2N
NN Ph
N H
NO
O 5c
438
588
NN
Ph
CHO
OO
3a4d
H2N
H2N
NO
2
NN
Ph
N H
N
OO
O 5d
N+ O
minus
432
586
International Journal of Medicinal Chemistry 5
Table1Con
tinued
3andashd
4andashf
5andashx
Con
ventional
Microwave
Time(hr)
Yield(
)Time(min)
Yield
NN
Ph
CHO
OO 3a
N
Br
4e
H2N
H2N
NN
Ph
N
N H
NO
OBr
5e
434
592
NN
Ph
CHO
OO 3a
4f
H2N
H2N
NN
Ph
N H
NO
O 5f
435
590
NN
Ph
CHO
OO
Cl3b
4a
H2N
H2N
NN
Ph
N H
NO
O
Cl
5g
540
694
NN
Ph
CHO
OO
Cl3b
Cl4b
H2N
H2N
NN
Ph
N H
NCl
OO
Cl
5h
532
685
6 International Journal of Medicinal Chemistry
Table1Con
tinued
3andashd
4andashf
5andashx
Con
ventional
Microwave
Time(hr)
Yield(
)Time(min)
Yield
NN
Ph
CHO
OO
Cl3b
4c
H2N
H2N
NN
Ph
N H
NO
O
Cl
5i
530
687
NN
Ph
CHO
OO
Cl3b
4d
H2N
H2N
NO
2N
N
Ph
N H
N
OO
O
Cl
5j
N+ O
minus
535
684
NN
Ph
CHO
OO
Cl3b
N
Br
4e
H2N
H2N
NN
Ph
N N H
NO
O
Cl
Br
5k
534
686
NN
Ph
CHO
OO
Cl3b
4f
H2N
H2N
NN
Ph
N H
NO
O
Cl
5l
537
690
International Journal of Medicinal Chemistry 7
Table1Con
tinued
3andashd
4andashf
5andashx
Con
ventional
Microwave
Time(hr)
Yield(
)Time(min)
Yield
NN
Ph
CHO
OO
Br3c
4a
H2N
H2N
NN
Ph
N H
NO
O
Br
5m
633
789
NN
Ph
CHO
OO
Br3c
Cl4b
H2N
H2N
NN
Ph
N H
NCl
OO
Br
5n
636
792
NN
Ph
CHO
OO
Br3c
4c
H2N
H2N
NN
Ph
N H
NO
O
Br
5o
639
794
NN
Ph
CHO
OO
Br3c
4d
H2N
H2N
NO
2
NN
Ph
N H
N
O
OO
Br
5p
N+
Ominus
636
784
8 International Journal of Medicinal Chemistry
Table1Con
tinued
3andashd
4andashf
5andashx
Con
ventional
Microwave
Time(hr)
Yield(
)Time(min)
Yield
NN
Ph
CHO
OO
Br3c
N
Br
4e
H2N
H2N
NN
Ph
N
N H
NO
O
Br
Br
5q
631
790
NN
Ph
CHO
OO
Br3c
4f
H2N
H2N
NN
Ph
N H
NO
O
Br
5r
634
792
NN
Ph
CHO
OO
O
3d4a
H2N
H2N
NN
Ph
N H
NO
O
O
5s
538
887
NN
Ph
CHO
OO
O
3d
Cl
4b
H2N
H2N
NN
Ph
N H
N
ClO
OO
5t
540
885
International Journal of Medicinal Chemistry 9
Table1Con
tinued
3andashd
4andashf
5andashx
Con
ventional
Microwave
Time(hr)
Yield(
)Time(min)
Yield
NN
Ph
CHO
OO
O
3d4c
H2N
H2N
NN
Ph
N H
NO
OO
5u
532
888
NN
Ph
CHO
OO
O
3d4d
H2N
H2N
NO
2
NN
Ph
N H
N
OO
OO
5v
N+
Ominus
536
886
NN
Ph
CHO
OO
O
3d
N
Br
4e
H2N
H2N
NN
Ph
N
N H
NO
OO
Br
5w
538
880
NN
Ph
CHO
OO
O
3d4f
H2N
H2N
NN
Ph
N H
NO
OO
5x
534
882
10 International Journal of Medicinal Chemistry
3 PDE Inhibition Study
31 Effect on Motility of Frozen-Thawed Human SpermatozoaTo assess the effect of title compounds on themotility patternhuman spermatozoa subjected to freeze-thaw process wasused It is well documented that freeze-thaw process canhave detrimental effect on the motility and survival of thespermatozoa [26] Therefore they can be an excellent modelto study the effect of any test compounds on themotility [27]
In the present study we assessed the motility patternin spermatozoa processed with media containing variouscompounds manually at 1 4 and 24 h after incubationin vitro Three arbitrary doses were selected (05 10 and50 120583gmL) for each compound to assess their effect on spermmotility Pentoxifylline (PTF) which is known to enhancethe human sperm motility by elevating intracellular cyclicadenosine monophosphate (cAMP) level [27] was taken asa positive control Based on the previous report PTF at 1mMconcentration was used [28] Since the test compounds werenot soluble in water dimethyl sulfoxide (DMSO 005) wasused as vehicle control
The sperm motility enhancement property of the com-pounds was categorized as below
(a) Marginal which resulted in lt5 increase in themotility (b) Good which resulted in 5ndash10 increase inmotility (c) Excellent which resulted in gt10 increase inmotility (d) Poor where there was no enhancement inmotility
At 1 h after the in vitro incubation the majority of thecompounds had marginal effect on the sperm motility com-pared to control At this interval PTF had higher percentageof total and progressivelymotile spermatozoa compared to allthe test compounds A large number of studies have shownthat PTF can trigger the motility in fresh or frozen-thawedspermatozoa as early as 1 h [29] even though the majorityof the studies indicate that this effect is mainly due to theelevated cAMP level in the spermatozoa [30] it can also bemediated through tyrosine phosphorylation [31]
Compounds 5a 5b 5c 5d 5e 5g 5h and 5k inducedmarginal enhancement in total motility while compounds5a 5b 5c and 5j resulted in similar effect on progressivemotility (Tables 2(a) and 2(b)) A 5ndash10 increase in totalmotility was induced by compound 5j However with respectto progressive motility compounds 5d 5e 5f and 5k cameunder compounds with good effect on motility More than10 increase in progressive motility was observed only forcompound 5g (at 05 120583gmL concentration) and compound5h (at 5 120583gmL concentration)
A time-dependent decrease in total and progressivemotility was observed in all the groups However the declinewasmore evident at 24 h intervalThis phenomenon has beendocumented by earlier studies [28] These changes may berelated to the oxidative stress induced by in vitro cultureconditions Spermatozoa are highly susceptible to reactiveoxygen species (ROS) due to high polyunsaturated fatty acidcontent in their plasma membrane and negligible amount ofcytoplasmic antioxidants
At 4 h interval compounds 5e 5f and 5g enhancedthe progressive motility of spermatozoa by 5ndash10 while
compound 5h with two chloro substituents on coumarinand benzimidazole resulted in almost 15 increase in theprogressive motility However the effect was lower than thatof pentoxifylline The rest of the compounds (5a 5b 5c5f and 5j) induced a marginal increase in motility exceptin compound 5e in which the motility was lower than thecontrol group
At 24 h interval the majority of the compounds (5a5b 5d 5f 5h and 5j) had marginally higher percentageof motility or poor motility compounds (5c 5e and 5g)compared to control Interestingly compound 5k with chlorosubstituent on coumarin and bromo substitution on ben-zimidazole had higher percentage of motile spermatozoacompared to control (14 higher) and even PTF (9 higher)This indicates that compound5k canhelp in prolonging the invitro survival of spermatozoa which has significant beneficialrole in assisted reproductive technologies such as in vitrofertilization Previous studies have shown that even thoughPTF has triggering effect on themotility it induces prematureacrosome reaction and drastic decrease in motility at laterintervals In this context the compounds which increasethe motility and longevity of spermatozoa under in vitroconditions are of clinical relevance
Based on the manual motility assessment we observedthat compounds 5d 5f 5g 5h and 5k have considerablebeneficial role in spermmotilityTherefore these compoundswere further assessed for their effect on the kinematicsof spermatozoa at 1 h However there was no significantdifference in the kinematics of spermatozoa compared tocontrol and PTF (Table 3) In conclusion the newly synthe-sized coumarin derivatives have sperm motility enhancingproperty However in this preliminary screening we havetaken three arbitrary doses to assess their effect on motilityCompounds 5g 5h and 5k show considerable beneficialeffect which can be of clinical significance where spermmotility enhancement is achieved under in vitro condi-tions Compounds 5g and 5h triggered the motility at earlyintervals which was similar to the PTF while compound5k improved the longevity of the spermatozoa Since thecoumarin is known to have PDE inhibitory function themotility enhancement could be due to the elevated cAMPlevel in sperm However further detailed studies are essentialto understand the mechanism of action of these compoundsand their clinical utility
4 Experimental
All the chemicals and reagents were purchased from theMerck and Aldrich chemical suppliers Melting points (mp)were determined in open capillaries and are uncorrectedThe IR spectra were recorded on a Nicolet Impact 410 FTIR spectrometer using KBr pellets (range 4000ndash500 cmminus1)The 1HNMR spectra were recorded at 400MHz on BrukerAvance FT NMR spectrometer in DMSO-d
6solvent with
TMS as internal standard 13CNMR spectra were recordedat 100MHz on Bruker Avance FT NMR spectrometer inDMSO-d
6solvent with TMS as internal standard The mass
spectra were recorded on Shimadzu GC-MS operating at70 eV Thin-layer chromatography (TLC) was performed on
International Journal of Medicinal Chemistry 11
Table 2 Effect of supplementation of coumarin derivatives (5andashj) to sperm wash medium on the motility pattern in frozen-thawed humanspermatozoa at various time intervals
(a)
Compounds Time (h)Total motility (mean plusmn SE of percentage)
020mm Aluchrosep silica gel 60 F254 plates (SD FineMumbai) Microwave irradiation experiments were carriedout using CEMDiscover SPMicrowave Synthesizer equippedwith IR sensor to monitor reaction temperatures
5 General Procedures for the Preparation ofTitle Compounds 5andashx
3-(2-Oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-car-baldehydes (3andashd 1 mmol) were prepared from 3-acetyl-coumarin (1andashd 1 mmol) phenylhydrazine (1mmol) andthe intermediate (Schiff base) (2andashd 1 mmol) followedby reaction with POCl
3in DMF by Vilsmeier Haack for-
mylation strategies [25] The target compounds 5andashxwere then achieved by stirring substituted carbaldehyde(3andashd 1 mmol) with different o-arylenediamine (4andashf1 mmol) under reflux for 4-5 hrs After completion (TLChexane ethylacetate 7 3) of the reaction the solid obtainedwas filtered washed with small quantity of ethanol anddried Further it was recrystallized in methanol to get thepure compound (5andashx)
51 Microwave Assisted Procedure for the Preparation of Com-pounds 5andashx A mixture of 3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde (3andashd 1 mmol) and o-phenylenediamine (4andashf 1 mmol) taken in ethanol (5mL)was introduced into a CEM microwave reaction vesselequipped with magnetic stirrer The reaction mixture was
prestirred for 1min at room temperature and irradiated at180W (150∘C) for about 6ndash8min (TLC using hexane ethylacetate (7 3 drops) as eluent) The reaction mixture wasthen quenched into crushed ice and the crude product wasfiltered washed and dried Recrystallization from methanolgave pure crystals of the compounds 5andashx
(Spectral as well as elemental analyses data are providedas electronic supplementary file in Supplementary Materialavailable online at httpdxdoiorg10115520169890630)
6 Assessment of Phosphodiesterase InhibitionUsing Human Spermatozoa as Model
Earlier studies have shown that PDE inhibitors can enhancethe spermmotility [32 33] Based on this in the present studywe have used an indirect approach to assess the inhibitoryeffect of the coumarin derivatives on PDE by assessing spermmotility pattern
61 Preparation of Test Solution Stock solution of the testcompounds (1mgmL) was prepared by dissolving them indimethyl sulfoxide (DMSO Sigma Cat number D5879)The stock solution was then diluted with Earlrsquos BalancedSalt Solution (EBSS) supplemented with 01 bovine serumalbumin (BSA) in a ratio of 1 200 1 1000 and 1 2000 to getworking solutions of 5 1 and 05 120583gmL concentrations Theworking solutions were preincubated at 37∘C and 5 CO
2
prior to use
International Journal of Medicinal Chemistry 13
Table 3 Effect of various coumarin derivatives on kinematics of frozen-thawed spermatozoa at one hour after incubation in vitro
Parameters Compounds Concentrations of compounds in 120583gmL VC PTFC 0 05 5 (005 DMSO) (1mM)
62 Study Subjects Infertile men who attended AndrologyLaboratory Kasturba Medical College Manipal UniversityManipal during the period of June-July 2015 for routinesemen evaluation were included in the study The subjects
with a sexual abstinence of 3ndash5 days were asked to providethe semen samples by masturbation in a sterile containerFollowing liquefaction the semen parameters were assessedas described by Kotdawala et al [34]The study was approved
14 International Journal of Medicinal Chemistry
by the Institutional Ethics Committee of Kasturba HospitalKasturba Medical College Manipal University (IEC number1552015)
63 Cryopreservation and Thawing The liquefied semensamples were cryopreserved by rapid freezing method asdescribed previously [34] with minor modifications Brieflythe semen samples were mixed with equal volume of freezingmedium (Sperm Freeze FertiPro Cat number 0344) in acryovial (Thermoscientific Nunc Cat number 138627) andkept at 4∘C for 10min in liquid nitrogen (LN2) vapor phasefor 5min and then finally plunged into liquid nitrogenThe samples were thawed after 1 week by rapid thawingmethod by placing themat 37∘C for 5minThe cryoprotectantmedium was completely removed by mixing the spermsuspension with EBSS medium and centrifuging at 1000 rpmfor 8min The resultant pellet was resuspended with freshEBSS medium and used for further analysis
64 Sample Preparation To screen the compounds withenhancing effect on themotility of spermatozoa under in vitroconditions the frozen-thawed semen samples were washedby mixing with equal volume of preincubated Earlrsquos BalancedSalt Solution (EBSS Sigma Cat number E2888) followed bycentrifugation at 1800 rpm for 8min The pellet was thengently mixed with 1mL of EBSS media containing 01bovine serum albumin (BSA Sigma Cat number A3311)and equally divided into control vehicle control and testgroups (with average sperm density of 5 times 104 spermatozoain each group) The motile sperm were extracted by swimup method as described earlier [35] The sperm suspensionwas centrifuged at 1200 rpm for 8min and the resultantpellet was overlaidwith EBSSmedium containing various testcompoundsThe sperm suspensions were incubated for 1 h at37∘Cand 5CO
2afterwhich themotile spermwere collected
from the supernatant
65 Motility Assessment Spermatozoa in the supernatantfraction were assessed for their motility patterns at 1 4 and24 h after incubation under light microscope as describedearlier [27] Sperm suspension was (10 120583L droplet) placed ona clean microscope slide and a coverslip was placed over itSpermatozoa with progressive motility and nonprogressivemotility and immotile spermatozoa were scored separatelyfrom a total of 200 spermatozoa in random fields at 400xmagnification
66 Sperm Kinematics Using Computer Assisted Semen Anal-ysis (CASA) The motion characteristics in the spermatozoawere assessed at 1 4 and 24 h after incubation using CASAsystem (ISAS Spain) Briefly 5 120583L of sperm suspension wasplaced on a clean microscope slide prewarmed at 37∘C Thesperm suspensionwas coveredwith prewarmed cover slip (22times 22mm) and observed under microscope (negative phasecontrast 10x objectives Proiser Spain) For each sample thekinematics was assessed randomly at ten different fields Theparameters such as curvilinear velocity (VCL) straight line
velocity (VSL) average path velocity (VAP) amplitude of lat-eral head displacement (ALH) linearity (LIN) straightness(STR) wobble (WOB) and beat cross frequency (BCF) wereanalyzed
7 Conclusions
In conclusion we have reported efficient and environmen-tally benignmethodologies for the synthesis of 3-[4-(1H-benzo[d]imidazol-2-yl)-1-phenyl-1H-pyrazol-3-yl]-2H-chromen-2-one derivatives 5andashx by using thermal and by microwaveirradiation under neat conditions in presence of ethanol Thereactions carried out under microwave irradiation affordedbenzimidazoles in short period of time with excellent yieldsHence this methodology would make an interesting strategyfor the synthesis of various substituted coumarin pyrazolebenzimidazoles Also compounds 5g 5h and 5k exhibitedimpressive sperm motility enhancing property whereas 5khas spermatozoa longevity property
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to University Scientific Instrumen-tation Centre (USIC) Karnatak University Dharwad forprovidingmass and IR spectral dataThe authors are thankfulto Indian Institute of Science (IISc) Bengaluru for carryingout NMR analyses and to UGC New Delhi for the financialassistance under UPE program vide no [F No 14-22012(NSPE) dated 22-01-2013] One of the authors (MNK)acknowledges University Grants Commission (UGC) NewDelhi for fellowship under UGC-UPE program
References
[1] P P Kattimani R R Kamble and G Y Meti ldquoExpedientsynthesis of benzimidazoles using amidesrdquo RSC Advances vol5 no 37 pp 29447ndash29455 2015
[2] L Li C Yee and J A Beavo ldquoCD3- and CD28-dependentinduction of PDE7 required for T cell activationrdquo Science vol283 no 5403 pp 848ndash849 1999
[3] T P Dousa ldquoCyclic-3101584051015840-nucleotide phosphodiesteraseisozymes in cell biology and pathophysiology of the kidneyrdquoKidney International vol 55 no 1 pp 29ndash62 1999
[4] C Pfister N Bennett F Bruckert et al ldquoInteractions of a G-protein with its effector transducin and cGMP phosphodi-esterase in retinal rodsrdquo Cell Signaling vol 5 no 3 pp 235ndash2411993
[5] S L C Jin F J Richard W-P Kuo A J DrsquoErcole andM Conti ldquoImpaired growth and fertility of cAMP-specificphosphodiesterase PDE4D-deficient micerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 21 pp 11998ndash12003 1999
[6] S H Francis I V Turko and J D Corbin ldquoCyclic nucleotidephosphodiesterases relating structure and functionrdquoProgress in
International Journal of Medicinal Chemistry 15
Nucleic Acid Research and Molecular Biology vol 65 pp 1ndash522000
[7] J M Hetman N Robas R Baxendale et al ldquoCloning and char-acterization of two splice variants of human phosphodiesterase11Ardquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 23 pp 12891ndash12895 2000
[8] K Yuasa T Ohgaru M Asahina and K Omori ldquoIdentificationof rat cyclic nucleotide phosphodiesterase 11A (PDE11A) com-parison of rat and human PDE11A splicing variantsrdquo EuropeanJournal of Biochemistry vol 268 no 16 pp 4440ndash4448 2001
[9] S K Sahu A Mishra and R K Behera ldquoSynthesis of thiazolebenzothiazole oxadiazole thiadiazole triazole and thiazolidi-none incorporated coumarinsrdquo Indian Journal of HeterocyclicChemistry vol 6 no 2 pp 91ndash94 1996
[10] G Roma M Di Braccio G Grossi et al ldquoSynthesis and in vitroantiplatelet activity of new 4-(1-piperazinyl) coumarin deriva-tives Human platelet phosphodiesterase 3 inhibitory propertiesof the two most effective compounds described and molecularmodeling study on their interactions with phosphodiesterase3A catalytic siterdquo Journal of Medicinal Chemistry vol 50 no 12pp 2886ndash2895 2007
[11] T Shyamsunder Reddy K Shiva Kumar C L T Medaet al ldquoConformationally restricted novel pyrazole deriva-tives synthesis of 18-disubstituted 55-dimethyl-45-dihydro-1H-benzo[g]indazoles as a new class of PDE4 inhibitorsrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 9 pp3248ndash3255 2012
[12] A Chino N Masuda Y Amano et al ldquoNovel benzimidazolederivatives as phosphodiesterase 10A (PDE10A) inhibitors withimproved metabolic stabilityrdquo Bioorganic and Medicinal Chem-istry vol 22 no 13 pp 3515ndash3526 2014
[13] H Yang FNMurigi ZWang J Li H Jin andZ Tu ldquoSynthesisand in vitro characterization of cinnoline and benzimidazoleanalogues as phosphodiesterase 10A inhibitorsrdquo Bioorganic andMedicinal Chemistry Letters vol 25 pp 919ndash924 2015
[14] J Nawrot-Modranka E Nawrot and J Graczyk ldquoIn vivo antitu-mor in vitro antibacterial activity and alkylating properties ofphosphorohydrazine derivatives of coumarin and chromonerdquoEuropean Journal of Medicinal Chemistry vol 41 no 11 pp1301ndash1309 2006
[15] A A Bekhit and T Abdel-Aziem ldquoDesign synthesis andbiological evaluation of some pyrazole derivatives as anti-inflammatory-antimicrobial agentsrdquo Bioorganic and MedicinalChemistry vol 12 no 8 pp 1935ndash1945 2004
[16] T Devji C Reddy CWoo S Awale S Kadota and D Carrico-Moniz ldquoPancreatic anticancer activity of a novel geranylgerany-lated coumarin derivativerdquo Bioorganic andMedicinal ChemistryLetters vol 21 no 19 pp 5770ndash5773 2011
[17] I Kostova I Manolov I Nicolova S Konstantinov andM Karaivanova ldquoNew lanthanide complexes of 4-methyl-7-hydroxycoumarin and their pharmacological activityrdquo Euro-pean Journal of Medicinal Chemistry vol 36 no 4 pp 339ndash3472001
[18] M Ghate R A Kusanur and M V Kulkarni ldquoSynthesis andin vivo analgesic and anti-inflammatory activity of some bi-heterocyclic coumarin derivativesrdquo European Journal of Medic-inal Chemistry vol 40 no 9 pp 882ndash887 2005
[19] PM RonadMN Noolvi S Sapkal S Dharbhamulla andV SMaddi ldquoSynthesis and antimicrobial activity of 7-(2-substitutedphenylthiazolidinyl)-benzopyran-2-one derivativesrdquo EuropeanJournal of Medicinal Chemistry vol 45 no 1 pp 85ndash89 2010
[20] GMelagraki A Afantitis O Igglessi-Markopoulou et al ldquoSyn-thesis and evaluation of the antioxidant and anti-inflammatoryactivity of novel coumarin-3-aminoamides and their alpha-lipoic acid adductsrdquo European Journal of Medicinal Chemistryvol 44 no 7 pp 3020ndash3026 2009
[21] J C Trivedi J B Bariwal K D Upadhyay et al ldquoImprovedand rapid synthesis of new coumarinyl chalcone derivatives andtheir antiviral activityrdquo Tetrahedron Letters vol 48 no 48 pp8472ndash8474 2007
[22] S Vilar E Quezada L Santana et al ldquoDesign synthe-sis and vasorelaxant and platelet antiaggregatory activitiesof coumarinndashresveratrol hybridsrdquo Bioorganic and MedicinalChemistry Letters vol 16 no 2 pp 257ndash261 2006
[23] W Hamaguchi N Masuda M Isomura et al ldquoDesign and syn-thesis of novel benzimidazole derivatives as phosphodiesterase10A inhibitors with reduced CYP1A2 inhibitionrdquo Bioorganicand Medicinal Chemistry vol 21 no 24 pp 7612ndash7623 2013
[24] A Dore B Asproni A Scampuddu et al ldquoSynthesis and SARstudy of novel tricyclic pyrazoles as potent phosphodiesterase10A inhibitorsrdquo European Journal of Medicinal Chemistry vol84 pp 181ndash193 2014
[25] S V Laxmi B S Kuarm and B Rajitha ldquoSynthesis and anti-microbial activity of coumarin pyrazole pyrimidine 246(1H3H5H)triones and thioxopyrimidine46(1H5H)dionesrdquo Me-dicinal Chemistry Research vol 22 no 2 pp 768ndash774 2013
[26] G Kalthur S Raj AThiyagarajan S Kumar P Kumar and S KAdiga ldquoVitamin E supplementation in semen-freezingmediumimproves the motility and protects sperm from freeze-thaw-induced DNA damagerdquo Fertility and Sterility vol 95 no 3 pp1149ndash1151 2011
[27] J Tesarik C Mendoza and A Carreras ldquoEffects of phosphodi-esterase inhibitors caffeine and pentoxifylline on spontaneousand stimulus-induced acrosome reactions in human spermrdquoFertility and Sterility vol 58 no 6 pp 1185ndash1190 1992
[28] G Kalthur S R Salian F Keyvanifard et al ldquoSupplementationof biotin to sperm preparation medium increases the motilityand longevity in cryopreserved human spermatozoardquo Journalof Assisted Reproduction and Genetics vol 29 no 7 pp 631ndash6352012
[29] A E Calogero S Fishel J Hall et al ldquoCorrelation betweenintracellular cAMP content kinematic parameters and hyper-activation of human spermatozoa after incubation with pentox-ifyllinerdquo Human Reproduction vol 13 no 4 pp 911ndash915 1998
[30] R Ain K Uma Devi S Shivaji and P B Seshagiri ldquoPen-toxifylline-stimulated capacitation and acrosome reaction inhamster spermatozoa involvement of intracellular signallingmoleculesrdquo Molecular Human Reproduction vol 5 no 7 pp618ndash626 1999
[31] M Bajpai andG F Doncel ldquoInvolvement of tyrosine kinase andcAMP-dependent kinase cross-talk in the regulation of humanspermmotilityrdquo Reproduction vol 126 no 2 pp 183ndash195 2003
[32] R Yunes P Fernandez G F Doncel and A A Acosta ldquoCyclicnucleotide phosphodiesterase inhibition increases tyrosinephosphorylation and hypermotility in normal and pathologicalhuman spermatozoardquo Biocell vol 29 no 3 pp 287ndash293 2005
[33] F Dimitriadis D Giannakis N Pardalidis et al ldquoEffectsof phosphodiesterase 5 inhibitors on sperm parameters andfertilizing capacityrdquo Asian Journal of Andrology vol 10 no 1pp 115ndash133 2008
[34] A P Kotdawala S Kumar S R Salian et al ldquoAddition of zincto human ejaculate prior to cryopreservation prevents freeze-thaw-induced DNA damage and preserves sperm functionrdquo
16 International Journal of Medicinal Chemistry
Journal of Assisted Reproduction and Genetics vol 29 no 12 pp1447ndash1453 2012
[35] S Mutalik S R Salian K Avadhani et al ldquoLiposome encapsu-lated soy lecithin and cholesterol can efficiently replace chickenegg yolk in human semen cryopreservation mediumrdquo SystemsBiology in Reproductive Medicine vol 60 no 3 pp 183ndash1882014
Figure 1 Some of the benzimidazole drugs and known PDE inhibitors
NO
NN
Ph NO N
N
Ph
RN
ON
O
Ph
VIII IX X
Figure 2 Representative drug molecules as PDE inhibitors VIIIndashX
Benzimidazole nucleus is the key building block fornumerous drugs that play beneficial roles in the functioningof biologically important molecules Specifically this nucleusis a constituent of Vit B
12and many currently existing
medications Almost all benzimidazoles with different het-erocyclic substituents led to essential modification in theirphysicochemical metabolic and pharmacokinetic properties[9] Benzimidazole is a core structural moiety found in someof the important drugs like albendazole (I)mebendazole (II)thiabendazole (III) rabeprazole (IV) and so forth Literaturesurvey revealed that coumarin (V) scaffolds were proved toincrease the cAMP levels through the specific inhibition ofPDE3 in accordance with their common structural features[10] and pyrazole (VI) derivatives have been explored for theidentification of phosphodiesterase (PDE4) inhibitors as isexemplified by the discovery and development of tofimilast[11] also most of the benzimidazole (VII) derivatives haveshown very prominent PDE (10A) activity [12] Coumarinpyrazole and benzimidazole core structuralmoiety are foundin some of the important PDE inhibition compounds by Yanget al [13] (Figure 1)
Also the scaffolds containing coumarin pyrazole andbenzimidazole are the key moieties in heterocyclic chemistryand are important structural units of various natural andsynthetic biologically active molecules They are known
to possess a wide range of pharmacological activities thatinclude antimicrobial and anti-inflammatory activitiesManycoumarin derivatives have shown anticancer anticoagulantanti-inflammatory antimicrobial antioxidant antiviral andcardiovascular activities [14ndash22]
During drug development of PDEs it was believed thatSAR of pyrazole and pyrazole replacements that remove thehydrogen bond donor were very promising The structuralanalogs viz compounds VIII and X contain benzoxazoleand benzimidazole attached to quinoline through oxygen aslinker groupThese have shown possess promising PDE inhi-bition as reported by Hamaguchi et al [23] Also substitutionof the pyrazole with simple alkyl groups retained potencywhile adding minimal molecular weight The methyl sub-stituted pyrazole IX (Figure 2) in an in vitro P-glycoprotein(PgP) overexpressing cell line had improved the efflux ratiosThese above drug development results and the structures ofvarious classes of clinically established PDEs conform to abroadly accepted pharmacophore (Figure 3) This suggeststhat three important structural requirements are to be presentto show PDE inhibition Those three important parametersare viz (i) fused heterocyclic ring for good oral bioavail-ability (ring A)and (ii) a five-membered heterocycle or alkylchain (B)which is connected to another fused five-memberedheterocycle (ring C) [24]
International Journal of Medicinal Chemistry 3
O O
NN
Ph
NH
N A B
C
Figure 3 Designed 3-(4-(1H-benzo[d]imidazol-2-yl)-1-phenyl-1H-pyrazol-3-yl)-2H-chromen-2-ones derivatives 5andashx
Reflux or MW
YX
1andashd 2andashd
3andashd
4andashf
5andashx
O O
NNPh
NH
N Y
O
O
O
R EtOHAcOH
RT stir
O
N
O
R
NH
Ph
O O
R
NN
CHO
Ph
R
X
DMFPOCl3
PhNHNH2
50ndash60∘C
4h
NH2
NH2
Scheme 1 Synthetic route for title compounds 5andashx where 5a R = -H X = -H Y = H 5b R = -H X = 6-Cl Y = H 5c R = -H X = 6-CH3
Y = H 5d R = -H X = 6-NO2 Y = H 5e R = -H X = 6-Br Y = N 5f R = -H X = 57-dimethyl Y = H 5g R = -6Cl X = -H Y = H 5h
R = -6Cl X = -4Cl Y = H 5i R = -6Cl X = -CH3 Y = H 5j R = -6Cl X = 6-NO
2 Y = H 5k R = -6Cl X = 6-Br Y = N 5l R = -6Cl X =
57-dimethyl Y = H 5m R = -6Br X = -H Y = H 5n R = -6Br X = -4Cl Y = H 5o R = -6Br X = -CH3 Y = H 5p R = -6Br X = 6-NO
2 Y =
H 5q R = -6Br X = 5-Br Y = N 5r R = -6Br X = 57-dimethyl Y = H 5s R = 8-OCH3 X = -H Y = H 5t R = 8-OCH
3 X = 4-Cl Y = H 5u
R = 8-OCH3 X = 6-CH
3 Y = H 5v R = 8-OCH
3 X = 6-NO
2 Y = H 5w R = 8-OCH
3 X = 5-Br Y = N 5x R = 8-OCH
3 X = 57-dimethyl
Y = H
In view of the above and in search of biologically potentnovel heterocyclic scaffolds herein we report facile and inex-pensive method for the synthesis of coumarin with pyrazolenucleus and functionalized benzimidazoles in a singlemoietywith an expectation to obtain potent PDE inhibitor
2 Results and Discussion
21 Chemistry The starting materials 3-acetyl-2H-chromen-2-one 1andashd and 3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde 3andashdwere obtained using literaturemethods [25]The synthetic protocols for the title compounds3-(4-(1H-benzo[d]imidazol-2-yl)-1-phenyl-1H-pyrazol-3-yl)-2H-chromen-2-ones 5andashx are outlined in Scheme 1 Alsoan optimum condition was established under microwaveirradiation for the synthesis of the title compounds 5andashxby condensation of o-arylenediamines 4andashf and 3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-carbaldehydes
3andashd in ethanol in 5ndash8min with excellent yields Comparedto conventional method (4ndash6 hrs) microwave irradiationgreatly reduced the reaction time from 3-4 h to 6ndash8min Theyield of the product was also increased up to 94 (Table 1)
Structures of all the synthesized compounds 5andashx wereconfirmed by various spectroscopic techniques viz IR 1H13CNMR MS and elemental analyses The compounds haveshown strong adsorption band for carbonyl of coumarin andN-H of benzimidazole ring at 1711ndash1737 and 3345ndash3368 cmminus1respectively In case of 1HNMR spectra all the compoundsexhibited a singlet in the range 1042ndash1336 ppm for benzim-idazole N-H ring and coumarin C
4H at 769ndash792 ppm The
aromatic protons of all pyrazole rings appeared as multipletsin the range 732ndash798 ppm In case of 13CNMR spectralstudy the numbers of signals are consistent with number ofmagnetically nonequivalent carbon atoms in the moleculeand in mass spectra all the synthesized title compounds haveshown the molecular ion peaks at their respectivemz values
4 International Journal of Medicinal Chemistry
Table1Com
paris
onof
conventio
naland
microwaves
ynthesisof
titlecompo
unds
5andashx
from
3andashd
and4andashf
inabsoluteethano
lund
erop
timized
cond
ition
s
3andashd
4andashf
5andashx
Con
ventional
Microwave
Time(hr)
Yield(
)Time(min)
Yield
NN
Ph
CHO
OO 3a
4a
H2N
H2N
NN
Ph
N H
NO
O 5a
439
594
NN
Ph
CHO
OO
3a
Cl4b
H2N
H2N
NN
Ph
N H
NCl
OO
5b
435
590
NN
Ph
CHO
OO
3a4c
H2N
H2N
NN Ph
N H
NO
O 5c
438
588
NN
Ph
CHO
OO
3a4d
H2N
H2N
NO
2
NN
Ph
N H
N
OO
O 5d
N+ O
minus
432
586
International Journal of Medicinal Chemistry 5
Table1Con
tinued
3andashd
4andashf
5andashx
Con
ventional
Microwave
Time(hr)
Yield(
)Time(min)
Yield
NN
Ph
CHO
OO 3a
N
Br
4e
H2N
H2N
NN
Ph
N
N H
NO
OBr
5e
434
592
NN
Ph
CHO
OO 3a
4f
H2N
H2N
NN
Ph
N H
NO
O 5f
435
590
NN
Ph
CHO
OO
Cl3b
4a
H2N
H2N
NN
Ph
N H
NO
O
Cl
5g
540
694
NN
Ph
CHO
OO
Cl3b
Cl4b
H2N
H2N
NN
Ph
N H
NCl
OO
Cl
5h
532
685
6 International Journal of Medicinal Chemistry
Table1Con
tinued
3andashd
4andashf
5andashx
Con
ventional
Microwave
Time(hr)
Yield(
)Time(min)
Yield
NN
Ph
CHO
OO
Cl3b
4c
H2N
H2N
NN
Ph
N H
NO
O
Cl
5i
530
687
NN
Ph
CHO
OO
Cl3b
4d
H2N
H2N
NO
2N
N
Ph
N H
N
OO
O
Cl
5j
N+ O
minus
535
684
NN
Ph
CHO
OO
Cl3b
N
Br
4e
H2N
H2N
NN
Ph
N N H
NO
O
Cl
Br
5k
534
686
NN
Ph
CHO
OO
Cl3b
4f
H2N
H2N
NN
Ph
N H
NO
O
Cl
5l
537
690
International Journal of Medicinal Chemistry 7
Table1Con
tinued
3andashd
4andashf
5andashx
Con
ventional
Microwave
Time(hr)
Yield(
)Time(min)
Yield
NN
Ph
CHO
OO
Br3c
4a
H2N
H2N
NN
Ph
N H
NO
O
Br
5m
633
789
NN
Ph
CHO
OO
Br3c
Cl4b
H2N
H2N
NN
Ph
N H
NCl
OO
Br
5n
636
792
NN
Ph
CHO
OO
Br3c
4c
H2N
H2N
NN
Ph
N H
NO
O
Br
5o
639
794
NN
Ph
CHO
OO
Br3c
4d
H2N
H2N
NO
2
NN
Ph
N H
N
O
OO
Br
5p
N+
Ominus
636
784
8 International Journal of Medicinal Chemistry
Table1Con
tinued
3andashd
4andashf
5andashx
Con
ventional
Microwave
Time(hr)
Yield(
)Time(min)
Yield
NN
Ph
CHO
OO
Br3c
N
Br
4e
H2N
H2N
NN
Ph
N
N H
NO
O
Br
Br
5q
631
790
NN
Ph
CHO
OO
Br3c
4f
H2N
H2N
NN
Ph
N H
NO
O
Br
5r
634
792
NN
Ph
CHO
OO
O
3d4a
H2N
H2N
NN
Ph
N H
NO
O
O
5s
538
887
NN
Ph
CHO
OO
O
3d
Cl
4b
H2N
H2N
NN
Ph
N H
N
ClO
OO
5t
540
885
International Journal of Medicinal Chemistry 9
Table1Con
tinued
3andashd
4andashf
5andashx
Con
ventional
Microwave
Time(hr)
Yield(
)Time(min)
Yield
NN
Ph
CHO
OO
O
3d4c
H2N
H2N
NN
Ph
N H
NO
OO
5u
532
888
NN
Ph
CHO
OO
O
3d4d
H2N
H2N
NO
2
NN
Ph
N H
N
OO
OO
5v
N+
Ominus
536
886
NN
Ph
CHO
OO
O
3d
N
Br
4e
H2N
H2N
NN
Ph
N
N H
NO
OO
Br
5w
538
880
NN
Ph
CHO
OO
O
3d4f
H2N
H2N
NN
Ph
N H
NO
OO
5x
534
882
10 International Journal of Medicinal Chemistry
3 PDE Inhibition Study
31 Effect on Motility of Frozen-Thawed Human SpermatozoaTo assess the effect of title compounds on themotility patternhuman spermatozoa subjected to freeze-thaw process wasused It is well documented that freeze-thaw process canhave detrimental effect on the motility and survival of thespermatozoa [26] Therefore they can be an excellent modelto study the effect of any test compounds on themotility [27]
In the present study we assessed the motility patternin spermatozoa processed with media containing variouscompounds manually at 1 4 and 24 h after incubationin vitro Three arbitrary doses were selected (05 10 and50 120583gmL) for each compound to assess their effect on spermmotility Pentoxifylline (PTF) which is known to enhancethe human sperm motility by elevating intracellular cyclicadenosine monophosphate (cAMP) level [27] was taken asa positive control Based on the previous report PTF at 1mMconcentration was used [28] Since the test compounds werenot soluble in water dimethyl sulfoxide (DMSO 005) wasused as vehicle control
The sperm motility enhancement property of the com-pounds was categorized as below
(a) Marginal which resulted in lt5 increase in themotility (b) Good which resulted in 5ndash10 increase inmotility (c) Excellent which resulted in gt10 increase inmotility (d) Poor where there was no enhancement inmotility
At 1 h after the in vitro incubation the majority of thecompounds had marginal effect on the sperm motility com-pared to control At this interval PTF had higher percentageof total and progressivelymotile spermatozoa compared to allthe test compounds A large number of studies have shownthat PTF can trigger the motility in fresh or frozen-thawedspermatozoa as early as 1 h [29] even though the majorityof the studies indicate that this effect is mainly due to theelevated cAMP level in the spermatozoa [30] it can also bemediated through tyrosine phosphorylation [31]
Compounds 5a 5b 5c 5d 5e 5g 5h and 5k inducedmarginal enhancement in total motility while compounds5a 5b 5c and 5j resulted in similar effect on progressivemotility (Tables 2(a) and 2(b)) A 5ndash10 increase in totalmotility was induced by compound 5j However with respectto progressive motility compounds 5d 5e 5f and 5k cameunder compounds with good effect on motility More than10 increase in progressive motility was observed only forcompound 5g (at 05 120583gmL concentration) and compound5h (at 5 120583gmL concentration)
A time-dependent decrease in total and progressivemotility was observed in all the groups However the declinewasmore evident at 24 h intervalThis phenomenon has beendocumented by earlier studies [28] These changes may berelated to the oxidative stress induced by in vitro cultureconditions Spermatozoa are highly susceptible to reactiveoxygen species (ROS) due to high polyunsaturated fatty acidcontent in their plasma membrane and negligible amount ofcytoplasmic antioxidants
At 4 h interval compounds 5e 5f and 5g enhancedthe progressive motility of spermatozoa by 5ndash10 while
compound 5h with two chloro substituents on coumarinand benzimidazole resulted in almost 15 increase in theprogressive motility However the effect was lower than thatof pentoxifylline The rest of the compounds (5a 5b 5c5f and 5j) induced a marginal increase in motility exceptin compound 5e in which the motility was lower than thecontrol group
At 24 h interval the majority of the compounds (5a5b 5d 5f 5h and 5j) had marginally higher percentageof motility or poor motility compounds (5c 5e and 5g)compared to control Interestingly compound 5k with chlorosubstituent on coumarin and bromo substitution on ben-zimidazole had higher percentage of motile spermatozoacompared to control (14 higher) and even PTF (9 higher)This indicates that compound5k canhelp in prolonging the invitro survival of spermatozoa which has significant beneficialrole in assisted reproductive technologies such as in vitrofertilization Previous studies have shown that even thoughPTF has triggering effect on themotility it induces prematureacrosome reaction and drastic decrease in motility at laterintervals In this context the compounds which increasethe motility and longevity of spermatozoa under in vitroconditions are of clinical relevance
Based on the manual motility assessment we observedthat compounds 5d 5f 5g 5h and 5k have considerablebeneficial role in spermmotilityTherefore these compoundswere further assessed for their effect on the kinematicsof spermatozoa at 1 h However there was no significantdifference in the kinematics of spermatozoa compared tocontrol and PTF (Table 3) In conclusion the newly synthe-sized coumarin derivatives have sperm motility enhancingproperty However in this preliminary screening we havetaken three arbitrary doses to assess their effect on motilityCompounds 5g 5h and 5k show considerable beneficialeffect which can be of clinical significance where spermmotility enhancement is achieved under in vitro condi-tions Compounds 5g and 5h triggered the motility at earlyintervals which was similar to the PTF while compound5k improved the longevity of the spermatozoa Since thecoumarin is known to have PDE inhibitory function themotility enhancement could be due to the elevated cAMPlevel in sperm However further detailed studies are essentialto understand the mechanism of action of these compoundsand their clinical utility
4 Experimental
All the chemicals and reagents were purchased from theMerck and Aldrich chemical suppliers Melting points (mp)were determined in open capillaries and are uncorrectedThe IR spectra were recorded on a Nicolet Impact 410 FTIR spectrometer using KBr pellets (range 4000ndash500 cmminus1)The 1HNMR spectra were recorded at 400MHz on BrukerAvance FT NMR spectrometer in DMSO-d
6solvent with
TMS as internal standard 13CNMR spectra were recordedat 100MHz on Bruker Avance FT NMR spectrometer inDMSO-d
6solvent with TMS as internal standard The mass
spectra were recorded on Shimadzu GC-MS operating at70 eV Thin-layer chromatography (TLC) was performed on
International Journal of Medicinal Chemistry 11
Table 2 Effect of supplementation of coumarin derivatives (5andashj) to sperm wash medium on the motility pattern in frozen-thawed humanspermatozoa at various time intervals
(a)
Compounds Time (h)Total motility (mean plusmn SE of percentage)
020mm Aluchrosep silica gel 60 F254 plates (SD FineMumbai) Microwave irradiation experiments were carriedout using CEMDiscover SPMicrowave Synthesizer equippedwith IR sensor to monitor reaction temperatures
5 General Procedures for the Preparation ofTitle Compounds 5andashx
3-(2-Oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-car-baldehydes (3andashd 1 mmol) were prepared from 3-acetyl-coumarin (1andashd 1 mmol) phenylhydrazine (1mmol) andthe intermediate (Schiff base) (2andashd 1 mmol) followedby reaction with POCl
3in DMF by Vilsmeier Haack for-
mylation strategies [25] The target compounds 5andashxwere then achieved by stirring substituted carbaldehyde(3andashd 1 mmol) with different o-arylenediamine (4andashf1 mmol) under reflux for 4-5 hrs After completion (TLChexane ethylacetate 7 3) of the reaction the solid obtainedwas filtered washed with small quantity of ethanol anddried Further it was recrystallized in methanol to get thepure compound (5andashx)
51 Microwave Assisted Procedure for the Preparation of Com-pounds 5andashx A mixture of 3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde (3andashd 1 mmol) and o-phenylenediamine (4andashf 1 mmol) taken in ethanol (5mL)was introduced into a CEM microwave reaction vesselequipped with magnetic stirrer The reaction mixture was
prestirred for 1min at room temperature and irradiated at180W (150∘C) for about 6ndash8min (TLC using hexane ethylacetate (7 3 drops) as eluent) The reaction mixture wasthen quenched into crushed ice and the crude product wasfiltered washed and dried Recrystallization from methanolgave pure crystals of the compounds 5andashx
(Spectral as well as elemental analyses data are providedas electronic supplementary file in Supplementary Materialavailable online at httpdxdoiorg10115520169890630)
6 Assessment of Phosphodiesterase InhibitionUsing Human Spermatozoa as Model
Earlier studies have shown that PDE inhibitors can enhancethe spermmotility [32 33] Based on this in the present studywe have used an indirect approach to assess the inhibitoryeffect of the coumarin derivatives on PDE by assessing spermmotility pattern
61 Preparation of Test Solution Stock solution of the testcompounds (1mgmL) was prepared by dissolving them indimethyl sulfoxide (DMSO Sigma Cat number D5879)The stock solution was then diluted with Earlrsquos BalancedSalt Solution (EBSS) supplemented with 01 bovine serumalbumin (BSA) in a ratio of 1 200 1 1000 and 1 2000 to getworking solutions of 5 1 and 05 120583gmL concentrations Theworking solutions were preincubated at 37∘C and 5 CO
2
prior to use
International Journal of Medicinal Chemistry 13
Table 3 Effect of various coumarin derivatives on kinematics of frozen-thawed spermatozoa at one hour after incubation in vitro
Parameters Compounds Concentrations of compounds in 120583gmL VC PTFC 0 05 5 (005 DMSO) (1mM)
62 Study Subjects Infertile men who attended AndrologyLaboratory Kasturba Medical College Manipal UniversityManipal during the period of June-July 2015 for routinesemen evaluation were included in the study The subjects
with a sexual abstinence of 3ndash5 days were asked to providethe semen samples by masturbation in a sterile containerFollowing liquefaction the semen parameters were assessedas described by Kotdawala et al [34]The study was approved
14 International Journal of Medicinal Chemistry
by the Institutional Ethics Committee of Kasturba HospitalKasturba Medical College Manipal University (IEC number1552015)
63 Cryopreservation and Thawing The liquefied semensamples were cryopreserved by rapid freezing method asdescribed previously [34] with minor modifications Brieflythe semen samples were mixed with equal volume of freezingmedium (Sperm Freeze FertiPro Cat number 0344) in acryovial (Thermoscientific Nunc Cat number 138627) andkept at 4∘C for 10min in liquid nitrogen (LN2) vapor phasefor 5min and then finally plunged into liquid nitrogenThe samples were thawed after 1 week by rapid thawingmethod by placing themat 37∘C for 5minThe cryoprotectantmedium was completely removed by mixing the spermsuspension with EBSS medium and centrifuging at 1000 rpmfor 8min The resultant pellet was resuspended with freshEBSS medium and used for further analysis
64 Sample Preparation To screen the compounds withenhancing effect on themotility of spermatozoa under in vitroconditions the frozen-thawed semen samples were washedby mixing with equal volume of preincubated Earlrsquos BalancedSalt Solution (EBSS Sigma Cat number E2888) followed bycentrifugation at 1800 rpm for 8min The pellet was thengently mixed with 1mL of EBSS media containing 01bovine serum albumin (BSA Sigma Cat number A3311)and equally divided into control vehicle control and testgroups (with average sperm density of 5 times 104 spermatozoain each group) The motile sperm were extracted by swimup method as described earlier [35] The sperm suspensionwas centrifuged at 1200 rpm for 8min and the resultantpellet was overlaidwith EBSSmedium containing various testcompoundsThe sperm suspensions were incubated for 1 h at37∘Cand 5CO
2afterwhich themotile spermwere collected
from the supernatant
65 Motility Assessment Spermatozoa in the supernatantfraction were assessed for their motility patterns at 1 4 and24 h after incubation under light microscope as describedearlier [27] Sperm suspension was (10 120583L droplet) placed ona clean microscope slide and a coverslip was placed over itSpermatozoa with progressive motility and nonprogressivemotility and immotile spermatozoa were scored separatelyfrom a total of 200 spermatozoa in random fields at 400xmagnification
66 Sperm Kinematics Using Computer Assisted Semen Anal-ysis (CASA) The motion characteristics in the spermatozoawere assessed at 1 4 and 24 h after incubation using CASAsystem (ISAS Spain) Briefly 5 120583L of sperm suspension wasplaced on a clean microscope slide prewarmed at 37∘C Thesperm suspensionwas coveredwith prewarmed cover slip (22times 22mm) and observed under microscope (negative phasecontrast 10x objectives Proiser Spain) For each sample thekinematics was assessed randomly at ten different fields Theparameters such as curvilinear velocity (VCL) straight line
velocity (VSL) average path velocity (VAP) amplitude of lat-eral head displacement (ALH) linearity (LIN) straightness(STR) wobble (WOB) and beat cross frequency (BCF) wereanalyzed
7 Conclusions
In conclusion we have reported efficient and environmen-tally benignmethodologies for the synthesis of 3-[4-(1H-benzo[d]imidazol-2-yl)-1-phenyl-1H-pyrazol-3-yl]-2H-chromen-2-one derivatives 5andashx by using thermal and by microwaveirradiation under neat conditions in presence of ethanol Thereactions carried out under microwave irradiation affordedbenzimidazoles in short period of time with excellent yieldsHence this methodology would make an interesting strategyfor the synthesis of various substituted coumarin pyrazolebenzimidazoles Also compounds 5g 5h and 5k exhibitedimpressive sperm motility enhancing property whereas 5khas spermatozoa longevity property
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to University Scientific Instrumen-tation Centre (USIC) Karnatak University Dharwad forprovidingmass and IR spectral dataThe authors are thankfulto Indian Institute of Science (IISc) Bengaluru for carryingout NMR analyses and to UGC New Delhi for the financialassistance under UPE program vide no [F No 14-22012(NSPE) dated 22-01-2013] One of the authors (MNK)acknowledges University Grants Commission (UGC) NewDelhi for fellowship under UGC-UPE program
References
[1] P P Kattimani R R Kamble and G Y Meti ldquoExpedientsynthesis of benzimidazoles using amidesrdquo RSC Advances vol5 no 37 pp 29447ndash29455 2015
[2] L Li C Yee and J A Beavo ldquoCD3- and CD28-dependentinduction of PDE7 required for T cell activationrdquo Science vol283 no 5403 pp 848ndash849 1999
[3] T P Dousa ldquoCyclic-3101584051015840-nucleotide phosphodiesteraseisozymes in cell biology and pathophysiology of the kidneyrdquoKidney International vol 55 no 1 pp 29ndash62 1999
[4] C Pfister N Bennett F Bruckert et al ldquoInteractions of a G-protein with its effector transducin and cGMP phosphodi-esterase in retinal rodsrdquo Cell Signaling vol 5 no 3 pp 235ndash2411993
[5] S L C Jin F J Richard W-P Kuo A J DrsquoErcole andM Conti ldquoImpaired growth and fertility of cAMP-specificphosphodiesterase PDE4D-deficient micerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 21 pp 11998ndash12003 1999
[6] S H Francis I V Turko and J D Corbin ldquoCyclic nucleotidephosphodiesterases relating structure and functionrdquoProgress in
International Journal of Medicinal Chemistry 15
Nucleic Acid Research and Molecular Biology vol 65 pp 1ndash522000
[7] J M Hetman N Robas R Baxendale et al ldquoCloning and char-acterization of two splice variants of human phosphodiesterase11Ardquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 23 pp 12891ndash12895 2000
[8] K Yuasa T Ohgaru M Asahina and K Omori ldquoIdentificationof rat cyclic nucleotide phosphodiesterase 11A (PDE11A) com-parison of rat and human PDE11A splicing variantsrdquo EuropeanJournal of Biochemistry vol 268 no 16 pp 4440ndash4448 2001
[9] S K Sahu A Mishra and R K Behera ldquoSynthesis of thiazolebenzothiazole oxadiazole thiadiazole triazole and thiazolidi-none incorporated coumarinsrdquo Indian Journal of HeterocyclicChemistry vol 6 no 2 pp 91ndash94 1996
[10] G Roma M Di Braccio G Grossi et al ldquoSynthesis and in vitroantiplatelet activity of new 4-(1-piperazinyl) coumarin deriva-tives Human platelet phosphodiesterase 3 inhibitory propertiesof the two most effective compounds described and molecularmodeling study on their interactions with phosphodiesterase3A catalytic siterdquo Journal of Medicinal Chemistry vol 50 no 12pp 2886ndash2895 2007
[11] T Shyamsunder Reddy K Shiva Kumar C L T Medaet al ldquoConformationally restricted novel pyrazole deriva-tives synthesis of 18-disubstituted 55-dimethyl-45-dihydro-1H-benzo[g]indazoles as a new class of PDE4 inhibitorsrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 9 pp3248ndash3255 2012
[12] A Chino N Masuda Y Amano et al ldquoNovel benzimidazolederivatives as phosphodiesterase 10A (PDE10A) inhibitors withimproved metabolic stabilityrdquo Bioorganic and Medicinal Chem-istry vol 22 no 13 pp 3515ndash3526 2014
[13] H Yang FNMurigi ZWang J Li H Jin andZ Tu ldquoSynthesisand in vitro characterization of cinnoline and benzimidazoleanalogues as phosphodiesterase 10A inhibitorsrdquo Bioorganic andMedicinal Chemistry Letters vol 25 pp 919ndash924 2015
[14] J Nawrot-Modranka E Nawrot and J Graczyk ldquoIn vivo antitu-mor in vitro antibacterial activity and alkylating properties ofphosphorohydrazine derivatives of coumarin and chromonerdquoEuropean Journal of Medicinal Chemistry vol 41 no 11 pp1301ndash1309 2006
[15] A A Bekhit and T Abdel-Aziem ldquoDesign synthesis andbiological evaluation of some pyrazole derivatives as anti-inflammatory-antimicrobial agentsrdquo Bioorganic and MedicinalChemistry vol 12 no 8 pp 1935ndash1945 2004
[16] T Devji C Reddy CWoo S Awale S Kadota and D Carrico-Moniz ldquoPancreatic anticancer activity of a novel geranylgerany-lated coumarin derivativerdquo Bioorganic andMedicinal ChemistryLetters vol 21 no 19 pp 5770ndash5773 2011
[17] I Kostova I Manolov I Nicolova S Konstantinov andM Karaivanova ldquoNew lanthanide complexes of 4-methyl-7-hydroxycoumarin and their pharmacological activityrdquo Euro-pean Journal of Medicinal Chemistry vol 36 no 4 pp 339ndash3472001
[18] M Ghate R A Kusanur and M V Kulkarni ldquoSynthesis andin vivo analgesic and anti-inflammatory activity of some bi-heterocyclic coumarin derivativesrdquo European Journal of Medic-inal Chemistry vol 40 no 9 pp 882ndash887 2005
[19] PM RonadMN Noolvi S Sapkal S Dharbhamulla andV SMaddi ldquoSynthesis and antimicrobial activity of 7-(2-substitutedphenylthiazolidinyl)-benzopyran-2-one derivativesrdquo EuropeanJournal of Medicinal Chemistry vol 45 no 1 pp 85ndash89 2010
[20] GMelagraki A Afantitis O Igglessi-Markopoulou et al ldquoSyn-thesis and evaluation of the antioxidant and anti-inflammatoryactivity of novel coumarin-3-aminoamides and their alpha-lipoic acid adductsrdquo European Journal of Medicinal Chemistryvol 44 no 7 pp 3020ndash3026 2009
[21] J C Trivedi J B Bariwal K D Upadhyay et al ldquoImprovedand rapid synthesis of new coumarinyl chalcone derivatives andtheir antiviral activityrdquo Tetrahedron Letters vol 48 no 48 pp8472ndash8474 2007
[22] S Vilar E Quezada L Santana et al ldquoDesign synthe-sis and vasorelaxant and platelet antiaggregatory activitiesof coumarinndashresveratrol hybridsrdquo Bioorganic and MedicinalChemistry Letters vol 16 no 2 pp 257ndash261 2006
[23] W Hamaguchi N Masuda M Isomura et al ldquoDesign and syn-thesis of novel benzimidazole derivatives as phosphodiesterase10A inhibitors with reduced CYP1A2 inhibitionrdquo Bioorganicand Medicinal Chemistry vol 21 no 24 pp 7612ndash7623 2013
[24] A Dore B Asproni A Scampuddu et al ldquoSynthesis and SARstudy of novel tricyclic pyrazoles as potent phosphodiesterase10A inhibitorsrdquo European Journal of Medicinal Chemistry vol84 pp 181ndash193 2014
[25] S V Laxmi B S Kuarm and B Rajitha ldquoSynthesis and anti-microbial activity of coumarin pyrazole pyrimidine 246(1H3H5H)triones and thioxopyrimidine46(1H5H)dionesrdquo Me-dicinal Chemistry Research vol 22 no 2 pp 768ndash774 2013
[26] G Kalthur S Raj AThiyagarajan S Kumar P Kumar and S KAdiga ldquoVitamin E supplementation in semen-freezingmediumimproves the motility and protects sperm from freeze-thaw-induced DNA damagerdquo Fertility and Sterility vol 95 no 3 pp1149ndash1151 2011
[27] J Tesarik C Mendoza and A Carreras ldquoEffects of phosphodi-esterase inhibitors caffeine and pentoxifylline on spontaneousand stimulus-induced acrosome reactions in human spermrdquoFertility and Sterility vol 58 no 6 pp 1185ndash1190 1992
[28] G Kalthur S R Salian F Keyvanifard et al ldquoSupplementationof biotin to sperm preparation medium increases the motilityand longevity in cryopreserved human spermatozoardquo Journalof Assisted Reproduction and Genetics vol 29 no 7 pp 631ndash6352012
[29] A E Calogero S Fishel J Hall et al ldquoCorrelation betweenintracellular cAMP content kinematic parameters and hyper-activation of human spermatozoa after incubation with pentox-ifyllinerdquo Human Reproduction vol 13 no 4 pp 911ndash915 1998
[30] R Ain K Uma Devi S Shivaji and P B Seshagiri ldquoPen-toxifylline-stimulated capacitation and acrosome reaction inhamster spermatozoa involvement of intracellular signallingmoleculesrdquo Molecular Human Reproduction vol 5 no 7 pp618ndash626 1999
[31] M Bajpai andG F Doncel ldquoInvolvement of tyrosine kinase andcAMP-dependent kinase cross-talk in the regulation of humanspermmotilityrdquo Reproduction vol 126 no 2 pp 183ndash195 2003
[32] R Yunes P Fernandez G F Doncel and A A Acosta ldquoCyclicnucleotide phosphodiesterase inhibition increases tyrosinephosphorylation and hypermotility in normal and pathologicalhuman spermatozoardquo Biocell vol 29 no 3 pp 287ndash293 2005
[33] F Dimitriadis D Giannakis N Pardalidis et al ldquoEffectsof phosphodiesterase 5 inhibitors on sperm parameters andfertilizing capacityrdquo Asian Journal of Andrology vol 10 no 1pp 115ndash133 2008
[34] A P Kotdawala S Kumar S R Salian et al ldquoAddition of zincto human ejaculate prior to cryopreservation prevents freeze-thaw-induced DNA damage and preserves sperm functionrdquo
16 International Journal of Medicinal Chemistry
Journal of Assisted Reproduction and Genetics vol 29 no 12 pp1447ndash1453 2012
[35] S Mutalik S R Salian K Avadhani et al ldquoLiposome encapsu-lated soy lecithin and cholesterol can efficiently replace chickenegg yolk in human semen cryopreservation mediumrdquo SystemsBiology in Reproductive Medicine vol 60 no 3 pp 183ndash1882014
Figure 3 Designed 3-(4-(1H-benzo[d]imidazol-2-yl)-1-phenyl-1H-pyrazol-3-yl)-2H-chromen-2-ones derivatives 5andashx
Reflux or MW
YX
1andashd 2andashd
3andashd
4andashf
5andashx
O O
NNPh
NH
N Y
O
O
O
R EtOHAcOH
RT stir
O
N
O
R
NH
Ph
O O
R
NN
CHO
Ph
R
X
DMFPOCl3
PhNHNH2
50ndash60∘C
4h
NH2
NH2
Scheme 1 Synthetic route for title compounds 5andashx where 5a R = -H X = -H Y = H 5b R = -H X = 6-Cl Y = H 5c R = -H X = 6-CH3
Y = H 5d R = -H X = 6-NO2 Y = H 5e R = -H X = 6-Br Y = N 5f R = -H X = 57-dimethyl Y = H 5g R = -6Cl X = -H Y = H 5h
R = -6Cl X = -4Cl Y = H 5i R = -6Cl X = -CH3 Y = H 5j R = -6Cl X = 6-NO
2 Y = H 5k R = -6Cl X = 6-Br Y = N 5l R = -6Cl X =
57-dimethyl Y = H 5m R = -6Br X = -H Y = H 5n R = -6Br X = -4Cl Y = H 5o R = -6Br X = -CH3 Y = H 5p R = -6Br X = 6-NO
2 Y =
H 5q R = -6Br X = 5-Br Y = N 5r R = -6Br X = 57-dimethyl Y = H 5s R = 8-OCH3 X = -H Y = H 5t R = 8-OCH
3 X = 4-Cl Y = H 5u
R = 8-OCH3 X = 6-CH
3 Y = H 5v R = 8-OCH
3 X = 6-NO
2 Y = H 5w R = 8-OCH
3 X = 5-Br Y = N 5x R = 8-OCH
3 X = 57-dimethyl
Y = H
In view of the above and in search of biologically potentnovel heterocyclic scaffolds herein we report facile and inex-pensive method for the synthesis of coumarin with pyrazolenucleus and functionalized benzimidazoles in a singlemoietywith an expectation to obtain potent PDE inhibitor
2 Results and Discussion
21 Chemistry The starting materials 3-acetyl-2H-chromen-2-one 1andashd and 3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde 3andashdwere obtained using literaturemethods [25]The synthetic protocols for the title compounds3-(4-(1H-benzo[d]imidazol-2-yl)-1-phenyl-1H-pyrazol-3-yl)-2H-chromen-2-ones 5andashx are outlined in Scheme 1 Alsoan optimum condition was established under microwaveirradiation for the synthesis of the title compounds 5andashxby condensation of o-arylenediamines 4andashf and 3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-carbaldehydes
3andashd in ethanol in 5ndash8min with excellent yields Comparedto conventional method (4ndash6 hrs) microwave irradiationgreatly reduced the reaction time from 3-4 h to 6ndash8min Theyield of the product was also increased up to 94 (Table 1)
Structures of all the synthesized compounds 5andashx wereconfirmed by various spectroscopic techniques viz IR 1H13CNMR MS and elemental analyses The compounds haveshown strong adsorption band for carbonyl of coumarin andN-H of benzimidazole ring at 1711ndash1737 and 3345ndash3368 cmminus1respectively In case of 1HNMR spectra all the compoundsexhibited a singlet in the range 1042ndash1336 ppm for benzim-idazole N-H ring and coumarin C
4H at 769ndash792 ppm The
aromatic protons of all pyrazole rings appeared as multipletsin the range 732ndash798 ppm In case of 13CNMR spectralstudy the numbers of signals are consistent with number ofmagnetically nonequivalent carbon atoms in the moleculeand in mass spectra all the synthesized title compounds haveshown the molecular ion peaks at their respectivemz values
4 International Journal of Medicinal Chemistry
Table1Com
paris
onof
conventio
naland
microwaves
ynthesisof
titlecompo
unds
5andashx
from
3andashd
and4andashf
inabsoluteethano
lund
erop
timized
cond
ition
s
3andashd
4andashf
5andashx
Con
ventional
Microwave
Time(hr)
Yield(
)Time(min)
Yield
NN
Ph
CHO
OO 3a
4a
H2N
H2N
NN
Ph
N H
NO
O 5a
439
594
NN
Ph
CHO
OO
3a
Cl4b
H2N
H2N
NN
Ph
N H
NCl
OO
5b
435
590
NN
Ph
CHO
OO
3a4c
H2N
H2N
NN Ph
N H
NO
O 5c
438
588
NN
Ph
CHO
OO
3a4d
H2N
H2N
NO
2
NN
Ph
N H
N
OO
O 5d
N+ O
minus
432
586
International Journal of Medicinal Chemistry 5
Table1Con
tinued
3andashd
4andashf
5andashx
Con
ventional
Microwave
Time(hr)
Yield(
)Time(min)
Yield
NN
Ph
CHO
OO 3a
N
Br
4e
H2N
H2N
NN
Ph
N
N H
NO
OBr
5e
434
592
NN
Ph
CHO
OO 3a
4f
H2N
H2N
NN
Ph
N H
NO
O 5f
435
590
NN
Ph
CHO
OO
Cl3b
4a
H2N
H2N
NN
Ph
N H
NO
O
Cl
5g
540
694
NN
Ph
CHO
OO
Cl3b
Cl4b
H2N
H2N
NN
Ph
N H
NCl
OO
Cl
5h
532
685
6 International Journal of Medicinal Chemistry
Table1Con
tinued
3andashd
4andashf
5andashx
Con
ventional
Microwave
Time(hr)
Yield(
)Time(min)
Yield
NN
Ph
CHO
OO
Cl3b
4c
H2N
H2N
NN
Ph
N H
NO
O
Cl
5i
530
687
NN
Ph
CHO
OO
Cl3b
4d
H2N
H2N
NO
2N
N
Ph
N H
N
OO
O
Cl
5j
N+ O
minus
535
684
NN
Ph
CHO
OO
Cl3b
N
Br
4e
H2N
H2N
NN
Ph
N N H
NO
O
Cl
Br
5k
534
686
NN
Ph
CHO
OO
Cl3b
4f
H2N
H2N
NN
Ph
N H
NO
O
Cl
5l
537
690
International Journal of Medicinal Chemistry 7
Table1Con
tinued
3andashd
4andashf
5andashx
Con
ventional
Microwave
Time(hr)
Yield(
)Time(min)
Yield
NN
Ph
CHO
OO
Br3c
4a
H2N
H2N
NN
Ph
N H
NO
O
Br
5m
633
789
NN
Ph
CHO
OO
Br3c
Cl4b
H2N
H2N
NN
Ph
N H
NCl
OO
Br
5n
636
792
NN
Ph
CHO
OO
Br3c
4c
H2N
H2N
NN
Ph
N H
NO
O
Br
5o
639
794
NN
Ph
CHO
OO
Br3c
4d
H2N
H2N
NO
2
NN
Ph
N H
N
O
OO
Br
5p
N+
Ominus
636
784
8 International Journal of Medicinal Chemistry
Table1Con
tinued
3andashd
4andashf
5andashx
Con
ventional
Microwave
Time(hr)
Yield(
)Time(min)
Yield
NN
Ph
CHO
OO
Br3c
N
Br
4e
H2N
H2N
NN
Ph
N
N H
NO
O
Br
Br
5q
631
790
NN
Ph
CHO
OO
Br3c
4f
H2N
H2N
NN
Ph
N H
NO
O
Br
5r
634
792
NN
Ph
CHO
OO
O
3d4a
H2N
H2N
NN
Ph
N H
NO
O
O
5s
538
887
NN
Ph
CHO
OO
O
3d
Cl
4b
H2N
H2N
NN
Ph
N H
N
ClO
OO
5t
540
885
International Journal of Medicinal Chemistry 9
Table1Con
tinued
3andashd
4andashf
5andashx
Con
ventional
Microwave
Time(hr)
Yield(
)Time(min)
Yield
NN
Ph
CHO
OO
O
3d4c
H2N
H2N
NN
Ph
N H
NO
OO
5u
532
888
NN
Ph
CHO
OO
O
3d4d
H2N
H2N
NO
2
NN
Ph
N H
N
OO
OO
5v
N+
Ominus
536
886
NN
Ph
CHO
OO
O
3d
N
Br
4e
H2N
H2N
NN
Ph
N
N H
NO
OO
Br
5w
538
880
NN
Ph
CHO
OO
O
3d4f
H2N
H2N
NN
Ph
N H
NO
OO
5x
534
882
10 International Journal of Medicinal Chemistry
3 PDE Inhibition Study
31 Effect on Motility of Frozen-Thawed Human SpermatozoaTo assess the effect of title compounds on themotility patternhuman spermatozoa subjected to freeze-thaw process wasused It is well documented that freeze-thaw process canhave detrimental effect on the motility and survival of thespermatozoa [26] Therefore they can be an excellent modelto study the effect of any test compounds on themotility [27]
In the present study we assessed the motility patternin spermatozoa processed with media containing variouscompounds manually at 1 4 and 24 h after incubationin vitro Three arbitrary doses were selected (05 10 and50 120583gmL) for each compound to assess their effect on spermmotility Pentoxifylline (PTF) which is known to enhancethe human sperm motility by elevating intracellular cyclicadenosine monophosphate (cAMP) level [27] was taken asa positive control Based on the previous report PTF at 1mMconcentration was used [28] Since the test compounds werenot soluble in water dimethyl sulfoxide (DMSO 005) wasused as vehicle control
The sperm motility enhancement property of the com-pounds was categorized as below
(a) Marginal which resulted in lt5 increase in themotility (b) Good which resulted in 5ndash10 increase inmotility (c) Excellent which resulted in gt10 increase inmotility (d) Poor where there was no enhancement inmotility
At 1 h after the in vitro incubation the majority of thecompounds had marginal effect on the sperm motility com-pared to control At this interval PTF had higher percentageof total and progressivelymotile spermatozoa compared to allthe test compounds A large number of studies have shownthat PTF can trigger the motility in fresh or frozen-thawedspermatozoa as early as 1 h [29] even though the majorityof the studies indicate that this effect is mainly due to theelevated cAMP level in the spermatozoa [30] it can also bemediated through tyrosine phosphorylation [31]
Compounds 5a 5b 5c 5d 5e 5g 5h and 5k inducedmarginal enhancement in total motility while compounds5a 5b 5c and 5j resulted in similar effect on progressivemotility (Tables 2(a) and 2(b)) A 5ndash10 increase in totalmotility was induced by compound 5j However with respectto progressive motility compounds 5d 5e 5f and 5k cameunder compounds with good effect on motility More than10 increase in progressive motility was observed only forcompound 5g (at 05 120583gmL concentration) and compound5h (at 5 120583gmL concentration)
A time-dependent decrease in total and progressivemotility was observed in all the groups However the declinewasmore evident at 24 h intervalThis phenomenon has beendocumented by earlier studies [28] These changes may berelated to the oxidative stress induced by in vitro cultureconditions Spermatozoa are highly susceptible to reactiveoxygen species (ROS) due to high polyunsaturated fatty acidcontent in their plasma membrane and negligible amount ofcytoplasmic antioxidants
At 4 h interval compounds 5e 5f and 5g enhancedthe progressive motility of spermatozoa by 5ndash10 while
compound 5h with two chloro substituents on coumarinand benzimidazole resulted in almost 15 increase in theprogressive motility However the effect was lower than thatof pentoxifylline The rest of the compounds (5a 5b 5c5f and 5j) induced a marginal increase in motility exceptin compound 5e in which the motility was lower than thecontrol group
At 24 h interval the majority of the compounds (5a5b 5d 5f 5h and 5j) had marginally higher percentageof motility or poor motility compounds (5c 5e and 5g)compared to control Interestingly compound 5k with chlorosubstituent on coumarin and bromo substitution on ben-zimidazole had higher percentage of motile spermatozoacompared to control (14 higher) and even PTF (9 higher)This indicates that compound5k canhelp in prolonging the invitro survival of spermatozoa which has significant beneficialrole in assisted reproductive technologies such as in vitrofertilization Previous studies have shown that even thoughPTF has triggering effect on themotility it induces prematureacrosome reaction and drastic decrease in motility at laterintervals In this context the compounds which increasethe motility and longevity of spermatozoa under in vitroconditions are of clinical relevance
Based on the manual motility assessment we observedthat compounds 5d 5f 5g 5h and 5k have considerablebeneficial role in spermmotilityTherefore these compoundswere further assessed for their effect on the kinematicsof spermatozoa at 1 h However there was no significantdifference in the kinematics of spermatozoa compared tocontrol and PTF (Table 3) In conclusion the newly synthe-sized coumarin derivatives have sperm motility enhancingproperty However in this preliminary screening we havetaken three arbitrary doses to assess their effect on motilityCompounds 5g 5h and 5k show considerable beneficialeffect which can be of clinical significance where spermmotility enhancement is achieved under in vitro condi-tions Compounds 5g and 5h triggered the motility at earlyintervals which was similar to the PTF while compound5k improved the longevity of the spermatozoa Since thecoumarin is known to have PDE inhibitory function themotility enhancement could be due to the elevated cAMPlevel in sperm However further detailed studies are essentialto understand the mechanism of action of these compoundsand their clinical utility
4 Experimental
All the chemicals and reagents were purchased from theMerck and Aldrich chemical suppliers Melting points (mp)were determined in open capillaries and are uncorrectedThe IR spectra were recorded on a Nicolet Impact 410 FTIR spectrometer using KBr pellets (range 4000ndash500 cmminus1)The 1HNMR spectra were recorded at 400MHz on BrukerAvance FT NMR spectrometer in DMSO-d
6solvent with
TMS as internal standard 13CNMR spectra were recordedat 100MHz on Bruker Avance FT NMR spectrometer inDMSO-d
6solvent with TMS as internal standard The mass
spectra were recorded on Shimadzu GC-MS operating at70 eV Thin-layer chromatography (TLC) was performed on
International Journal of Medicinal Chemistry 11
Table 2 Effect of supplementation of coumarin derivatives (5andashj) to sperm wash medium on the motility pattern in frozen-thawed humanspermatozoa at various time intervals
(a)
Compounds Time (h)Total motility (mean plusmn SE of percentage)
020mm Aluchrosep silica gel 60 F254 plates (SD FineMumbai) Microwave irradiation experiments were carriedout using CEMDiscover SPMicrowave Synthesizer equippedwith IR sensor to monitor reaction temperatures
5 General Procedures for the Preparation ofTitle Compounds 5andashx
3-(2-Oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-car-baldehydes (3andashd 1 mmol) were prepared from 3-acetyl-coumarin (1andashd 1 mmol) phenylhydrazine (1mmol) andthe intermediate (Schiff base) (2andashd 1 mmol) followedby reaction with POCl
3in DMF by Vilsmeier Haack for-
mylation strategies [25] The target compounds 5andashxwere then achieved by stirring substituted carbaldehyde(3andashd 1 mmol) with different o-arylenediamine (4andashf1 mmol) under reflux for 4-5 hrs After completion (TLChexane ethylacetate 7 3) of the reaction the solid obtainedwas filtered washed with small quantity of ethanol anddried Further it was recrystallized in methanol to get thepure compound (5andashx)
51 Microwave Assisted Procedure for the Preparation of Com-pounds 5andashx A mixture of 3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde (3andashd 1 mmol) and o-phenylenediamine (4andashf 1 mmol) taken in ethanol (5mL)was introduced into a CEM microwave reaction vesselequipped with magnetic stirrer The reaction mixture was
prestirred for 1min at room temperature and irradiated at180W (150∘C) for about 6ndash8min (TLC using hexane ethylacetate (7 3 drops) as eluent) The reaction mixture wasthen quenched into crushed ice and the crude product wasfiltered washed and dried Recrystallization from methanolgave pure crystals of the compounds 5andashx
(Spectral as well as elemental analyses data are providedas electronic supplementary file in Supplementary Materialavailable online at httpdxdoiorg10115520169890630)
6 Assessment of Phosphodiesterase InhibitionUsing Human Spermatozoa as Model
Earlier studies have shown that PDE inhibitors can enhancethe spermmotility [32 33] Based on this in the present studywe have used an indirect approach to assess the inhibitoryeffect of the coumarin derivatives on PDE by assessing spermmotility pattern
61 Preparation of Test Solution Stock solution of the testcompounds (1mgmL) was prepared by dissolving them indimethyl sulfoxide (DMSO Sigma Cat number D5879)The stock solution was then diluted with Earlrsquos BalancedSalt Solution (EBSS) supplemented with 01 bovine serumalbumin (BSA) in a ratio of 1 200 1 1000 and 1 2000 to getworking solutions of 5 1 and 05 120583gmL concentrations Theworking solutions were preincubated at 37∘C and 5 CO
2
prior to use
International Journal of Medicinal Chemistry 13
Table 3 Effect of various coumarin derivatives on kinematics of frozen-thawed spermatozoa at one hour after incubation in vitro
Parameters Compounds Concentrations of compounds in 120583gmL VC PTFC 0 05 5 (005 DMSO) (1mM)
62 Study Subjects Infertile men who attended AndrologyLaboratory Kasturba Medical College Manipal UniversityManipal during the period of June-July 2015 for routinesemen evaluation were included in the study The subjects
with a sexual abstinence of 3ndash5 days were asked to providethe semen samples by masturbation in a sterile containerFollowing liquefaction the semen parameters were assessedas described by Kotdawala et al [34]The study was approved
14 International Journal of Medicinal Chemistry
by the Institutional Ethics Committee of Kasturba HospitalKasturba Medical College Manipal University (IEC number1552015)
63 Cryopreservation and Thawing The liquefied semensamples were cryopreserved by rapid freezing method asdescribed previously [34] with minor modifications Brieflythe semen samples were mixed with equal volume of freezingmedium (Sperm Freeze FertiPro Cat number 0344) in acryovial (Thermoscientific Nunc Cat number 138627) andkept at 4∘C for 10min in liquid nitrogen (LN2) vapor phasefor 5min and then finally plunged into liquid nitrogenThe samples were thawed after 1 week by rapid thawingmethod by placing themat 37∘C for 5minThe cryoprotectantmedium was completely removed by mixing the spermsuspension with EBSS medium and centrifuging at 1000 rpmfor 8min The resultant pellet was resuspended with freshEBSS medium and used for further analysis
64 Sample Preparation To screen the compounds withenhancing effect on themotility of spermatozoa under in vitroconditions the frozen-thawed semen samples were washedby mixing with equal volume of preincubated Earlrsquos BalancedSalt Solution (EBSS Sigma Cat number E2888) followed bycentrifugation at 1800 rpm for 8min The pellet was thengently mixed with 1mL of EBSS media containing 01bovine serum albumin (BSA Sigma Cat number A3311)and equally divided into control vehicle control and testgroups (with average sperm density of 5 times 104 spermatozoain each group) The motile sperm were extracted by swimup method as described earlier [35] The sperm suspensionwas centrifuged at 1200 rpm for 8min and the resultantpellet was overlaidwith EBSSmedium containing various testcompoundsThe sperm suspensions were incubated for 1 h at37∘Cand 5CO
2afterwhich themotile spermwere collected
from the supernatant
65 Motility Assessment Spermatozoa in the supernatantfraction were assessed for their motility patterns at 1 4 and24 h after incubation under light microscope as describedearlier [27] Sperm suspension was (10 120583L droplet) placed ona clean microscope slide and a coverslip was placed over itSpermatozoa with progressive motility and nonprogressivemotility and immotile spermatozoa were scored separatelyfrom a total of 200 spermatozoa in random fields at 400xmagnification
66 Sperm Kinematics Using Computer Assisted Semen Anal-ysis (CASA) The motion characteristics in the spermatozoawere assessed at 1 4 and 24 h after incubation using CASAsystem (ISAS Spain) Briefly 5 120583L of sperm suspension wasplaced on a clean microscope slide prewarmed at 37∘C Thesperm suspensionwas coveredwith prewarmed cover slip (22times 22mm) and observed under microscope (negative phasecontrast 10x objectives Proiser Spain) For each sample thekinematics was assessed randomly at ten different fields Theparameters such as curvilinear velocity (VCL) straight line
velocity (VSL) average path velocity (VAP) amplitude of lat-eral head displacement (ALH) linearity (LIN) straightness(STR) wobble (WOB) and beat cross frequency (BCF) wereanalyzed
7 Conclusions
In conclusion we have reported efficient and environmen-tally benignmethodologies for the synthesis of 3-[4-(1H-benzo[d]imidazol-2-yl)-1-phenyl-1H-pyrazol-3-yl]-2H-chromen-2-one derivatives 5andashx by using thermal and by microwaveirradiation under neat conditions in presence of ethanol Thereactions carried out under microwave irradiation affordedbenzimidazoles in short period of time with excellent yieldsHence this methodology would make an interesting strategyfor the synthesis of various substituted coumarin pyrazolebenzimidazoles Also compounds 5g 5h and 5k exhibitedimpressive sperm motility enhancing property whereas 5khas spermatozoa longevity property
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to University Scientific Instrumen-tation Centre (USIC) Karnatak University Dharwad forprovidingmass and IR spectral dataThe authors are thankfulto Indian Institute of Science (IISc) Bengaluru for carryingout NMR analyses and to UGC New Delhi for the financialassistance under UPE program vide no [F No 14-22012(NSPE) dated 22-01-2013] One of the authors (MNK)acknowledges University Grants Commission (UGC) NewDelhi for fellowship under UGC-UPE program
References
[1] P P Kattimani R R Kamble and G Y Meti ldquoExpedientsynthesis of benzimidazoles using amidesrdquo RSC Advances vol5 no 37 pp 29447ndash29455 2015
[2] L Li C Yee and J A Beavo ldquoCD3- and CD28-dependentinduction of PDE7 required for T cell activationrdquo Science vol283 no 5403 pp 848ndash849 1999
[3] T P Dousa ldquoCyclic-3101584051015840-nucleotide phosphodiesteraseisozymes in cell biology and pathophysiology of the kidneyrdquoKidney International vol 55 no 1 pp 29ndash62 1999
[4] C Pfister N Bennett F Bruckert et al ldquoInteractions of a G-protein with its effector transducin and cGMP phosphodi-esterase in retinal rodsrdquo Cell Signaling vol 5 no 3 pp 235ndash2411993
[5] S L C Jin F J Richard W-P Kuo A J DrsquoErcole andM Conti ldquoImpaired growth and fertility of cAMP-specificphosphodiesterase PDE4D-deficient micerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 21 pp 11998ndash12003 1999
[6] S H Francis I V Turko and J D Corbin ldquoCyclic nucleotidephosphodiesterases relating structure and functionrdquoProgress in
International Journal of Medicinal Chemistry 15
Nucleic Acid Research and Molecular Biology vol 65 pp 1ndash522000
[7] J M Hetman N Robas R Baxendale et al ldquoCloning and char-acterization of two splice variants of human phosphodiesterase11Ardquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 23 pp 12891ndash12895 2000
[8] K Yuasa T Ohgaru M Asahina and K Omori ldquoIdentificationof rat cyclic nucleotide phosphodiesterase 11A (PDE11A) com-parison of rat and human PDE11A splicing variantsrdquo EuropeanJournal of Biochemistry vol 268 no 16 pp 4440ndash4448 2001
[9] S K Sahu A Mishra and R K Behera ldquoSynthesis of thiazolebenzothiazole oxadiazole thiadiazole triazole and thiazolidi-none incorporated coumarinsrdquo Indian Journal of HeterocyclicChemistry vol 6 no 2 pp 91ndash94 1996
[10] G Roma M Di Braccio G Grossi et al ldquoSynthesis and in vitroantiplatelet activity of new 4-(1-piperazinyl) coumarin deriva-tives Human platelet phosphodiesterase 3 inhibitory propertiesof the two most effective compounds described and molecularmodeling study on their interactions with phosphodiesterase3A catalytic siterdquo Journal of Medicinal Chemistry vol 50 no 12pp 2886ndash2895 2007
[11] T Shyamsunder Reddy K Shiva Kumar C L T Medaet al ldquoConformationally restricted novel pyrazole deriva-tives synthesis of 18-disubstituted 55-dimethyl-45-dihydro-1H-benzo[g]indazoles as a new class of PDE4 inhibitorsrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 9 pp3248ndash3255 2012
[12] A Chino N Masuda Y Amano et al ldquoNovel benzimidazolederivatives as phosphodiesterase 10A (PDE10A) inhibitors withimproved metabolic stabilityrdquo Bioorganic and Medicinal Chem-istry vol 22 no 13 pp 3515ndash3526 2014
[13] H Yang FNMurigi ZWang J Li H Jin andZ Tu ldquoSynthesisand in vitro characterization of cinnoline and benzimidazoleanalogues as phosphodiesterase 10A inhibitorsrdquo Bioorganic andMedicinal Chemistry Letters vol 25 pp 919ndash924 2015
[14] J Nawrot-Modranka E Nawrot and J Graczyk ldquoIn vivo antitu-mor in vitro antibacterial activity and alkylating properties ofphosphorohydrazine derivatives of coumarin and chromonerdquoEuropean Journal of Medicinal Chemistry vol 41 no 11 pp1301ndash1309 2006
[15] A A Bekhit and T Abdel-Aziem ldquoDesign synthesis andbiological evaluation of some pyrazole derivatives as anti-inflammatory-antimicrobial agentsrdquo Bioorganic and MedicinalChemistry vol 12 no 8 pp 1935ndash1945 2004
[16] T Devji C Reddy CWoo S Awale S Kadota and D Carrico-Moniz ldquoPancreatic anticancer activity of a novel geranylgerany-lated coumarin derivativerdquo Bioorganic andMedicinal ChemistryLetters vol 21 no 19 pp 5770ndash5773 2011
[17] I Kostova I Manolov I Nicolova S Konstantinov andM Karaivanova ldquoNew lanthanide complexes of 4-methyl-7-hydroxycoumarin and their pharmacological activityrdquo Euro-pean Journal of Medicinal Chemistry vol 36 no 4 pp 339ndash3472001
[18] M Ghate R A Kusanur and M V Kulkarni ldquoSynthesis andin vivo analgesic and anti-inflammatory activity of some bi-heterocyclic coumarin derivativesrdquo European Journal of Medic-inal Chemistry vol 40 no 9 pp 882ndash887 2005
[19] PM RonadMN Noolvi S Sapkal S Dharbhamulla andV SMaddi ldquoSynthesis and antimicrobial activity of 7-(2-substitutedphenylthiazolidinyl)-benzopyran-2-one derivativesrdquo EuropeanJournal of Medicinal Chemistry vol 45 no 1 pp 85ndash89 2010
[20] GMelagraki A Afantitis O Igglessi-Markopoulou et al ldquoSyn-thesis and evaluation of the antioxidant and anti-inflammatoryactivity of novel coumarin-3-aminoamides and their alpha-lipoic acid adductsrdquo European Journal of Medicinal Chemistryvol 44 no 7 pp 3020ndash3026 2009
[21] J C Trivedi J B Bariwal K D Upadhyay et al ldquoImprovedand rapid synthesis of new coumarinyl chalcone derivatives andtheir antiviral activityrdquo Tetrahedron Letters vol 48 no 48 pp8472ndash8474 2007
[22] S Vilar E Quezada L Santana et al ldquoDesign synthe-sis and vasorelaxant and platelet antiaggregatory activitiesof coumarinndashresveratrol hybridsrdquo Bioorganic and MedicinalChemistry Letters vol 16 no 2 pp 257ndash261 2006
[23] W Hamaguchi N Masuda M Isomura et al ldquoDesign and syn-thesis of novel benzimidazole derivatives as phosphodiesterase10A inhibitors with reduced CYP1A2 inhibitionrdquo Bioorganicand Medicinal Chemistry vol 21 no 24 pp 7612ndash7623 2013
[24] A Dore B Asproni A Scampuddu et al ldquoSynthesis and SARstudy of novel tricyclic pyrazoles as potent phosphodiesterase10A inhibitorsrdquo European Journal of Medicinal Chemistry vol84 pp 181ndash193 2014
[25] S V Laxmi B S Kuarm and B Rajitha ldquoSynthesis and anti-microbial activity of coumarin pyrazole pyrimidine 246(1H3H5H)triones and thioxopyrimidine46(1H5H)dionesrdquo Me-dicinal Chemistry Research vol 22 no 2 pp 768ndash774 2013
[26] G Kalthur S Raj AThiyagarajan S Kumar P Kumar and S KAdiga ldquoVitamin E supplementation in semen-freezingmediumimproves the motility and protects sperm from freeze-thaw-induced DNA damagerdquo Fertility and Sterility vol 95 no 3 pp1149ndash1151 2011
[27] J Tesarik C Mendoza and A Carreras ldquoEffects of phosphodi-esterase inhibitors caffeine and pentoxifylline on spontaneousand stimulus-induced acrosome reactions in human spermrdquoFertility and Sterility vol 58 no 6 pp 1185ndash1190 1992
[28] G Kalthur S R Salian F Keyvanifard et al ldquoSupplementationof biotin to sperm preparation medium increases the motilityand longevity in cryopreserved human spermatozoardquo Journalof Assisted Reproduction and Genetics vol 29 no 7 pp 631ndash6352012
[29] A E Calogero S Fishel J Hall et al ldquoCorrelation betweenintracellular cAMP content kinematic parameters and hyper-activation of human spermatozoa after incubation with pentox-ifyllinerdquo Human Reproduction vol 13 no 4 pp 911ndash915 1998
[30] R Ain K Uma Devi S Shivaji and P B Seshagiri ldquoPen-toxifylline-stimulated capacitation and acrosome reaction inhamster spermatozoa involvement of intracellular signallingmoleculesrdquo Molecular Human Reproduction vol 5 no 7 pp618ndash626 1999
[31] M Bajpai andG F Doncel ldquoInvolvement of tyrosine kinase andcAMP-dependent kinase cross-talk in the regulation of humanspermmotilityrdquo Reproduction vol 126 no 2 pp 183ndash195 2003
[32] R Yunes P Fernandez G F Doncel and A A Acosta ldquoCyclicnucleotide phosphodiesterase inhibition increases tyrosinephosphorylation and hypermotility in normal and pathologicalhuman spermatozoardquo Biocell vol 29 no 3 pp 287ndash293 2005
[33] F Dimitriadis D Giannakis N Pardalidis et al ldquoEffectsof phosphodiesterase 5 inhibitors on sperm parameters andfertilizing capacityrdquo Asian Journal of Andrology vol 10 no 1pp 115ndash133 2008
[34] A P Kotdawala S Kumar S R Salian et al ldquoAddition of zincto human ejaculate prior to cryopreservation prevents freeze-thaw-induced DNA damage and preserves sperm functionrdquo
16 International Journal of Medicinal Chemistry
Journal of Assisted Reproduction and Genetics vol 29 no 12 pp1447ndash1453 2012
[35] S Mutalik S R Salian K Avadhani et al ldquoLiposome encapsu-lated soy lecithin and cholesterol can efficiently replace chickenegg yolk in human semen cryopreservation mediumrdquo SystemsBiology in Reproductive Medicine vol 60 no 3 pp 183ndash1882014
31 Effect on Motility of Frozen-Thawed Human SpermatozoaTo assess the effect of title compounds on themotility patternhuman spermatozoa subjected to freeze-thaw process wasused It is well documented that freeze-thaw process canhave detrimental effect on the motility and survival of thespermatozoa [26] Therefore they can be an excellent modelto study the effect of any test compounds on themotility [27]
In the present study we assessed the motility patternin spermatozoa processed with media containing variouscompounds manually at 1 4 and 24 h after incubationin vitro Three arbitrary doses were selected (05 10 and50 120583gmL) for each compound to assess their effect on spermmotility Pentoxifylline (PTF) which is known to enhancethe human sperm motility by elevating intracellular cyclicadenosine monophosphate (cAMP) level [27] was taken asa positive control Based on the previous report PTF at 1mMconcentration was used [28] Since the test compounds werenot soluble in water dimethyl sulfoxide (DMSO 005) wasused as vehicle control
The sperm motility enhancement property of the com-pounds was categorized as below
(a) Marginal which resulted in lt5 increase in themotility (b) Good which resulted in 5ndash10 increase inmotility (c) Excellent which resulted in gt10 increase inmotility (d) Poor where there was no enhancement inmotility
At 1 h after the in vitro incubation the majority of thecompounds had marginal effect on the sperm motility com-pared to control At this interval PTF had higher percentageof total and progressivelymotile spermatozoa compared to allthe test compounds A large number of studies have shownthat PTF can trigger the motility in fresh or frozen-thawedspermatozoa as early as 1 h [29] even though the majorityof the studies indicate that this effect is mainly due to theelevated cAMP level in the spermatozoa [30] it can also bemediated through tyrosine phosphorylation [31]
Compounds 5a 5b 5c 5d 5e 5g 5h and 5k inducedmarginal enhancement in total motility while compounds5a 5b 5c and 5j resulted in similar effect on progressivemotility (Tables 2(a) and 2(b)) A 5ndash10 increase in totalmotility was induced by compound 5j However with respectto progressive motility compounds 5d 5e 5f and 5k cameunder compounds with good effect on motility More than10 increase in progressive motility was observed only forcompound 5g (at 05 120583gmL concentration) and compound5h (at 5 120583gmL concentration)
A time-dependent decrease in total and progressivemotility was observed in all the groups However the declinewasmore evident at 24 h intervalThis phenomenon has beendocumented by earlier studies [28] These changes may berelated to the oxidative stress induced by in vitro cultureconditions Spermatozoa are highly susceptible to reactiveoxygen species (ROS) due to high polyunsaturated fatty acidcontent in their plasma membrane and negligible amount ofcytoplasmic antioxidants
At 4 h interval compounds 5e 5f and 5g enhancedthe progressive motility of spermatozoa by 5ndash10 while
compound 5h with two chloro substituents on coumarinand benzimidazole resulted in almost 15 increase in theprogressive motility However the effect was lower than thatof pentoxifylline The rest of the compounds (5a 5b 5c5f and 5j) induced a marginal increase in motility exceptin compound 5e in which the motility was lower than thecontrol group
At 24 h interval the majority of the compounds (5a5b 5d 5f 5h and 5j) had marginally higher percentageof motility or poor motility compounds (5c 5e and 5g)compared to control Interestingly compound 5k with chlorosubstituent on coumarin and bromo substitution on ben-zimidazole had higher percentage of motile spermatozoacompared to control (14 higher) and even PTF (9 higher)This indicates that compound5k canhelp in prolonging the invitro survival of spermatozoa which has significant beneficialrole in assisted reproductive technologies such as in vitrofertilization Previous studies have shown that even thoughPTF has triggering effect on themotility it induces prematureacrosome reaction and drastic decrease in motility at laterintervals In this context the compounds which increasethe motility and longevity of spermatozoa under in vitroconditions are of clinical relevance
Based on the manual motility assessment we observedthat compounds 5d 5f 5g 5h and 5k have considerablebeneficial role in spermmotilityTherefore these compoundswere further assessed for their effect on the kinematicsof spermatozoa at 1 h However there was no significantdifference in the kinematics of spermatozoa compared tocontrol and PTF (Table 3) In conclusion the newly synthe-sized coumarin derivatives have sperm motility enhancingproperty However in this preliminary screening we havetaken three arbitrary doses to assess their effect on motilityCompounds 5g 5h and 5k show considerable beneficialeffect which can be of clinical significance where spermmotility enhancement is achieved under in vitro condi-tions Compounds 5g and 5h triggered the motility at earlyintervals which was similar to the PTF while compound5k improved the longevity of the spermatozoa Since thecoumarin is known to have PDE inhibitory function themotility enhancement could be due to the elevated cAMPlevel in sperm However further detailed studies are essentialto understand the mechanism of action of these compoundsand their clinical utility
4 Experimental
All the chemicals and reagents were purchased from theMerck and Aldrich chemical suppliers Melting points (mp)were determined in open capillaries and are uncorrectedThe IR spectra were recorded on a Nicolet Impact 410 FTIR spectrometer using KBr pellets (range 4000ndash500 cmminus1)The 1HNMR spectra were recorded at 400MHz on BrukerAvance FT NMR spectrometer in DMSO-d
6solvent with
TMS as internal standard 13CNMR spectra were recordedat 100MHz on Bruker Avance FT NMR spectrometer inDMSO-d
6solvent with TMS as internal standard The mass
spectra were recorded on Shimadzu GC-MS operating at70 eV Thin-layer chromatography (TLC) was performed on
International Journal of Medicinal Chemistry 11
Table 2 Effect of supplementation of coumarin derivatives (5andashj) to sperm wash medium on the motility pattern in frozen-thawed humanspermatozoa at various time intervals
(a)
Compounds Time (h)Total motility (mean plusmn SE of percentage)
020mm Aluchrosep silica gel 60 F254 plates (SD FineMumbai) Microwave irradiation experiments were carriedout using CEMDiscover SPMicrowave Synthesizer equippedwith IR sensor to monitor reaction temperatures
5 General Procedures for the Preparation ofTitle Compounds 5andashx
3-(2-Oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-car-baldehydes (3andashd 1 mmol) were prepared from 3-acetyl-coumarin (1andashd 1 mmol) phenylhydrazine (1mmol) andthe intermediate (Schiff base) (2andashd 1 mmol) followedby reaction with POCl
3in DMF by Vilsmeier Haack for-
mylation strategies [25] The target compounds 5andashxwere then achieved by stirring substituted carbaldehyde(3andashd 1 mmol) with different o-arylenediamine (4andashf1 mmol) under reflux for 4-5 hrs After completion (TLChexane ethylacetate 7 3) of the reaction the solid obtainedwas filtered washed with small quantity of ethanol anddried Further it was recrystallized in methanol to get thepure compound (5andashx)
51 Microwave Assisted Procedure for the Preparation of Com-pounds 5andashx A mixture of 3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde (3andashd 1 mmol) and o-phenylenediamine (4andashf 1 mmol) taken in ethanol (5mL)was introduced into a CEM microwave reaction vesselequipped with magnetic stirrer The reaction mixture was
prestirred for 1min at room temperature and irradiated at180W (150∘C) for about 6ndash8min (TLC using hexane ethylacetate (7 3 drops) as eluent) The reaction mixture wasthen quenched into crushed ice and the crude product wasfiltered washed and dried Recrystallization from methanolgave pure crystals of the compounds 5andashx
(Spectral as well as elemental analyses data are providedas electronic supplementary file in Supplementary Materialavailable online at httpdxdoiorg10115520169890630)
6 Assessment of Phosphodiesterase InhibitionUsing Human Spermatozoa as Model
Earlier studies have shown that PDE inhibitors can enhancethe spermmotility [32 33] Based on this in the present studywe have used an indirect approach to assess the inhibitoryeffect of the coumarin derivatives on PDE by assessing spermmotility pattern
61 Preparation of Test Solution Stock solution of the testcompounds (1mgmL) was prepared by dissolving them indimethyl sulfoxide (DMSO Sigma Cat number D5879)The stock solution was then diluted with Earlrsquos BalancedSalt Solution (EBSS) supplemented with 01 bovine serumalbumin (BSA) in a ratio of 1 200 1 1000 and 1 2000 to getworking solutions of 5 1 and 05 120583gmL concentrations Theworking solutions were preincubated at 37∘C and 5 CO
2
prior to use
International Journal of Medicinal Chemistry 13
Table 3 Effect of various coumarin derivatives on kinematics of frozen-thawed spermatozoa at one hour after incubation in vitro
Parameters Compounds Concentrations of compounds in 120583gmL VC PTFC 0 05 5 (005 DMSO) (1mM)
62 Study Subjects Infertile men who attended AndrologyLaboratory Kasturba Medical College Manipal UniversityManipal during the period of June-July 2015 for routinesemen evaluation were included in the study The subjects
with a sexual abstinence of 3ndash5 days were asked to providethe semen samples by masturbation in a sterile containerFollowing liquefaction the semen parameters were assessedas described by Kotdawala et al [34]The study was approved
14 International Journal of Medicinal Chemistry
by the Institutional Ethics Committee of Kasturba HospitalKasturba Medical College Manipal University (IEC number1552015)
63 Cryopreservation and Thawing The liquefied semensamples were cryopreserved by rapid freezing method asdescribed previously [34] with minor modifications Brieflythe semen samples were mixed with equal volume of freezingmedium (Sperm Freeze FertiPro Cat number 0344) in acryovial (Thermoscientific Nunc Cat number 138627) andkept at 4∘C for 10min in liquid nitrogen (LN2) vapor phasefor 5min and then finally plunged into liquid nitrogenThe samples were thawed after 1 week by rapid thawingmethod by placing themat 37∘C for 5minThe cryoprotectantmedium was completely removed by mixing the spermsuspension with EBSS medium and centrifuging at 1000 rpmfor 8min The resultant pellet was resuspended with freshEBSS medium and used for further analysis
64 Sample Preparation To screen the compounds withenhancing effect on themotility of spermatozoa under in vitroconditions the frozen-thawed semen samples were washedby mixing with equal volume of preincubated Earlrsquos BalancedSalt Solution (EBSS Sigma Cat number E2888) followed bycentrifugation at 1800 rpm for 8min The pellet was thengently mixed with 1mL of EBSS media containing 01bovine serum albumin (BSA Sigma Cat number A3311)and equally divided into control vehicle control and testgroups (with average sperm density of 5 times 104 spermatozoain each group) The motile sperm were extracted by swimup method as described earlier [35] The sperm suspensionwas centrifuged at 1200 rpm for 8min and the resultantpellet was overlaidwith EBSSmedium containing various testcompoundsThe sperm suspensions were incubated for 1 h at37∘Cand 5CO
2afterwhich themotile spermwere collected
from the supernatant
65 Motility Assessment Spermatozoa in the supernatantfraction were assessed for their motility patterns at 1 4 and24 h after incubation under light microscope as describedearlier [27] Sperm suspension was (10 120583L droplet) placed ona clean microscope slide and a coverslip was placed over itSpermatozoa with progressive motility and nonprogressivemotility and immotile spermatozoa were scored separatelyfrom a total of 200 spermatozoa in random fields at 400xmagnification
66 Sperm Kinematics Using Computer Assisted Semen Anal-ysis (CASA) The motion characteristics in the spermatozoawere assessed at 1 4 and 24 h after incubation using CASAsystem (ISAS Spain) Briefly 5 120583L of sperm suspension wasplaced on a clean microscope slide prewarmed at 37∘C Thesperm suspensionwas coveredwith prewarmed cover slip (22times 22mm) and observed under microscope (negative phasecontrast 10x objectives Proiser Spain) For each sample thekinematics was assessed randomly at ten different fields Theparameters such as curvilinear velocity (VCL) straight line
velocity (VSL) average path velocity (VAP) amplitude of lat-eral head displacement (ALH) linearity (LIN) straightness(STR) wobble (WOB) and beat cross frequency (BCF) wereanalyzed
7 Conclusions
In conclusion we have reported efficient and environmen-tally benignmethodologies for the synthesis of 3-[4-(1H-benzo[d]imidazol-2-yl)-1-phenyl-1H-pyrazol-3-yl]-2H-chromen-2-one derivatives 5andashx by using thermal and by microwaveirradiation under neat conditions in presence of ethanol Thereactions carried out under microwave irradiation affordedbenzimidazoles in short period of time with excellent yieldsHence this methodology would make an interesting strategyfor the synthesis of various substituted coumarin pyrazolebenzimidazoles Also compounds 5g 5h and 5k exhibitedimpressive sperm motility enhancing property whereas 5khas spermatozoa longevity property
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to University Scientific Instrumen-tation Centre (USIC) Karnatak University Dharwad forprovidingmass and IR spectral dataThe authors are thankfulto Indian Institute of Science (IISc) Bengaluru for carryingout NMR analyses and to UGC New Delhi for the financialassistance under UPE program vide no [F No 14-22012(NSPE) dated 22-01-2013] One of the authors (MNK)acknowledges University Grants Commission (UGC) NewDelhi for fellowship under UGC-UPE program
References
[1] P P Kattimani R R Kamble and G Y Meti ldquoExpedientsynthesis of benzimidazoles using amidesrdquo RSC Advances vol5 no 37 pp 29447ndash29455 2015
[2] L Li C Yee and J A Beavo ldquoCD3- and CD28-dependentinduction of PDE7 required for T cell activationrdquo Science vol283 no 5403 pp 848ndash849 1999
[3] T P Dousa ldquoCyclic-3101584051015840-nucleotide phosphodiesteraseisozymes in cell biology and pathophysiology of the kidneyrdquoKidney International vol 55 no 1 pp 29ndash62 1999
[4] C Pfister N Bennett F Bruckert et al ldquoInteractions of a G-protein with its effector transducin and cGMP phosphodi-esterase in retinal rodsrdquo Cell Signaling vol 5 no 3 pp 235ndash2411993
[5] S L C Jin F J Richard W-P Kuo A J DrsquoErcole andM Conti ldquoImpaired growth and fertility of cAMP-specificphosphodiesterase PDE4D-deficient micerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 21 pp 11998ndash12003 1999
[6] S H Francis I V Turko and J D Corbin ldquoCyclic nucleotidephosphodiesterases relating structure and functionrdquoProgress in
International Journal of Medicinal Chemistry 15
Nucleic Acid Research and Molecular Biology vol 65 pp 1ndash522000
[7] J M Hetman N Robas R Baxendale et al ldquoCloning and char-acterization of two splice variants of human phosphodiesterase11Ardquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 23 pp 12891ndash12895 2000
[8] K Yuasa T Ohgaru M Asahina and K Omori ldquoIdentificationof rat cyclic nucleotide phosphodiesterase 11A (PDE11A) com-parison of rat and human PDE11A splicing variantsrdquo EuropeanJournal of Biochemistry vol 268 no 16 pp 4440ndash4448 2001
[9] S K Sahu A Mishra and R K Behera ldquoSynthesis of thiazolebenzothiazole oxadiazole thiadiazole triazole and thiazolidi-none incorporated coumarinsrdquo Indian Journal of HeterocyclicChemistry vol 6 no 2 pp 91ndash94 1996
[10] G Roma M Di Braccio G Grossi et al ldquoSynthesis and in vitroantiplatelet activity of new 4-(1-piperazinyl) coumarin deriva-tives Human platelet phosphodiesterase 3 inhibitory propertiesof the two most effective compounds described and molecularmodeling study on their interactions with phosphodiesterase3A catalytic siterdquo Journal of Medicinal Chemistry vol 50 no 12pp 2886ndash2895 2007
[11] T Shyamsunder Reddy K Shiva Kumar C L T Medaet al ldquoConformationally restricted novel pyrazole deriva-tives synthesis of 18-disubstituted 55-dimethyl-45-dihydro-1H-benzo[g]indazoles as a new class of PDE4 inhibitorsrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 9 pp3248ndash3255 2012
[12] A Chino N Masuda Y Amano et al ldquoNovel benzimidazolederivatives as phosphodiesterase 10A (PDE10A) inhibitors withimproved metabolic stabilityrdquo Bioorganic and Medicinal Chem-istry vol 22 no 13 pp 3515ndash3526 2014
[13] H Yang FNMurigi ZWang J Li H Jin andZ Tu ldquoSynthesisand in vitro characterization of cinnoline and benzimidazoleanalogues as phosphodiesterase 10A inhibitorsrdquo Bioorganic andMedicinal Chemistry Letters vol 25 pp 919ndash924 2015
[14] J Nawrot-Modranka E Nawrot and J Graczyk ldquoIn vivo antitu-mor in vitro antibacterial activity and alkylating properties ofphosphorohydrazine derivatives of coumarin and chromonerdquoEuropean Journal of Medicinal Chemistry vol 41 no 11 pp1301ndash1309 2006
[15] A A Bekhit and T Abdel-Aziem ldquoDesign synthesis andbiological evaluation of some pyrazole derivatives as anti-inflammatory-antimicrobial agentsrdquo Bioorganic and MedicinalChemistry vol 12 no 8 pp 1935ndash1945 2004
[16] T Devji C Reddy CWoo S Awale S Kadota and D Carrico-Moniz ldquoPancreatic anticancer activity of a novel geranylgerany-lated coumarin derivativerdquo Bioorganic andMedicinal ChemistryLetters vol 21 no 19 pp 5770ndash5773 2011
[17] I Kostova I Manolov I Nicolova S Konstantinov andM Karaivanova ldquoNew lanthanide complexes of 4-methyl-7-hydroxycoumarin and their pharmacological activityrdquo Euro-pean Journal of Medicinal Chemistry vol 36 no 4 pp 339ndash3472001
[18] M Ghate R A Kusanur and M V Kulkarni ldquoSynthesis andin vivo analgesic and anti-inflammatory activity of some bi-heterocyclic coumarin derivativesrdquo European Journal of Medic-inal Chemistry vol 40 no 9 pp 882ndash887 2005
[19] PM RonadMN Noolvi S Sapkal S Dharbhamulla andV SMaddi ldquoSynthesis and antimicrobial activity of 7-(2-substitutedphenylthiazolidinyl)-benzopyran-2-one derivativesrdquo EuropeanJournal of Medicinal Chemistry vol 45 no 1 pp 85ndash89 2010
[20] GMelagraki A Afantitis O Igglessi-Markopoulou et al ldquoSyn-thesis and evaluation of the antioxidant and anti-inflammatoryactivity of novel coumarin-3-aminoamides and their alpha-lipoic acid adductsrdquo European Journal of Medicinal Chemistryvol 44 no 7 pp 3020ndash3026 2009
[21] J C Trivedi J B Bariwal K D Upadhyay et al ldquoImprovedand rapid synthesis of new coumarinyl chalcone derivatives andtheir antiviral activityrdquo Tetrahedron Letters vol 48 no 48 pp8472ndash8474 2007
[22] S Vilar E Quezada L Santana et al ldquoDesign synthe-sis and vasorelaxant and platelet antiaggregatory activitiesof coumarinndashresveratrol hybridsrdquo Bioorganic and MedicinalChemistry Letters vol 16 no 2 pp 257ndash261 2006
[23] W Hamaguchi N Masuda M Isomura et al ldquoDesign and syn-thesis of novel benzimidazole derivatives as phosphodiesterase10A inhibitors with reduced CYP1A2 inhibitionrdquo Bioorganicand Medicinal Chemistry vol 21 no 24 pp 7612ndash7623 2013
[24] A Dore B Asproni A Scampuddu et al ldquoSynthesis and SARstudy of novel tricyclic pyrazoles as potent phosphodiesterase10A inhibitorsrdquo European Journal of Medicinal Chemistry vol84 pp 181ndash193 2014
[25] S V Laxmi B S Kuarm and B Rajitha ldquoSynthesis and anti-microbial activity of coumarin pyrazole pyrimidine 246(1H3H5H)triones and thioxopyrimidine46(1H5H)dionesrdquo Me-dicinal Chemistry Research vol 22 no 2 pp 768ndash774 2013
[26] G Kalthur S Raj AThiyagarajan S Kumar P Kumar and S KAdiga ldquoVitamin E supplementation in semen-freezingmediumimproves the motility and protects sperm from freeze-thaw-induced DNA damagerdquo Fertility and Sterility vol 95 no 3 pp1149ndash1151 2011
[27] J Tesarik C Mendoza and A Carreras ldquoEffects of phosphodi-esterase inhibitors caffeine and pentoxifylline on spontaneousand stimulus-induced acrosome reactions in human spermrdquoFertility and Sterility vol 58 no 6 pp 1185ndash1190 1992
[28] G Kalthur S R Salian F Keyvanifard et al ldquoSupplementationof biotin to sperm preparation medium increases the motilityand longevity in cryopreserved human spermatozoardquo Journalof Assisted Reproduction and Genetics vol 29 no 7 pp 631ndash6352012
[29] A E Calogero S Fishel J Hall et al ldquoCorrelation betweenintracellular cAMP content kinematic parameters and hyper-activation of human spermatozoa after incubation with pentox-ifyllinerdquo Human Reproduction vol 13 no 4 pp 911ndash915 1998
[30] R Ain K Uma Devi S Shivaji and P B Seshagiri ldquoPen-toxifylline-stimulated capacitation and acrosome reaction inhamster spermatozoa involvement of intracellular signallingmoleculesrdquo Molecular Human Reproduction vol 5 no 7 pp618ndash626 1999
[31] M Bajpai andG F Doncel ldquoInvolvement of tyrosine kinase andcAMP-dependent kinase cross-talk in the regulation of humanspermmotilityrdquo Reproduction vol 126 no 2 pp 183ndash195 2003
[32] R Yunes P Fernandez G F Doncel and A A Acosta ldquoCyclicnucleotide phosphodiesterase inhibition increases tyrosinephosphorylation and hypermotility in normal and pathologicalhuman spermatozoardquo Biocell vol 29 no 3 pp 287ndash293 2005
[33] F Dimitriadis D Giannakis N Pardalidis et al ldquoEffectsof phosphodiesterase 5 inhibitors on sperm parameters andfertilizing capacityrdquo Asian Journal of Andrology vol 10 no 1pp 115ndash133 2008
[34] A P Kotdawala S Kumar S R Salian et al ldquoAddition of zincto human ejaculate prior to cryopreservation prevents freeze-thaw-induced DNA damage and preserves sperm functionrdquo
16 International Journal of Medicinal Chemistry
Journal of Assisted Reproduction and Genetics vol 29 no 12 pp1447ndash1453 2012
[35] S Mutalik S R Salian K Avadhani et al ldquoLiposome encapsu-lated soy lecithin and cholesterol can efficiently replace chickenegg yolk in human semen cryopreservation mediumrdquo SystemsBiology in Reproductive Medicine vol 60 no 3 pp 183ndash1882014
31 Effect on Motility of Frozen-Thawed Human SpermatozoaTo assess the effect of title compounds on themotility patternhuman spermatozoa subjected to freeze-thaw process wasused It is well documented that freeze-thaw process canhave detrimental effect on the motility and survival of thespermatozoa [26] Therefore they can be an excellent modelto study the effect of any test compounds on themotility [27]
In the present study we assessed the motility patternin spermatozoa processed with media containing variouscompounds manually at 1 4 and 24 h after incubationin vitro Three arbitrary doses were selected (05 10 and50 120583gmL) for each compound to assess their effect on spermmotility Pentoxifylline (PTF) which is known to enhancethe human sperm motility by elevating intracellular cyclicadenosine monophosphate (cAMP) level [27] was taken asa positive control Based on the previous report PTF at 1mMconcentration was used [28] Since the test compounds werenot soluble in water dimethyl sulfoxide (DMSO 005) wasused as vehicle control
The sperm motility enhancement property of the com-pounds was categorized as below
(a) Marginal which resulted in lt5 increase in themotility (b) Good which resulted in 5ndash10 increase inmotility (c) Excellent which resulted in gt10 increase inmotility (d) Poor where there was no enhancement inmotility
At 1 h after the in vitro incubation the majority of thecompounds had marginal effect on the sperm motility com-pared to control At this interval PTF had higher percentageof total and progressivelymotile spermatozoa compared to allthe test compounds A large number of studies have shownthat PTF can trigger the motility in fresh or frozen-thawedspermatozoa as early as 1 h [29] even though the majorityof the studies indicate that this effect is mainly due to theelevated cAMP level in the spermatozoa [30] it can also bemediated through tyrosine phosphorylation [31]
Compounds 5a 5b 5c 5d 5e 5g 5h and 5k inducedmarginal enhancement in total motility while compounds5a 5b 5c and 5j resulted in similar effect on progressivemotility (Tables 2(a) and 2(b)) A 5ndash10 increase in totalmotility was induced by compound 5j However with respectto progressive motility compounds 5d 5e 5f and 5k cameunder compounds with good effect on motility More than10 increase in progressive motility was observed only forcompound 5g (at 05 120583gmL concentration) and compound5h (at 5 120583gmL concentration)
A time-dependent decrease in total and progressivemotility was observed in all the groups However the declinewasmore evident at 24 h intervalThis phenomenon has beendocumented by earlier studies [28] These changes may berelated to the oxidative stress induced by in vitro cultureconditions Spermatozoa are highly susceptible to reactiveoxygen species (ROS) due to high polyunsaturated fatty acidcontent in their plasma membrane and negligible amount ofcytoplasmic antioxidants
At 4 h interval compounds 5e 5f and 5g enhancedthe progressive motility of spermatozoa by 5ndash10 while
compound 5h with two chloro substituents on coumarinand benzimidazole resulted in almost 15 increase in theprogressive motility However the effect was lower than thatof pentoxifylline The rest of the compounds (5a 5b 5c5f and 5j) induced a marginal increase in motility exceptin compound 5e in which the motility was lower than thecontrol group
At 24 h interval the majority of the compounds (5a5b 5d 5f 5h and 5j) had marginally higher percentageof motility or poor motility compounds (5c 5e and 5g)compared to control Interestingly compound 5k with chlorosubstituent on coumarin and bromo substitution on ben-zimidazole had higher percentage of motile spermatozoacompared to control (14 higher) and even PTF (9 higher)This indicates that compound5k canhelp in prolonging the invitro survival of spermatozoa which has significant beneficialrole in assisted reproductive technologies such as in vitrofertilization Previous studies have shown that even thoughPTF has triggering effect on themotility it induces prematureacrosome reaction and drastic decrease in motility at laterintervals In this context the compounds which increasethe motility and longevity of spermatozoa under in vitroconditions are of clinical relevance
Based on the manual motility assessment we observedthat compounds 5d 5f 5g 5h and 5k have considerablebeneficial role in spermmotilityTherefore these compoundswere further assessed for their effect on the kinematicsof spermatozoa at 1 h However there was no significantdifference in the kinematics of spermatozoa compared tocontrol and PTF (Table 3) In conclusion the newly synthe-sized coumarin derivatives have sperm motility enhancingproperty However in this preliminary screening we havetaken three arbitrary doses to assess their effect on motilityCompounds 5g 5h and 5k show considerable beneficialeffect which can be of clinical significance where spermmotility enhancement is achieved under in vitro condi-tions Compounds 5g and 5h triggered the motility at earlyintervals which was similar to the PTF while compound5k improved the longevity of the spermatozoa Since thecoumarin is known to have PDE inhibitory function themotility enhancement could be due to the elevated cAMPlevel in sperm However further detailed studies are essentialto understand the mechanism of action of these compoundsand their clinical utility
4 Experimental
All the chemicals and reagents were purchased from theMerck and Aldrich chemical suppliers Melting points (mp)were determined in open capillaries and are uncorrectedThe IR spectra were recorded on a Nicolet Impact 410 FTIR spectrometer using KBr pellets (range 4000ndash500 cmminus1)The 1HNMR spectra were recorded at 400MHz on BrukerAvance FT NMR spectrometer in DMSO-d
6solvent with
TMS as internal standard 13CNMR spectra were recordedat 100MHz on Bruker Avance FT NMR spectrometer inDMSO-d
6solvent with TMS as internal standard The mass
spectra were recorded on Shimadzu GC-MS operating at70 eV Thin-layer chromatography (TLC) was performed on
International Journal of Medicinal Chemistry 11
Table 2 Effect of supplementation of coumarin derivatives (5andashj) to sperm wash medium on the motility pattern in frozen-thawed humanspermatozoa at various time intervals
(a)
Compounds Time (h)Total motility (mean plusmn SE of percentage)
020mm Aluchrosep silica gel 60 F254 plates (SD FineMumbai) Microwave irradiation experiments were carriedout using CEMDiscover SPMicrowave Synthesizer equippedwith IR sensor to monitor reaction temperatures
5 General Procedures for the Preparation ofTitle Compounds 5andashx
3-(2-Oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-car-baldehydes (3andashd 1 mmol) were prepared from 3-acetyl-coumarin (1andashd 1 mmol) phenylhydrazine (1mmol) andthe intermediate (Schiff base) (2andashd 1 mmol) followedby reaction with POCl
3in DMF by Vilsmeier Haack for-
mylation strategies [25] The target compounds 5andashxwere then achieved by stirring substituted carbaldehyde(3andashd 1 mmol) with different o-arylenediamine (4andashf1 mmol) under reflux for 4-5 hrs After completion (TLChexane ethylacetate 7 3) of the reaction the solid obtainedwas filtered washed with small quantity of ethanol anddried Further it was recrystallized in methanol to get thepure compound (5andashx)
51 Microwave Assisted Procedure for the Preparation of Com-pounds 5andashx A mixture of 3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde (3andashd 1 mmol) and o-phenylenediamine (4andashf 1 mmol) taken in ethanol (5mL)was introduced into a CEM microwave reaction vesselequipped with magnetic stirrer The reaction mixture was
prestirred for 1min at room temperature and irradiated at180W (150∘C) for about 6ndash8min (TLC using hexane ethylacetate (7 3 drops) as eluent) The reaction mixture wasthen quenched into crushed ice and the crude product wasfiltered washed and dried Recrystallization from methanolgave pure crystals of the compounds 5andashx
(Spectral as well as elemental analyses data are providedas electronic supplementary file in Supplementary Materialavailable online at httpdxdoiorg10115520169890630)
6 Assessment of Phosphodiesterase InhibitionUsing Human Spermatozoa as Model
Earlier studies have shown that PDE inhibitors can enhancethe spermmotility [32 33] Based on this in the present studywe have used an indirect approach to assess the inhibitoryeffect of the coumarin derivatives on PDE by assessing spermmotility pattern
61 Preparation of Test Solution Stock solution of the testcompounds (1mgmL) was prepared by dissolving them indimethyl sulfoxide (DMSO Sigma Cat number D5879)The stock solution was then diluted with Earlrsquos BalancedSalt Solution (EBSS) supplemented with 01 bovine serumalbumin (BSA) in a ratio of 1 200 1 1000 and 1 2000 to getworking solutions of 5 1 and 05 120583gmL concentrations Theworking solutions were preincubated at 37∘C and 5 CO
2
prior to use
International Journal of Medicinal Chemistry 13
Table 3 Effect of various coumarin derivatives on kinematics of frozen-thawed spermatozoa at one hour after incubation in vitro
Parameters Compounds Concentrations of compounds in 120583gmL VC PTFC 0 05 5 (005 DMSO) (1mM)
62 Study Subjects Infertile men who attended AndrologyLaboratory Kasturba Medical College Manipal UniversityManipal during the period of June-July 2015 for routinesemen evaluation were included in the study The subjects
with a sexual abstinence of 3ndash5 days were asked to providethe semen samples by masturbation in a sterile containerFollowing liquefaction the semen parameters were assessedas described by Kotdawala et al [34]The study was approved
14 International Journal of Medicinal Chemistry
by the Institutional Ethics Committee of Kasturba HospitalKasturba Medical College Manipal University (IEC number1552015)
63 Cryopreservation and Thawing The liquefied semensamples were cryopreserved by rapid freezing method asdescribed previously [34] with minor modifications Brieflythe semen samples were mixed with equal volume of freezingmedium (Sperm Freeze FertiPro Cat number 0344) in acryovial (Thermoscientific Nunc Cat number 138627) andkept at 4∘C for 10min in liquid nitrogen (LN2) vapor phasefor 5min and then finally plunged into liquid nitrogenThe samples were thawed after 1 week by rapid thawingmethod by placing themat 37∘C for 5minThe cryoprotectantmedium was completely removed by mixing the spermsuspension with EBSS medium and centrifuging at 1000 rpmfor 8min The resultant pellet was resuspended with freshEBSS medium and used for further analysis
64 Sample Preparation To screen the compounds withenhancing effect on themotility of spermatozoa under in vitroconditions the frozen-thawed semen samples were washedby mixing with equal volume of preincubated Earlrsquos BalancedSalt Solution (EBSS Sigma Cat number E2888) followed bycentrifugation at 1800 rpm for 8min The pellet was thengently mixed with 1mL of EBSS media containing 01bovine serum albumin (BSA Sigma Cat number A3311)and equally divided into control vehicle control and testgroups (with average sperm density of 5 times 104 spermatozoain each group) The motile sperm were extracted by swimup method as described earlier [35] The sperm suspensionwas centrifuged at 1200 rpm for 8min and the resultantpellet was overlaidwith EBSSmedium containing various testcompoundsThe sperm suspensions were incubated for 1 h at37∘Cand 5CO
2afterwhich themotile spermwere collected
from the supernatant
65 Motility Assessment Spermatozoa in the supernatantfraction were assessed for their motility patterns at 1 4 and24 h after incubation under light microscope as describedearlier [27] Sperm suspension was (10 120583L droplet) placed ona clean microscope slide and a coverslip was placed over itSpermatozoa with progressive motility and nonprogressivemotility and immotile spermatozoa were scored separatelyfrom a total of 200 spermatozoa in random fields at 400xmagnification
66 Sperm Kinematics Using Computer Assisted Semen Anal-ysis (CASA) The motion characteristics in the spermatozoawere assessed at 1 4 and 24 h after incubation using CASAsystem (ISAS Spain) Briefly 5 120583L of sperm suspension wasplaced on a clean microscope slide prewarmed at 37∘C Thesperm suspensionwas coveredwith prewarmed cover slip (22times 22mm) and observed under microscope (negative phasecontrast 10x objectives Proiser Spain) For each sample thekinematics was assessed randomly at ten different fields Theparameters such as curvilinear velocity (VCL) straight line
velocity (VSL) average path velocity (VAP) amplitude of lat-eral head displacement (ALH) linearity (LIN) straightness(STR) wobble (WOB) and beat cross frequency (BCF) wereanalyzed
7 Conclusions
In conclusion we have reported efficient and environmen-tally benignmethodologies for the synthesis of 3-[4-(1H-benzo[d]imidazol-2-yl)-1-phenyl-1H-pyrazol-3-yl]-2H-chromen-2-one derivatives 5andashx by using thermal and by microwaveirradiation under neat conditions in presence of ethanol Thereactions carried out under microwave irradiation affordedbenzimidazoles in short period of time with excellent yieldsHence this methodology would make an interesting strategyfor the synthesis of various substituted coumarin pyrazolebenzimidazoles Also compounds 5g 5h and 5k exhibitedimpressive sperm motility enhancing property whereas 5khas spermatozoa longevity property
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to University Scientific Instrumen-tation Centre (USIC) Karnatak University Dharwad forprovidingmass and IR spectral dataThe authors are thankfulto Indian Institute of Science (IISc) Bengaluru for carryingout NMR analyses and to UGC New Delhi for the financialassistance under UPE program vide no [F No 14-22012(NSPE) dated 22-01-2013] One of the authors (MNK)acknowledges University Grants Commission (UGC) NewDelhi for fellowship under UGC-UPE program
References
[1] P P Kattimani R R Kamble and G Y Meti ldquoExpedientsynthesis of benzimidazoles using amidesrdquo RSC Advances vol5 no 37 pp 29447ndash29455 2015
[2] L Li C Yee and J A Beavo ldquoCD3- and CD28-dependentinduction of PDE7 required for T cell activationrdquo Science vol283 no 5403 pp 848ndash849 1999
[3] T P Dousa ldquoCyclic-3101584051015840-nucleotide phosphodiesteraseisozymes in cell biology and pathophysiology of the kidneyrdquoKidney International vol 55 no 1 pp 29ndash62 1999
[4] C Pfister N Bennett F Bruckert et al ldquoInteractions of a G-protein with its effector transducin and cGMP phosphodi-esterase in retinal rodsrdquo Cell Signaling vol 5 no 3 pp 235ndash2411993
[5] S L C Jin F J Richard W-P Kuo A J DrsquoErcole andM Conti ldquoImpaired growth and fertility of cAMP-specificphosphodiesterase PDE4D-deficient micerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 21 pp 11998ndash12003 1999
[6] S H Francis I V Turko and J D Corbin ldquoCyclic nucleotidephosphodiesterases relating structure and functionrdquoProgress in
International Journal of Medicinal Chemistry 15
Nucleic Acid Research and Molecular Biology vol 65 pp 1ndash522000
[7] J M Hetman N Robas R Baxendale et al ldquoCloning and char-acterization of two splice variants of human phosphodiesterase11Ardquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 23 pp 12891ndash12895 2000
[8] K Yuasa T Ohgaru M Asahina and K Omori ldquoIdentificationof rat cyclic nucleotide phosphodiesterase 11A (PDE11A) com-parison of rat and human PDE11A splicing variantsrdquo EuropeanJournal of Biochemistry vol 268 no 16 pp 4440ndash4448 2001
[9] S K Sahu A Mishra and R K Behera ldquoSynthesis of thiazolebenzothiazole oxadiazole thiadiazole triazole and thiazolidi-none incorporated coumarinsrdquo Indian Journal of HeterocyclicChemistry vol 6 no 2 pp 91ndash94 1996
[10] G Roma M Di Braccio G Grossi et al ldquoSynthesis and in vitroantiplatelet activity of new 4-(1-piperazinyl) coumarin deriva-tives Human platelet phosphodiesterase 3 inhibitory propertiesof the two most effective compounds described and molecularmodeling study on their interactions with phosphodiesterase3A catalytic siterdquo Journal of Medicinal Chemistry vol 50 no 12pp 2886ndash2895 2007
[11] T Shyamsunder Reddy K Shiva Kumar C L T Medaet al ldquoConformationally restricted novel pyrazole deriva-tives synthesis of 18-disubstituted 55-dimethyl-45-dihydro-1H-benzo[g]indazoles as a new class of PDE4 inhibitorsrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 9 pp3248ndash3255 2012
[12] A Chino N Masuda Y Amano et al ldquoNovel benzimidazolederivatives as phosphodiesterase 10A (PDE10A) inhibitors withimproved metabolic stabilityrdquo Bioorganic and Medicinal Chem-istry vol 22 no 13 pp 3515ndash3526 2014
[13] H Yang FNMurigi ZWang J Li H Jin andZ Tu ldquoSynthesisand in vitro characterization of cinnoline and benzimidazoleanalogues as phosphodiesterase 10A inhibitorsrdquo Bioorganic andMedicinal Chemistry Letters vol 25 pp 919ndash924 2015
[14] J Nawrot-Modranka E Nawrot and J Graczyk ldquoIn vivo antitu-mor in vitro antibacterial activity and alkylating properties ofphosphorohydrazine derivatives of coumarin and chromonerdquoEuropean Journal of Medicinal Chemistry vol 41 no 11 pp1301ndash1309 2006
[15] A A Bekhit and T Abdel-Aziem ldquoDesign synthesis andbiological evaluation of some pyrazole derivatives as anti-inflammatory-antimicrobial agentsrdquo Bioorganic and MedicinalChemistry vol 12 no 8 pp 1935ndash1945 2004
[16] T Devji C Reddy CWoo S Awale S Kadota and D Carrico-Moniz ldquoPancreatic anticancer activity of a novel geranylgerany-lated coumarin derivativerdquo Bioorganic andMedicinal ChemistryLetters vol 21 no 19 pp 5770ndash5773 2011
[17] I Kostova I Manolov I Nicolova S Konstantinov andM Karaivanova ldquoNew lanthanide complexes of 4-methyl-7-hydroxycoumarin and their pharmacological activityrdquo Euro-pean Journal of Medicinal Chemistry vol 36 no 4 pp 339ndash3472001
[18] M Ghate R A Kusanur and M V Kulkarni ldquoSynthesis andin vivo analgesic and anti-inflammatory activity of some bi-heterocyclic coumarin derivativesrdquo European Journal of Medic-inal Chemistry vol 40 no 9 pp 882ndash887 2005
[19] PM RonadMN Noolvi S Sapkal S Dharbhamulla andV SMaddi ldquoSynthesis and antimicrobial activity of 7-(2-substitutedphenylthiazolidinyl)-benzopyran-2-one derivativesrdquo EuropeanJournal of Medicinal Chemistry vol 45 no 1 pp 85ndash89 2010
[20] GMelagraki A Afantitis O Igglessi-Markopoulou et al ldquoSyn-thesis and evaluation of the antioxidant and anti-inflammatoryactivity of novel coumarin-3-aminoamides and their alpha-lipoic acid adductsrdquo European Journal of Medicinal Chemistryvol 44 no 7 pp 3020ndash3026 2009
[21] J C Trivedi J B Bariwal K D Upadhyay et al ldquoImprovedand rapid synthesis of new coumarinyl chalcone derivatives andtheir antiviral activityrdquo Tetrahedron Letters vol 48 no 48 pp8472ndash8474 2007
[22] S Vilar E Quezada L Santana et al ldquoDesign synthe-sis and vasorelaxant and platelet antiaggregatory activitiesof coumarinndashresveratrol hybridsrdquo Bioorganic and MedicinalChemistry Letters vol 16 no 2 pp 257ndash261 2006
[23] W Hamaguchi N Masuda M Isomura et al ldquoDesign and syn-thesis of novel benzimidazole derivatives as phosphodiesterase10A inhibitors with reduced CYP1A2 inhibitionrdquo Bioorganicand Medicinal Chemistry vol 21 no 24 pp 7612ndash7623 2013
[24] A Dore B Asproni A Scampuddu et al ldquoSynthesis and SARstudy of novel tricyclic pyrazoles as potent phosphodiesterase10A inhibitorsrdquo European Journal of Medicinal Chemistry vol84 pp 181ndash193 2014
[25] S V Laxmi B S Kuarm and B Rajitha ldquoSynthesis and anti-microbial activity of coumarin pyrazole pyrimidine 246(1H3H5H)triones and thioxopyrimidine46(1H5H)dionesrdquo Me-dicinal Chemistry Research vol 22 no 2 pp 768ndash774 2013
[26] G Kalthur S Raj AThiyagarajan S Kumar P Kumar and S KAdiga ldquoVitamin E supplementation in semen-freezingmediumimproves the motility and protects sperm from freeze-thaw-induced DNA damagerdquo Fertility and Sterility vol 95 no 3 pp1149ndash1151 2011
[27] J Tesarik C Mendoza and A Carreras ldquoEffects of phosphodi-esterase inhibitors caffeine and pentoxifylline on spontaneousand stimulus-induced acrosome reactions in human spermrdquoFertility and Sterility vol 58 no 6 pp 1185ndash1190 1992
[28] G Kalthur S R Salian F Keyvanifard et al ldquoSupplementationof biotin to sperm preparation medium increases the motilityand longevity in cryopreserved human spermatozoardquo Journalof Assisted Reproduction and Genetics vol 29 no 7 pp 631ndash6352012
[29] A E Calogero S Fishel J Hall et al ldquoCorrelation betweenintracellular cAMP content kinematic parameters and hyper-activation of human spermatozoa after incubation with pentox-ifyllinerdquo Human Reproduction vol 13 no 4 pp 911ndash915 1998
[30] R Ain K Uma Devi S Shivaji and P B Seshagiri ldquoPen-toxifylline-stimulated capacitation and acrosome reaction inhamster spermatozoa involvement of intracellular signallingmoleculesrdquo Molecular Human Reproduction vol 5 no 7 pp618ndash626 1999
[31] M Bajpai andG F Doncel ldquoInvolvement of tyrosine kinase andcAMP-dependent kinase cross-talk in the regulation of humanspermmotilityrdquo Reproduction vol 126 no 2 pp 183ndash195 2003
[32] R Yunes P Fernandez G F Doncel and A A Acosta ldquoCyclicnucleotide phosphodiesterase inhibition increases tyrosinephosphorylation and hypermotility in normal and pathologicalhuman spermatozoardquo Biocell vol 29 no 3 pp 287ndash293 2005
[33] F Dimitriadis D Giannakis N Pardalidis et al ldquoEffectsof phosphodiesterase 5 inhibitors on sperm parameters andfertilizing capacityrdquo Asian Journal of Andrology vol 10 no 1pp 115ndash133 2008
[34] A P Kotdawala S Kumar S R Salian et al ldquoAddition of zincto human ejaculate prior to cryopreservation prevents freeze-thaw-induced DNA damage and preserves sperm functionrdquo
16 International Journal of Medicinal Chemistry
Journal of Assisted Reproduction and Genetics vol 29 no 12 pp1447ndash1453 2012
[35] S Mutalik S R Salian K Avadhani et al ldquoLiposome encapsu-lated soy lecithin and cholesterol can efficiently replace chickenegg yolk in human semen cryopreservation mediumrdquo SystemsBiology in Reproductive Medicine vol 60 no 3 pp 183ndash1882014
31 Effect on Motility of Frozen-Thawed Human SpermatozoaTo assess the effect of title compounds on themotility patternhuman spermatozoa subjected to freeze-thaw process wasused It is well documented that freeze-thaw process canhave detrimental effect on the motility and survival of thespermatozoa [26] Therefore they can be an excellent modelto study the effect of any test compounds on themotility [27]
In the present study we assessed the motility patternin spermatozoa processed with media containing variouscompounds manually at 1 4 and 24 h after incubationin vitro Three arbitrary doses were selected (05 10 and50 120583gmL) for each compound to assess their effect on spermmotility Pentoxifylline (PTF) which is known to enhancethe human sperm motility by elevating intracellular cyclicadenosine monophosphate (cAMP) level [27] was taken asa positive control Based on the previous report PTF at 1mMconcentration was used [28] Since the test compounds werenot soluble in water dimethyl sulfoxide (DMSO 005) wasused as vehicle control
The sperm motility enhancement property of the com-pounds was categorized as below
(a) Marginal which resulted in lt5 increase in themotility (b) Good which resulted in 5ndash10 increase inmotility (c) Excellent which resulted in gt10 increase inmotility (d) Poor where there was no enhancement inmotility
At 1 h after the in vitro incubation the majority of thecompounds had marginal effect on the sperm motility com-pared to control At this interval PTF had higher percentageof total and progressivelymotile spermatozoa compared to allthe test compounds A large number of studies have shownthat PTF can trigger the motility in fresh or frozen-thawedspermatozoa as early as 1 h [29] even though the majorityof the studies indicate that this effect is mainly due to theelevated cAMP level in the spermatozoa [30] it can also bemediated through tyrosine phosphorylation [31]
Compounds 5a 5b 5c 5d 5e 5g 5h and 5k inducedmarginal enhancement in total motility while compounds5a 5b 5c and 5j resulted in similar effect on progressivemotility (Tables 2(a) and 2(b)) A 5ndash10 increase in totalmotility was induced by compound 5j However with respectto progressive motility compounds 5d 5e 5f and 5k cameunder compounds with good effect on motility More than10 increase in progressive motility was observed only forcompound 5g (at 05 120583gmL concentration) and compound5h (at 5 120583gmL concentration)
A time-dependent decrease in total and progressivemotility was observed in all the groups However the declinewasmore evident at 24 h intervalThis phenomenon has beendocumented by earlier studies [28] These changes may berelated to the oxidative stress induced by in vitro cultureconditions Spermatozoa are highly susceptible to reactiveoxygen species (ROS) due to high polyunsaturated fatty acidcontent in their plasma membrane and negligible amount ofcytoplasmic antioxidants
At 4 h interval compounds 5e 5f and 5g enhancedthe progressive motility of spermatozoa by 5ndash10 while
compound 5h with two chloro substituents on coumarinand benzimidazole resulted in almost 15 increase in theprogressive motility However the effect was lower than thatof pentoxifylline The rest of the compounds (5a 5b 5c5f and 5j) induced a marginal increase in motility exceptin compound 5e in which the motility was lower than thecontrol group
At 24 h interval the majority of the compounds (5a5b 5d 5f 5h and 5j) had marginally higher percentageof motility or poor motility compounds (5c 5e and 5g)compared to control Interestingly compound 5k with chlorosubstituent on coumarin and bromo substitution on ben-zimidazole had higher percentage of motile spermatozoacompared to control (14 higher) and even PTF (9 higher)This indicates that compound5k canhelp in prolonging the invitro survival of spermatozoa which has significant beneficialrole in assisted reproductive technologies such as in vitrofertilization Previous studies have shown that even thoughPTF has triggering effect on themotility it induces prematureacrosome reaction and drastic decrease in motility at laterintervals In this context the compounds which increasethe motility and longevity of spermatozoa under in vitroconditions are of clinical relevance
Based on the manual motility assessment we observedthat compounds 5d 5f 5g 5h and 5k have considerablebeneficial role in spermmotilityTherefore these compoundswere further assessed for their effect on the kinematicsof spermatozoa at 1 h However there was no significantdifference in the kinematics of spermatozoa compared tocontrol and PTF (Table 3) In conclusion the newly synthe-sized coumarin derivatives have sperm motility enhancingproperty However in this preliminary screening we havetaken three arbitrary doses to assess their effect on motilityCompounds 5g 5h and 5k show considerable beneficialeffect which can be of clinical significance where spermmotility enhancement is achieved under in vitro condi-tions Compounds 5g and 5h triggered the motility at earlyintervals which was similar to the PTF while compound5k improved the longevity of the spermatozoa Since thecoumarin is known to have PDE inhibitory function themotility enhancement could be due to the elevated cAMPlevel in sperm However further detailed studies are essentialto understand the mechanism of action of these compoundsand their clinical utility
4 Experimental
All the chemicals and reagents were purchased from theMerck and Aldrich chemical suppliers Melting points (mp)were determined in open capillaries and are uncorrectedThe IR spectra were recorded on a Nicolet Impact 410 FTIR spectrometer using KBr pellets (range 4000ndash500 cmminus1)The 1HNMR spectra were recorded at 400MHz on BrukerAvance FT NMR spectrometer in DMSO-d
6solvent with
TMS as internal standard 13CNMR spectra were recordedat 100MHz on Bruker Avance FT NMR spectrometer inDMSO-d
6solvent with TMS as internal standard The mass
spectra were recorded on Shimadzu GC-MS operating at70 eV Thin-layer chromatography (TLC) was performed on
International Journal of Medicinal Chemistry 11
Table 2 Effect of supplementation of coumarin derivatives (5andashj) to sperm wash medium on the motility pattern in frozen-thawed humanspermatozoa at various time intervals
(a)
Compounds Time (h)Total motility (mean plusmn SE of percentage)
020mm Aluchrosep silica gel 60 F254 plates (SD FineMumbai) Microwave irradiation experiments were carriedout using CEMDiscover SPMicrowave Synthesizer equippedwith IR sensor to monitor reaction temperatures
5 General Procedures for the Preparation ofTitle Compounds 5andashx
3-(2-Oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-car-baldehydes (3andashd 1 mmol) were prepared from 3-acetyl-coumarin (1andashd 1 mmol) phenylhydrazine (1mmol) andthe intermediate (Schiff base) (2andashd 1 mmol) followedby reaction with POCl
3in DMF by Vilsmeier Haack for-
mylation strategies [25] The target compounds 5andashxwere then achieved by stirring substituted carbaldehyde(3andashd 1 mmol) with different o-arylenediamine (4andashf1 mmol) under reflux for 4-5 hrs After completion (TLChexane ethylacetate 7 3) of the reaction the solid obtainedwas filtered washed with small quantity of ethanol anddried Further it was recrystallized in methanol to get thepure compound (5andashx)
51 Microwave Assisted Procedure for the Preparation of Com-pounds 5andashx A mixture of 3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde (3andashd 1 mmol) and o-phenylenediamine (4andashf 1 mmol) taken in ethanol (5mL)was introduced into a CEM microwave reaction vesselequipped with magnetic stirrer The reaction mixture was
prestirred for 1min at room temperature and irradiated at180W (150∘C) for about 6ndash8min (TLC using hexane ethylacetate (7 3 drops) as eluent) The reaction mixture wasthen quenched into crushed ice and the crude product wasfiltered washed and dried Recrystallization from methanolgave pure crystals of the compounds 5andashx
(Spectral as well as elemental analyses data are providedas electronic supplementary file in Supplementary Materialavailable online at httpdxdoiorg10115520169890630)
6 Assessment of Phosphodiesterase InhibitionUsing Human Spermatozoa as Model
Earlier studies have shown that PDE inhibitors can enhancethe spermmotility [32 33] Based on this in the present studywe have used an indirect approach to assess the inhibitoryeffect of the coumarin derivatives on PDE by assessing spermmotility pattern
61 Preparation of Test Solution Stock solution of the testcompounds (1mgmL) was prepared by dissolving them indimethyl sulfoxide (DMSO Sigma Cat number D5879)The stock solution was then diluted with Earlrsquos BalancedSalt Solution (EBSS) supplemented with 01 bovine serumalbumin (BSA) in a ratio of 1 200 1 1000 and 1 2000 to getworking solutions of 5 1 and 05 120583gmL concentrations Theworking solutions were preincubated at 37∘C and 5 CO
2
prior to use
International Journal of Medicinal Chemistry 13
Table 3 Effect of various coumarin derivatives on kinematics of frozen-thawed spermatozoa at one hour after incubation in vitro
Parameters Compounds Concentrations of compounds in 120583gmL VC PTFC 0 05 5 (005 DMSO) (1mM)
62 Study Subjects Infertile men who attended AndrologyLaboratory Kasturba Medical College Manipal UniversityManipal during the period of June-July 2015 for routinesemen evaluation were included in the study The subjects
with a sexual abstinence of 3ndash5 days were asked to providethe semen samples by masturbation in a sterile containerFollowing liquefaction the semen parameters were assessedas described by Kotdawala et al [34]The study was approved
14 International Journal of Medicinal Chemistry
by the Institutional Ethics Committee of Kasturba HospitalKasturba Medical College Manipal University (IEC number1552015)
63 Cryopreservation and Thawing The liquefied semensamples were cryopreserved by rapid freezing method asdescribed previously [34] with minor modifications Brieflythe semen samples were mixed with equal volume of freezingmedium (Sperm Freeze FertiPro Cat number 0344) in acryovial (Thermoscientific Nunc Cat number 138627) andkept at 4∘C for 10min in liquid nitrogen (LN2) vapor phasefor 5min and then finally plunged into liquid nitrogenThe samples were thawed after 1 week by rapid thawingmethod by placing themat 37∘C for 5minThe cryoprotectantmedium was completely removed by mixing the spermsuspension with EBSS medium and centrifuging at 1000 rpmfor 8min The resultant pellet was resuspended with freshEBSS medium and used for further analysis
64 Sample Preparation To screen the compounds withenhancing effect on themotility of spermatozoa under in vitroconditions the frozen-thawed semen samples were washedby mixing with equal volume of preincubated Earlrsquos BalancedSalt Solution (EBSS Sigma Cat number E2888) followed bycentrifugation at 1800 rpm for 8min The pellet was thengently mixed with 1mL of EBSS media containing 01bovine serum albumin (BSA Sigma Cat number A3311)and equally divided into control vehicle control and testgroups (with average sperm density of 5 times 104 spermatozoain each group) The motile sperm were extracted by swimup method as described earlier [35] The sperm suspensionwas centrifuged at 1200 rpm for 8min and the resultantpellet was overlaidwith EBSSmedium containing various testcompoundsThe sperm suspensions were incubated for 1 h at37∘Cand 5CO
2afterwhich themotile spermwere collected
from the supernatant
65 Motility Assessment Spermatozoa in the supernatantfraction were assessed for their motility patterns at 1 4 and24 h after incubation under light microscope as describedearlier [27] Sperm suspension was (10 120583L droplet) placed ona clean microscope slide and a coverslip was placed over itSpermatozoa with progressive motility and nonprogressivemotility and immotile spermatozoa were scored separatelyfrom a total of 200 spermatozoa in random fields at 400xmagnification
66 Sperm Kinematics Using Computer Assisted Semen Anal-ysis (CASA) The motion characteristics in the spermatozoawere assessed at 1 4 and 24 h after incubation using CASAsystem (ISAS Spain) Briefly 5 120583L of sperm suspension wasplaced on a clean microscope slide prewarmed at 37∘C Thesperm suspensionwas coveredwith prewarmed cover slip (22times 22mm) and observed under microscope (negative phasecontrast 10x objectives Proiser Spain) For each sample thekinematics was assessed randomly at ten different fields Theparameters such as curvilinear velocity (VCL) straight line
velocity (VSL) average path velocity (VAP) amplitude of lat-eral head displacement (ALH) linearity (LIN) straightness(STR) wobble (WOB) and beat cross frequency (BCF) wereanalyzed
7 Conclusions
In conclusion we have reported efficient and environmen-tally benignmethodologies for the synthesis of 3-[4-(1H-benzo[d]imidazol-2-yl)-1-phenyl-1H-pyrazol-3-yl]-2H-chromen-2-one derivatives 5andashx by using thermal and by microwaveirradiation under neat conditions in presence of ethanol Thereactions carried out under microwave irradiation affordedbenzimidazoles in short period of time with excellent yieldsHence this methodology would make an interesting strategyfor the synthesis of various substituted coumarin pyrazolebenzimidazoles Also compounds 5g 5h and 5k exhibitedimpressive sperm motility enhancing property whereas 5khas spermatozoa longevity property
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to University Scientific Instrumen-tation Centre (USIC) Karnatak University Dharwad forprovidingmass and IR spectral dataThe authors are thankfulto Indian Institute of Science (IISc) Bengaluru for carryingout NMR analyses and to UGC New Delhi for the financialassistance under UPE program vide no [F No 14-22012(NSPE) dated 22-01-2013] One of the authors (MNK)acknowledges University Grants Commission (UGC) NewDelhi for fellowship under UGC-UPE program
References
[1] P P Kattimani R R Kamble and G Y Meti ldquoExpedientsynthesis of benzimidazoles using amidesrdquo RSC Advances vol5 no 37 pp 29447ndash29455 2015
[2] L Li C Yee and J A Beavo ldquoCD3- and CD28-dependentinduction of PDE7 required for T cell activationrdquo Science vol283 no 5403 pp 848ndash849 1999
[3] T P Dousa ldquoCyclic-3101584051015840-nucleotide phosphodiesteraseisozymes in cell biology and pathophysiology of the kidneyrdquoKidney International vol 55 no 1 pp 29ndash62 1999
[4] C Pfister N Bennett F Bruckert et al ldquoInteractions of a G-protein with its effector transducin and cGMP phosphodi-esterase in retinal rodsrdquo Cell Signaling vol 5 no 3 pp 235ndash2411993
[5] S L C Jin F J Richard W-P Kuo A J DrsquoErcole andM Conti ldquoImpaired growth and fertility of cAMP-specificphosphodiesterase PDE4D-deficient micerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 21 pp 11998ndash12003 1999
[6] S H Francis I V Turko and J D Corbin ldquoCyclic nucleotidephosphodiesterases relating structure and functionrdquoProgress in
International Journal of Medicinal Chemistry 15
Nucleic Acid Research and Molecular Biology vol 65 pp 1ndash522000
[7] J M Hetman N Robas R Baxendale et al ldquoCloning and char-acterization of two splice variants of human phosphodiesterase11Ardquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 23 pp 12891ndash12895 2000
[8] K Yuasa T Ohgaru M Asahina and K Omori ldquoIdentificationof rat cyclic nucleotide phosphodiesterase 11A (PDE11A) com-parison of rat and human PDE11A splicing variantsrdquo EuropeanJournal of Biochemistry vol 268 no 16 pp 4440ndash4448 2001
[9] S K Sahu A Mishra and R K Behera ldquoSynthesis of thiazolebenzothiazole oxadiazole thiadiazole triazole and thiazolidi-none incorporated coumarinsrdquo Indian Journal of HeterocyclicChemistry vol 6 no 2 pp 91ndash94 1996
[10] G Roma M Di Braccio G Grossi et al ldquoSynthesis and in vitroantiplatelet activity of new 4-(1-piperazinyl) coumarin deriva-tives Human platelet phosphodiesterase 3 inhibitory propertiesof the two most effective compounds described and molecularmodeling study on their interactions with phosphodiesterase3A catalytic siterdquo Journal of Medicinal Chemistry vol 50 no 12pp 2886ndash2895 2007
[11] T Shyamsunder Reddy K Shiva Kumar C L T Medaet al ldquoConformationally restricted novel pyrazole deriva-tives synthesis of 18-disubstituted 55-dimethyl-45-dihydro-1H-benzo[g]indazoles as a new class of PDE4 inhibitorsrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 9 pp3248ndash3255 2012
[12] A Chino N Masuda Y Amano et al ldquoNovel benzimidazolederivatives as phosphodiesterase 10A (PDE10A) inhibitors withimproved metabolic stabilityrdquo Bioorganic and Medicinal Chem-istry vol 22 no 13 pp 3515ndash3526 2014
[13] H Yang FNMurigi ZWang J Li H Jin andZ Tu ldquoSynthesisand in vitro characterization of cinnoline and benzimidazoleanalogues as phosphodiesterase 10A inhibitorsrdquo Bioorganic andMedicinal Chemistry Letters vol 25 pp 919ndash924 2015
[14] J Nawrot-Modranka E Nawrot and J Graczyk ldquoIn vivo antitu-mor in vitro antibacterial activity and alkylating properties ofphosphorohydrazine derivatives of coumarin and chromonerdquoEuropean Journal of Medicinal Chemistry vol 41 no 11 pp1301ndash1309 2006
[15] A A Bekhit and T Abdel-Aziem ldquoDesign synthesis andbiological evaluation of some pyrazole derivatives as anti-inflammatory-antimicrobial agentsrdquo Bioorganic and MedicinalChemistry vol 12 no 8 pp 1935ndash1945 2004
[16] T Devji C Reddy CWoo S Awale S Kadota and D Carrico-Moniz ldquoPancreatic anticancer activity of a novel geranylgerany-lated coumarin derivativerdquo Bioorganic andMedicinal ChemistryLetters vol 21 no 19 pp 5770ndash5773 2011
[17] I Kostova I Manolov I Nicolova S Konstantinov andM Karaivanova ldquoNew lanthanide complexes of 4-methyl-7-hydroxycoumarin and their pharmacological activityrdquo Euro-pean Journal of Medicinal Chemistry vol 36 no 4 pp 339ndash3472001
[18] M Ghate R A Kusanur and M V Kulkarni ldquoSynthesis andin vivo analgesic and anti-inflammatory activity of some bi-heterocyclic coumarin derivativesrdquo European Journal of Medic-inal Chemistry vol 40 no 9 pp 882ndash887 2005
[19] PM RonadMN Noolvi S Sapkal S Dharbhamulla andV SMaddi ldquoSynthesis and antimicrobial activity of 7-(2-substitutedphenylthiazolidinyl)-benzopyran-2-one derivativesrdquo EuropeanJournal of Medicinal Chemistry vol 45 no 1 pp 85ndash89 2010
[20] GMelagraki A Afantitis O Igglessi-Markopoulou et al ldquoSyn-thesis and evaluation of the antioxidant and anti-inflammatoryactivity of novel coumarin-3-aminoamides and their alpha-lipoic acid adductsrdquo European Journal of Medicinal Chemistryvol 44 no 7 pp 3020ndash3026 2009
[21] J C Trivedi J B Bariwal K D Upadhyay et al ldquoImprovedand rapid synthesis of new coumarinyl chalcone derivatives andtheir antiviral activityrdquo Tetrahedron Letters vol 48 no 48 pp8472ndash8474 2007
[22] S Vilar E Quezada L Santana et al ldquoDesign synthe-sis and vasorelaxant and platelet antiaggregatory activitiesof coumarinndashresveratrol hybridsrdquo Bioorganic and MedicinalChemistry Letters vol 16 no 2 pp 257ndash261 2006
[23] W Hamaguchi N Masuda M Isomura et al ldquoDesign and syn-thesis of novel benzimidazole derivatives as phosphodiesterase10A inhibitors with reduced CYP1A2 inhibitionrdquo Bioorganicand Medicinal Chemistry vol 21 no 24 pp 7612ndash7623 2013
[24] A Dore B Asproni A Scampuddu et al ldquoSynthesis and SARstudy of novel tricyclic pyrazoles as potent phosphodiesterase10A inhibitorsrdquo European Journal of Medicinal Chemistry vol84 pp 181ndash193 2014
[25] S V Laxmi B S Kuarm and B Rajitha ldquoSynthesis and anti-microbial activity of coumarin pyrazole pyrimidine 246(1H3H5H)triones and thioxopyrimidine46(1H5H)dionesrdquo Me-dicinal Chemistry Research vol 22 no 2 pp 768ndash774 2013
[26] G Kalthur S Raj AThiyagarajan S Kumar P Kumar and S KAdiga ldquoVitamin E supplementation in semen-freezingmediumimproves the motility and protects sperm from freeze-thaw-induced DNA damagerdquo Fertility and Sterility vol 95 no 3 pp1149ndash1151 2011
[27] J Tesarik C Mendoza and A Carreras ldquoEffects of phosphodi-esterase inhibitors caffeine and pentoxifylline on spontaneousand stimulus-induced acrosome reactions in human spermrdquoFertility and Sterility vol 58 no 6 pp 1185ndash1190 1992
[28] G Kalthur S R Salian F Keyvanifard et al ldquoSupplementationof biotin to sperm preparation medium increases the motilityand longevity in cryopreserved human spermatozoardquo Journalof Assisted Reproduction and Genetics vol 29 no 7 pp 631ndash6352012
[29] A E Calogero S Fishel J Hall et al ldquoCorrelation betweenintracellular cAMP content kinematic parameters and hyper-activation of human spermatozoa after incubation with pentox-ifyllinerdquo Human Reproduction vol 13 no 4 pp 911ndash915 1998
[30] R Ain K Uma Devi S Shivaji and P B Seshagiri ldquoPen-toxifylline-stimulated capacitation and acrosome reaction inhamster spermatozoa involvement of intracellular signallingmoleculesrdquo Molecular Human Reproduction vol 5 no 7 pp618ndash626 1999
[31] M Bajpai andG F Doncel ldquoInvolvement of tyrosine kinase andcAMP-dependent kinase cross-talk in the regulation of humanspermmotilityrdquo Reproduction vol 126 no 2 pp 183ndash195 2003
[32] R Yunes P Fernandez G F Doncel and A A Acosta ldquoCyclicnucleotide phosphodiesterase inhibition increases tyrosinephosphorylation and hypermotility in normal and pathologicalhuman spermatozoardquo Biocell vol 29 no 3 pp 287ndash293 2005
[33] F Dimitriadis D Giannakis N Pardalidis et al ldquoEffectsof phosphodiesterase 5 inhibitors on sperm parameters andfertilizing capacityrdquo Asian Journal of Andrology vol 10 no 1pp 115ndash133 2008
[34] A P Kotdawala S Kumar S R Salian et al ldquoAddition of zincto human ejaculate prior to cryopreservation prevents freeze-thaw-induced DNA damage and preserves sperm functionrdquo
16 International Journal of Medicinal Chemistry
Journal of Assisted Reproduction and Genetics vol 29 no 12 pp1447ndash1453 2012
[35] S Mutalik S R Salian K Avadhani et al ldquoLiposome encapsu-lated soy lecithin and cholesterol can efficiently replace chickenegg yolk in human semen cryopreservation mediumrdquo SystemsBiology in Reproductive Medicine vol 60 no 3 pp 183ndash1882014
31 Effect on Motility of Frozen-Thawed Human SpermatozoaTo assess the effect of title compounds on themotility patternhuman spermatozoa subjected to freeze-thaw process wasused It is well documented that freeze-thaw process canhave detrimental effect on the motility and survival of thespermatozoa [26] Therefore they can be an excellent modelto study the effect of any test compounds on themotility [27]
In the present study we assessed the motility patternin spermatozoa processed with media containing variouscompounds manually at 1 4 and 24 h after incubationin vitro Three arbitrary doses were selected (05 10 and50 120583gmL) for each compound to assess their effect on spermmotility Pentoxifylline (PTF) which is known to enhancethe human sperm motility by elevating intracellular cyclicadenosine monophosphate (cAMP) level [27] was taken asa positive control Based on the previous report PTF at 1mMconcentration was used [28] Since the test compounds werenot soluble in water dimethyl sulfoxide (DMSO 005) wasused as vehicle control
The sperm motility enhancement property of the com-pounds was categorized as below
(a) Marginal which resulted in lt5 increase in themotility (b) Good which resulted in 5ndash10 increase inmotility (c) Excellent which resulted in gt10 increase inmotility (d) Poor where there was no enhancement inmotility
At 1 h after the in vitro incubation the majority of thecompounds had marginal effect on the sperm motility com-pared to control At this interval PTF had higher percentageof total and progressivelymotile spermatozoa compared to allthe test compounds A large number of studies have shownthat PTF can trigger the motility in fresh or frozen-thawedspermatozoa as early as 1 h [29] even though the majorityof the studies indicate that this effect is mainly due to theelevated cAMP level in the spermatozoa [30] it can also bemediated through tyrosine phosphorylation [31]
Compounds 5a 5b 5c 5d 5e 5g 5h and 5k inducedmarginal enhancement in total motility while compounds5a 5b 5c and 5j resulted in similar effect on progressivemotility (Tables 2(a) and 2(b)) A 5ndash10 increase in totalmotility was induced by compound 5j However with respectto progressive motility compounds 5d 5e 5f and 5k cameunder compounds with good effect on motility More than10 increase in progressive motility was observed only forcompound 5g (at 05 120583gmL concentration) and compound5h (at 5 120583gmL concentration)
A time-dependent decrease in total and progressivemotility was observed in all the groups However the declinewasmore evident at 24 h intervalThis phenomenon has beendocumented by earlier studies [28] These changes may berelated to the oxidative stress induced by in vitro cultureconditions Spermatozoa are highly susceptible to reactiveoxygen species (ROS) due to high polyunsaturated fatty acidcontent in their plasma membrane and negligible amount ofcytoplasmic antioxidants
At 4 h interval compounds 5e 5f and 5g enhancedthe progressive motility of spermatozoa by 5ndash10 while
compound 5h with two chloro substituents on coumarinand benzimidazole resulted in almost 15 increase in theprogressive motility However the effect was lower than thatof pentoxifylline The rest of the compounds (5a 5b 5c5f and 5j) induced a marginal increase in motility exceptin compound 5e in which the motility was lower than thecontrol group
At 24 h interval the majority of the compounds (5a5b 5d 5f 5h and 5j) had marginally higher percentageof motility or poor motility compounds (5c 5e and 5g)compared to control Interestingly compound 5k with chlorosubstituent on coumarin and bromo substitution on ben-zimidazole had higher percentage of motile spermatozoacompared to control (14 higher) and even PTF (9 higher)This indicates that compound5k canhelp in prolonging the invitro survival of spermatozoa which has significant beneficialrole in assisted reproductive technologies such as in vitrofertilization Previous studies have shown that even thoughPTF has triggering effect on themotility it induces prematureacrosome reaction and drastic decrease in motility at laterintervals In this context the compounds which increasethe motility and longevity of spermatozoa under in vitroconditions are of clinical relevance
Based on the manual motility assessment we observedthat compounds 5d 5f 5g 5h and 5k have considerablebeneficial role in spermmotilityTherefore these compoundswere further assessed for their effect on the kinematicsof spermatozoa at 1 h However there was no significantdifference in the kinematics of spermatozoa compared tocontrol and PTF (Table 3) In conclusion the newly synthe-sized coumarin derivatives have sperm motility enhancingproperty However in this preliminary screening we havetaken three arbitrary doses to assess their effect on motilityCompounds 5g 5h and 5k show considerable beneficialeffect which can be of clinical significance where spermmotility enhancement is achieved under in vitro condi-tions Compounds 5g and 5h triggered the motility at earlyintervals which was similar to the PTF while compound5k improved the longevity of the spermatozoa Since thecoumarin is known to have PDE inhibitory function themotility enhancement could be due to the elevated cAMPlevel in sperm However further detailed studies are essentialto understand the mechanism of action of these compoundsand their clinical utility
4 Experimental
All the chemicals and reagents were purchased from theMerck and Aldrich chemical suppliers Melting points (mp)were determined in open capillaries and are uncorrectedThe IR spectra were recorded on a Nicolet Impact 410 FTIR spectrometer using KBr pellets (range 4000ndash500 cmminus1)The 1HNMR spectra were recorded at 400MHz on BrukerAvance FT NMR spectrometer in DMSO-d
6solvent with
TMS as internal standard 13CNMR spectra were recordedat 100MHz on Bruker Avance FT NMR spectrometer inDMSO-d
6solvent with TMS as internal standard The mass
spectra were recorded on Shimadzu GC-MS operating at70 eV Thin-layer chromatography (TLC) was performed on
International Journal of Medicinal Chemistry 11
Table 2 Effect of supplementation of coumarin derivatives (5andashj) to sperm wash medium on the motility pattern in frozen-thawed humanspermatozoa at various time intervals
(a)
Compounds Time (h)Total motility (mean plusmn SE of percentage)
020mm Aluchrosep silica gel 60 F254 plates (SD FineMumbai) Microwave irradiation experiments were carriedout using CEMDiscover SPMicrowave Synthesizer equippedwith IR sensor to monitor reaction temperatures
5 General Procedures for the Preparation ofTitle Compounds 5andashx
3-(2-Oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-car-baldehydes (3andashd 1 mmol) were prepared from 3-acetyl-coumarin (1andashd 1 mmol) phenylhydrazine (1mmol) andthe intermediate (Schiff base) (2andashd 1 mmol) followedby reaction with POCl
3in DMF by Vilsmeier Haack for-
mylation strategies [25] The target compounds 5andashxwere then achieved by stirring substituted carbaldehyde(3andashd 1 mmol) with different o-arylenediamine (4andashf1 mmol) under reflux for 4-5 hrs After completion (TLChexane ethylacetate 7 3) of the reaction the solid obtainedwas filtered washed with small quantity of ethanol anddried Further it was recrystallized in methanol to get thepure compound (5andashx)
51 Microwave Assisted Procedure for the Preparation of Com-pounds 5andashx A mixture of 3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde (3andashd 1 mmol) and o-phenylenediamine (4andashf 1 mmol) taken in ethanol (5mL)was introduced into a CEM microwave reaction vesselequipped with magnetic stirrer The reaction mixture was
prestirred for 1min at room temperature and irradiated at180W (150∘C) for about 6ndash8min (TLC using hexane ethylacetate (7 3 drops) as eluent) The reaction mixture wasthen quenched into crushed ice and the crude product wasfiltered washed and dried Recrystallization from methanolgave pure crystals of the compounds 5andashx
(Spectral as well as elemental analyses data are providedas electronic supplementary file in Supplementary Materialavailable online at httpdxdoiorg10115520169890630)
6 Assessment of Phosphodiesterase InhibitionUsing Human Spermatozoa as Model
Earlier studies have shown that PDE inhibitors can enhancethe spermmotility [32 33] Based on this in the present studywe have used an indirect approach to assess the inhibitoryeffect of the coumarin derivatives on PDE by assessing spermmotility pattern
61 Preparation of Test Solution Stock solution of the testcompounds (1mgmL) was prepared by dissolving them indimethyl sulfoxide (DMSO Sigma Cat number D5879)The stock solution was then diluted with Earlrsquos BalancedSalt Solution (EBSS) supplemented with 01 bovine serumalbumin (BSA) in a ratio of 1 200 1 1000 and 1 2000 to getworking solutions of 5 1 and 05 120583gmL concentrations Theworking solutions were preincubated at 37∘C and 5 CO
2
prior to use
International Journal of Medicinal Chemistry 13
Table 3 Effect of various coumarin derivatives on kinematics of frozen-thawed spermatozoa at one hour after incubation in vitro
Parameters Compounds Concentrations of compounds in 120583gmL VC PTFC 0 05 5 (005 DMSO) (1mM)
62 Study Subjects Infertile men who attended AndrologyLaboratory Kasturba Medical College Manipal UniversityManipal during the period of June-July 2015 for routinesemen evaluation were included in the study The subjects
with a sexual abstinence of 3ndash5 days were asked to providethe semen samples by masturbation in a sterile containerFollowing liquefaction the semen parameters were assessedas described by Kotdawala et al [34]The study was approved
14 International Journal of Medicinal Chemistry
by the Institutional Ethics Committee of Kasturba HospitalKasturba Medical College Manipal University (IEC number1552015)
63 Cryopreservation and Thawing The liquefied semensamples were cryopreserved by rapid freezing method asdescribed previously [34] with minor modifications Brieflythe semen samples were mixed with equal volume of freezingmedium (Sperm Freeze FertiPro Cat number 0344) in acryovial (Thermoscientific Nunc Cat number 138627) andkept at 4∘C for 10min in liquid nitrogen (LN2) vapor phasefor 5min and then finally plunged into liquid nitrogenThe samples were thawed after 1 week by rapid thawingmethod by placing themat 37∘C for 5minThe cryoprotectantmedium was completely removed by mixing the spermsuspension with EBSS medium and centrifuging at 1000 rpmfor 8min The resultant pellet was resuspended with freshEBSS medium and used for further analysis
64 Sample Preparation To screen the compounds withenhancing effect on themotility of spermatozoa under in vitroconditions the frozen-thawed semen samples were washedby mixing with equal volume of preincubated Earlrsquos BalancedSalt Solution (EBSS Sigma Cat number E2888) followed bycentrifugation at 1800 rpm for 8min The pellet was thengently mixed with 1mL of EBSS media containing 01bovine serum albumin (BSA Sigma Cat number A3311)and equally divided into control vehicle control and testgroups (with average sperm density of 5 times 104 spermatozoain each group) The motile sperm were extracted by swimup method as described earlier [35] The sperm suspensionwas centrifuged at 1200 rpm for 8min and the resultantpellet was overlaidwith EBSSmedium containing various testcompoundsThe sperm suspensions were incubated for 1 h at37∘Cand 5CO
2afterwhich themotile spermwere collected
from the supernatant
65 Motility Assessment Spermatozoa in the supernatantfraction were assessed for their motility patterns at 1 4 and24 h after incubation under light microscope as describedearlier [27] Sperm suspension was (10 120583L droplet) placed ona clean microscope slide and a coverslip was placed over itSpermatozoa with progressive motility and nonprogressivemotility and immotile spermatozoa were scored separatelyfrom a total of 200 spermatozoa in random fields at 400xmagnification
66 Sperm Kinematics Using Computer Assisted Semen Anal-ysis (CASA) The motion characteristics in the spermatozoawere assessed at 1 4 and 24 h after incubation using CASAsystem (ISAS Spain) Briefly 5 120583L of sperm suspension wasplaced on a clean microscope slide prewarmed at 37∘C Thesperm suspensionwas coveredwith prewarmed cover slip (22times 22mm) and observed under microscope (negative phasecontrast 10x objectives Proiser Spain) For each sample thekinematics was assessed randomly at ten different fields Theparameters such as curvilinear velocity (VCL) straight line
velocity (VSL) average path velocity (VAP) amplitude of lat-eral head displacement (ALH) linearity (LIN) straightness(STR) wobble (WOB) and beat cross frequency (BCF) wereanalyzed
7 Conclusions
In conclusion we have reported efficient and environmen-tally benignmethodologies for the synthesis of 3-[4-(1H-benzo[d]imidazol-2-yl)-1-phenyl-1H-pyrazol-3-yl]-2H-chromen-2-one derivatives 5andashx by using thermal and by microwaveirradiation under neat conditions in presence of ethanol Thereactions carried out under microwave irradiation affordedbenzimidazoles in short period of time with excellent yieldsHence this methodology would make an interesting strategyfor the synthesis of various substituted coumarin pyrazolebenzimidazoles Also compounds 5g 5h and 5k exhibitedimpressive sperm motility enhancing property whereas 5khas spermatozoa longevity property
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to University Scientific Instrumen-tation Centre (USIC) Karnatak University Dharwad forprovidingmass and IR spectral dataThe authors are thankfulto Indian Institute of Science (IISc) Bengaluru for carryingout NMR analyses and to UGC New Delhi for the financialassistance under UPE program vide no [F No 14-22012(NSPE) dated 22-01-2013] One of the authors (MNK)acknowledges University Grants Commission (UGC) NewDelhi for fellowship under UGC-UPE program
References
[1] P P Kattimani R R Kamble and G Y Meti ldquoExpedientsynthesis of benzimidazoles using amidesrdquo RSC Advances vol5 no 37 pp 29447ndash29455 2015
[2] L Li C Yee and J A Beavo ldquoCD3- and CD28-dependentinduction of PDE7 required for T cell activationrdquo Science vol283 no 5403 pp 848ndash849 1999
[3] T P Dousa ldquoCyclic-3101584051015840-nucleotide phosphodiesteraseisozymes in cell biology and pathophysiology of the kidneyrdquoKidney International vol 55 no 1 pp 29ndash62 1999
[4] C Pfister N Bennett F Bruckert et al ldquoInteractions of a G-protein with its effector transducin and cGMP phosphodi-esterase in retinal rodsrdquo Cell Signaling vol 5 no 3 pp 235ndash2411993
[5] S L C Jin F J Richard W-P Kuo A J DrsquoErcole andM Conti ldquoImpaired growth and fertility of cAMP-specificphosphodiesterase PDE4D-deficient micerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 21 pp 11998ndash12003 1999
[6] S H Francis I V Turko and J D Corbin ldquoCyclic nucleotidephosphodiesterases relating structure and functionrdquoProgress in
International Journal of Medicinal Chemistry 15
Nucleic Acid Research and Molecular Biology vol 65 pp 1ndash522000
[7] J M Hetman N Robas R Baxendale et al ldquoCloning and char-acterization of two splice variants of human phosphodiesterase11Ardquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 23 pp 12891ndash12895 2000
[8] K Yuasa T Ohgaru M Asahina and K Omori ldquoIdentificationof rat cyclic nucleotide phosphodiesterase 11A (PDE11A) com-parison of rat and human PDE11A splicing variantsrdquo EuropeanJournal of Biochemistry vol 268 no 16 pp 4440ndash4448 2001
[9] S K Sahu A Mishra and R K Behera ldquoSynthesis of thiazolebenzothiazole oxadiazole thiadiazole triazole and thiazolidi-none incorporated coumarinsrdquo Indian Journal of HeterocyclicChemistry vol 6 no 2 pp 91ndash94 1996
[10] G Roma M Di Braccio G Grossi et al ldquoSynthesis and in vitroantiplatelet activity of new 4-(1-piperazinyl) coumarin deriva-tives Human platelet phosphodiesterase 3 inhibitory propertiesof the two most effective compounds described and molecularmodeling study on their interactions with phosphodiesterase3A catalytic siterdquo Journal of Medicinal Chemistry vol 50 no 12pp 2886ndash2895 2007
[11] T Shyamsunder Reddy K Shiva Kumar C L T Medaet al ldquoConformationally restricted novel pyrazole deriva-tives synthesis of 18-disubstituted 55-dimethyl-45-dihydro-1H-benzo[g]indazoles as a new class of PDE4 inhibitorsrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 9 pp3248ndash3255 2012
[12] A Chino N Masuda Y Amano et al ldquoNovel benzimidazolederivatives as phosphodiesterase 10A (PDE10A) inhibitors withimproved metabolic stabilityrdquo Bioorganic and Medicinal Chem-istry vol 22 no 13 pp 3515ndash3526 2014
[13] H Yang FNMurigi ZWang J Li H Jin andZ Tu ldquoSynthesisand in vitro characterization of cinnoline and benzimidazoleanalogues as phosphodiesterase 10A inhibitorsrdquo Bioorganic andMedicinal Chemistry Letters vol 25 pp 919ndash924 2015
[14] J Nawrot-Modranka E Nawrot and J Graczyk ldquoIn vivo antitu-mor in vitro antibacterial activity and alkylating properties ofphosphorohydrazine derivatives of coumarin and chromonerdquoEuropean Journal of Medicinal Chemistry vol 41 no 11 pp1301ndash1309 2006
[15] A A Bekhit and T Abdel-Aziem ldquoDesign synthesis andbiological evaluation of some pyrazole derivatives as anti-inflammatory-antimicrobial agentsrdquo Bioorganic and MedicinalChemistry vol 12 no 8 pp 1935ndash1945 2004
[16] T Devji C Reddy CWoo S Awale S Kadota and D Carrico-Moniz ldquoPancreatic anticancer activity of a novel geranylgerany-lated coumarin derivativerdquo Bioorganic andMedicinal ChemistryLetters vol 21 no 19 pp 5770ndash5773 2011
[17] I Kostova I Manolov I Nicolova S Konstantinov andM Karaivanova ldquoNew lanthanide complexes of 4-methyl-7-hydroxycoumarin and their pharmacological activityrdquo Euro-pean Journal of Medicinal Chemistry vol 36 no 4 pp 339ndash3472001
[18] M Ghate R A Kusanur and M V Kulkarni ldquoSynthesis andin vivo analgesic and anti-inflammatory activity of some bi-heterocyclic coumarin derivativesrdquo European Journal of Medic-inal Chemistry vol 40 no 9 pp 882ndash887 2005
[19] PM RonadMN Noolvi S Sapkal S Dharbhamulla andV SMaddi ldquoSynthesis and antimicrobial activity of 7-(2-substitutedphenylthiazolidinyl)-benzopyran-2-one derivativesrdquo EuropeanJournal of Medicinal Chemistry vol 45 no 1 pp 85ndash89 2010
[20] GMelagraki A Afantitis O Igglessi-Markopoulou et al ldquoSyn-thesis and evaluation of the antioxidant and anti-inflammatoryactivity of novel coumarin-3-aminoamides and their alpha-lipoic acid adductsrdquo European Journal of Medicinal Chemistryvol 44 no 7 pp 3020ndash3026 2009
[21] J C Trivedi J B Bariwal K D Upadhyay et al ldquoImprovedand rapid synthesis of new coumarinyl chalcone derivatives andtheir antiviral activityrdquo Tetrahedron Letters vol 48 no 48 pp8472ndash8474 2007
[22] S Vilar E Quezada L Santana et al ldquoDesign synthe-sis and vasorelaxant and platelet antiaggregatory activitiesof coumarinndashresveratrol hybridsrdquo Bioorganic and MedicinalChemistry Letters vol 16 no 2 pp 257ndash261 2006
[23] W Hamaguchi N Masuda M Isomura et al ldquoDesign and syn-thesis of novel benzimidazole derivatives as phosphodiesterase10A inhibitors with reduced CYP1A2 inhibitionrdquo Bioorganicand Medicinal Chemistry vol 21 no 24 pp 7612ndash7623 2013
[24] A Dore B Asproni A Scampuddu et al ldquoSynthesis and SARstudy of novel tricyclic pyrazoles as potent phosphodiesterase10A inhibitorsrdquo European Journal of Medicinal Chemistry vol84 pp 181ndash193 2014
[25] S V Laxmi B S Kuarm and B Rajitha ldquoSynthesis and anti-microbial activity of coumarin pyrazole pyrimidine 246(1H3H5H)triones and thioxopyrimidine46(1H5H)dionesrdquo Me-dicinal Chemistry Research vol 22 no 2 pp 768ndash774 2013
[26] G Kalthur S Raj AThiyagarajan S Kumar P Kumar and S KAdiga ldquoVitamin E supplementation in semen-freezingmediumimproves the motility and protects sperm from freeze-thaw-induced DNA damagerdquo Fertility and Sterility vol 95 no 3 pp1149ndash1151 2011
[27] J Tesarik C Mendoza and A Carreras ldquoEffects of phosphodi-esterase inhibitors caffeine and pentoxifylline on spontaneousand stimulus-induced acrosome reactions in human spermrdquoFertility and Sterility vol 58 no 6 pp 1185ndash1190 1992
[28] G Kalthur S R Salian F Keyvanifard et al ldquoSupplementationof biotin to sperm preparation medium increases the motilityand longevity in cryopreserved human spermatozoardquo Journalof Assisted Reproduction and Genetics vol 29 no 7 pp 631ndash6352012
[29] A E Calogero S Fishel J Hall et al ldquoCorrelation betweenintracellular cAMP content kinematic parameters and hyper-activation of human spermatozoa after incubation with pentox-ifyllinerdquo Human Reproduction vol 13 no 4 pp 911ndash915 1998
[30] R Ain K Uma Devi S Shivaji and P B Seshagiri ldquoPen-toxifylline-stimulated capacitation and acrosome reaction inhamster spermatozoa involvement of intracellular signallingmoleculesrdquo Molecular Human Reproduction vol 5 no 7 pp618ndash626 1999
[31] M Bajpai andG F Doncel ldquoInvolvement of tyrosine kinase andcAMP-dependent kinase cross-talk in the regulation of humanspermmotilityrdquo Reproduction vol 126 no 2 pp 183ndash195 2003
[32] R Yunes P Fernandez G F Doncel and A A Acosta ldquoCyclicnucleotide phosphodiesterase inhibition increases tyrosinephosphorylation and hypermotility in normal and pathologicalhuman spermatozoardquo Biocell vol 29 no 3 pp 287ndash293 2005
[33] F Dimitriadis D Giannakis N Pardalidis et al ldquoEffectsof phosphodiesterase 5 inhibitors on sperm parameters andfertilizing capacityrdquo Asian Journal of Andrology vol 10 no 1pp 115ndash133 2008
[34] A P Kotdawala S Kumar S R Salian et al ldquoAddition of zincto human ejaculate prior to cryopreservation prevents freeze-thaw-induced DNA damage and preserves sperm functionrdquo
16 International Journal of Medicinal Chemistry
Journal of Assisted Reproduction and Genetics vol 29 no 12 pp1447ndash1453 2012
[35] S Mutalik S R Salian K Avadhani et al ldquoLiposome encapsu-lated soy lecithin and cholesterol can efficiently replace chickenegg yolk in human semen cryopreservation mediumrdquo SystemsBiology in Reproductive Medicine vol 60 no 3 pp 183ndash1882014
31 Effect on Motility of Frozen-Thawed Human SpermatozoaTo assess the effect of title compounds on themotility patternhuman spermatozoa subjected to freeze-thaw process wasused It is well documented that freeze-thaw process canhave detrimental effect on the motility and survival of thespermatozoa [26] Therefore they can be an excellent modelto study the effect of any test compounds on themotility [27]
In the present study we assessed the motility patternin spermatozoa processed with media containing variouscompounds manually at 1 4 and 24 h after incubationin vitro Three arbitrary doses were selected (05 10 and50 120583gmL) for each compound to assess their effect on spermmotility Pentoxifylline (PTF) which is known to enhancethe human sperm motility by elevating intracellular cyclicadenosine monophosphate (cAMP) level [27] was taken asa positive control Based on the previous report PTF at 1mMconcentration was used [28] Since the test compounds werenot soluble in water dimethyl sulfoxide (DMSO 005) wasused as vehicle control
The sperm motility enhancement property of the com-pounds was categorized as below
(a) Marginal which resulted in lt5 increase in themotility (b) Good which resulted in 5ndash10 increase inmotility (c) Excellent which resulted in gt10 increase inmotility (d) Poor where there was no enhancement inmotility
At 1 h after the in vitro incubation the majority of thecompounds had marginal effect on the sperm motility com-pared to control At this interval PTF had higher percentageof total and progressivelymotile spermatozoa compared to allthe test compounds A large number of studies have shownthat PTF can trigger the motility in fresh or frozen-thawedspermatozoa as early as 1 h [29] even though the majorityof the studies indicate that this effect is mainly due to theelevated cAMP level in the spermatozoa [30] it can also bemediated through tyrosine phosphorylation [31]
Compounds 5a 5b 5c 5d 5e 5g 5h and 5k inducedmarginal enhancement in total motility while compounds5a 5b 5c and 5j resulted in similar effect on progressivemotility (Tables 2(a) and 2(b)) A 5ndash10 increase in totalmotility was induced by compound 5j However with respectto progressive motility compounds 5d 5e 5f and 5k cameunder compounds with good effect on motility More than10 increase in progressive motility was observed only forcompound 5g (at 05 120583gmL concentration) and compound5h (at 5 120583gmL concentration)
A time-dependent decrease in total and progressivemotility was observed in all the groups However the declinewasmore evident at 24 h intervalThis phenomenon has beendocumented by earlier studies [28] These changes may berelated to the oxidative stress induced by in vitro cultureconditions Spermatozoa are highly susceptible to reactiveoxygen species (ROS) due to high polyunsaturated fatty acidcontent in their plasma membrane and negligible amount ofcytoplasmic antioxidants
At 4 h interval compounds 5e 5f and 5g enhancedthe progressive motility of spermatozoa by 5ndash10 while
compound 5h with two chloro substituents on coumarinand benzimidazole resulted in almost 15 increase in theprogressive motility However the effect was lower than thatof pentoxifylline The rest of the compounds (5a 5b 5c5f and 5j) induced a marginal increase in motility exceptin compound 5e in which the motility was lower than thecontrol group
At 24 h interval the majority of the compounds (5a5b 5d 5f 5h and 5j) had marginally higher percentageof motility or poor motility compounds (5c 5e and 5g)compared to control Interestingly compound 5k with chlorosubstituent on coumarin and bromo substitution on ben-zimidazole had higher percentage of motile spermatozoacompared to control (14 higher) and even PTF (9 higher)This indicates that compound5k canhelp in prolonging the invitro survival of spermatozoa which has significant beneficialrole in assisted reproductive technologies such as in vitrofertilization Previous studies have shown that even thoughPTF has triggering effect on themotility it induces prematureacrosome reaction and drastic decrease in motility at laterintervals In this context the compounds which increasethe motility and longevity of spermatozoa under in vitroconditions are of clinical relevance
Based on the manual motility assessment we observedthat compounds 5d 5f 5g 5h and 5k have considerablebeneficial role in spermmotilityTherefore these compoundswere further assessed for their effect on the kinematicsof spermatozoa at 1 h However there was no significantdifference in the kinematics of spermatozoa compared tocontrol and PTF (Table 3) In conclusion the newly synthe-sized coumarin derivatives have sperm motility enhancingproperty However in this preliminary screening we havetaken three arbitrary doses to assess their effect on motilityCompounds 5g 5h and 5k show considerable beneficialeffect which can be of clinical significance where spermmotility enhancement is achieved under in vitro condi-tions Compounds 5g and 5h triggered the motility at earlyintervals which was similar to the PTF while compound5k improved the longevity of the spermatozoa Since thecoumarin is known to have PDE inhibitory function themotility enhancement could be due to the elevated cAMPlevel in sperm However further detailed studies are essentialto understand the mechanism of action of these compoundsand their clinical utility
4 Experimental
All the chemicals and reagents were purchased from theMerck and Aldrich chemical suppliers Melting points (mp)were determined in open capillaries and are uncorrectedThe IR spectra were recorded on a Nicolet Impact 410 FTIR spectrometer using KBr pellets (range 4000ndash500 cmminus1)The 1HNMR spectra were recorded at 400MHz on BrukerAvance FT NMR spectrometer in DMSO-d
6solvent with
TMS as internal standard 13CNMR spectra were recordedat 100MHz on Bruker Avance FT NMR spectrometer inDMSO-d
6solvent with TMS as internal standard The mass
spectra were recorded on Shimadzu GC-MS operating at70 eV Thin-layer chromatography (TLC) was performed on
International Journal of Medicinal Chemistry 11
Table 2 Effect of supplementation of coumarin derivatives (5andashj) to sperm wash medium on the motility pattern in frozen-thawed humanspermatozoa at various time intervals
(a)
Compounds Time (h)Total motility (mean plusmn SE of percentage)
020mm Aluchrosep silica gel 60 F254 plates (SD FineMumbai) Microwave irradiation experiments were carriedout using CEMDiscover SPMicrowave Synthesizer equippedwith IR sensor to monitor reaction temperatures
5 General Procedures for the Preparation ofTitle Compounds 5andashx
3-(2-Oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-car-baldehydes (3andashd 1 mmol) were prepared from 3-acetyl-coumarin (1andashd 1 mmol) phenylhydrazine (1mmol) andthe intermediate (Schiff base) (2andashd 1 mmol) followedby reaction with POCl
3in DMF by Vilsmeier Haack for-
mylation strategies [25] The target compounds 5andashxwere then achieved by stirring substituted carbaldehyde(3andashd 1 mmol) with different o-arylenediamine (4andashf1 mmol) under reflux for 4-5 hrs After completion (TLChexane ethylacetate 7 3) of the reaction the solid obtainedwas filtered washed with small quantity of ethanol anddried Further it was recrystallized in methanol to get thepure compound (5andashx)
51 Microwave Assisted Procedure for the Preparation of Com-pounds 5andashx A mixture of 3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde (3andashd 1 mmol) and o-phenylenediamine (4andashf 1 mmol) taken in ethanol (5mL)was introduced into a CEM microwave reaction vesselequipped with magnetic stirrer The reaction mixture was
prestirred for 1min at room temperature and irradiated at180W (150∘C) for about 6ndash8min (TLC using hexane ethylacetate (7 3 drops) as eluent) The reaction mixture wasthen quenched into crushed ice and the crude product wasfiltered washed and dried Recrystallization from methanolgave pure crystals of the compounds 5andashx
(Spectral as well as elemental analyses data are providedas electronic supplementary file in Supplementary Materialavailable online at httpdxdoiorg10115520169890630)
6 Assessment of Phosphodiesterase InhibitionUsing Human Spermatozoa as Model
Earlier studies have shown that PDE inhibitors can enhancethe spermmotility [32 33] Based on this in the present studywe have used an indirect approach to assess the inhibitoryeffect of the coumarin derivatives on PDE by assessing spermmotility pattern
61 Preparation of Test Solution Stock solution of the testcompounds (1mgmL) was prepared by dissolving them indimethyl sulfoxide (DMSO Sigma Cat number D5879)The stock solution was then diluted with Earlrsquos BalancedSalt Solution (EBSS) supplemented with 01 bovine serumalbumin (BSA) in a ratio of 1 200 1 1000 and 1 2000 to getworking solutions of 5 1 and 05 120583gmL concentrations Theworking solutions were preincubated at 37∘C and 5 CO
2
prior to use
International Journal of Medicinal Chemistry 13
Table 3 Effect of various coumarin derivatives on kinematics of frozen-thawed spermatozoa at one hour after incubation in vitro
Parameters Compounds Concentrations of compounds in 120583gmL VC PTFC 0 05 5 (005 DMSO) (1mM)
62 Study Subjects Infertile men who attended AndrologyLaboratory Kasturba Medical College Manipal UniversityManipal during the period of June-July 2015 for routinesemen evaluation were included in the study The subjects
with a sexual abstinence of 3ndash5 days were asked to providethe semen samples by masturbation in a sterile containerFollowing liquefaction the semen parameters were assessedas described by Kotdawala et al [34]The study was approved
14 International Journal of Medicinal Chemistry
by the Institutional Ethics Committee of Kasturba HospitalKasturba Medical College Manipal University (IEC number1552015)
63 Cryopreservation and Thawing The liquefied semensamples were cryopreserved by rapid freezing method asdescribed previously [34] with minor modifications Brieflythe semen samples were mixed with equal volume of freezingmedium (Sperm Freeze FertiPro Cat number 0344) in acryovial (Thermoscientific Nunc Cat number 138627) andkept at 4∘C for 10min in liquid nitrogen (LN2) vapor phasefor 5min and then finally plunged into liquid nitrogenThe samples were thawed after 1 week by rapid thawingmethod by placing themat 37∘C for 5minThe cryoprotectantmedium was completely removed by mixing the spermsuspension with EBSS medium and centrifuging at 1000 rpmfor 8min The resultant pellet was resuspended with freshEBSS medium and used for further analysis
64 Sample Preparation To screen the compounds withenhancing effect on themotility of spermatozoa under in vitroconditions the frozen-thawed semen samples were washedby mixing with equal volume of preincubated Earlrsquos BalancedSalt Solution (EBSS Sigma Cat number E2888) followed bycentrifugation at 1800 rpm for 8min The pellet was thengently mixed with 1mL of EBSS media containing 01bovine serum albumin (BSA Sigma Cat number A3311)and equally divided into control vehicle control and testgroups (with average sperm density of 5 times 104 spermatozoain each group) The motile sperm were extracted by swimup method as described earlier [35] The sperm suspensionwas centrifuged at 1200 rpm for 8min and the resultantpellet was overlaidwith EBSSmedium containing various testcompoundsThe sperm suspensions were incubated for 1 h at37∘Cand 5CO
2afterwhich themotile spermwere collected
from the supernatant
65 Motility Assessment Spermatozoa in the supernatantfraction were assessed for their motility patterns at 1 4 and24 h after incubation under light microscope as describedearlier [27] Sperm suspension was (10 120583L droplet) placed ona clean microscope slide and a coverslip was placed over itSpermatozoa with progressive motility and nonprogressivemotility and immotile spermatozoa were scored separatelyfrom a total of 200 spermatozoa in random fields at 400xmagnification
66 Sperm Kinematics Using Computer Assisted Semen Anal-ysis (CASA) The motion characteristics in the spermatozoawere assessed at 1 4 and 24 h after incubation using CASAsystem (ISAS Spain) Briefly 5 120583L of sperm suspension wasplaced on a clean microscope slide prewarmed at 37∘C Thesperm suspensionwas coveredwith prewarmed cover slip (22times 22mm) and observed under microscope (negative phasecontrast 10x objectives Proiser Spain) For each sample thekinematics was assessed randomly at ten different fields Theparameters such as curvilinear velocity (VCL) straight line
velocity (VSL) average path velocity (VAP) amplitude of lat-eral head displacement (ALH) linearity (LIN) straightness(STR) wobble (WOB) and beat cross frequency (BCF) wereanalyzed
7 Conclusions
In conclusion we have reported efficient and environmen-tally benignmethodologies for the synthesis of 3-[4-(1H-benzo[d]imidazol-2-yl)-1-phenyl-1H-pyrazol-3-yl]-2H-chromen-2-one derivatives 5andashx by using thermal and by microwaveirradiation under neat conditions in presence of ethanol Thereactions carried out under microwave irradiation affordedbenzimidazoles in short period of time with excellent yieldsHence this methodology would make an interesting strategyfor the synthesis of various substituted coumarin pyrazolebenzimidazoles Also compounds 5g 5h and 5k exhibitedimpressive sperm motility enhancing property whereas 5khas spermatozoa longevity property
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to University Scientific Instrumen-tation Centre (USIC) Karnatak University Dharwad forprovidingmass and IR spectral dataThe authors are thankfulto Indian Institute of Science (IISc) Bengaluru for carryingout NMR analyses and to UGC New Delhi for the financialassistance under UPE program vide no [F No 14-22012(NSPE) dated 22-01-2013] One of the authors (MNK)acknowledges University Grants Commission (UGC) NewDelhi for fellowship under UGC-UPE program
References
[1] P P Kattimani R R Kamble and G Y Meti ldquoExpedientsynthesis of benzimidazoles using amidesrdquo RSC Advances vol5 no 37 pp 29447ndash29455 2015
[2] L Li C Yee and J A Beavo ldquoCD3- and CD28-dependentinduction of PDE7 required for T cell activationrdquo Science vol283 no 5403 pp 848ndash849 1999
[3] T P Dousa ldquoCyclic-3101584051015840-nucleotide phosphodiesteraseisozymes in cell biology and pathophysiology of the kidneyrdquoKidney International vol 55 no 1 pp 29ndash62 1999
[4] C Pfister N Bennett F Bruckert et al ldquoInteractions of a G-protein with its effector transducin and cGMP phosphodi-esterase in retinal rodsrdquo Cell Signaling vol 5 no 3 pp 235ndash2411993
[5] S L C Jin F J Richard W-P Kuo A J DrsquoErcole andM Conti ldquoImpaired growth and fertility of cAMP-specificphosphodiesterase PDE4D-deficient micerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 21 pp 11998ndash12003 1999
[6] S H Francis I V Turko and J D Corbin ldquoCyclic nucleotidephosphodiesterases relating structure and functionrdquoProgress in
International Journal of Medicinal Chemistry 15
Nucleic Acid Research and Molecular Biology vol 65 pp 1ndash522000
[7] J M Hetman N Robas R Baxendale et al ldquoCloning and char-acterization of two splice variants of human phosphodiesterase11Ardquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 23 pp 12891ndash12895 2000
[8] K Yuasa T Ohgaru M Asahina and K Omori ldquoIdentificationof rat cyclic nucleotide phosphodiesterase 11A (PDE11A) com-parison of rat and human PDE11A splicing variantsrdquo EuropeanJournal of Biochemistry vol 268 no 16 pp 4440ndash4448 2001
[9] S K Sahu A Mishra and R K Behera ldquoSynthesis of thiazolebenzothiazole oxadiazole thiadiazole triazole and thiazolidi-none incorporated coumarinsrdquo Indian Journal of HeterocyclicChemistry vol 6 no 2 pp 91ndash94 1996
[10] G Roma M Di Braccio G Grossi et al ldquoSynthesis and in vitroantiplatelet activity of new 4-(1-piperazinyl) coumarin deriva-tives Human platelet phosphodiesterase 3 inhibitory propertiesof the two most effective compounds described and molecularmodeling study on their interactions with phosphodiesterase3A catalytic siterdquo Journal of Medicinal Chemistry vol 50 no 12pp 2886ndash2895 2007
[11] T Shyamsunder Reddy K Shiva Kumar C L T Medaet al ldquoConformationally restricted novel pyrazole deriva-tives synthesis of 18-disubstituted 55-dimethyl-45-dihydro-1H-benzo[g]indazoles as a new class of PDE4 inhibitorsrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 9 pp3248ndash3255 2012
[12] A Chino N Masuda Y Amano et al ldquoNovel benzimidazolederivatives as phosphodiesterase 10A (PDE10A) inhibitors withimproved metabolic stabilityrdquo Bioorganic and Medicinal Chem-istry vol 22 no 13 pp 3515ndash3526 2014
[13] H Yang FNMurigi ZWang J Li H Jin andZ Tu ldquoSynthesisand in vitro characterization of cinnoline and benzimidazoleanalogues as phosphodiesterase 10A inhibitorsrdquo Bioorganic andMedicinal Chemistry Letters vol 25 pp 919ndash924 2015
[14] J Nawrot-Modranka E Nawrot and J Graczyk ldquoIn vivo antitu-mor in vitro antibacterial activity and alkylating properties ofphosphorohydrazine derivatives of coumarin and chromonerdquoEuropean Journal of Medicinal Chemistry vol 41 no 11 pp1301ndash1309 2006
[15] A A Bekhit and T Abdel-Aziem ldquoDesign synthesis andbiological evaluation of some pyrazole derivatives as anti-inflammatory-antimicrobial agentsrdquo Bioorganic and MedicinalChemistry vol 12 no 8 pp 1935ndash1945 2004
[16] T Devji C Reddy CWoo S Awale S Kadota and D Carrico-Moniz ldquoPancreatic anticancer activity of a novel geranylgerany-lated coumarin derivativerdquo Bioorganic andMedicinal ChemistryLetters vol 21 no 19 pp 5770ndash5773 2011
[17] I Kostova I Manolov I Nicolova S Konstantinov andM Karaivanova ldquoNew lanthanide complexes of 4-methyl-7-hydroxycoumarin and their pharmacological activityrdquo Euro-pean Journal of Medicinal Chemistry vol 36 no 4 pp 339ndash3472001
[18] M Ghate R A Kusanur and M V Kulkarni ldquoSynthesis andin vivo analgesic and anti-inflammatory activity of some bi-heterocyclic coumarin derivativesrdquo European Journal of Medic-inal Chemistry vol 40 no 9 pp 882ndash887 2005
[19] PM RonadMN Noolvi S Sapkal S Dharbhamulla andV SMaddi ldquoSynthesis and antimicrobial activity of 7-(2-substitutedphenylthiazolidinyl)-benzopyran-2-one derivativesrdquo EuropeanJournal of Medicinal Chemistry vol 45 no 1 pp 85ndash89 2010
[20] GMelagraki A Afantitis O Igglessi-Markopoulou et al ldquoSyn-thesis and evaluation of the antioxidant and anti-inflammatoryactivity of novel coumarin-3-aminoamides and their alpha-lipoic acid adductsrdquo European Journal of Medicinal Chemistryvol 44 no 7 pp 3020ndash3026 2009
[21] J C Trivedi J B Bariwal K D Upadhyay et al ldquoImprovedand rapid synthesis of new coumarinyl chalcone derivatives andtheir antiviral activityrdquo Tetrahedron Letters vol 48 no 48 pp8472ndash8474 2007
[22] S Vilar E Quezada L Santana et al ldquoDesign synthe-sis and vasorelaxant and platelet antiaggregatory activitiesof coumarinndashresveratrol hybridsrdquo Bioorganic and MedicinalChemistry Letters vol 16 no 2 pp 257ndash261 2006
[23] W Hamaguchi N Masuda M Isomura et al ldquoDesign and syn-thesis of novel benzimidazole derivatives as phosphodiesterase10A inhibitors with reduced CYP1A2 inhibitionrdquo Bioorganicand Medicinal Chemistry vol 21 no 24 pp 7612ndash7623 2013
[24] A Dore B Asproni A Scampuddu et al ldquoSynthesis and SARstudy of novel tricyclic pyrazoles as potent phosphodiesterase10A inhibitorsrdquo European Journal of Medicinal Chemistry vol84 pp 181ndash193 2014
[25] S V Laxmi B S Kuarm and B Rajitha ldquoSynthesis and anti-microbial activity of coumarin pyrazole pyrimidine 246(1H3H5H)triones and thioxopyrimidine46(1H5H)dionesrdquo Me-dicinal Chemistry Research vol 22 no 2 pp 768ndash774 2013
[26] G Kalthur S Raj AThiyagarajan S Kumar P Kumar and S KAdiga ldquoVitamin E supplementation in semen-freezingmediumimproves the motility and protects sperm from freeze-thaw-induced DNA damagerdquo Fertility and Sterility vol 95 no 3 pp1149ndash1151 2011
[27] J Tesarik C Mendoza and A Carreras ldquoEffects of phosphodi-esterase inhibitors caffeine and pentoxifylline on spontaneousand stimulus-induced acrosome reactions in human spermrdquoFertility and Sterility vol 58 no 6 pp 1185ndash1190 1992
[28] G Kalthur S R Salian F Keyvanifard et al ldquoSupplementationof biotin to sperm preparation medium increases the motilityand longevity in cryopreserved human spermatozoardquo Journalof Assisted Reproduction and Genetics vol 29 no 7 pp 631ndash6352012
[29] A E Calogero S Fishel J Hall et al ldquoCorrelation betweenintracellular cAMP content kinematic parameters and hyper-activation of human spermatozoa after incubation with pentox-ifyllinerdquo Human Reproduction vol 13 no 4 pp 911ndash915 1998
[30] R Ain K Uma Devi S Shivaji and P B Seshagiri ldquoPen-toxifylline-stimulated capacitation and acrosome reaction inhamster spermatozoa involvement of intracellular signallingmoleculesrdquo Molecular Human Reproduction vol 5 no 7 pp618ndash626 1999
[31] M Bajpai andG F Doncel ldquoInvolvement of tyrosine kinase andcAMP-dependent kinase cross-talk in the regulation of humanspermmotilityrdquo Reproduction vol 126 no 2 pp 183ndash195 2003
[32] R Yunes P Fernandez G F Doncel and A A Acosta ldquoCyclicnucleotide phosphodiesterase inhibition increases tyrosinephosphorylation and hypermotility in normal and pathologicalhuman spermatozoardquo Biocell vol 29 no 3 pp 287ndash293 2005
[33] F Dimitriadis D Giannakis N Pardalidis et al ldquoEffectsof phosphodiesterase 5 inhibitors on sperm parameters andfertilizing capacityrdquo Asian Journal of Andrology vol 10 no 1pp 115ndash133 2008
[34] A P Kotdawala S Kumar S R Salian et al ldquoAddition of zincto human ejaculate prior to cryopreservation prevents freeze-thaw-induced DNA damage and preserves sperm functionrdquo
16 International Journal of Medicinal Chemistry
Journal of Assisted Reproduction and Genetics vol 29 no 12 pp1447ndash1453 2012
[35] S Mutalik S R Salian K Avadhani et al ldquoLiposome encapsu-lated soy lecithin and cholesterol can efficiently replace chickenegg yolk in human semen cryopreservation mediumrdquo SystemsBiology in Reproductive Medicine vol 60 no 3 pp 183ndash1882014
31 Effect on Motility of Frozen-Thawed Human SpermatozoaTo assess the effect of title compounds on themotility patternhuman spermatozoa subjected to freeze-thaw process wasused It is well documented that freeze-thaw process canhave detrimental effect on the motility and survival of thespermatozoa [26] Therefore they can be an excellent modelto study the effect of any test compounds on themotility [27]
In the present study we assessed the motility patternin spermatozoa processed with media containing variouscompounds manually at 1 4 and 24 h after incubationin vitro Three arbitrary doses were selected (05 10 and50 120583gmL) for each compound to assess their effect on spermmotility Pentoxifylline (PTF) which is known to enhancethe human sperm motility by elevating intracellular cyclicadenosine monophosphate (cAMP) level [27] was taken asa positive control Based on the previous report PTF at 1mMconcentration was used [28] Since the test compounds werenot soluble in water dimethyl sulfoxide (DMSO 005) wasused as vehicle control
The sperm motility enhancement property of the com-pounds was categorized as below
(a) Marginal which resulted in lt5 increase in themotility (b) Good which resulted in 5ndash10 increase inmotility (c) Excellent which resulted in gt10 increase inmotility (d) Poor where there was no enhancement inmotility
At 1 h after the in vitro incubation the majority of thecompounds had marginal effect on the sperm motility com-pared to control At this interval PTF had higher percentageof total and progressivelymotile spermatozoa compared to allthe test compounds A large number of studies have shownthat PTF can trigger the motility in fresh or frozen-thawedspermatozoa as early as 1 h [29] even though the majorityof the studies indicate that this effect is mainly due to theelevated cAMP level in the spermatozoa [30] it can also bemediated through tyrosine phosphorylation [31]
Compounds 5a 5b 5c 5d 5e 5g 5h and 5k inducedmarginal enhancement in total motility while compounds5a 5b 5c and 5j resulted in similar effect on progressivemotility (Tables 2(a) and 2(b)) A 5ndash10 increase in totalmotility was induced by compound 5j However with respectto progressive motility compounds 5d 5e 5f and 5k cameunder compounds with good effect on motility More than10 increase in progressive motility was observed only forcompound 5g (at 05 120583gmL concentration) and compound5h (at 5 120583gmL concentration)
A time-dependent decrease in total and progressivemotility was observed in all the groups However the declinewasmore evident at 24 h intervalThis phenomenon has beendocumented by earlier studies [28] These changes may berelated to the oxidative stress induced by in vitro cultureconditions Spermatozoa are highly susceptible to reactiveoxygen species (ROS) due to high polyunsaturated fatty acidcontent in their plasma membrane and negligible amount ofcytoplasmic antioxidants
At 4 h interval compounds 5e 5f and 5g enhancedthe progressive motility of spermatozoa by 5ndash10 while
compound 5h with two chloro substituents on coumarinand benzimidazole resulted in almost 15 increase in theprogressive motility However the effect was lower than thatof pentoxifylline The rest of the compounds (5a 5b 5c5f and 5j) induced a marginal increase in motility exceptin compound 5e in which the motility was lower than thecontrol group
At 24 h interval the majority of the compounds (5a5b 5d 5f 5h and 5j) had marginally higher percentageof motility or poor motility compounds (5c 5e and 5g)compared to control Interestingly compound 5k with chlorosubstituent on coumarin and bromo substitution on ben-zimidazole had higher percentage of motile spermatozoacompared to control (14 higher) and even PTF (9 higher)This indicates that compound5k canhelp in prolonging the invitro survival of spermatozoa which has significant beneficialrole in assisted reproductive technologies such as in vitrofertilization Previous studies have shown that even thoughPTF has triggering effect on themotility it induces prematureacrosome reaction and drastic decrease in motility at laterintervals In this context the compounds which increasethe motility and longevity of spermatozoa under in vitroconditions are of clinical relevance
Based on the manual motility assessment we observedthat compounds 5d 5f 5g 5h and 5k have considerablebeneficial role in spermmotilityTherefore these compoundswere further assessed for their effect on the kinematicsof spermatozoa at 1 h However there was no significantdifference in the kinematics of spermatozoa compared tocontrol and PTF (Table 3) In conclusion the newly synthe-sized coumarin derivatives have sperm motility enhancingproperty However in this preliminary screening we havetaken three arbitrary doses to assess their effect on motilityCompounds 5g 5h and 5k show considerable beneficialeffect which can be of clinical significance where spermmotility enhancement is achieved under in vitro condi-tions Compounds 5g and 5h triggered the motility at earlyintervals which was similar to the PTF while compound5k improved the longevity of the spermatozoa Since thecoumarin is known to have PDE inhibitory function themotility enhancement could be due to the elevated cAMPlevel in sperm However further detailed studies are essentialto understand the mechanism of action of these compoundsand their clinical utility
4 Experimental
All the chemicals and reagents were purchased from theMerck and Aldrich chemical suppliers Melting points (mp)were determined in open capillaries and are uncorrectedThe IR spectra were recorded on a Nicolet Impact 410 FTIR spectrometer using KBr pellets (range 4000ndash500 cmminus1)The 1HNMR spectra were recorded at 400MHz on BrukerAvance FT NMR spectrometer in DMSO-d
6solvent with
TMS as internal standard 13CNMR spectra were recordedat 100MHz on Bruker Avance FT NMR spectrometer inDMSO-d
6solvent with TMS as internal standard The mass
spectra were recorded on Shimadzu GC-MS operating at70 eV Thin-layer chromatography (TLC) was performed on
International Journal of Medicinal Chemistry 11
Table 2 Effect of supplementation of coumarin derivatives (5andashj) to sperm wash medium on the motility pattern in frozen-thawed humanspermatozoa at various time intervals
(a)
Compounds Time (h)Total motility (mean plusmn SE of percentage)
020mm Aluchrosep silica gel 60 F254 plates (SD FineMumbai) Microwave irradiation experiments were carriedout using CEMDiscover SPMicrowave Synthesizer equippedwith IR sensor to monitor reaction temperatures
5 General Procedures for the Preparation ofTitle Compounds 5andashx
3-(2-Oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-car-baldehydes (3andashd 1 mmol) were prepared from 3-acetyl-coumarin (1andashd 1 mmol) phenylhydrazine (1mmol) andthe intermediate (Schiff base) (2andashd 1 mmol) followedby reaction with POCl
3in DMF by Vilsmeier Haack for-
mylation strategies [25] The target compounds 5andashxwere then achieved by stirring substituted carbaldehyde(3andashd 1 mmol) with different o-arylenediamine (4andashf1 mmol) under reflux for 4-5 hrs After completion (TLChexane ethylacetate 7 3) of the reaction the solid obtainedwas filtered washed with small quantity of ethanol anddried Further it was recrystallized in methanol to get thepure compound (5andashx)
51 Microwave Assisted Procedure for the Preparation of Com-pounds 5andashx A mixture of 3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde (3andashd 1 mmol) and o-phenylenediamine (4andashf 1 mmol) taken in ethanol (5mL)was introduced into a CEM microwave reaction vesselequipped with magnetic stirrer The reaction mixture was
prestirred for 1min at room temperature and irradiated at180W (150∘C) for about 6ndash8min (TLC using hexane ethylacetate (7 3 drops) as eluent) The reaction mixture wasthen quenched into crushed ice and the crude product wasfiltered washed and dried Recrystallization from methanolgave pure crystals of the compounds 5andashx
(Spectral as well as elemental analyses data are providedas electronic supplementary file in Supplementary Materialavailable online at httpdxdoiorg10115520169890630)
6 Assessment of Phosphodiesterase InhibitionUsing Human Spermatozoa as Model
Earlier studies have shown that PDE inhibitors can enhancethe spermmotility [32 33] Based on this in the present studywe have used an indirect approach to assess the inhibitoryeffect of the coumarin derivatives on PDE by assessing spermmotility pattern
61 Preparation of Test Solution Stock solution of the testcompounds (1mgmL) was prepared by dissolving them indimethyl sulfoxide (DMSO Sigma Cat number D5879)The stock solution was then diluted with Earlrsquos BalancedSalt Solution (EBSS) supplemented with 01 bovine serumalbumin (BSA) in a ratio of 1 200 1 1000 and 1 2000 to getworking solutions of 5 1 and 05 120583gmL concentrations Theworking solutions were preincubated at 37∘C and 5 CO
2
prior to use
International Journal of Medicinal Chemistry 13
Table 3 Effect of various coumarin derivatives on kinematics of frozen-thawed spermatozoa at one hour after incubation in vitro
Parameters Compounds Concentrations of compounds in 120583gmL VC PTFC 0 05 5 (005 DMSO) (1mM)
62 Study Subjects Infertile men who attended AndrologyLaboratory Kasturba Medical College Manipal UniversityManipal during the period of June-July 2015 for routinesemen evaluation were included in the study The subjects
with a sexual abstinence of 3ndash5 days were asked to providethe semen samples by masturbation in a sterile containerFollowing liquefaction the semen parameters were assessedas described by Kotdawala et al [34]The study was approved
14 International Journal of Medicinal Chemistry
by the Institutional Ethics Committee of Kasturba HospitalKasturba Medical College Manipal University (IEC number1552015)
63 Cryopreservation and Thawing The liquefied semensamples were cryopreserved by rapid freezing method asdescribed previously [34] with minor modifications Brieflythe semen samples were mixed with equal volume of freezingmedium (Sperm Freeze FertiPro Cat number 0344) in acryovial (Thermoscientific Nunc Cat number 138627) andkept at 4∘C for 10min in liquid nitrogen (LN2) vapor phasefor 5min and then finally plunged into liquid nitrogenThe samples were thawed after 1 week by rapid thawingmethod by placing themat 37∘C for 5minThe cryoprotectantmedium was completely removed by mixing the spermsuspension with EBSS medium and centrifuging at 1000 rpmfor 8min The resultant pellet was resuspended with freshEBSS medium and used for further analysis
64 Sample Preparation To screen the compounds withenhancing effect on themotility of spermatozoa under in vitroconditions the frozen-thawed semen samples were washedby mixing with equal volume of preincubated Earlrsquos BalancedSalt Solution (EBSS Sigma Cat number E2888) followed bycentrifugation at 1800 rpm for 8min The pellet was thengently mixed with 1mL of EBSS media containing 01bovine serum albumin (BSA Sigma Cat number A3311)and equally divided into control vehicle control and testgroups (with average sperm density of 5 times 104 spermatozoain each group) The motile sperm were extracted by swimup method as described earlier [35] The sperm suspensionwas centrifuged at 1200 rpm for 8min and the resultantpellet was overlaidwith EBSSmedium containing various testcompoundsThe sperm suspensions were incubated for 1 h at37∘Cand 5CO
2afterwhich themotile spermwere collected
from the supernatant
65 Motility Assessment Spermatozoa in the supernatantfraction were assessed for their motility patterns at 1 4 and24 h after incubation under light microscope as describedearlier [27] Sperm suspension was (10 120583L droplet) placed ona clean microscope slide and a coverslip was placed over itSpermatozoa with progressive motility and nonprogressivemotility and immotile spermatozoa were scored separatelyfrom a total of 200 spermatozoa in random fields at 400xmagnification
66 Sperm Kinematics Using Computer Assisted Semen Anal-ysis (CASA) The motion characteristics in the spermatozoawere assessed at 1 4 and 24 h after incubation using CASAsystem (ISAS Spain) Briefly 5 120583L of sperm suspension wasplaced on a clean microscope slide prewarmed at 37∘C Thesperm suspensionwas coveredwith prewarmed cover slip (22times 22mm) and observed under microscope (negative phasecontrast 10x objectives Proiser Spain) For each sample thekinematics was assessed randomly at ten different fields Theparameters such as curvilinear velocity (VCL) straight line
velocity (VSL) average path velocity (VAP) amplitude of lat-eral head displacement (ALH) linearity (LIN) straightness(STR) wobble (WOB) and beat cross frequency (BCF) wereanalyzed
7 Conclusions
In conclusion we have reported efficient and environmen-tally benignmethodologies for the synthesis of 3-[4-(1H-benzo[d]imidazol-2-yl)-1-phenyl-1H-pyrazol-3-yl]-2H-chromen-2-one derivatives 5andashx by using thermal and by microwaveirradiation under neat conditions in presence of ethanol Thereactions carried out under microwave irradiation affordedbenzimidazoles in short period of time with excellent yieldsHence this methodology would make an interesting strategyfor the synthesis of various substituted coumarin pyrazolebenzimidazoles Also compounds 5g 5h and 5k exhibitedimpressive sperm motility enhancing property whereas 5khas spermatozoa longevity property
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to University Scientific Instrumen-tation Centre (USIC) Karnatak University Dharwad forprovidingmass and IR spectral dataThe authors are thankfulto Indian Institute of Science (IISc) Bengaluru for carryingout NMR analyses and to UGC New Delhi for the financialassistance under UPE program vide no [F No 14-22012(NSPE) dated 22-01-2013] One of the authors (MNK)acknowledges University Grants Commission (UGC) NewDelhi for fellowship under UGC-UPE program
References
[1] P P Kattimani R R Kamble and G Y Meti ldquoExpedientsynthesis of benzimidazoles using amidesrdquo RSC Advances vol5 no 37 pp 29447ndash29455 2015
[2] L Li C Yee and J A Beavo ldquoCD3- and CD28-dependentinduction of PDE7 required for T cell activationrdquo Science vol283 no 5403 pp 848ndash849 1999
[3] T P Dousa ldquoCyclic-3101584051015840-nucleotide phosphodiesteraseisozymes in cell biology and pathophysiology of the kidneyrdquoKidney International vol 55 no 1 pp 29ndash62 1999
[4] C Pfister N Bennett F Bruckert et al ldquoInteractions of a G-protein with its effector transducin and cGMP phosphodi-esterase in retinal rodsrdquo Cell Signaling vol 5 no 3 pp 235ndash2411993
[5] S L C Jin F J Richard W-P Kuo A J DrsquoErcole andM Conti ldquoImpaired growth and fertility of cAMP-specificphosphodiesterase PDE4D-deficient micerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 21 pp 11998ndash12003 1999
[6] S H Francis I V Turko and J D Corbin ldquoCyclic nucleotidephosphodiesterases relating structure and functionrdquoProgress in
International Journal of Medicinal Chemistry 15
Nucleic Acid Research and Molecular Biology vol 65 pp 1ndash522000
[7] J M Hetman N Robas R Baxendale et al ldquoCloning and char-acterization of two splice variants of human phosphodiesterase11Ardquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 23 pp 12891ndash12895 2000
[8] K Yuasa T Ohgaru M Asahina and K Omori ldquoIdentificationof rat cyclic nucleotide phosphodiesterase 11A (PDE11A) com-parison of rat and human PDE11A splicing variantsrdquo EuropeanJournal of Biochemistry vol 268 no 16 pp 4440ndash4448 2001
[9] S K Sahu A Mishra and R K Behera ldquoSynthesis of thiazolebenzothiazole oxadiazole thiadiazole triazole and thiazolidi-none incorporated coumarinsrdquo Indian Journal of HeterocyclicChemistry vol 6 no 2 pp 91ndash94 1996
[10] G Roma M Di Braccio G Grossi et al ldquoSynthesis and in vitroantiplatelet activity of new 4-(1-piperazinyl) coumarin deriva-tives Human platelet phosphodiesterase 3 inhibitory propertiesof the two most effective compounds described and molecularmodeling study on their interactions with phosphodiesterase3A catalytic siterdquo Journal of Medicinal Chemistry vol 50 no 12pp 2886ndash2895 2007
[11] T Shyamsunder Reddy K Shiva Kumar C L T Medaet al ldquoConformationally restricted novel pyrazole deriva-tives synthesis of 18-disubstituted 55-dimethyl-45-dihydro-1H-benzo[g]indazoles as a new class of PDE4 inhibitorsrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 9 pp3248ndash3255 2012
[12] A Chino N Masuda Y Amano et al ldquoNovel benzimidazolederivatives as phosphodiesterase 10A (PDE10A) inhibitors withimproved metabolic stabilityrdquo Bioorganic and Medicinal Chem-istry vol 22 no 13 pp 3515ndash3526 2014
[13] H Yang FNMurigi ZWang J Li H Jin andZ Tu ldquoSynthesisand in vitro characterization of cinnoline and benzimidazoleanalogues as phosphodiesterase 10A inhibitorsrdquo Bioorganic andMedicinal Chemistry Letters vol 25 pp 919ndash924 2015
[14] J Nawrot-Modranka E Nawrot and J Graczyk ldquoIn vivo antitu-mor in vitro antibacterial activity and alkylating properties ofphosphorohydrazine derivatives of coumarin and chromonerdquoEuropean Journal of Medicinal Chemistry vol 41 no 11 pp1301ndash1309 2006
[15] A A Bekhit and T Abdel-Aziem ldquoDesign synthesis andbiological evaluation of some pyrazole derivatives as anti-inflammatory-antimicrobial agentsrdquo Bioorganic and MedicinalChemistry vol 12 no 8 pp 1935ndash1945 2004
[16] T Devji C Reddy CWoo S Awale S Kadota and D Carrico-Moniz ldquoPancreatic anticancer activity of a novel geranylgerany-lated coumarin derivativerdquo Bioorganic andMedicinal ChemistryLetters vol 21 no 19 pp 5770ndash5773 2011
[17] I Kostova I Manolov I Nicolova S Konstantinov andM Karaivanova ldquoNew lanthanide complexes of 4-methyl-7-hydroxycoumarin and their pharmacological activityrdquo Euro-pean Journal of Medicinal Chemistry vol 36 no 4 pp 339ndash3472001
[18] M Ghate R A Kusanur and M V Kulkarni ldquoSynthesis andin vivo analgesic and anti-inflammatory activity of some bi-heterocyclic coumarin derivativesrdquo European Journal of Medic-inal Chemistry vol 40 no 9 pp 882ndash887 2005
[19] PM RonadMN Noolvi S Sapkal S Dharbhamulla andV SMaddi ldquoSynthesis and antimicrobial activity of 7-(2-substitutedphenylthiazolidinyl)-benzopyran-2-one derivativesrdquo EuropeanJournal of Medicinal Chemistry vol 45 no 1 pp 85ndash89 2010
[20] GMelagraki A Afantitis O Igglessi-Markopoulou et al ldquoSyn-thesis and evaluation of the antioxidant and anti-inflammatoryactivity of novel coumarin-3-aminoamides and their alpha-lipoic acid adductsrdquo European Journal of Medicinal Chemistryvol 44 no 7 pp 3020ndash3026 2009
[21] J C Trivedi J B Bariwal K D Upadhyay et al ldquoImprovedand rapid synthesis of new coumarinyl chalcone derivatives andtheir antiviral activityrdquo Tetrahedron Letters vol 48 no 48 pp8472ndash8474 2007
[22] S Vilar E Quezada L Santana et al ldquoDesign synthe-sis and vasorelaxant and platelet antiaggregatory activitiesof coumarinndashresveratrol hybridsrdquo Bioorganic and MedicinalChemistry Letters vol 16 no 2 pp 257ndash261 2006
[23] W Hamaguchi N Masuda M Isomura et al ldquoDesign and syn-thesis of novel benzimidazole derivatives as phosphodiesterase10A inhibitors with reduced CYP1A2 inhibitionrdquo Bioorganicand Medicinal Chemistry vol 21 no 24 pp 7612ndash7623 2013
[24] A Dore B Asproni A Scampuddu et al ldquoSynthesis and SARstudy of novel tricyclic pyrazoles as potent phosphodiesterase10A inhibitorsrdquo European Journal of Medicinal Chemistry vol84 pp 181ndash193 2014
[25] S V Laxmi B S Kuarm and B Rajitha ldquoSynthesis and anti-microbial activity of coumarin pyrazole pyrimidine 246(1H3H5H)triones and thioxopyrimidine46(1H5H)dionesrdquo Me-dicinal Chemistry Research vol 22 no 2 pp 768ndash774 2013
[26] G Kalthur S Raj AThiyagarajan S Kumar P Kumar and S KAdiga ldquoVitamin E supplementation in semen-freezingmediumimproves the motility and protects sperm from freeze-thaw-induced DNA damagerdquo Fertility and Sterility vol 95 no 3 pp1149ndash1151 2011
[27] J Tesarik C Mendoza and A Carreras ldquoEffects of phosphodi-esterase inhibitors caffeine and pentoxifylline on spontaneousand stimulus-induced acrosome reactions in human spermrdquoFertility and Sterility vol 58 no 6 pp 1185ndash1190 1992
[28] G Kalthur S R Salian F Keyvanifard et al ldquoSupplementationof biotin to sperm preparation medium increases the motilityand longevity in cryopreserved human spermatozoardquo Journalof Assisted Reproduction and Genetics vol 29 no 7 pp 631ndash6352012
[29] A E Calogero S Fishel J Hall et al ldquoCorrelation betweenintracellular cAMP content kinematic parameters and hyper-activation of human spermatozoa after incubation with pentox-ifyllinerdquo Human Reproduction vol 13 no 4 pp 911ndash915 1998
[30] R Ain K Uma Devi S Shivaji and P B Seshagiri ldquoPen-toxifylline-stimulated capacitation and acrosome reaction inhamster spermatozoa involvement of intracellular signallingmoleculesrdquo Molecular Human Reproduction vol 5 no 7 pp618ndash626 1999
[31] M Bajpai andG F Doncel ldquoInvolvement of tyrosine kinase andcAMP-dependent kinase cross-talk in the regulation of humanspermmotilityrdquo Reproduction vol 126 no 2 pp 183ndash195 2003
[32] R Yunes P Fernandez G F Doncel and A A Acosta ldquoCyclicnucleotide phosphodiesterase inhibition increases tyrosinephosphorylation and hypermotility in normal and pathologicalhuman spermatozoardquo Biocell vol 29 no 3 pp 287ndash293 2005
[33] F Dimitriadis D Giannakis N Pardalidis et al ldquoEffectsof phosphodiesterase 5 inhibitors on sperm parameters andfertilizing capacityrdquo Asian Journal of Andrology vol 10 no 1pp 115ndash133 2008
[34] A P Kotdawala S Kumar S R Salian et al ldquoAddition of zincto human ejaculate prior to cryopreservation prevents freeze-thaw-induced DNA damage and preserves sperm functionrdquo
16 International Journal of Medicinal Chemistry
Journal of Assisted Reproduction and Genetics vol 29 no 12 pp1447ndash1453 2012
[35] S Mutalik S R Salian K Avadhani et al ldquoLiposome encapsu-lated soy lecithin and cholesterol can efficiently replace chickenegg yolk in human semen cryopreservation mediumrdquo SystemsBiology in Reproductive Medicine vol 60 no 3 pp 183ndash1882014
31 Effect on Motility of Frozen-Thawed Human SpermatozoaTo assess the effect of title compounds on themotility patternhuman spermatozoa subjected to freeze-thaw process wasused It is well documented that freeze-thaw process canhave detrimental effect on the motility and survival of thespermatozoa [26] Therefore they can be an excellent modelto study the effect of any test compounds on themotility [27]
In the present study we assessed the motility patternin spermatozoa processed with media containing variouscompounds manually at 1 4 and 24 h after incubationin vitro Three arbitrary doses were selected (05 10 and50 120583gmL) for each compound to assess their effect on spermmotility Pentoxifylline (PTF) which is known to enhancethe human sperm motility by elevating intracellular cyclicadenosine monophosphate (cAMP) level [27] was taken asa positive control Based on the previous report PTF at 1mMconcentration was used [28] Since the test compounds werenot soluble in water dimethyl sulfoxide (DMSO 005) wasused as vehicle control
The sperm motility enhancement property of the com-pounds was categorized as below
(a) Marginal which resulted in lt5 increase in themotility (b) Good which resulted in 5ndash10 increase inmotility (c) Excellent which resulted in gt10 increase inmotility (d) Poor where there was no enhancement inmotility
At 1 h after the in vitro incubation the majority of thecompounds had marginal effect on the sperm motility com-pared to control At this interval PTF had higher percentageof total and progressivelymotile spermatozoa compared to allthe test compounds A large number of studies have shownthat PTF can trigger the motility in fresh or frozen-thawedspermatozoa as early as 1 h [29] even though the majorityof the studies indicate that this effect is mainly due to theelevated cAMP level in the spermatozoa [30] it can also bemediated through tyrosine phosphorylation [31]
Compounds 5a 5b 5c 5d 5e 5g 5h and 5k inducedmarginal enhancement in total motility while compounds5a 5b 5c and 5j resulted in similar effect on progressivemotility (Tables 2(a) and 2(b)) A 5ndash10 increase in totalmotility was induced by compound 5j However with respectto progressive motility compounds 5d 5e 5f and 5k cameunder compounds with good effect on motility More than10 increase in progressive motility was observed only forcompound 5g (at 05 120583gmL concentration) and compound5h (at 5 120583gmL concentration)
A time-dependent decrease in total and progressivemotility was observed in all the groups However the declinewasmore evident at 24 h intervalThis phenomenon has beendocumented by earlier studies [28] These changes may berelated to the oxidative stress induced by in vitro cultureconditions Spermatozoa are highly susceptible to reactiveoxygen species (ROS) due to high polyunsaturated fatty acidcontent in their plasma membrane and negligible amount ofcytoplasmic antioxidants
At 4 h interval compounds 5e 5f and 5g enhancedthe progressive motility of spermatozoa by 5ndash10 while
compound 5h with two chloro substituents on coumarinand benzimidazole resulted in almost 15 increase in theprogressive motility However the effect was lower than thatof pentoxifylline The rest of the compounds (5a 5b 5c5f and 5j) induced a marginal increase in motility exceptin compound 5e in which the motility was lower than thecontrol group
At 24 h interval the majority of the compounds (5a5b 5d 5f 5h and 5j) had marginally higher percentageof motility or poor motility compounds (5c 5e and 5g)compared to control Interestingly compound 5k with chlorosubstituent on coumarin and bromo substitution on ben-zimidazole had higher percentage of motile spermatozoacompared to control (14 higher) and even PTF (9 higher)This indicates that compound5k canhelp in prolonging the invitro survival of spermatozoa which has significant beneficialrole in assisted reproductive technologies such as in vitrofertilization Previous studies have shown that even thoughPTF has triggering effect on themotility it induces prematureacrosome reaction and drastic decrease in motility at laterintervals In this context the compounds which increasethe motility and longevity of spermatozoa under in vitroconditions are of clinical relevance
Based on the manual motility assessment we observedthat compounds 5d 5f 5g 5h and 5k have considerablebeneficial role in spermmotilityTherefore these compoundswere further assessed for their effect on the kinematicsof spermatozoa at 1 h However there was no significantdifference in the kinematics of spermatozoa compared tocontrol and PTF (Table 3) In conclusion the newly synthe-sized coumarin derivatives have sperm motility enhancingproperty However in this preliminary screening we havetaken three arbitrary doses to assess their effect on motilityCompounds 5g 5h and 5k show considerable beneficialeffect which can be of clinical significance where spermmotility enhancement is achieved under in vitro condi-tions Compounds 5g and 5h triggered the motility at earlyintervals which was similar to the PTF while compound5k improved the longevity of the spermatozoa Since thecoumarin is known to have PDE inhibitory function themotility enhancement could be due to the elevated cAMPlevel in sperm However further detailed studies are essentialto understand the mechanism of action of these compoundsand their clinical utility
4 Experimental
All the chemicals and reagents were purchased from theMerck and Aldrich chemical suppliers Melting points (mp)were determined in open capillaries and are uncorrectedThe IR spectra were recorded on a Nicolet Impact 410 FTIR spectrometer using KBr pellets (range 4000ndash500 cmminus1)The 1HNMR spectra were recorded at 400MHz on BrukerAvance FT NMR spectrometer in DMSO-d
6solvent with
TMS as internal standard 13CNMR spectra were recordedat 100MHz on Bruker Avance FT NMR spectrometer inDMSO-d
6solvent with TMS as internal standard The mass
spectra were recorded on Shimadzu GC-MS operating at70 eV Thin-layer chromatography (TLC) was performed on
International Journal of Medicinal Chemistry 11
Table 2 Effect of supplementation of coumarin derivatives (5andashj) to sperm wash medium on the motility pattern in frozen-thawed humanspermatozoa at various time intervals
(a)
Compounds Time (h)Total motility (mean plusmn SE of percentage)
020mm Aluchrosep silica gel 60 F254 plates (SD FineMumbai) Microwave irradiation experiments were carriedout using CEMDiscover SPMicrowave Synthesizer equippedwith IR sensor to monitor reaction temperatures
5 General Procedures for the Preparation ofTitle Compounds 5andashx
3-(2-Oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-car-baldehydes (3andashd 1 mmol) were prepared from 3-acetyl-coumarin (1andashd 1 mmol) phenylhydrazine (1mmol) andthe intermediate (Schiff base) (2andashd 1 mmol) followedby reaction with POCl
3in DMF by Vilsmeier Haack for-
mylation strategies [25] The target compounds 5andashxwere then achieved by stirring substituted carbaldehyde(3andashd 1 mmol) with different o-arylenediamine (4andashf1 mmol) under reflux for 4-5 hrs After completion (TLChexane ethylacetate 7 3) of the reaction the solid obtainedwas filtered washed with small quantity of ethanol anddried Further it was recrystallized in methanol to get thepure compound (5andashx)
51 Microwave Assisted Procedure for the Preparation of Com-pounds 5andashx A mixture of 3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde (3andashd 1 mmol) and o-phenylenediamine (4andashf 1 mmol) taken in ethanol (5mL)was introduced into a CEM microwave reaction vesselequipped with magnetic stirrer The reaction mixture was
prestirred for 1min at room temperature and irradiated at180W (150∘C) for about 6ndash8min (TLC using hexane ethylacetate (7 3 drops) as eluent) The reaction mixture wasthen quenched into crushed ice and the crude product wasfiltered washed and dried Recrystallization from methanolgave pure crystals of the compounds 5andashx
(Spectral as well as elemental analyses data are providedas electronic supplementary file in Supplementary Materialavailable online at httpdxdoiorg10115520169890630)
6 Assessment of Phosphodiesterase InhibitionUsing Human Spermatozoa as Model
Earlier studies have shown that PDE inhibitors can enhancethe spermmotility [32 33] Based on this in the present studywe have used an indirect approach to assess the inhibitoryeffect of the coumarin derivatives on PDE by assessing spermmotility pattern
61 Preparation of Test Solution Stock solution of the testcompounds (1mgmL) was prepared by dissolving them indimethyl sulfoxide (DMSO Sigma Cat number D5879)The stock solution was then diluted with Earlrsquos BalancedSalt Solution (EBSS) supplemented with 01 bovine serumalbumin (BSA) in a ratio of 1 200 1 1000 and 1 2000 to getworking solutions of 5 1 and 05 120583gmL concentrations Theworking solutions were preincubated at 37∘C and 5 CO
2
prior to use
International Journal of Medicinal Chemistry 13
Table 3 Effect of various coumarin derivatives on kinematics of frozen-thawed spermatozoa at one hour after incubation in vitro
Parameters Compounds Concentrations of compounds in 120583gmL VC PTFC 0 05 5 (005 DMSO) (1mM)
62 Study Subjects Infertile men who attended AndrologyLaboratory Kasturba Medical College Manipal UniversityManipal during the period of June-July 2015 for routinesemen evaluation were included in the study The subjects
with a sexual abstinence of 3ndash5 days were asked to providethe semen samples by masturbation in a sterile containerFollowing liquefaction the semen parameters were assessedas described by Kotdawala et al [34]The study was approved
14 International Journal of Medicinal Chemistry
by the Institutional Ethics Committee of Kasturba HospitalKasturba Medical College Manipal University (IEC number1552015)
63 Cryopreservation and Thawing The liquefied semensamples were cryopreserved by rapid freezing method asdescribed previously [34] with minor modifications Brieflythe semen samples were mixed with equal volume of freezingmedium (Sperm Freeze FertiPro Cat number 0344) in acryovial (Thermoscientific Nunc Cat number 138627) andkept at 4∘C for 10min in liquid nitrogen (LN2) vapor phasefor 5min and then finally plunged into liquid nitrogenThe samples were thawed after 1 week by rapid thawingmethod by placing themat 37∘C for 5minThe cryoprotectantmedium was completely removed by mixing the spermsuspension with EBSS medium and centrifuging at 1000 rpmfor 8min The resultant pellet was resuspended with freshEBSS medium and used for further analysis
64 Sample Preparation To screen the compounds withenhancing effect on themotility of spermatozoa under in vitroconditions the frozen-thawed semen samples were washedby mixing with equal volume of preincubated Earlrsquos BalancedSalt Solution (EBSS Sigma Cat number E2888) followed bycentrifugation at 1800 rpm for 8min The pellet was thengently mixed with 1mL of EBSS media containing 01bovine serum albumin (BSA Sigma Cat number A3311)and equally divided into control vehicle control and testgroups (with average sperm density of 5 times 104 spermatozoain each group) The motile sperm were extracted by swimup method as described earlier [35] The sperm suspensionwas centrifuged at 1200 rpm for 8min and the resultantpellet was overlaidwith EBSSmedium containing various testcompoundsThe sperm suspensions were incubated for 1 h at37∘Cand 5CO
2afterwhich themotile spermwere collected
from the supernatant
65 Motility Assessment Spermatozoa in the supernatantfraction were assessed for their motility patterns at 1 4 and24 h after incubation under light microscope as describedearlier [27] Sperm suspension was (10 120583L droplet) placed ona clean microscope slide and a coverslip was placed over itSpermatozoa with progressive motility and nonprogressivemotility and immotile spermatozoa were scored separatelyfrom a total of 200 spermatozoa in random fields at 400xmagnification
66 Sperm Kinematics Using Computer Assisted Semen Anal-ysis (CASA) The motion characteristics in the spermatozoawere assessed at 1 4 and 24 h after incubation using CASAsystem (ISAS Spain) Briefly 5 120583L of sperm suspension wasplaced on a clean microscope slide prewarmed at 37∘C Thesperm suspensionwas coveredwith prewarmed cover slip (22times 22mm) and observed under microscope (negative phasecontrast 10x objectives Proiser Spain) For each sample thekinematics was assessed randomly at ten different fields Theparameters such as curvilinear velocity (VCL) straight line
velocity (VSL) average path velocity (VAP) amplitude of lat-eral head displacement (ALH) linearity (LIN) straightness(STR) wobble (WOB) and beat cross frequency (BCF) wereanalyzed
7 Conclusions
In conclusion we have reported efficient and environmen-tally benignmethodologies for the synthesis of 3-[4-(1H-benzo[d]imidazol-2-yl)-1-phenyl-1H-pyrazol-3-yl]-2H-chromen-2-one derivatives 5andashx by using thermal and by microwaveirradiation under neat conditions in presence of ethanol Thereactions carried out under microwave irradiation affordedbenzimidazoles in short period of time with excellent yieldsHence this methodology would make an interesting strategyfor the synthesis of various substituted coumarin pyrazolebenzimidazoles Also compounds 5g 5h and 5k exhibitedimpressive sperm motility enhancing property whereas 5khas spermatozoa longevity property
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to University Scientific Instrumen-tation Centre (USIC) Karnatak University Dharwad forprovidingmass and IR spectral dataThe authors are thankfulto Indian Institute of Science (IISc) Bengaluru for carryingout NMR analyses and to UGC New Delhi for the financialassistance under UPE program vide no [F No 14-22012(NSPE) dated 22-01-2013] One of the authors (MNK)acknowledges University Grants Commission (UGC) NewDelhi for fellowship under UGC-UPE program
References
[1] P P Kattimani R R Kamble and G Y Meti ldquoExpedientsynthesis of benzimidazoles using amidesrdquo RSC Advances vol5 no 37 pp 29447ndash29455 2015
[2] L Li C Yee and J A Beavo ldquoCD3- and CD28-dependentinduction of PDE7 required for T cell activationrdquo Science vol283 no 5403 pp 848ndash849 1999
[3] T P Dousa ldquoCyclic-3101584051015840-nucleotide phosphodiesteraseisozymes in cell biology and pathophysiology of the kidneyrdquoKidney International vol 55 no 1 pp 29ndash62 1999
[4] C Pfister N Bennett F Bruckert et al ldquoInteractions of a G-protein with its effector transducin and cGMP phosphodi-esterase in retinal rodsrdquo Cell Signaling vol 5 no 3 pp 235ndash2411993
[5] S L C Jin F J Richard W-P Kuo A J DrsquoErcole andM Conti ldquoImpaired growth and fertility of cAMP-specificphosphodiesterase PDE4D-deficient micerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 21 pp 11998ndash12003 1999
[6] S H Francis I V Turko and J D Corbin ldquoCyclic nucleotidephosphodiesterases relating structure and functionrdquoProgress in
International Journal of Medicinal Chemistry 15
Nucleic Acid Research and Molecular Biology vol 65 pp 1ndash522000
[7] J M Hetman N Robas R Baxendale et al ldquoCloning and char-acterization of two splice variants of human phosphodiesterase11Ardquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 23 pp 12891ndash12895 2000
[8] K Yuasa T Ohgaru M Asahina and K Omori ldquoIdentificationof rat cyclic nucleotide phosphodiesterase 11A (PDE11A) com-parison of rat and human PDE11A splicing variantsrdquo EuropeanJournal of Biochemistry vol 268 no 16 pp 4440ndash4448 2001
[9] S K Sahu A Mishra and R K Behera ldquoSynthesis of thiazolebenzothiazole oxadiazole thiadiazole triazole and thiazolidi-none incorporated coumarinsrdquo Indian Journal of HeterocyclicChemistry vol 6 no 2 pp 91ndash94 1996
[10] G Roma M Di Braccio G Grossi et al ldquoSynthesis and in vitroantiplatelet activity of new 4-(1-piperazinyl) coumarin deriva-tives Human platelet phosphodiesterase 3 inhibitory propertiesof the two most effective compounds described and molecularmodeling study on their interactions with phosphodiesterase3A catalytic siterdquo Journal of Medicinal Chemistry vol 50 no 12pp 2886ndash2895 2007
[11] T Shyamsunder Reddy K Shiva Kumar C L T Medaet al ldquoConformationally restricted novel pyrazole deriva-tives synthesis of 18-disubstituted 55-dimethyl-45-dihydro-1H-benzo[g]indazoles as a new class of PDE4 inhibitorsrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 9 pp3248ndash3255 2012
[12] A Chino N Masuda Y Amano et al ldquoNovel benzimidazolederivatives as phosphodiesterase 10A (PDE10A) inhibitors withimproved metabolic stabilityrdquo Bioorganic and Medicinal Chem-istry vol 22 no 13 pp 3515ndash3526 2014
[13] H Yang FNMurigi ZWang J Li H Jin andZ Tu ldquoSynthesisand in vitro characterization of cinnoline and benzimidazoleanalogues as phosphodiesterase 10A inhibitorsrdquo Bioorganic andMedicinal Chemistry Letters vol 25 pp 919ndash924 2015
[14] J Nawrot-Modranka E Nawrot and J Graczyk ldquoIn vivo antitu-mor in vitro antibacterial activity and alkylating properties ofphosphorohydrazine derivatives of coumarin and chromonerdquoEuropean Journal of Medicinal Chemistry vol 41 no 11 pp1301ndash1309 2006
[15] A A Bekhit and T Abdel-Aziem ldquoDesign synthesis andbiological evaluation of some pyrazole derivatives as anti-inflammatory-antimicrobial agentsrdquo Bioorganic and MedicinalChemistry vol 12 no 8 pp 1935ndash1945 2004
[16] T Devji C Reddy CWoo S Awale S Kadota and D Carrico-Moniz ldquoPancreatic anticancer activity of a novel geranylgerany-lated coumarin derivativerdquo Bioorganic andMedicinal ChemistryLetters vol 21 no 19 pp 5770ndash5773 2011
[17] I Kostova I Manolov I Nicolova S Konstantinov andM Karaivanova ldquoNew lanthanide complexes of 4-methyl-7-hydroxycoumarin and their pharmacological activityrdquo Euro-pean Journal of Medicinal Chemistry vol 36 no 4 pp 339ndash3472001
[18] M Ghate R A Kusanur and M V Kulkarni ldquoSynthesis andin vivo analgesic and anti-inflammatory activity of some bi-heterocyclic coumarin derivativesrdquo European Journal of Medic-inal Chemistry vol 40 no 9 pp 882ndash887 2005
[19] PM RonadMN Noolvi S Sapkal S Dharbhamulla andV SMaddi ldquoSynthesis and antimicrobial activity of 7-(2-substitutedphenylthiazolidinyl)-benzopyran-2-one derivativesrdquo EuropeanJournal of Medicinal Chemistry vol 45 no 1 pp 85ndash89 2010
[20] GMelagraki A Afantitis O Igglessi-Markopoulou et al ldquoSyn-thesis and evaluation of the antioxidant and anti-inflammatoryactivity of novel coumarin-3-aminoamides and their alpha-lipoic acid adductsrdquo European Journal of Medicinal Chemistryvol 44 no 7 pp 3020ndash3026 2009
[21] J C Trivedi J B Bariwal K D Upadhyay et al ldquoImprovedand rapid synthesis of new coumarinyl chalcone derivatives andtheir antiviral activityrdquo Tetrahedron Letters vol 48 no 48 pp8472ndash8474 2007
[22] S Vilar E Quezada L Santana et al ldquoDesign synthe-sis and vasorelaxant and platelet antiaggregatory activitiesof coumarinndashresveratrol hybridsrdquo Bioorganic and MedicinalChemistry Letters vol 16 no 2 pp 257ndash261 2006
[23] W Hamaguchi N Masuda M Isomura et al ldquoDesign and syn-thesis of novel benzimidazole derivatives as phosphodiesterase10A inhibitors with reduced CYP1A2 inhibitionrdquo Bioorganicand Medicinal Chemistry vol 21 no 24 pp 7612ndash7623 2013
[24] A Dore B Asproni A Scampuddu et al ldquoSynthesis and SARstudy of novel tricyclic pyrazoles as potent phosphodiesterase10A inhibitorsrdquo European Journal of Medicinal Chemistry vol84 pp 181ndash193 2014
[25] S V Laxmi B S Kuarm and B Rajitha ldquoSynthesis and anti-microbial activity of coumarin pyrazole pyrimidine 246(1H3H5H)triones and thioxopyrimidine46(1H5H)dionesrdquo Me-dicinal Chemistry Research vol 22 no 2 pp 768ndash774 2013
[26] G Kalthur S Raj AThiyagarajan S Kumar P Kumar and S KAdiga ldquoVitamin E supplementation in semen-freezingmediumimproves the motility and protects sperm from freeze-thaw-induced DNA damagerdquo Fertility and Sterility vol 95 no 3 pp1149ndash1151 2011
[27] J Tesarik C Mendoza and A Carreras ldquoEffects of phosphodi-esterase inhibitors caffeine and pentoxifylline on spontaneousand stimulus-induced acrosome reactions in human spermrdquoFertility and Sterility vol 58 no 6 pp 1185ndash1190 1992
[28] G Kalthur S R Salian F Keyvanifard et al ldquoSupplementationof biotin to sperm preparation medium increases the motilityand longevity in cryopreserved human spermatozoardquo Journalof Assisted Reproduction and Genetics vol 29 no 7 pp 631ndash6352012
[29] A E Calogero S Fishel J Hall et al ldquoCorrelation betweenintracellular cAMP content kinematic parameters and hyper-activation of human spermatozoa after incubation with pentox-ifyllinerdquo Human Reproduction vol 13 no 4 pp 911ndash915 1998
[30] R Ain K Uma Devi S Shivaji and P B Seshagiri ldquoPen-toxifylline-stimulated capacitation and acrosome reaction inhamster spermatozoa involvement of intracellular signallingmoleculesrdquo Molecular Human Reproduction vol 5 no 7 pp618ndash626 1999
[31] M Bajpai andG F Doncel ldquoInvolvement of tyrosine kinase andcAMP-dependent kinase cross-talk in the regulation of humanspermmotilityrdquo Reproduction vol 126 no 2 pp 183ndash195 2003
[32] R Yunes P Fernandez G F Doncel and A A Acosta ldquoCyclicnucleotide phosphodiesterase inhibition increases tyrosinephosphorylation and hypermotility in normal and pathologicalhuman spermatozoardquo Biocell vol 29 no 3 pp 287ndash293 2005
[33] F Dimitriadis D Giannakis N Pardalidis et al ldquoEffectsof phosphodiesterase 5 inhibitors on sperm parameters andfertilizing capacityrdquo Asian Journal of Andrology vol 10 no 1pp 115ndash133 2008
[34] A P Kotdawala S Kumar S R Salian et al ldquoAddition of zincto human ejaculate prior to cryopreservation prevents freeze-thaw-induced DNA damage and preserves sperm functionrdquo
16 International Journal of Medicinal Chemistry
Journal of Assisted Reproduction and Genetics vol 29 no 12 pp1447ndash1453 2012
[35] S Mutalik S R Salian K Avadhani et al ldquoLiposome encapsu-lated soy lecithin and cholesterol can efficiently replace chickenegg yolk in human semen cryopreservation mediumrdquo SystemsBiology in Reproductive Medicine vol 60 no 3 pp 183ndash1882014
Table 2 Effect of supplementation of coumarin derivatives (5andashj) to sperm wash medium on the motility pattern in frozen-thawed humanspermatozoa at various time intervals
(a)
Compounds Time (h)Total motility (mean plusmn SE of percentage)
020mm Aluchrosep silica gel 60 F254 plates (SD FineMumbai) Microwave irradiation experiments were carriedout using CEMDiscover SPMicrowave Synthesizer equippedwith IR sensor to monitor reaction temperatures
5 General Procedures for the Preparation ofTitle Compounds 5andashx
3-(2-Oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-car-baldehydes (3andashd 1 mmol) were prepared from 3-acetyl-coumarin (1andashd 1 mmol) phenylhydrazine (1mmol) andthe intermediate (Schiff base) (2andashd 1 mmol) followedby reaction with POCl
3in DMF by Vilsmeier Haack for-
mylation strategies [25] The target compounds 5andashxwere then achieved by stirring substituted carbaldehyde(3andashd 1 mmol) with different o-arylenediamine (4andashf1 mmol) under reflux for 4-5 hrs After completion (TLChexane ethylacetate 7 3) of the reaction the solid obtainedwas filtered washed with small quantity of ethanol anddried Further it was recrystallized in methanol to get thepure compound (5andashx)
51 Microwave Assisted Procedure for the Preparation of Com-pounds 5andashx A mixture of 3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde (3andashd 1 mmol) and o-phenylenediamine (4andashf 1 mmol) taken in ethanol (5mL)was introduced into a CEM microwave reaction vesselequipped with magnetic stirrer The reaction mixture was
prestirred for 1min at room temperature and irradiated at180W (150∘C) for about 6ndash8min (TLC using hexane ethylacetate (7 3 drops) as eluent) The reaction mixture wasthen quenched into crushed ice and the crude product wasfiltered washed and dried Recrystallization from methanolgave pure crystals of the compounds 5andashx
(Spectral as well as elemental analyses data are providedas electronic supplementary file in Supplementary Materialavailable online at httpdxdoiorg10115520169890630)
6 Assessment of Phosphodiesterase InhibitionUsing Human Spermatozoa as Model
Earlier studies have shown that PDE inhibitors can enhancethe spermmotility [32 33] Based on this in the present studywe have used an indirect approach to assess the inhibitoryeffect of the coumarin derivatives on PDE by assessing spermmotility pattern
61 Preparation of Test Solution Stock solution of the testcompounds (1mgmL) was prepared by dissolving them indimethyl sulfoxide (DMSO Sigma Cat number D5879)The stock solution was then diluted with Earlrsquos BalancedSalt Solution (EBSS) supplemented with 01 bovine serumalbumin (BSA) in a ratio of 1 200 1 1000 and 1 2000 to getworking solutions of 5 1 and 05 120583gmL concentrations Theworking solutions were preincubated at 37∘C and 5 CO
2
prior to use
International Journal of Medicinal Chemistry 13
Table 3 Effect of various coumarin derivatives on kinematics of frozen-thawed spermatozoa at one hour after incubation in vitro
Parameters Compounds Concentrations of compounds in 120583gmL VC PTFC 0 05 5 (005 DMSO) (1mM)
62 Study Subjects Infertile men who attended AndrologyLaboratory Kasturba Medical College Manipal UniversityManipal during the period of June-July 2015 for routinesemen evaluation were included in the study The subjects
with a sexual abstinence of 3ndash5 days were asked to providethe semen samples by masturbation in a sterile containerFollowing liquefaction the semen parameters were assessedas described by Kotdawala et al [34]The study was approved
14 International Journal of Medicinal Chemistry
by the Institutional Ethics Committee of Kasturba HospitalKasturba Medical College Manipal University (IEC number1552015)
63 Cryopreservation and Thawing The liquefied semensamples were cryopreserved by rapid freezing method asdescribed previously [34] with minor modifications Brieflythe semen samples were mixed with equal volume of freezingmedium (Sperm Freeze FertiPro Cat number 0344) in acryovial (Thermoscientific Nunc Cat number 138627) andkept at 4∘C for 10min in liquid nitrogen (LN2) vapor phasefor 5min and then finally plunged into liquid nitrogenThe samples were thawed after 1 week by rapid thawingmethod by placing themat 37∘C for 5minThe cryoprotectantmedium was completely removed by mixing the spermsuspension with EBSS medium and centrifuging at 1000 rpmfor 8min The resultant pellet was resuspended with freshEBSS medium and used for further analysis
64 Sample Preparation To screen the compounds withenhancing effect on themotility of spermatozoa under in vitroconditions the frozen-thawed semen samples were washedby mixing with equal volume of preincubated Earlrsquos BalancedSalt Solution (EBSS Sigma Cat number E2888) followed bycentrifugation at 1800 rpm for 8min The pellet was thengently mixed with 1mL of EBSS media containing 01bovine serum albumin (BSA Sigma Cat number A3311)and equally divided into control vehicle control and testgroups (with average sperm density of 5 times 104 spermatozoain each group) The motile sperm were extracted by swimup method as described earlier [35] The sperm suspensionwas centrifuged at 1200 rpm for 8min and the resultantpellet was overlaidwith EBSSmedium containing various testcompoundsThe sperm suspensions were incubated for 1 h at37∘Cand 5CO
2afterwhich themotile spermwere collected
from the supernatant
65 Motility Assessment Spermatozoa in the supernatantfraction were assessed for their motility patterns at 1 4 and24 h after incubation under light microscope as describedearlier [27] Sperm suspension was (10 120583L droplet) placed ona clean microscope slide and a coverslip was placed over itSpermatozoa with progressive motility and nonprogressivemotility and immotile spermatozoa were scored separatelyfrom a total of 200 spermatozoa in random fields at 400xmagnification
66 Sperm Kinematics Using Computer Assisted Semen Anal-ysis (CASA) The motion characteristics in the spermatozoawere assessed at 1 4 and 24 h after incubation using CASAsystem (ISAS Spain) Briefly 5 120583L of sperm suspension wasplaced on a clean microscope slide prewarmed at 37∘C Thesperm suspensionwas coveredwith prewarmed cover slip (22times 22mm) and observed under microscope (negative phasecontrast 10x objectives Proiser Spain) For each sample thekinematics was assessed randomly at ten different fields Theparameters such as curvilinear velocity (VCL) straight line
velocity (VSL) average path velocity (VAP) amplitude of lat-eral head displacement (ALH) linearity (LIN) straightness(STR) wobble (WOB) and beat cross frequency (BCF) wereanalyzed
7 Conclusions
In conclusion we have reported efficient and environmen-tally benignmethodologies for the synthesis of 3-[4-(1H-benzo[d]imidazol-2-yl)-1-phenyl-1H-pyrazol-3-yl]-2H-chromen-2-one derivatives 5andashx by using thermal and by microwaveirradiation under neat conditions in presence of ethanol Thereactions carried out under microwave irradiation affordedbenzimidazoles in short period of time with excellent yieldsHence this methodology would make an interesting strategyfor the synthesis of various substituted coumarin pyrazolebenzimidazoles Also compounds 5g 5h and 5k exhibitedimpressive sperm motility enhancing property whereas 5khas spermatozoa longevity property
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to University Scientific Instrumen-tation Centre (USIC) Karnatak University Dharwad forprovidingmass and IR spectral dataThe authors are thankfulto Indian Institute of Science (IISc) Bengaluru for carryingout NMR analyses and to UGC New Delhi for the financialassistance under UPE program vide no [F No 14-22012(NSPE) dated 22-01-2013] One of the authors (MNK)acknowledges University Grants Commission (UGC) NewDelhi for fellowship under UGC-UPE program
References
[1] P P Kattimani R R Kamble and G Y Meti ldquoExpedientsynthesis of benzimidazoles using amidesrdquo RSC Advances vol5 no 37 pp 29447ndash29455 2015
[2] L Li C Yee and J A Beavo ldquoCD3- and CD28-dependentinduction of PDE7 required for T cell activationrdquo Science vol283 no 5403 pp 848ndash849 1999
[3] T P Dousa ldquoCyclic-3101584051015840-nucleotide phosphodiesteraseisozymes in cell biology and pathophysiology of the kidneyrdquoKidney International vol 55 no 1 pp 29ndash62 1999
[4] C Pfister N Bennett F Bruckert et al ldquoInteractions of a G-protein with its effector transducin and cGMP phosphodi-esterase in retinal rodsrdquo Cell Signaling vol 5 no 3 pp 235ndash2411993
[5] S L C Jin F J Richard W-P Kuo A J DrsquoErcole andM Conti ldquoImpaired growth and fertility of cAMP-specificphosphodiesterase PDE4D-deficient micerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 21 pp 11998ndash12003 1999
[6] S H Francis I V Turko and J D Corbin ldquoCyclic nucleotidephosphodiesterases relating structure and functionrdquoProgress in
International Journal of Medicinal Chemistry 15
Nucleic Acid Research and Molecular Biology vol 65 pp 1ndash522000
[7] J M Hetman N Robas R Baxendale et al ldquoCloning and char-acterization of two splice variants of human phosphodiesterase11Ardquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 23 pp 12891ndash12895 2000
[8] K Yuasa T Ohgaru M Asahina and K Omori ldquoIdentificationof rat cyclic nucleotide phosphodiesterase 11A (PDE11A) com-parison of rat and human PDE11A splicing variantsrdquo EuropeanJournal of Biochemistry vol 268 no 16 pp 4440ndash4448 2001
[9] S K Sahu A Mishra and R K Behera ldquoSynthesis of thiazolebenzothiazole oxadiazole thiadiazole triazole and thiazolidi-none incorporated coumarinsrdquo Indian Journal of HeterocyclicChemistry vol 6 no 2 pp 91ndash94 1996
[10] G Roma M Di Braccio G Grossi et al ldquoSynthesis and in vitroantiplatelet activity of new 4-(1-piperazinyl) coumarin deriva-tives Human platelet phosphodiesterase 3 inhibitory propertiesof the two most effective compounds described and molecularmodeling study on their interactions with phosphodiesterase3A catalytic siterdquo Journal of Medicinal Chemistry vol 50 no 12pp 2886ndash2895 2007
[11] T Shyamsunder Reddy K Shiva Kumar C L T Medaet al ldquoConformationally restricted novel pyrazole deriva-tives synthesis of 18-disubstituted 55-dimethyl-45-dihydro-1H-benzo[g]indazoles as a new class of PDE4 inhibitorsrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 9 pp3248ndash3255 2012
[12] A Chino N Masuda Y Amano et al ldquoNovel benzimidazolederivatives as phosphodiesterase 10A (PDE10A) inhibitors withimproved metabolic stabilityrdquo Bioorganic and Medicinal Chem-istry vol 22 no 13 pp 3515ndash3526 2014
[13] H Yang FNMurigi ZWang J Li H Jin andZ Tu ldquoSynthesisand in vitro characterization of cinnoline and benzimidazoleanalogues as phosphodiesterase 10A inhibitorsrdquo Bioorganic andMedicinal Chemistry Letters vol 25 pp 919ndash924 2015
[14] J Nawrot-Modranka E Nawrot and J Graczyk ldquoIn vivo antitu-mor in vitro antibacterial activity and alkylating properties ofphosphorohydrazine derivatives of coumarin and chromonerdquoEuropean Journal of Medicinal Chemistry vol 41 no 11 pp1301ndash1309 2006
[15] A A Bekhit and T Abdel-Aziem ldquoDesign synthesis andbiological evaluation of some pyrazole derivatives as anti-inflammatory-antimicrobial agentsrdquo Bioorganic and MedicinalChemistry vol 12 no 8 pp 1935ndash1945 2004
[16] T Devji C Reddy CWoo S Awale S Kadota and D Carrico-Moniz ldquoPancreatic anticancer activity of a novel geranylgerany-lated coumarin derivativerdquo Bioorganic andMedicinal ChemistryLetters vol 21 no 19 pp 5770ndash5773 2011
[17] I Kostova I Manolov I Nicolova S Konstantinov andM Karaivanova ldquoNew lanthanide complexes of 4-methyl-7-hydroxycoumarin and their pharmacological activityrdquo Euro-pean Journal of Medicinal Chemistry vol 36 no 4 pp 339ndash3472001
[18] M Ghate R A Kusanur and M V Kulkarni ldquoSynthesis andin vivo analgesic and anti-inflammatory activity of some bi-heterocyclic coumarin derivativesrdquo European Journal of Medic-inal Chemistry vol 40 no 9 pp 882ndash887 2005
[19] PM RonadMN Noolvi S Sapkal S Dharbhamulla andV SMaddi ldquoSynthesis and antimicrobial activity of 7-(2-substitutedphenylthiazolidinyl)-benzopyran-2-one derivativesrdquo EuropeanJournal of Medicinal Chemistry vol 45 no 1 pp 85ndash89 2010
[20] GMelagraki A Afantitis O Igglessi-Markopoulou et al ldquoSyn-thesis and evaluation of the antioxidant and anti-inflammatoryactivity of novel coumarin-3-aminoamides and their alpha-lipoic acid adductsrdquo European Journal of Medicinal Chemistryvol 44 no 7 pp 3020ndash3026 2009
[21] J C Trivedi J B Bariwal K D Upadhyay et al ldquoImprovedand rapid synthesis of new coumarinyl chalcone derivatives andtheir antiviral activityrdquo Tetrahedron Letters vol 48 no 48 pp8472ndash8474 2007
[22] S Vilar E Quezada L Santana et al ldquoDesign synthe-sis and vasorelaxant and platelet antiaggregatory activitiesof coumarinndashresveratrol hybridsrdquo Bioorganic and MedicinalChemistry Letters vol 16 no 2 pp 257ndash261 2006
[23] W Hamaguchi N Masuda M Isomura et al ldquoDesign and syn-thesis of novel benzimidazole derivatives as phosphodiesterase10A inhibitors with reduced CYP1A2 inhibitionrdquo Bioorganicand Medicinal Chemistry vol 21 no 24 pp 7612ndash7623 2013
[24] A Dore B Asproni A Scampuddu et al ldquoSynthesis and SARstudy of novel tricyclic pyrazoles as potent phosphodiesterase10A inhibitorsrdquo European Journal of Medicinal Chemistry vol84 pp 181ndash193 2014
[25] S V Laxmi B S Kuarm and B Rajitha ldquoSynthesis and anti-microbial activity of coumarin pyrazole pyrimidine 246(1H3H5H)triones and thioxopyrimidine46(1H5H)dionesrdquo Me-dicinal Chemistry Research vol 22 no 2 pp 768ndash774 2013
[26] G Kalthur S Raj AThiyagarajan S Kumar P Kumar and S KAdiga ldquoVitamin E supplementation in semen-freezingmediumimproves the motility and protects sperm from freeze-thaw-induced DNA damagerdquo Fertility and Sterility vol 95 no 3 pp1149ndash1151 2011
[27] J Tesarik C Mendoza and A Carreras ldquoEffects of phosphodi-esterase inhibitors caffeine and pentoxifylline on spontaneousand stimulus-induced acrosome reactions in human spermrdquoFertility and Sterility vol 58 no 6 pp 1185ndash1190 1992
[28] G Kalthur S R Salian F Keyvanifard et al ldquoSupplementationof biotin to sperm preparation medium increases the motilityand longevity in cryopreserved human spermatozoardquo Journalof Assisted Reproduction and Genetics vol 29 no 7 pp 631ndash6352012
[29] A E Calogero S Fishel J Hall et al ldquoCorrelation betweenintracellular cAMP content kinematic parameters and hyper-activation of human spermatozoa after incubation with pentox-ifyllinerdquo Human Reproduction vol 13 no 4 pp 911ndash915 1998
[30] R Ain K Uma Devi S Shivaji and P B Seshagiri ldquoPen-toxifylline-stimulated capacitation and acrosome reaction inhamster spermatozoa involvement of intracellular signallingmoleculesrdquo Molecular Human Reproduction vol 5 no 7 pp618ndash626 1999
[31] M Bajpai andG F Doncel ldquoInvolvement of tyrosine kinase andcAMP-dependent kinase cross-talk in the regulation of humanspermmotilityrdquo Reproduction vol 126 no 2 pp 183ndash195 2003
[32] R Yunes P Fernandez G F Doncel and A A Acosta ldquoCyclicnucleotide phosphodiesterase inhibition increases tyrosinephosphorylation and hypermotility in normal and pathologicalhuman spermatozoardquo Biocell vol 29 no 3 pp 287ndash293 2005
[33] F Dimitriadis D Giannakis N Pardalidis et al ldquoEffectsof phosphodiesterase 5 inhibitors on sperm parameters andfertilizing capacityrdquo Asian Journal of Andrology vol 10 no 1pp 115ndash133 2008
[34] A P Kotdawala S Kumar S R Salian et al ldquoAddition of zincto human ejaculate prior to cryopreservation prevents freeze-thaw-induced DNA damage and preserves sperm functionrdquo
16 International Journal of Medicinal Chemistry
Journal of Assisted Reproduction and Genetics vol 29 no 12 pp1447ndash1453 2012
[35] S Mutalik S R Salian K Avadhani et al ldquoLiposome encapsu-lated soy lecithin and cholesterol can efficiently replace chickenegg yolk in human semen cryopreservation mediumrdquo SystemsBiology in Reproductive Medicine vol 60 no 3 pp 183ndash1882014
020mm Aluchrosep silica gel 60 F254 plates (SD FineMumbai) Microwave irradiation experiments were carriedout using CEMDiscover SPMicrowave Synthesizer equippedwith IR sensor to monitor reaction temperatures
5 General Procedures for the Preparation ofTitle Compounds 5andashx
3-(2-Oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-car-baldehydes (3andashd 1 mmol) were prepared from 3-acetyl-coumarin (1andashd 1 mmol) phenylhydrazine (1mmol) andthe intermediate (Schiff base) (2andashd 1 mmol) followedby reaction with POCl
3in DMF by Vilsmeier Haack for-
mylation strategies [25] The target compounds 5andashxwere then achieved by stirring substituted carbaldehyde(3andashd 1 mmol) with different o-arylenediamine (4andashf1 mmol) under reflux for 4-5 hrs After completion (TLChexane ethylacetate 7 3) of the reaction the solid obtainedwas filtered washed with small quantity of ethanol anddried Further it was recrystallized in methanol to get thepure compound (5andashx)
51 Microwave Assisted Procedure for the Preparation of Com-pounds 5andashx A mixture of 3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde (3andashd 1 mmol) and o-phenylenediamine (4andashf 1 mmol) taken in ethanol (5mL)was introduced into a CEM microwave reaction vesselequipped with magnetic stirrer The reaction mixture was
prestirred for 1min at room temperature and irradiated at180W (150∘C) for about 6ndash8min (TLC using hexane ethylacetate (7 3 drops) as eluent) The reaction mixture wasthen quenched into crushed ice and the crude product wasfiltered washed and dried Recrystallization from methanolgave pure crystals of the compounds 5andashx
(Spectral as well as elemental analyses data are providedas electronic supplementary file in Supplementary Materialavailable online at httpdxdoiorg10115520169890630)
6 Assessment of Phosphodiesterase InhibitionUsing Human Spermatozoa as Model
Earlier studies have shown that PDE inhibitors can enhancethe spermmotility [32 33] Based on this in the present studywe have used an indirect approach to assess the inhibitoryeffect of the coumarin derivatives on PDE by assessing spermmotility pattern
61 Preparation of Test Solution Stock solution of the testcompounds (1mgmL) was prepared by dissolving them indimethyl sulfoxide (DMSO Sigma Cat number D5879)The stock solution was then diluted with Earlrsquos BalancedSalt Solution (EBSS) supplemented with 01 bovine serumalbumin (BSA) in a ratio of 1 200 1 1000 and 1 2000 to getworking solutions of 5 1 and 05 120583gmL concentrations Theworking solutions were preincubated at 37∘C and 5 CO
2
prior to use
International Journal of Medicinal Chemistry 13
Table 3 Effect of various coumarin derivatives on kinematics of frozen-thawed spermatozoa at one hour after incubation in vitro
Parameters Compounds Concentrations of compounds in 120583gmL VC PTFC 0 05 5 (005 DMSO) (1mM)
62 Study Subjects Infertile men who attended AndrologyLaboratory Kasturba Medical College Manipal UniversityManipal during the period of June-July 2015 for routinesemen evaluation were included in the study The subjects
with a sexual abstinence of 3ndash5 days were asked to providethe semen samples by masturbation in a sterile containerFollowing liquefaction the semen parameters were assessedas described by Kotdawala et al [34]The study was approved
14 International Journal of Medicinal Chemistry
by the Institutional Ethics Committee of Kasturba HospitalKasturba Medical College Manipal University (IEC number1552015)
63 Cryopreservation and Thawing The liquefied semensamples were cryopreserved by rapid freezing method asdescribed previously [34] with minor modifications Brieflythe semen samples were mixed with equal volume of freezingmedium (Sperm Freeze FertiPro Cat number 0344) in acryovial (Thermoscientific Nunc Cat number 138627) andkept at 4∘C for 10min in liquid nitrogen (LN2) vapor phasefor 5min and then finally plunged into liquid nitrogenThe samples were thawed after 1 week by rapid thawingmethod by placing themat 37∘C for 5minThe cryoprotectantmedium was completely removed by mixing the spermsuspension with EBSS medium and centrifuging at 1000 rpmfor 8min The resultant pellet was resuspended with freshEBSS medium and used for further analysis
64 Sample Preparation To screen the compounds withenhancing effect on themotility of spermatozoa under in vitroconditions the frozen-thawed semen samples were washedby mixing with equal volume of preincubated Earlrsquos BalancedSalt Solution (EBSS Sigma Cat number E2888) followed bycentrifugation at 1800 rpm for 8min The pellet was thengently mixed with 1mL of EBSS media containing 01bovine serum albumin (BSA Sigma Cat number A3311)and equally divided into control vehicle control and testgroups (with average sperm density of 5 times 104 spermatozoain each group) The motile sperm were extracted by swimup method as described earlier [35] The sperm suspensionwas centrifuged at 1200 rpm for 8min and the resultantpellet was overlaidwith EBSSmedium containing various testcompoundsThe sperm suspensions were incubated for 1 h at37∘Cand 5CO
2afterwhich themotile spermwere collected
from the supernatant
65 Motility Assessment Spermatozoa in the supernatantfraction were assessed for their motility patterns at 1 4 and24 h after incubation under light microscope as describedearlier [27] Sperm suspension was (10 120583L droplet) placed ona clean microscope slide and a coverslip was placed over itSpermatozoa with progressive motility and nonprogressivemotility and immotile spermatozoa were scored separatelyfrom a total of 200 spermatozoa in random fields at 400xmagnification
66 Sperm Kinematics Using Computer Assisted Semen Anal-ysis (CASA) The motion characteristics in the spermatozoawere assessed at 1 4 and 24 h after incubation using CASAsystem (ISAS Spain) Briefly 5 120583L of sperm suspension wasplaced on a clean microscope slide prewarmed at 37∘C Thesperm suspensionwas coveredwith prewarmed cover slip (22times 22mm) and observed under microscope (negative phasecontrast 10x objectives Proiser Spain) For each sample thekinematics was assessed randomly at ten different fields Theparameters such as curvilinear velocity (VCL) straight line
velocity (VSL) average path velocity (VAP) amplitude of lat-eral head displacement (ALH) linearity (LIN) straightness(STR) wobble (WOB) and beat cross frequency (BCF) wereanalyzed
7 Conclusions
In conclusion we have reported efficient and environmen-tally benignmethodologies for the synthesis of 3-[4-(1H-benzo[d]imidazol-2-yl)-1-phenyl-1H-pyrazol-3-yl]-2H-chromen-2-one derivatives 5andashx by using thermal and by microwaveirradiation under neat conditions in presence of ethanol Thereactions carried out under microwave irradiation affordedbenzimidazoles in short period of time with excellent yieldsHence this methodology would make an interesting strategyfor the synthesis of various substituted coumarin pyrazolebenzimidazoles Also compounds 5g 5h and 5k exhibitedimpressive sperm motility enhancing property whereas 5khas spermatozoa longevity property
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to University Scientific Instrumen-tation Centre (USIC) Karnatak University Dharwad forprovidingmass and IR spectral dataThe authors are thankfulto Indian Institute of Science (IISc) Bengaluru for carryingout NMR analyses and to UGC New Delhi for the financialassistance under UPE program vide no [F No 14-22012(NSPE) dated 22-01-2013] One of the authors (MNK)acknowledges University Grants Commission (UGC) NewDelhi for fellowship under UGC-UPE program
References
[1] P P Kattimani R R Kamble and G Y Meti ldquoExpedientsynthesis of benzimidazoles using amidesrdquo RSC Advances vol5 no 37 pp 29447ndash29455 2015
[2] L Li C Yee and J A Beavo ldquoCD3- and CD28-dependentinduction of PDE7 required for T cell activationrdquo Science vol283 no 5403 pp 848ndash849 1999
[3] T P Dousa ldquoCyclic-3101584051015840-nucleotide phosphodiesteraseisozymes in cell biology and pathophysiology of the kidneyrdquoKidney International vol 55 no 1 pp 29ndash62 1999
[4] C Pfister N Bennett F Bruckert et al ldquoInteractions of a G-protein with its effector transducin and cGMP phosphodi-esterase in retinal rodsrdquo Cell Signaling vol 5 no 3 pp 235ndash2411993
[5] S L C Jin F J Richard W-P Kuo A J DrsquoErcole andM Conti ldquoImpaired growth and fertility of cAMP-specificphosphodiesterase PDE4D-deficient micerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 21 pp 11998ndash12003 1999
[6] S H Francis I V Turko and J D Corbin ldquoCyclic nucleotidephosphodiesterases relating structure and functionrdquoProgress in
International Journal of Medicinal Chemistry 15
Nucleic Acid Research and Molecular Biology vol 65 pp 1ndash522000
[7] J M Hetman N Robas R Baxendale et al ldquoCloning and char-acterization of two splice variants of human phosphodiesterase11Ardquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 23 pp 12891ndash12895 2000
[8] K Yuasa T Ohgaru M Asahina and K Omori ldquoIdentificationof rat cyclic nucleotide phosphodiesterase 11A (PDE11A) com-parison of rat and human PDE11A splicing variantsrdquo EuropeanJournal of Biochemistry vol 268 no 16 pp 4440ndash4448 2001
[9] S K Sahu A Mishra and R K Behera ldquoSynthesis of thiazolebenzothiazole oxadiazole thiadiazole triazole and thiazolidi-none incorporated coumarinsrdquo Indian Journal of HeterocyclicChemistry vol 6 no 2 pp 91ndash94 1996
[10] G Roma M Di Braccio G Grossi et al ldquoSynthesis and in vitroantiplatelet activity of new 4-(1-piperazinyl) coumarin deriva-tives Human platelet phosphodiesterase 3 inhibitory propertiesof the two most effective compounds described and molecularmodeling study on their interactions with phosphodiesterase3A catalytic siterdquo Journal of Medicinal Chemistry vol 50 no 12pp 2886ndash2895 2007
[11] T Shyamsunder Reddy K Shiva Kumar C L T Medaet al ldquoConformationally restricted novel pyrazole deriva-tives synthesis of 18-disubstituted 55-dimethyl-45-dihydro-1H-benzo[g]indazoles as a new class of PDE4 inhibitorsrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 9 pp3248ndash3255 2012
[12] A Chino N Masuda Y Amano et al ldquoNovel benzimidazolederivatives as phosphodiesterase 10A (PDE10A) inhibitors withimproved metabolic stabilityrdquo Bioorganic and Medicinal Chem-istry vol 22 no 13 pp 3515ndash3526 2014
[13] H Yang FNMurigi ZWang J Li H Jin andZ Tu ldquoSynthesisand in vitro characterization of cinnoline and benzimidazoleanalogues as phosphodiesterase 10A inhibitorsrdquo Bioorganic andMedicinal Chemistry Letters vol 25 pp 919ndash924 2015
[14] J Nawrot-Modranka E Nawrot and J Graczyk ldquoIn vivo antitu-mor in vitro antibacterial activity and alkylating properties ofphosphorohydrazine derivatives of coumarin and chromonerdquoEuropean Journal of Medicinal Chemistry vol 41 no 11 pp1301ndash1309 2006
[15] A A Bekhit and T Abdel-Aziem ldquoDesign synthesis andbiological evaluation of some pyrazole derivatives as anti-inflammatory-antimicrobial agentsrdquo Bioorganic and MedicinalChemistry vol 12 no 8 pp 1935ndash1945 2004
[16] T Devji C Reddy CWoo S Awale S Kadota and D Carrico-Moniz ldquoPancreatic anticancer activity of a novel geranylgerany-lated coumarin derivativerdquo Bioorganic andMedicinal ChemistryLetters vol 21 no 19 pp 5770ndash5773 2011
[17] I Kostova I Manolov I Nicolova S Konstantinov andM Karaivanova ldquoNew lanthanide complexes of 4-methyl-7-hydroxycoumarin and their pharmacological activityrdquo Euro-pean Journal of Medicinal Chemistry vol 36 no 4 pp 339ndash3472001
[18] M Ghate R A Kusanur and M V Kulkarni ldquoSynthesis andin vivo analgesic and anti-inflammatory activity of some bi-heterocyclic coumarin derivativesrdquo European Journal of Medic-inal Chemistry vol 40 no 9 pp 882ndash887 2005
[19] PM RonadMN Noolvi S Sapkal S Dharbhamulla andV SMaddi ldquoSynthesis and antimicrobial activity of 7-(2-substitutedphenylthiazolidinyl)-benzopyran-2-one derivativesrdquo EuropeanJournal of Medicinal Chemistry vol 45 no 1 pp 85ndash89 2010
[20] GMelagraki A Afantitis O Igglessi-Markopoulou et al ldquoSyn-thesis and evaluation of the antioxidant and anti-inflammatoryactivity of novel coumarin-3-aminoamides and their alpha-lipoic acid adductsrdquo European Journal of Medicinal Chemistryvol 44 no 7 pp 3020ndash3026 2009
[21] J C Trivedi J B Bariwal K D Upadhyay et al ldquoImprovedand rapid synthesis of new coumarinyl chalcone derivatives andtheir antiviral activityrdquo Tetrahedron Letters vol 48 no 48 pp8472ndash8474 2007
[22] S Vilar E Quezada L Santana et al ldquoDesign synthe-sis and vasorelaxant and platelet antiaggregatory activitiesof coumarinndashresveratrol hybridsrdquo Bioorganic and MedicinalChemistry Letters vol 16 no 2 pp 257ndash261 2006
[23] W Hamaguchi N Masuda M Isomura et al ldquoDesign and syn-thesis of novel benzimidazole derivatives as phosphodiesterase10A inhibitors with reduced CYP1A2 inhibitionrdquo Bioorganicand Medicinal Chemistry vol 21 no 24 pp 7612ndash7623 2013
[24] A Dore B Asproni A Scampuddu et al ldquoSynthesis and SARstudy of novel tricyclic pyrazoles as potent phosphodiesterase10A inhibitorsrdquo European Journal of Medicinal Chemistry vol84 pp 181ndash193 2014
[25] S V Laxmi B S Kuarm and B Rajitha ldquoSynthesis and anti-microbial activity of coumarin pyrazole pyrimidine 246(1H3H5H)triones and thioxopyrimidine46(1H5H)dionesrdquo Me-dicinal Chemistry Research vol 22 no 2 pp 768ndash774 2013
[26] G Kalthur S Raj AThiyagarajan S Kumar P Kumar and S KAdiga ldquoVitamin E supplementation in semen-freezingmediumimproves the motility and protects sperm from freeze-thaw-induced DNA damagerdquo Fertility and Sterility vol 95 no 3 pp1149ndash1151 2011
[27] J Tesarik C Mendoza and A Carreras ldquoEffects of phosphodi-esterase inhibitors caffeine and pentoxifylline on spontaneousand stimulus-induced acrosome reactions in human spermrdquoFertility and Sterility vol 58 no 6 pp 1185ndash1190 1992
[28] G Kalthur S R Salian F Keyvanifard et al ldquoSupplementationof biotin to sperm preparation medium increases the motilityand longevity in cryopreserved human spermatozoardquo Journalof Assisted Reproduction and Genetics vol 29 no 7 pp 631ndash6352012
[29] A E Calogero S Fishel J Hall et al ldquoCorrelation betweenintracellular cAMP content kinematic parameters and hyper-activation of human spermatozoa after incubation with pentox-ifyllinerdquo Human Reproduction vol 13 no 4 pp 911ndash915 1998
[30] R Ain K Uma Devi S Shivaji and P B Seshagiri ldquoPen-toxifylline-stimulated capacitation and acrosome reaction inhamster spermatozoa involvement of intracellular signallingmoleculesrdquo Molecular Human Reproduction vol 5 no 7 pp618ndash626 1999
[31] M Bajpai andG F Doncel ldquoInvolvement of tyrosine kinase andcAMP-dependent kinase cross-talk in the regulation of humanspermmotilityrdquo Reproduction vol 126 no 2 pp 183ndash195 2003
[32] R Yunes P Fernandez G F Doncel and A A Acosta ldquoCyclicnucleotide phosphodiesterase inhibition increases tyrosinephosphorylation and hypermotility in normal and pathologicalhuman spermatozoardquo Biocell vol 29 no 3 pp 287ndash293 2005
[33] F Dimitriadis D Giannakis N Pardalidis et al ldquoEffectsof phosphodiesterase 5 inhibitors on sperm parameters andfertilizing capacityrdquo Asian Journal of Andrology vol 10 no 1pp 115ndash133 2008
[34] A P Kotdawala S Kumar S R Salian et al ldquoAddition of zincto human ejaculate prior to cryopreservation prevents freeze-thaw-induced DNA damage and preserves sperm functionrdquo
16 International Journal of Medicinal Chemistry
Journal of Assisted Reproduction and Genetics vol 29 no 12 pp1447ndash1453 2012
[35] S Mutalik S R Salian K Avadhani et al ldquoLiposome encapsu-lated soy lecithin and cholesterol can efficiently replace chickenegg yolk in human semen cryopreservation mediumrdquo SystemsBiology in Reproductive Medicine vol 60 no 3 pp 183ndash1882014
62 Study Subjects Infertile men who attended AndrologyLaboratory Kasturba Medical College Manipal UniversityManipal during the period of June-July 2015 for routinesemen evaluation were included in the study The subjects
with a sexual abstinence of 3ndash5 days were asked to providethe semen samples by masturbation in a sterile containerFollowing liquefaction the semen parameters were assessedas described by Kotdawala et al [34]The study was approved
14 International Journal of Medicinal Chemistry
by the Institutional Ethics Committee of Kasturba HospitalKasturba Medical College Manipal University (IEC number1552015)
63 Cryopreservation and Thawing The liquefied semensamples were cryopreserved by rapid freezing method asdescribed previously [34] with minor modifications Brieflythe semen samples were mixed with equal volume of freezingmedium (Sperm Freeze FertiPro Cat number 0344) in acryovial (Thermoscientific Nunc Cat number 138627) andkept at 4∘C for 10min in liquid nitrogen (LN2) vapor phasefor 5min and then finally plunged into liquid nitrogenThe samples were thawed after 1 week by rapid thawingmethod by placing themat 37∘C for 5minThe cryoprotectantmedium was completely removed by mixing the spermsuspension with EBSS medium and centrifuging at 1000 rpmfor 8min The resultant pellet was resuspended with freshEBSS medium and used for further analysis
64 Sample Preparation To screen the compounds withenhancing effect on themotility of spermatozoa under in vitroconditions the frozen-thawed semen samples were washedby mixing with equal volume of preincubated Earlrsquos BalancedSalt Solution (EBSS Sigma Cat number E2888) followed bycentrifugation at 1800 rpm for 8min The pellet was thengently mixed with 1mL of EBSS media containing 01bovine serum albumin (BSA Sigma Cat number A3311)and equally divided into control vehicle control and testgroups (with average sperm density of 5 times 104 spermatozoain each group) The motile sperm were extracted by swimup method as described earlier [35] The sperm suspensionwas centrifuged at 1200 rpm for 8min and the resultantpellet was overlaidwith EBSSmedium containing various testcompoundsThe sperm suspensions were incubated for 1 h at37∘Cand 5CO
2afterwhich themotile spermwere collected
from the supernatant
65 Motility Assessment Spermatozoa in the supernatantfraction were assessed for their motility patterns at 1 4 and24 h after incubation under light microscope as describedearlier [27] Sperm suspension was (10 120583L droplet) placed ona clean microscope slide and a coverslip was placed over itSpermatozoa with progressive motility and nonprogressivemotility and immotile spermatozoa were scored separatelyfrom a total of 200 spermatozoa in random fields at 400xmagnification
66 Sperm Kinematics Using Computer Assisted Semen Anal-ysis (CASA) The motion characteristics in the spermatozoawere assessed at 1 4 and 24 h after incubation using CASAsystem (ISAS Spain) Briefly 5 120583L of sperm suspension wasplaced on a clean microscope slide prewarmed at 37∘C Thesperm suspensionwas coveredwith prewarmed cover slip (22times 22mm) and observed under microscope (negative phasecontrast 10x objectives Proiser Spain) For each sample thekinematics was assessed randomly at ten different fields Theparameters such as curvilinear velocity (VCL) straight line
velocity (VSL) average path velocity (VAP) amplitude of lat-eral head displacement (ALH) linearity (LIN) straightness(STR) wobble (WOB) and beat cross frequency (BCF) wereanalyzed
7 Conclusions
In conclusion we have reported efficient and environmen-tally benignmethodologies for the synthesis of 3-[4-(1H-benzo[d]imidazol-2-yl)-1-phenyl-1H-pyrazol-3-yl]-2H-chromen-2-one derivatives 5andashx by using thermal and by microwaveirradiation under neat conditions in presence of ethanol Thereactions carried out under microwave irradiation affordedbenzimidazoles in short period of time with excellent yieldsHence this methodology would make an interesting strategyfor the synthesis of various substituted coumarin pyrazolebenzimidazoles Also compounds 5g 5h and 5k exhibitedimpressive sperm motility enhancing property whereas 5khas spermatozoa longevity property
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to University Scientific Instrumen-tation Centre (USIC) Karnatak University Dharwad forprovidingmass and IR spectral dataThe authors are thankfulto Indian Institute of Science (IISc) Bengaluru for carryingout NMR analyses and to UGC New Delhi for the financialassistance under UPE program vide no [F No 14-22012(NSPE) dated 22-01-2013] One of the authors (MNK)acknowledges University Grants Commission (UGC) NewDelhi for fellowship under UGC-UPE program
References
[1] P P Kattimani R R Kamble and G Y Meti ldquoExpedientsynthesis of benzimidazoles using amidesrdquo RSC Advances vol5 no 37 pp 29447ndash29455 2015
[2] L Li C Yee and J A Beavo ldquoCD3- and CD28-dependentinduction of PDE7 required for T cell activationrdquo Science vol283 no 5403 pp 848ndash849 1999
[3] T P Dousa ldquoCyclic-3101584051015840-nucleotide phosphodiesteraseisozymes in cell biology and pathophysiology of the kidneyrdquoKidney International vol 55 no 1 pp 29ndash62 1999
[4] C Pfister N Bennett F Bruckert et al ldquoInteractions of a G-protein with its effector transducin and cGMP phosphodi-esterase in retinal rodsrdquo Cell Signaling vol 5 no 3 pp 235ndash2411993
[5] S L C Jin F J Richard W-P Kuo A J DrsquoErcole andM Conti ldquoImpaired growth and fertility of cAMP-specificphosphodiesterase PDE4D-deficient micerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 21 pp 11998ndash12003 1999
[6] S H Francis I V Turko and J D Corbin ldquoCyclic nucleotidephosphodiesterases relating structure and functionrdquoProgress in
International Journal of Medicinal Chemistry 15
Nucleic Acid Research and Molecular Biology vol 65 pp 1ndash522000
[7] J M Hetman N Robas R Baxendale et al ldquoCloning and char-acterization of two splice variants of human phosphodiesterase11Ardquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 23 pp 12891ndash12895 2000
[8] K Yuasa T Ohgaru M Asahina and K Omori ldquoIdentificationof rat cyclic nucleotide phosphodiesterase 11A (PDE11A) com-parison of rat and human PDE11A splicing variantsrdquo EuropeanJournal of Biochemistry vol 268 no 16 pp 4440ndash4448 2001
[9] S K Sahu A Mishra and R K Behera ldquoSynthesis of thiazolebenzothiazole oxadiazole thiadiazole triazole and thiazolidi-none incorporated coumarinsrdquo Indian Journal of HeterocyclicChemistry vol 6 no 2 pp 91ndash94 1996
[10] G Roma M Di Braccio G Grossi et al ldquoSynthesis and in vitroantiplatelet activity of new 4-(1-piperazinyl) coumarin deriva-tives Human platelet phosphodiesterase 3 inhibitory propertiesof the two most effective compounds described and molecularmodeling study on their interactions with phosphodiesterase3A catalytic siterdquo Journal of Medicinal Chemistry vol 50 no 12pp 2886ndash2895 2007
[11] T Shyamsunder Reddy K Shiva Kumar C L T Medaet al ldquoConformationally restricted novel pyrazole deriva-tives synthesis of 18-disubstituted 55-dimethyl-45-dihydro-1H-benzo[g]indazoles as a new class of PDE4 inhibitorsrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 9 pp3248ndash3255 2012
[12] A Chino N Masuda Y Amano et al ldquoNovel benzimidazolederivatives as phosphodiesterase 10A (PDE10A) inhibitors withimproved metabolic stabilityrdquo Bioorganic and Medicinal Chem-istry vol 22 no 13 pp 3515ndash3526 2014
[13] H Yang FNMurigi ZWang J Li H Jin andZ Tu ldquoSynthesisand in vitro characterization of cinnoline and benzimidazoleanalogues as phosphodiesterase 10A inhibitorsrdquo Bioorganic andMedicinal Chemistry Letters vol 25 pp 919ndash924 2015
[14] J Nawrot-Modranka E Nawrot and J Graczyk ldquoIn vivo antitu-mor in vitro antibacterial activity and alkylating properties ofphosphorohydrazine derivatives of coumarin and chromonerdquoEuropean Journal of Medicinal Chemistry vol 41 no 11 pp1301ndash1309 2006
[15] A A Bekhit and T Abdel-Aziem ldquoDesign synthesis andbiological evaluation of some pyrazole derivatives as anti-inflammatory-antimicrobial agentsrdquo Bioorganic and MedicinalChemistry vol 12 no 8 pp 1935ndash1945 2004
[16] T Devji C Reddy CWoo S Awale S Kadota and D Carrico-Moniz ldquoPancreatic anticancer activity of a novel geranylgerany-lated coumarin derivativerdquo Bioorganic andMedicinal ChemistryLetters vol 21 no 19 pp 5770ndash5773 2011
[17] I Kostova I Manolov I Nicolova S Konstantinov andM Karaivanova ldquoNew lanthanide complexes of 4-methyl-7-hydroxycoumarin and their pharmacological activityrdquo Euro-pean Journal of Medicinal Chemistry vol 36 no 4 pp 339ndash3472001
[18] M Ghate R A Kusanur and M V Kulkarni ldquoSynthesis andin vivo analgesic and anti-inflammatory activity of some bi-heterocyclic coumarin derivativesrdquo European Journal of Medic-inal Chemistry vol 40 no 9 pp 882ndash887 2005
[19] PM RonadMN Noolvi S Sapkal S Dharbhamulla andV SMaddi ldquoSynthesis and antimicrobial activity of 7-(2-substitutedphenylthiazolidinyl)-benzopyran-2-one derivativesrdquo EuropeanJournal of Medicinal Chemistry vol 45 no 1 pp 85ndash89 2010
[20] GMelagraki A Afantitis O Igglessi-Markopoulou et al ldquoSyn-thesis and evaluation of the antioxidant and anti-inflammatoryactivity of novel coumarin-3-aminoamides and their alpha-lipoic acid adductsrdquo European Journal of Medicinal Chemistryvol 44 no 7 pp 3020ndash3026 2009
[21] J C Trivedi J B Bariwal K D Upadhyay et al ldquoImprovedand rapid synthesis of new coumarinyl chalcone derivatives andtheir antiviral activityrdquo Tetrahedron Letters vol 48 no 48 pp8472ndash8474 2007
[22] S Vilar E Quezada L Santana et al ldquoDesign synthe-sis and vasorelaxant and platelet antiaggregatory activitiesof coumarinndashresveratrol hybridsrdquo Bioorganic and MedicinalChemistry Letters vol 16 no 2 pp 257ndash261 2006
[23] W Hamaguchi N Masuda M Isomura et al ldquoDesign and syn-thesis of novel benzimidazole derivatives as phosphodiesterase10A inhibitors with reduced CYP1A2 inhibitionrdquo Bioorganicand Medicinal Chemistry vol 21 no 24 pp 7612ndash7623 2013
[24] A Dore B Asproni A Scampuddu et al ldquoSynthesis and SARstudy of novel tricyclic pyrazoles as potent phosphodiesterase10A inhibitorsrdquo European Journal of Medicinal Chemistry vol84 pp 181ndash193 2014
[25] S V Laxmi B S Kuarm and B Rajitha ldquoSynthesis and anti-microbial activity of coumarin pyrazole pyrimidine 246(1H3H5H)triones and thioxopyrimidine46(1H5H)dionesrdquo Me-dicinal Chemistry Research vol 22 no 2 pp 768ndash774 2013
[26] G Kalthur S Raj AThiyagarajan S Kumar P Kumar and S KAdiga ldquoVitamin E supplementation in semen-freezingmediumimproves the motility and protects sperm from freeze-thaw-induced DNA damagerdquo Fertility and Sterility vol 95 no 3 pp1149ndash1151 2011
[27] J Tesarik C Mendoza and A Carreras ldquoEffects of phosphodi-esterase inhibitors caffeine and pentoxifylline on spontaneousand stimulus-induced acrosome reactions in human spermrdquoFertility and Sterility vol 58 no 6 pp 1185ndash1190 1992
[28] G Kalthur S R Salian F Keyvanifard et al ldquoSupplementationof biotin to sperm preparation medium increases the motilityand longevity in cryopreserved human spermatozoardquo Journalof Assisted Reproduction and Genetics vol 29 no 7 pp 631ndash6352012
[29] A E Calogero S Fishel J Hall et al ldquoCorrelation betweenintracellular cAMP content kinematic parameters and hyper-activation of human spermatozoa after incubation with pentox-ifyllinerdquo Human Reproduction vol 13 no 4 pp 911ndash915 1998
[30] R Ain K Uma Devi S Shivaji and P B Seshagiri ldquoPen-toxifylline-stimulated capacitation and acrosome reaction inhamster spermatozoa involvement of intracellular signallingmoleculesrdquo Molecular Human Reproduction vol 5 no 7 pp618ndash626 1999
[31] M Bajpai andG F Doncel ldquoInvolvement of tyrosine kinase andcAMP-dependent kinase cross-talk in the regulation of humanspermmotilityrdquo Reproduction vol 126 no 2 pp 183ndash195 2003
[32] R Yunes P Fernandez G F Doncel and A A Acosta ldquoCyclicnucleotide phosphodiesterase inhibition increases tyrosinephosphorylation and hypermotility in normal and pathologicalhuman spermatozoardquo Biocell vol 29 no 3 pp 287ndash293 2005
[33] F Dimitriadis D Giannakis N Pardalidis et al ldquoEffectsof phosphodiesterase 5 inhibitors on sperm parameters andfertilizing capacityrdquo Asian Journal of Andrology vol 10 no 1pp 115ndash133 2008
[34] A P Kotdawala S Kumar S R Salian et al ldquoAddition of zincto human ejaculate prior to cryopreservation prevents freeze-thaw-induced DNA damage and preserves sperm functionrdquo
16 International Journal of Medicinal Chemistry
Journal of Assisted Reproduction and Genetics vol 29 no 12 pp1447ndash1453 2012
[35] S Mutalik S R Salian K Avadhani et al ldquoLiposome encapsu-lated soy lecithin and cholesterol can efficiently replace chickenegg yolk in human semen cryopreservation mediumrdquo SystemsBiology in Reproductive Medicine vol 60 no 3 pp 183ndash1882014
by the Institutional Ethics Committee of Kasturba HospitalKasturba Medical College Manipal University (IEC number1552015)
63 Cryopreservation and Thawing The liquefied semensamples were cryopreserved by rapid freezing method asdescribed previously [34] with minor modifications Brieflythe semen samples were mixed with equal volume of freezingmedium (Sperm Freeze FertiPro Cat number 0344) in acryovial (Thermoscientific Nunc Cat number 138627) andkept at 4∘C for 10min in liquid nitrogen (LN2) vapor phasefor 5min and then finally plunged into liquid nitrogenThe samples were thawed after 1 week by rapid thawingmethod by placing themat 37∘C for 5minThe cryoprotectantmedium was completely removed by mixing the spermsuspension with EBSS medium and centrifuging at 1000 rpmfor 8min The resultant pellet was resuspended with freshEBSS medium and used for further analysis
64 Sample Preparation To screen the compounds withenhancing effect on themotility of spermatozoa under in vitroconditions the frozen-thawed semen samples were washedby mixing with equal volume of preincubated Earlrsquos BalancedSalt Solution (EBSS Sigma Cat number E2888) followed bycentrifugation at 1800 rpm for 8min The pellet was thengently mixed with 1mL of EBSS media containing 01bovine serum albumin (BSA Sigma Cat number A3311)and equally divided into control vehicle control and testgroups (with average sperm density of 5 times 104 spermatozoain each group) The motile sperm were extracted by swimup method as described earlier [35] The sperm suspensionwas centrifuged at 1200 rpm for 8min and the resultantpellet was overlaidwith EBSSmedium containing various testcompoundsThe sperm suspensions were incubated for 1 h at37∘Cand 5CO
2afterwhich themotile spermwere collected
from the supernatant
65 Motility Assessment Spermatozoa in the supernatantfraction were assessed for their motility patterns at 1 4 and24 h after incubation under light microscope as describedearlier [27] Sperm suspension was (10 120583L droplet) placed ona clean microscope slide and a coverslip was placed over itSpermatozoa with progressive motility and nonprogressivemotility and immotile spermatozoa were scored separatelyfrom a total of 200 spermatozoa in random fields at 400xmagnification
66 Sperm Kinematics Using Computer Assisted Semen Anal-ysis (CASA) The motion characteristics in the spermatozoawere assessed at 1 4 and 24 h after incubation using CASAsystem (ISAS Spain) Briefly 5 120583L of sperm suspension wasplaced on a clean microscope slide prewarmed at 37∘C Thesperm suspensionwas coveredwith prewarmed cover slip (22times 22mm) and observed under microscope (negative phasecontrast 10x objectives Proiser Spain) For each sample thekinematics was assessed randomly at ten different fields Theparameters such as curvilinear velocity (VCL) straight line
velocity (VSL) average path velocity (VAP) amplitude of lat-eral head displacement (ALH) linearity (LIN) straightness(STR) wobble (WOB) and beat cross frequency (BCF) wereanalyzed
7 Conclusions
In conclusion we have reported efficient and environmen-tally benignmethodologies for the synthesis of 3-[4-(1H-benzo[d]imidazol-2-yl)-1-phenyl-1H-pyrazol-3-yl]-2H-chromen-2-one derivatives 5andashx by using thermal and by microwaveirradiation under neat conditions in presence of ethanol Thereactions carried out under microwave irradiation affordedbenzimidazoles in short period of time with excellent yieldsHence this methodology would make an interesting strategyfor the synthesis of various substituted coumarin pyrazolebenzimidazoles Also compounds 5g 5h and 5k exhibitedimpressive sperm motility enhancing property whereas 5khas spermatozoa longevity property
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to University Scientific Instrumen-tation Centre (USIC) Karnatak University Dharwad forprovidingmass and IR spectral dataThe authors are thankfulto Indian Institute of Science (IISc) Bengaluru for carryingout NMR analyses and to UGC New Delhi for the financialassistance under UPE program vide no [F No 14-22012(NSPE) dated 22-01-2013] One of the authors (MNK)acknowledges University Grants Commission (UGC) NewDelhi for fellowship under UGC-UPE program
References
[1] P P Kattimani R R Kamble and G Y Meti ldquoExpedientsynthesis of benzimidazoles using amidesrdquo RSC Advances vol5 no 37 pp 29447ndash29455 2015
[2] L Li C Yee and J A Beavo ldquoCD3- and CD28-dependentinduction of PDE7 required for T cell activationrdquo Science vol283 no 5403 pp 848ndash849 1999
[3] T P Dousa ldquoCyclic-3101584051015840-nucleotide phosphodiesteraseisozymes in cell biology and pathophysiology of the kidneyrdquoKidney International vol 55 no 1 pp 29ndash62 1999
[4] C Pfister N Bennett F Bruckert et al ldquoInteractions of a G-protein with its effector transducin and cGMP phosphodi-esterase in retinal rodsrdquo Cell Signaling vol 5 no 3 pp 235ndash2411993
[5] S L C Jin F J Richard W-P Kuo A J DrsquoErcole andM Conti ldquoImpaired growth and fertility of cAMP-specificphosphodiesterase PDE4D-deficient micerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 21 pp 11998ndash12003 1999
[6] S H Francis I V Turko and J D Corbin ldquoCyclic nucleotidephosphodiesterases relating structure and functionrdquoProgress in
International Journal of Medicinal Chemistry 15
Nucleic Acid Research and Molecular Biology vol 65 pp 1ndash522000
[7] J M Hetman N Robas R Baxendale et al ldquoCloning and char-acterization of two splice variants of human phosphodiesterase11Ardquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 23 pp 12891ndash12895 2000
[8] K Yuasa T Ohgaru M Asahina and K Omori ldquoIdentificationof rat cyclic nucleotide phosphodiesterase 11A (PDE11A) com-parison of rat and human PDE11A splicing variantsrdquo EuropeanJournal of Biochemistry vol 268 no 16 pp 4440ndash4448 2001
[9] S K Sahu A Mishra and R K Behera ldquoSynthesis of thiazolebenzothiazole oxadiazole thiadiazole triazole and thiazolidi-none incorporated coumarinsrdquo Indian Journal of HeterocyclicChemistry vol 6 no 2 pp 91ndash94 1996
[10] G Roma M Di Braccio G Grossi et al ldquoSynthesis and in vitroantiplatelet activity of new 4-(1-piperazinyl) coumarin deriva-tives Human platelet phosphodiesterase 3 inhibitory propertiesof the two most effective compounds described and molecularmodeling study on their interactions with phosphodiesterase3A catalytic siterdquo Journal of Medicinal Chemistry vol 50 no 12pp 2886ndash2895 2007
[11] T Shyamsunder Reddy K Shiva Kumar C L T Medaet al ldquoConformationally restricted novel pyrazole deriva-tives synthesis of 18-disubstituted 55-dimethyl-45-dihydro-1H-benzo[g]indazoles as a new class of PDE4 inhibitorsrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 9 pp3248ndash3255 2012
[12] A Chino N Masuda Y Amano et al ldquoNovel benzimidazolederivatives as phosphodiesterase 10A (PDE10A) inhibitors withimproved metabolic stabilityrdquo Bioorganic and Medicinal Chem-istry vol 22 no 13 pp 3515ndash3526 2014
[13] H Yang FNMurigi ZWang J Li H Jin andZ Tu ldquoSynthesisand in vitro characterization of cinnoline and benzimidazoleanalogues as phosphodiesterase 10A inhibitorsrdquo Bioorganic andMedicinal Chemistry Letters vol 25 pp 919ndash924 2015
[14] J Nawrot-Modranka E Nawrot and J Graczyk ldquoIn vivo antitu-mor in vitro antibacterial activity and alkylating properties ofphosphorohydrazine derivatives of coumarin and chromonerdquoEuropean Journal of Medicinal Chemistry vol 41 no 11 pp1301ndash1309 2006
[15] A A Bekhit and T Abdel-Aziem ldquoDesign synthesis andbiological evaluation of some pyrazole derivatives as anti-inflammatory-antimicrobial agentsrdquo Bioorganic and MedicinalChemistry vol 12 no 8 pp 1935ndash1945 2004
[16] T Devji C Reddy CWoo S Awale S Kadota and D Carrico-Moniz ldquoPancreatic anticancer activity of a novel geranylgerany-lated coumarin derivativerdquo Bioorganic andMedicinal ChemistryLetters vol 21 no 19 pp 5770ndash5773 2011
[17] I Kostova I Manolov I Nicolova S Konstantinov andM Karaivanova ldquoNew lanthanide complexes of 4-methyl-7-hydroxycoumarin and their pharmacological activityrdquo Euro-pean Journal of Medicinal Chemistry vol 36 no 4 pp 339ndash3472001
[18] M Ghate R A Kusanur and M V Kulkarni ldquoSynthesis andin vivo analgesic and anti-inflammatory activity of some bi-heterocyclic coumarin derivativesrdquo European Journal of Medic-inal Chemistry vol 40 no 9 pp 882ndash887 2005
[19] PM RonadMN Noolvi S Sapkal S Dharbhamulla andV SMaddi ldquoSynthesis and antimicrobial activity of 7-(2-substitutedphenylthiazolidinyl)-benzopyran-2-one derivativesrdquo EuropeanJournal of Medicinal Chemistry vol 45 no 1 pp 85ndash89 2010
[20] GMelagraki A Afantitis O Igglessi-Markopoulou et al ldquoSyn-thesis and evaluation of the antioxidant and anti-inflammatoryactivity of novel coumarin-3-aminoamides and their alpha-lipoic acid adductsrdquo European Journal of Medicinal Chemistryvol 44 no 7 pp 3020ndash3026 2009
[21] J C Trivedi J B Bariwal K D Upadhyay et al ldquoImprovedand rapid synthesis of new coumarinyl chalcone derivatives andtheir antiviral activityrdquo Tetrahedron Letters vol 48 no 48 pp8472ndash8474 2007
[22] S Vilar E Quezada L Santana et al ldquoDesign synthe-sis and vasorelaxant and platelet antiaggregatory activitiesof coumarinndashresveratrol hybridsrdquo Bioorganic and MedicinalChemistry Letters vol 16 no 2 pp 257ndash261 2006
[23] W Hamaguchi N Masuda M Isomura et al ldquoDesign and syn-thesis of novel benzimidazole derivatives as phosphodiesterase10A inhibitors with reduced CYP1A2 inhibitionrdquo Bioorganicand Medicinal Chemistry vol 21 no 24 pp 7612ndash7623 2013
[24] A Dore B Asproni A Scampuddu et al ldquoSynthesis and SARstudy of novel tricyclic pyrazoles as potent phosphodiesterase10A inhibitorsrdquo European Journal of Medicinal Chemistry vol84 pp 181ndash193 2014
[25] S V Laxmi B S Kuarm and B Rajitha ldquoSynthesis and anti-microbial activity of coumarin pyrazole pyrimidine 246(1H3H5H)triones and thioxopyrimidine46(1H5H)dionesrdquo Me-dicinal Chemistry Research vol 22 no 2 pp 768ndash774 2013
[26] G Kalthur S Raj AThiyagarajan S Kumar P Kumar and S KAdiga ldquoVitamin E supplementation in semen-freezingmediumimproves the motility and protects sperm from freeze-thaw-induced DNA damagerdquo Fertility and Sterility vol 95 no 3 pp1149ndash1151 2011
[27] J Tesarik C Mendoza and A Carreras ldquoEffects of phosphodi-esterase inhibitors caffeine and pentoxifylline on spontaneousand stimulus-induced acrosome reactions in human spermrdquoFertility and Sterility vol 58 no 6 pp 1185ndash1190 1992
[28] G Kalthur S R Salian F Keyvanifard et al ldquoSupplementationof biotin to sperm preparation medium increases the motilityand longevity in cryopreserved human spermatozoardquo Journalof Assisted Reproduction and Genetics vol 29 no 7 pp 631ndash6352012
[29] A E Calogero S Fishel J Hall et al ldquoCorrelation betweenintracellular cAMP content kinematic parameters and hyper-activation of human spermatozoa after incubation with pentox-ifyllinerdquo Human Reproduction vol 13 no 4 pp 911ndash915 1998
[30] R Ain K Uma Devi S Shivaji and P B Seshagiri ldquoPen-toxifylline-stimulated capacitation and acrosome reaction inhamster spermatozoa involvement of intracellular signallingmoleculesrdquo Molecular Human Reproduction vol 5 no 7 pp618ndash626 1999
[31] M Bajpai andG F Doncel ldquoInvolvement of tyrosine kinase andcAMP-dependent kinase cross-talk in the regulation of humanspermmotilityrdquo Reproduction vol 126 no 2 pp 183ndash195 2003
[32] R Yunes P Fernandez G F Doncel and A A Acosta ldquoCyclicnucleotide phosphodiesterase inhibition increases tyrosinephosphorylation and hypermotility in normal and pathologicalhuman spermatozoardquo Biocell vol 29 no 3 pp 287ndash293 2005
[33] F Dimitriadis D Giannakis N Pardalidis et al ldquoEffectsof phosphodiesterase 5 inhibitors on sperm parameters andfertilizing capacityrdquo Asian Journal of Andrology vol 10 no 1pp 115ndash133 2008
[34] A P Kotdawala S Kumar S R Salian et al ldquoAddition of zincto human ejaculate prior to cryopreservation prevents freeze-thaw-induced DNA damage and preserves sperm functionrdquo
16 International Journal of Medicinal Chemistry
Journal of Assisted Reproduction and Genetics vol 29 no 12 pp1447ndash1453 2012
[35] S Mutalik S R Salian K Avadhani et al ldquoLiposome encapsu-lated soy lecithin and cholesterol can efficiently replace chickenegg yolk in human semen cryopreservation mediumrdquo SystemsBiology in Reproductive Medicine vol 60 no 3 pp 183ndash1882014
Nucleic Acid Research and Molecular Biology vol 65 pp 1ndash522000
[7] J M Hetman N Robas R Baxendale et al ldquoCloning and char-acterization of two splice variants of human phosphodiesterase11Ardquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 23 pp 12891ndash12895 2000
[8] K Yuasa T Ohgaru M Asahina and K Omori ldquoIdentificationof rat cyclic nucleotide phosphodiesterase 11A (PDE11A) com-parison of rat and human PDE11A splicing variantsrdquo EuropeanJournal of Biochemistry vol 268 no 16 pp 4440ndash4448 2001
[9] S K Sahu A Mishra and R K Behera ldquoSynthesis of thiazolebenzothiazole oxadiazole thiadiazole triazole and thiazolidi-none incorporated coumarinsrdquo Indian Journal of HeterocyclicChemistry vol 6 no 2 pp 91ndash94 1996
[10] G Roma M Di Braccio G Grossi et al ldquoSynthesis and in vitroantiplatelet activity of new 4-(1-piperazinyl) coumarin deriva-tives Human platelet phosphodiesterase 3 inhibitory propertiesof the two most effective compounds described and molecularmodeling study on their interactions with phosphodiesterase3A catalytic siterdquo Journal of Medicinal Chemistry vol 50 no 12pp 2886ndash2895 2007
[11] T Shyamsunder Reddy K Shiva Kumar C L T Medaet al ldquoConformationally restricted novel pyrazole deriva-tives synthesis of 18-disubstituted 55-dimethyl-45-dihydro-1H-benzo[g]indazoles as a new class of PDE4 inhibitorsrdquoBioorganic and Medicinal Chemistry Letters vol 22 no 9 pp3248ndash3255 2012
[12] A Chino N Masuda Y Amano et al ldquoNovel benzimidazolederivatives as phosphodiesterase 10A (PDE10A) inhibitors withimproved metabolic stabilityrdquo Bioorganic and Medicinal Chem-istry vol 22 no 13 pp 3515ndash3526 2014
[13] H Yang FNMurigi ZWang J Li H Jin andZ Tu ldquoSynthesisand in vitro characterization of cinnoline and benzimidazoleanalogues as phosphodiesterase 10A inhibitorsrdquo Bioorganic andMedicinal Chemistry Letters vol 25 pp 919ndash924 2015
[14] J Nawrot-Modranka E Nawrot and J Graczyk ldquoIn vivo antitu-mor in vitro antibacterial activity and alkylating properties ofphosphorohydrazine derivatives of coumarin and chromonerdquoEuropean Journal of Medicinal Chemistry vol 41 no 11 pp1301ndash1309 2006
[15] A A Bekhit and T Abdel-Aziem ldquoDesign synthesis andbiological evaluation of some pyrazole derivatives as anti-inflammatory-antimicrobial agentsrdquo Bioorganic and MedicinalChemistry vol 12 no 8 pp 1935ndash1945 2004
[16] T Devji C Reddy CWoo S Awale S Kadota and D Carrico-Moniz ldquoPancreatic anticancer activity of a novel geranylgerany-lated coumarin derivativerdquo Bioorganic andMedicinal ChemistryLetters vol 21 no 19 pp 5770ndash5773 2011
[17] I Kostova I Manolov I Nicolova S Konstantinov andM Karaivanova ldquoNew lanthanide complexes of 4-methyl-7-hydroxycoumarin and their pharmacological activityrdquo Euro-pean Journal of Medicinal Chemistry vol 36 no 4 pp 339ndash3472001
[18] M Ghate R A Kusanur and M V Kulkarni ldquoSynthesis andin vivo analgesic and anti-inflammatory activity of some bi-heterocyclic coumarin derivativesrdquo European Journal of Medic-inal Chemistry vol 40 no 9 pp 882ndash887 2005
[19] PM RonadMN Noolvi S Sapkal S Dharbhamulla andV SMaddi ldquoSynthesis and antimicrobial activity of 7-(2-substitutedphenylthiazolidinyl)-benzopyran-2-one derivativesrdquo EuropeanJournal of Medicinal Chemistry vol 45 no 1 pp 85ndash89 2010
[20] GMelagraki A Afantitis O Igglessi-Markopoulou et al ldquoSyn-thesis and evaluation of the antioxidant and anti-inflammatoryactivity of novel coumarin-3-aminoamides and their alpha-lipoic acid adductsrdquo European Journal of Medicinal Chemistryvol 44 no 7 pp 3020ndash3026 2009
[21] J C Trivedi J B Bariwal K D Upadhyay et al ldquoImprovedand rapid synthesis of new coumarinyl chalcone derivatives andtheir antiviral activityrdquo Tetrahedron Letters vol 48 no 48 pp8472ndash8474 2007
[22] S Vilar E Quezada L Santana et al ldquoDesign synthe-sis and vasorelaxant and platelet antiaggregatory activitiesof coumarinndashresveratrol hybridsrdquo Bioorganic and MedicinalChemistry Letters vol 16 no 2 pp 257ndash261 2006
[23] W Hamaguchi N Masuda M Isomura et al ldquoDesign and syn-thesis of novel benzimidazole derivatives as phosphodiesterase10A inhibitors with reduced CYP1A2 inhibitionrdquo Bioorganicand Medicinal Chemistry vol 21 no 24 pp 7612ndash7623 2013
[24] A Dore B Asproni A Scampuddu et al ldquoSynthesis and SARstudy of novel tricyclic pyrazoles as potent phosphodiesterase10A inhibitorsrdquo European Journal of Medicinal Chemistry vol84 pp 181ndash193 2014
[25] S V Laxmi B S Kuarm and B Rajitha ldquoSynthesis and anti-microbial activity of coumarin pyrazole pyrimidine 246(1H3H5H)triones and thioxopyrimidine46(1H5H)dionesrdquo Me-dicinal Chemistry Research vol 22 no 2 pp 768ndash774 2013
[26] G Kalthur S Raj AThiyagarajan S Kumar P Kumar and S KAdiga ldquoVitamin E supplementation in semen-freezingmediumimproves the motility and protects sperm from freeze-thaw-induced DNA damagerdquo Fertility and Sterility vol 95 no 3 pp1149ndash1151 2011
[27] J Tesarik C Mendoza and A Carreras ldquoEffects of phosphodi-esterase inhibitors caffeine and pentoxifylline on spontaneousand stimulus-induced acrosome reactions in human spermrdquoFertility and Sterility vol 58 no 6 pp 1185ndash1190 1992
[28] G Kalthur S R Salian F Keyvanifard et al ldquoSupplementationof biotin to sperm preparation medium increases the motilityand longevity in cryopreserved human spermatozoardquo Journalof Assisted Reproduction and Genetics vol 29 no 7 pp 631ndash6352012
[29] A E Calogero S Fishel J Hall et al ldquoCorrelation betweenintracellular cAMP content kinematic parameters and hyper-activation of human spermatozoa after incubation with pentox-ifyllinerdquo Human Reproduction vol 13 no 4 pp 911ndash915 1998
[30] R Ain K Uma Devi S Shivaji and P B Seshagiri ldquoPen-toxifylline-stimulated capacitation and acrosome reaction inhamster spermatozoa involvement of intracellular signallingmoleculesrdquo Molecular Human Reproduction vol 5 no 7 pp618ndash626 1999
[31] M Bajpai andG F Doncel ldquoInvolvement of tyrosine kinase andcAMP-dependent kinase cross-talk in the regulation of humanspermmotilityrdquo Reproduction vol 126 no 2 pp 183ndash195 2003
[32] R Yunes P Fernandez G F Doncel and A A Acosta ldquoCyclicnucleotide phosphodiesterase inhibition increases tyrosinephosphorylation and hypermotility in normal and pathologicalhuman spermatozoardquo Biocell vol 29 no 3 pp 287ndash293 2005
[33] F Dimitriadis D Giannakis N Pardalidis et al ldquoEffectsof phosphodiesterase 5 inhibitors on sperm parameters andfertilizing capacityrdquo Asian Journal of Andrology vol 10 no 1pp 115ndash133 2008
[34] A P Kotdawala S Kumar S R Salian et al ldquoAddition of zincto human ejaculate prior to cryopreservation prevents freeze-thaw-induced DNA damage and preserves sperm functionrdquo
16 International Journal of Medicinal Chemistry
Journal of Assisted Reproduction and Genetics vol 29 no 12 pp1447ndash1453 2012
[35] S Mutalik S R Salian K Avadhani et al ldquoLiposome encapsu-lated soy lecithin and cholesterol can efficiently replace chickenegg yolk in human semen cryopreservation mediumrdquo SystemsBiology in Reproductive Medicine vol 60 no 3 pp 183ndash1882014
Journal of Assisted Reproduction and Genetics vol 29 no 12 pp1447ndash1453 2012
[35] S Mutalik S R Salian K Avadhani et al ldquoLiposome encapsu-lated soy lecithin and cholesterol can efficiently replace chickenegg yolk in human semen cryopreservation mediumrdquo SystemsBiology in Reproductive Medicine vol 60 no 3 pp 183ndash1882014