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Nucleosides, Nucleotides & Nucleic Acids
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Synthesis of new uracil derivatives and their sugarhydrazones
with potent antimicrobial, antioxidantand anticancer activities
Ibrahim F. Nassar, Ahmed F. El Farargy, Fathy M. Abdelrazek
& Zeinab Hamza
To cite this article: Ibrahim F. Nassar, Ahmed F. El Farargy,
Fathy M. Abdelrazek & ZeinabHamza (2020) Synthesis of new
uracil derivatives and their sugar hydrazones with
potentantimicrobial, antioxidant and anticancer activities,
Nucleosides, Nucleotides & Nucleic Acids, 39:7,991-1010, DOI:
10.1080/15257770.2020.1736300
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https://doi.org/10.1080/15257770.2020.1736300
Published online: 04 Mar 2020.
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Synthesis of new uracil derivatives and their sugarhydrazones
with potent antimicrobial, antioxidantand anticancer activities
Ibrahim F. Nassara, Ahmed F. El Farargyb, Fathy M. Abdelrazekc,
andZeinab Hamzad
aFaculty of Specific Education, Ain Shams University, Abassia,
Cairo, Egypt; bDepartment ofChemistry, Faculty of Science, Zagazig
University, Zagazig, Egypt; cDepartment of Chemistry,Faculty of
Science, Cairo University, Giza, Egypt; dFood Toxicology and
ContaminantsDepartment, National Research Centre, Dokki, Giza,
Egypt
ABSTRACT6-(4-Chloro-3-nitrophenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyr-imidine-5-carbonitrile
(4) was prepared and was reactedwith ethyl chloroacetate, hydrazine
hydrate, 4-chloroaniline,formaldehyde, acetic anhydride, formic
acid, carbon disulfide,4-cyanobenzaldehyde, triethyl orthoformate,
D-sugars, 4-aminoacetophenone, benzoyl choride and cyclohexanone
toafford a series of new uracil derivatives (5–18). Examination
ofsome of the prepared compounds for their antimicrobial,
anti-oxidant and anticancer activities was conducted. Among
thetested samples, compound 17 was the most active substanceagainst
the gram-positive bacteria and was more potent thanthe reference
drug Cefoperazone. Moreover, the antibacterialactivity of 17 was
higher against gram-negative bacteria.Compounds 6 and 13 reached a
higher scavenging abilitytoward DPPH radicals and are better
candidates for antioxi-dant activity. Also, compounds 6 and 13 had
no significantanticancer activity toward liver cancer (Hep G2) and
breastcancer (MCF-7) cell lines.
GRAPHICAL ABSTRACT
ARTICLE HISTORYReceived 27 October 2019Accepted 25 February
2020
KEYWORDSUracil; derivatives;antimicrobial;
antioxidant;anticancer; activity
CONTACT Ibrahim F. Nassar [email protected]
Faculty of Specific Education, Ain ShamsUniversity, 365 Ramsis
Street, Abassia, Cairo, Egypt.� 2020 Taylor & Francis Group,
LLC
NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS2020, VOL. 39, NO. 7,
991–1010https://doi.org/10.1080/15257770.2020.1736300
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1. Introduction
Pyrimidines chemistry is of interest because these ring systems
representthe main skeleton in alkaloids and nucleic bases in
addition to their higherbiological activities. Many pyrimidine
derivatives possess anticancer,[1,2]
antiviral,[2,3] antibacterial[4,5] antifungal,[6]
anti-inflammatory[7] and centralnervous activities.[8] The
pyrimidine derivatives have been investigatedfor their medicinal
interest, for example the antithyroid activity of
the5-fluoro-2-thiouracil.[9] Pyrimidine derivatives ethirimoland
methirimolwere some of the earliest fungicides. Furthermore
pyrimidine derivativesalso show the different pharmacological
activities like antitumor,[10]
analgesic,[11] antineoplastic,[12] cardiovascular,[13]
antiallergic.[14]
In general, nucleoside analogs are structurally, metabolically,
andpharmacodynamically related agents that nevertheless have
diversebiological actions and therapeutic effects. This class of
agents affects thestructural integrity of DNA, usually after
incorporation during replicationor DNA excision repair synthesis,
leading to stalled replication forks andchain termination. One of
the remarkable features that is still unexplainedabout nucleic
antagonists is how drugs with such similar structural
features,which share metabolite pathways and elements of their
mechanismsof action, show such diversity in their clinical
activities.[15] One of themost frequently used approaches to new
antitumor drugs is a designof antimetabolites based on the
similarity of structure to the naturallyoccurring pyrimidine and
purines involved in the biosynthesis of DNA.Novel compounds should
interfere with more biological applications ofnaturally occurring
analogs. Since Carbon discovered in 1965 the presenceof
2-thiouracil tRNA of Escherichia coli[16] the role of
thiopyrimidine nucle-obase and their biological activity has been
investigated, although they havenot been studied as frequently as
their oxygen analogs. It was foundthat also 4-thiouracil[17] and
2-thiocytosine[18–20] are present in tRNA ofseveral sources. These
thionucleobases and their derivatives show biologicalactivity.
Whereas the thionucleobases possess antiviral
properties,[21,22]
derivatives of their nucleosides are potential antitumor
agents.[23]
Thiopyrimidines show pronounced in vitro bacteriostatic
activity[24] andthey are basic constituents of some t-RNAs.[25]
They exhibit antitumor andantithyroidal activities because they are
readily incorporated intonucleic acids.[26] Thiopyrimidine
derivatives also act as potential inhibi-tors of protein and
nucleic acid syntheses[27] as antimetabolites.[28]
From the above findings and our interest in the design and
synthesis ofnew heterocyclic compounds with high impact as
agrochemicals[29–35]
we synthesized new derivatives of a uracil ring linked to sugars
andheterocyclic moieties and evaluating them for antimicrobial,
antioxidantand anticancer activities.
992 I. F. NASSAR ET AL.
-
2. Results and discussion
Reaction of 4-chloro-3-nitrobenzaldehyde 1 with thiourea 2 and
ethylcyanoacetate 3 in ethanol under reflux afforded
6-(4-chloro-3-nitrophenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile
(4). The IRspectrum of compound 4 showed strong absorption bands at
3171 and1670 cm�1 assignable to NH and C¼O groups respectively. The
1H NMRspectrum of the same compound revealed signals at d 12.0,
13.10 ppmfor two NH groups (D2O exchangeable), in addition to the
peaks of thearomatic protons which appeared as two douplets and one
singlet. Also,the 13C NMR of 4 gave another proof of the structure
(cf. Section 3).Stirring a solution of 4 and ethyl chloroacetate in
dry acetone and potas-sium carbonate yielded ethyl
2-((4-(4-chloro-3-nitrophenyl)-5-cyano-6-oxo-1,6-dihydro-pyrimidin-2-yl)thio)acetate
(5) which in turn was con-verted to the hydrazinyl derivative 6
when refluxed with hydrazine hydratein absolute ethanol. Compound 6
also was afforded via refluxing ofcompound 4 with hydrazine hydrate
in absolute ethanol. The 1H NMRspectrum of 5 showed signals at d
1.2 and 4.2 ppm attributed to the CH3(t)and the CH2(q) of the ethyl
acetate ester group beside another singlet
Scheme 1. Synthesis of compounds 4–8.
NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS 993
-
signal for the other CH2 group at 4.0 ppm. The13C NMR of 5
revealed two
signals at d 15 and 60 ppm for CH3 and CH2 groups,
respectively.The 1H NMR spectrum of 6 revealed signals at d 5.9 and
8.3 ppm for
NH2 and NH groups, respectively. Reaction of 4 with
4-chloroanilinein the presence of formaldehyde under Mannich
conditions yielded
(((4-chlorophenyl)amino)methyl)thio)-6-oxo-1,6-dihydropyrimidine-5-carbonitrilederivative
7. Acetylation of 4 by refluxing with acetic anhydride
afforded1-acetyl derivative 8. The 1H NMR spectrum of 7 showed
signals at d 4.7,8.6 and 12 ppm attributable for one CH2 and two NH
groups, respectively.The 13C NMR of 7 revealed signal at d 45ppm
for CH2 group. The1H NMR spectrum of compound 8 revealed signals at
d 1.9 ppm attributedto the acetyl CH3 group. Also, the
13C NMR of 8 showed a signal at d25 ppm for the CH3 group (cf.
Section 3 and Scheme 1).The hydrazinyl derivative 6 was used as a
key starting material for preparing
some interesting heterocyclic compounds. Thus, when the
hydrazinyl derivative 6was refluxed with formic acid the [1,2,4]
triazolo[4,3-a]pyrimidine derivative 9was produced. The 1H NMR
spectrum of 9 showed signals at d 2.0 and 7.92ppmattributable to an
exchangeable NH and triazole CH groups respectively.Compound 6 was
reacted with carbon disulfide in pyridine and ethanol to
yield3-thioxo-1,2,3,5-tetrahydro-[1,2,4]triazolo[4,3-a]pyrimidine
derivative 10. The13C NMR of 10 showed a signal at d 180ppm for the
triazole C¼S group.
Scheme 2. Synthesis of compounds 9–13.
994 I. F. NASSAR ET AL.
-
Acetylation of 6 with acetic anhydride afforded
1,6-dihydropyrimidin-2-yl)acetohydrazide 11. The 1H NMR spectrum of
11 revealed signals at d1.9, 7.6, 10.6 and 11.0 ppm for one CH3 and
three NH groups (D2Oexchangeable), respectively.Refluxing compound
6 with 4-cyanobenzaldehyde in ethanol and glacial
acetic acid afforded
2-(2-(4-cyanobenzylidene)hydrazinyl)-6-oxo-1,6-dihy-dropyrimidine-5-carbonitrile
derivative 12. The 1H NMR spectrum of 12showed signals at d 8.2,
8.7 and 11.10 ppm for N¼CH and NH groups(D2O exchangeable),
respectively. The
13C NMR of 12 showed signals forN¼CH group at d 161 ppm.
Reaction of 6 with triethyl orthoformate inacetic anhydride yielded
5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)formo-hydrazonate 13. The
1H NMR spectrum of 13 inferred signals at d 1.2, 3.5and 8.0 ppm for
CH3, CH2 and N¼CH groups respectively. The 13C NMRspectrum of 13
showed signals for the same groups, respectively (cf.Section 3 and
Scheme 2).Similarly, the hydrazinyl derivative 6 was allowed to
react with D-glucose
and D-xylose under the same conditions to afford the sugar
hydrazonederivatives 14 and 15, respectively. The 1H NMR spectra of
compounds 14and 15 revealed the signals of the sugar moiety at
their specific regionsindicating the presence of the sugar parts.
Also, the 13C NMR spectra
Scheme 3. Synthesis of compounds 14–18.
NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS 995
-
proved the presence of the sugar carbons and the C-1 of the
sugar. Similarly,reaction of 6 with 4-aminoacetophenone or
cyclohexanone under thesame conditions yielded
2-(2-(1-(4-aminophenyl)ethylidene)hydrazinyl)-4-(4-chloro-3-nitrophenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
16,and
4-(4-chloro-3-nitrophenyl)-2-(2-cyclohexylidenehydrazinyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
17, respectively.The 1H NMR spectrum of 16 revealed signals for CH3
and NH2 groups
at d 2.3 and 5.4 ppm, respectively. The 13C NMR spectrum of the
samecompound proved the presence of CH3 group at 15.5 ppm. In
addition, the1H NMR spectrum of compound 17 showed two multiplet
signals at d 1.59and 2.3 ppm for ten protons of the cyclohexyl
moiety. The 13C NMR spec-trum of the same compound showed signals
at d 25–34 ppm for the samemoiety. Finally, refluxing of compound 6
with benzoyl chloride
yieldedN0-(4-(4-chloro-3-nitrophenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)benzohydrazide
18 (cf. Section 3 and Scheme 3).
2.1. In vitro antimicrobial activity
The antibacterial activity of compounds 4, 6, 8, 10, 12, 13, 15,
16, 17 and18 was evaluated against gram positive bacteria S.
aureus, B. cereus,L. monocytogenes and gram negative bacteria as,
E. coli, S. typhimurium andY. enterocolitica in terms of the
diameter of the inhibition zone diameterin mm relative to
Cefoperazone, which was used as a reference drug.The results of the
antimicrobial activity showed that the compounds 4, 6,
8, 10, 12, 13, 15, 16, 17 and 18 had antibacterial activities
that variedamong the tested microbial strains (Table 1).
Antibacterial and antifungalactivity of the synthesized derivatives
was done in comparison withCefoperazone as standard to reveal the
potency of the synthesizedcompounds. Compound 17 was the most
active substance among the testedsamples against the following
gram-positive bacteria L. monocytogenes
Table 1. In vitro antibacterial activity of compounds 4, 6, 8,
10, 12, 13, 15, 16, 17 and 18against gram positive and gram
negative bacteria in terms of the diameter of the inhibitionzone
diameter in mm.
Test microorganisms
Zone of inhibition (mm)a antibacterial activity (1mg/mL)
4 6 8 10 13 12 15 17 16 18Cefoperazone(100 mg/mL)
Gram1 ve bacteriaS. aureus – – – – 7.5 – – – – – 12B. cereus – –
– – – – – 9 – – 12L. monocytogenes 12 14 13 12 14 13 11 11Gram2 ve
bacteriaE. coli 11 13 8 12 9 8.5 11 13 12 13 15S. typhimurium – – –
– – – – – – – 11Y. enterocolitica 16 – – 7.5 9 – – 11 – – 12aThe
values (average of triplicate) are diameter of zone of inhibition
at 1mg/mL
996 I. F. NASSAR ET AL.
-
(14mm) and was more potent than the reference drug. Moreover,
its anti-bacterial activity was significant against gram-negative
bacteria E. coli, withan inhibition zone of 13mm comparing to the
other compounds (Table 1).Compound 4 showed a high inhibition zone
(16mm) against Y. enterocoli-tica as compared to the other
compounds that showed considerable inhibi-tory zones 7.5–12mm
(Figure 1a). The synthesized compounds showed noactivity against S.
typhimurium, also all compounds are inactive against S.aureus and
B. cereus except 13 and 17, respectively. An entirely
differenttrend in antifungal activity was observed, with all
compounds beinginactive against A. flavus and A. niger. The
findings of the present studyshowed that there were differences
between the antimicrobial activitiesamong the compounds, and the
nature of the substituent on the benzenering and the heterocyclic
skeleton attached to the pyrimidine molecules incompounds 4, 17 and
16 has a strong influence on the extent of antimicro-bial activity.
The mechanism of bactericidal action is thought to be due
todisruption of intermolecular interactions. This can cause
dissociation of cel-lular membrane lipid bilayers, which
compromises cellular permeabilitycontrols and induces leakage of
cellular contents. The antimicrobial activityof the synthesized
compounds was related to the inactivation of cellularenzymes, which
depend on the penetration of the compounds into the cellor caused
by membrane permeability changes. The antibacterial activityseemed
to be dependent on the nature of substituents that had a
prominenteffect on the activity profile of compounds rather the
basic skeleton of themolecules. Our findings are in line with Fang
et al.[36] who tested the
Figure 1. The effect of the synthesized compounds on the growth
of pathogenic bacteria bydisc diffusion assay. (a) E. coli. (b) Y.
enterocolitica, (c) L. monocytogenes.
NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS 997
-
antibacterial activity of a new
2,4-disubstituted-6-thiophenyl-pyrimidinederivative against
Gram-positive bacteria through different biological assays.
2.2. In vitro antioxidant activity
The antioxidant activity of compounds (4, 6, 8, 10, 12, 13, 15,
16, 17 and18) was determined against DPPH, which is a free radical
compound thathas been widely used to determine the free
radical-scavenging ability forthe compounds.
2.2.1. The DPPH assay for evaluation of antioxidant activityFree
radicals are involved in the normal physiology of living
organisms.The excess of free radicals and reactive oxygen species
have been proposedto induce cellular oxidative damage, which
results in a variety of chronicdiseases such as cancer,
arteriosclerosis, inflammatory disorders as hereffects on the aging
process.[37] DPPH is a free radical compound that hasbeen widely
used to determine the free radical-scavenging ability of
varioussamples and decreases significantly depending on the
exposure to protonradical scavengers.[38] The reduction capability
of DPPH radicals are deter-mined by the decrease in the absorbance
at 517 nm, which is induced byantioxidants.[39] In Figure 2, the
lowest antioxidant activity was observedwith 15, 17 and 16, while 6
and 13 recorded the highest scavenging results.Both 6 and 13
reached the maximum scavenging ability against DPPH rad-icals of
99.6% and 99.5%, respectively at 75 mg/mL. There are
noticeableeffects on the scavenging of free radical in a
concentration-dependentmode with higher scavenging activity upon
increasing the concentration ofthe other compounds. The results are
higher activity than those of com-mercial phenolic antioxidants
such as BHA and TBHQ. The increasing
0
10
20
30
40
50
60
70
80
90
100
25 ug/ml 50 ug/ml 75 ug/ml
)%(
ytiv itcat nadi xoitnA
4 6 8 10 13 12 15 17 16 18 BHA TBHQTreatment
Figure 2. DPPH radical Scavenging activity of compounds 4, 6, 8,
10, 12, 13, 15, 16, 17 and 18.Values are expressed as mean (n¼ 3)
of the percent inhibition of the absorbance of DPPH radicals.
998 I. F. NASSAR ET AL.
-
scavenging activity upon increasing concentration of the
compounds 4–18from 25mg/mL to 75 mg/mL is due to the decrease in
the concentrationof the DPPH radical, which indicates the reduction
capability of DPPHradicals by the antioxidant activity.The DPPH
antioxidant assay measures the hydrogen-donating capacity of
the molecules in the sample. On the other hand, the
chemiluminescencemethod is based on the light emission produced by
a chemical reaction. TheDPPH radical loses its chromophore upon
receiving a proton from a hydro-gen donor. The scavenging potential
of the synthesized compounds 6 and 13was appraised through
investigating their DPPH reduction against positivecontrol BHA and
TBHQ. The compounds that had a high antioxidant activityexert their
effects via several basic mechanisms which include scavenging
thespecies that initiate peroxidation, quenching singlet oxygen,
breaking the freeradical chain reaction and reducing the
concentration of O2.
[40]
2.3. Anti-tumor activity
The anti-tumor activity of compounds 6 and 13 was determined
againstLiver Hep G2 and Breast MCF-7 cancer cell lines using the
MTT assay(n¼ 3). We found that there is no activity for the tested
compounds againsteither Hep G2 or MCF-7 cell lines and so we cannot
calculated the IC50values for the two tested compounds.
3. Experimental
3.1. Chemistry
All melting points were measured using a Reichert Thermovar
apparatus and areuncorrected. Yields listed are of isolated
compounds. The IR spectra wererecorded on a Perkin-Elmer model 1720
FTIR spectrometer for KBr disc.Routine NMR measurements were made
on a Bruker AC-300 or DPX-300 spec-trometer. Chemical shifts were
reported in d scale (ppm) relative to TMS as a ref-erence standard
and the coupling constants J values are given in Hz. Theprogress of
the reactions was monitored by TLC using aluminum silica gel
plates60 F245. Spectral measurements and Elemental analyses were
performed at theMicro-analytical center at the Faculty of science,
Cairo University, Cairo, Egypt.
3.1.1.
6-(4-Chloro-3-nitrophenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile
(4)
An equimolar mixture of 4-chloro-3-nitrobenzaldehyde 1, thiourea
2, ethylcyano-acetate 3 and anhydrous K2CO3 in absolute ethanol
(30mL) wasrefluxed for 5 h. The mixture was cooled, filtered off;
the precipitate formedwas dissolved in water and neutralized with
dill HCl. The formed
NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS 999
-
precipitate was filtered off, washed with water, dried and
crystallized fromethanol to give 4 as a Brown powder; Yield (75%),
mp �C; IR (KBr cm�1)mmax: 3171 (NH), 1670 (C¼O); 1H NMR (DMSO-d6,
300MHz): d 7.74,7.96 (2d, 2H, Ar-H), 8.05 (s, 1H, Ar-H), 12.0,
13.10 (2s, 2H, 2NH, D2Oexchangeable), 13C NMR (75MHz): d 74 (C-5),
115–145 (CN, Ar-C), 166(C¼O), 170 (C-NH), 175 (C¼S); Analysis
Calcd. for C11H5ClN4O3S(308.70) C, 42.80; H, 1.63; N, 18.15; Found
C, 42.75, H, 1.80, N, 18.0.
3.1.2. Ethyl
2-((4-(4-chloro-3-nitrophenyl)-5-cyano-6-oxo-1,6-dihydro-pyrimidin-2-yl)thio)acetate
(5)
A mixture of 4 (0.01mol) and anhydrous K2CO3 (0.01mol) in dry
acetone(30mL) was stirred at room temperature for 1 h, ethyl
chloroacetate (2mL)was added and the stirring was continued for 25
h at the same temperature.The solvent was removed under reduced
pressure and water was added tothe residue. The formed solid was
filtered off and crystallized from benzeneto form 5. Yield (65%),
mp �C; IR (KBr cm�1) mmax: 3171 (NH), 1735(C¼O); 1H NMR (DMSO-d6,
300MHz): d 1.2 (t, 3H, J¼ 5.2, CH3), 4.0(s, 2H, CH2), 4.2 (q, 2H,
J¼ 5.2, CH2), 7.74, 7.96 (2d, 2H, Ar-H), 8.05(s, 1H, Ar-H), 12.0
(s, 1H, NH, D2O exchangeable);
13C NMR (75MHz): d15 (CH3), 30, 60 (2CH2), 95–145 (pyriminine-C,
CN, Ar-C), 160, 170(2CN), 165, 169 (2CO), 180 (CS); Analysis Calcd.
for C15H11ClN4O5S(394.79) C, 45.64; H, 2.81; N, 14.19; Found C,
45.70, H, 2.80, N, 14.20.
3.1.3.
4-(4-Chloro-3-nitrophenyl)-2-hydrazinyl-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
(6)
A solution of compound 5 (0.01mol) and hydrazine hydrate
(0.150mol) inabsolute ethanol (30mL) was refluxed for 4 h. The
reaction mixture wasallowed to cool at room temperature and the
solvent was evaporated underreduced pressure, the solid that formed
was collected and recrystallizedfrom benzene to afford 6. Yield
(85%), mp �C; IR (KBr cm�1) mmax: 3420,3171 (NH2, NH), 1670 (C¼O);
1H NMR (DMSO-d6, 300MHz): d 5.9 (bs,2H, NH2, D2O exchangeable),
7.74, 7.96 (2d, 2H, Ar-H), 8.05 (s, 1H, Ar-H), 8.3 (s, 1H, NH, D2O
exchangeable), 11.5 (s, 1H, NH, D2O exchange-able); 13C NMR
(75MHz): d 95–145 (pyriminine-C, CN, Ar-C), 160, 170(2CN), 165
(CO); Analysis Calcd. for C11H7ClN6O3 (306.67) C, 43.08; H,2.30; N,
27.41; Found C, 43.0, H, 2.35, N, 27.45.
3.1.4.
4-(4-Chloro-3-nitrophenyl)-2-((((4-chlorophenyl)amino)methyl)thio)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
(7)
A mixture of 4 (0.01mol), primary amine such as
4-chloroaniline(0.01mol) and formaldehyde (2mL) was stirred in
acetonitrile (20mL) at
1000 I. F. NASSAR ET AL.
-
room temperature for 2–3h. The formed precipitate was filtered
off, dried andcrystallized from methanol to form 7. Dark brown
solid; Yield (55%), mp144–146 �C; IR (KBr cm�1) mmax: 3150 (NH),
1670 (CO);
1H NMR (DMSO-d6,300MHz): d 4.7 (s, 2H, CH2), 6.54, 6.57 (2d, 4H,
Ar-H), 7.74, 7.96 (2d, 2H, Ar-H), 8.05 (s, 1H, Ar-H), 8.6 (s, 1H,
NH, D2O exchangeable), 12.0 (s, 1H, NH, D2Oexchangeable); 13C NMR
(75MHz): d 45 (CH2), 95–145 (pyriminine-C, CN, Ar-C), 160 (C-S),
165 (CO), 170 (C¼N), Analysis Calcd. for C18H11Cl2N5O3S(448.28) C,
48.23; H, 2.47; N, 15.62; Found C, 48.10, H, 2.45, N, 15.60.
3.1.5.
1-Acetyl-6-(4-chloro-3-nitrophenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile
(8)
A mixture of 4 (0.01mol), Acetic anhydride (20mL) and few drops
of pyridinewas refluxed for 5–8 h. The reaction mixture was poured
in dilute HCl, theformed precipitate was filtered off, dried and
crystallized from methanol toafford 8. Dark brown solid; Yield
(60%), mp above 300 �C; IR (KBr cm�1)mmax: 3150 (NH), 1670
(CO);
1H NMR (DMSO-d6, 300MHz): d 1.9 (s, 3H,CH3), 7.74, 7.96 (2d, 2H,
Ar-H), 8.05 (s, 1H, Ar-H), 10.9 (s, 1H, NH exchange-able); 13C NMR
(75MHz): d 25 (CH3), 74 (C-5), 115–145 (CN, Ar-C), 160 (C-4), 166,
167 (2C¼O), 175 (C¼S); Analysis Calcd. for C13H7ClN4O4S (350.73)C,
44.52; H, 2.01; N, 15.97; Found C, 44.50, H, 2.0, N, 15.99.
3.1.6.
7-(4-Chloro-3-nitrophenyl)-5-oxo-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carbonitrile
(9)
A mixture of 6 (0.01mol) and formic acid (20mL) was refluxed for
8–10 h,the formed precipitate was filtered off, dried and
crystallized from ethanolto form 9. Yield (70%), mp 100–102 �C; 1H
NMR (DMSO-d6, 300MHz): d2.0 (s, 1H, NH exchangeable); 7.74, 7.91
(2d, 2H, Ar-H), 7.92 (s, 1H, CH),8.05 (s, 1H, Ar-H), 13C NMR
(75MHz): d 94 (C-5), 120–145 (CN, Ar-C,tetrazole-C3,5), 166 (C¼O),
170 (C-N), Analysis Calcd. for C12H5ClN6O3(316.66) C, 45.52; H,
1.59; N, 26.54; Found C, 45.50, H, 1.55, N, 26.55.
3.1.7.
7-(4-Chloro-3-nitrophenyl)-5-oxo-3-thioxo-3,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carbonitrile
(10)
A solution of compound 6 (0.01mol) in pyridine (15mL), carbon
disulfide(5mL) was added with stirring for 10min., the reaction
mixture wasrefluxed for 18 h on water bath, then cooled to room
temperature andacidified with ice cold HCl. The formed precipitate
was filtered off, driedand crystallized from methanol to afford 10
as dark brown solid. Yield(50%), mp 170–172 �C; 1H NMR (DMSO-d6,
300MHz): d 7.74, 7.96 (2d,2H, Ar-H), 8.05 (s, 1H, Ar-H), 13C NMR
(75MHz): d 94 (C-5), 120–145(CN, Ar-C, tetrazole-C5), 165 (C¼O),
180 (C¼S), Analysis Calcd. for
NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS 1001
-
C12H3ClN6O3S (346.71) C, 41.57; H, 0.87; N, 24.24; Found C,
41.55, H,0.80, N, 24.25.
3.1.8.
N0-(4-(4-Chloro-3-nitrophenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)acetohydrazide
(11)
A mixture of 6 (0.01mol) and acetic anhydride (20mL) was
refluxed for 8–10hthe formed solid was filtered, dried and
crystallized from ethanol to yield 11.Brown solid, Yield (65%), mp
120–122 �C; 1H NMR (DMSO-d6, 300MHz): d 1.9(s, 3H, CH3), 7.60 (s,
1H, NH, D2O exchangeable), 7.74, 7.96 (2d, 2H, Ar-H),8.05 (s, 1H,
Ar-H), 10.60 (s, 1H, NH, D2O exchangeable), 11.10 (s, 1H, NH,
D2Oexchangeable); 13C NMR (75MHz): d 20 (CH3), 94 (C-5), 120–145
(CN, Ar-C,),153, 170 (C-2,4), 165, 168 (2C¼O); Analysis Calcd. for
C13H9ClN6O4 (348.70) C,44.78; H, 2.60; N, 24.10; Found C, 44.75, H,
2.60, N, 24.15.
3.1.9.
4-(4-Chloro-3-nitrophenyl)-2-(2-(4-cyanobenzylidene)hydrazinyl)-6-oxo-1,6-dihydro-
pyrimidine-5-carbonitrile (12)
A mixture of compound 6 (0.01mol) and 4-cyanobezaldehyde
(0.01mol) inethanol (20mL) and few drops of glacial acetic acid was
refluxed for 6 hand cooled to room temperature. The solvent was
evaporated underreduced pressure; the formed solid was crystallized
from methanol to yieldcompound 12. Brown solid; Yield (70%), mp
285–287 �C; 1H NMR(DMSO-d6, 300MHz): d 7.50, 7.64 (2d, 2H, Ar-H),
7.7, 7.9 (2d, 4H, Ar-H),8.05 (s, 1H, Ar-H), 8.2 (s, 1H, N¼CH), 8.7
(s, 1H, NH, D2O exchangeable),11.10 (s, 1H, NH, D2O
exchangeable);
13C NMR (75MHz): d 94 (C-5),115–145 (2CN, Ar-C,), 153, 170
(C-2,4), 161 (N¼CH), 166 (C¼O);Analysis Calcd. for C19H10ClN7O3
(419.79) C, 54.36; H, 2.40; N, 23.36;Found C, 54.33, H, 2.40, N,
23.22.
3.1.10. Ethyl
N-(4-(4-chloro-3-nitrophenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)formo
hydrazonate (13)
A mixture of 6 (0.01mol), triethylorthoformate (10mL) and acetic
anhyd-ride (10mL) was refluxed for 7 hr .The reaction mixture was
poured tocold water, the formed precipitate was washed with water,
filtered off andcrystallized from ethyl alcohol to yield compound
13. Brown solid; Yield(66%), mp 83–85 �C; 1H NMR (DMSO-d6, 300MHz):
d 1.2 (t, J¼ 5.3, 3H,CH3), 3.5 (q, J¼ 5.3 2H, CH2), 7.7, 7.9 (2d,
4H, Ar-H), 7.96 (s, 1H, Ar-H),8.0 (s, 1H, N¼CH), 8.7 (s, 1H, NH,
D2O exchangeable), 11.10 (s, 1H, NH,D2O exchangeable);
13C NMR (75MHz): d 15 (CH3), 63 (CH2), 94 (C-5),120–150 (CN,
Ar-C, N¼CH), 153, 170 (C-2,4), 166 (C¼O), 161 (N¼CH);Analysis
Calcd. for C14H11ClN6O4 (362.73) C, 46.36; H, 3.06; N, 23.17Found
C, 46.33, H, 3.0, N, 23.15.
1002 I. F. NASSAR ET AL.
-
3.2. General procedure to prepare compounds 14 and 15
To a well stirred solution of the respective monosaccharide
(5mmol) inwater (1mL), and glacial acetic acid (1mL) was added
compound 6(5mmol) in ethanol (15mL). The mixture was heated under
reflux for 3 hand the resulting solution was concentrated and left
to cool. The formedprecipitate was filtered off, washed with water
and ethanol, then dried andcrystallized from ethanol.
3.2.1.
4-(4-Chloro-3-nitrophenyl)-6-oxo-2-(2-D-Galactopentitolylidenehydrazinyl)-1,6-dihydropyrimidine-5-carbonitrile
(14)
Dark brown solid; Yield (55%), mp 102–104 �C; 1H NMR (DMSO-d6):
d3.30–3.39 (m, 2H, H-60,600), 3.73 (m, 1H, H-50), 4.12 (m, 1H,
H-40), 4.25 (t,J¼ 7.4Hz, 1H, H-30), 4.36 (dd, J¼ 7.4Hz, J¼ 7.8Hz,
1H, H-20), 4.45 (m, 1H,OH), 4.48 (d, J¼ 6.4Hz, 1H, OH), 5.19 (m,
1H, OH), 5.63 (t, J¼ 4.6Hz, 1H,OH), 5.79 (t, J¼ 4.6Hz, 1H, OH),
7.7, 7.9 (2d, 4H, Ar-H), 7.94 (d, 1H, J10,20 ¼7.8Hz, H-10), 7.96
(s, 1H, Ar-H), 8.7 (s, 1H, NH, D2O exchangeable), 11.10(s, 1H, NH,
D2O exchangeable);
13C NMR (75MHz): d 62.10 (C-60), 63.05(C-50), 69.21 (C-40),
74.44 (C-20), 75.71 (C-30), 94 (C-5), 120–135 (CN, Ar-C),152
(C-10), 153, 170 (C-2,4), 166 (C¼O); Analysis Calcd. for
C17H17ClN6O8(468.81) C, 43.55; H, 3.66; N, 17.93; Found C, 43.53,
H, 3.60, N, 14.95.
3.2.2.
4-(4-Chloro-3-nitrophenyl)-6-oxo-2-(2-D-Xylotetritolylidenehydrazinyl)-1,6-dihydropyrimidine-5-carbonitrile
(15)
Dark brown solid; Yield (65%), mp 100–102 �C; 1H NMR (DMSO-d6):
d3.49–3.53 (m, 2H, H-50,500), 3.73 (m, 1H, H-40), 4.12 (m, 1H,
H-30), 4.36 (dd,J¼ 7.4Hz, J¼ 7.8Hz, 1H, H-20), 4.45 (m, 1H, OH),
4.88 (d, J¼ 6.4Hz, 1H,OH), 5.60 (t, J¼ 4.6Hz, 1H, OH), 5.72 (t, J¼
4.6Hz, 1H, OH), 7.28 (m, 3H,Ar-H), 7.36 (m, 3H, Ar-H), 7.42 (m, 2H,
Ar-H), 7.52 (d, 1H, J10,20 ¼ 7.8Hz,H-10), 7.7, 7.9 (2d, 4H, Ar-H),
7.96 (s, 1H, Ar-H), 8.7 (s, 1H, NH, D2Oexchangeable), 11.10 (s, 1H,
NH, D2O exchangeable);
13C NMR (DMSO-d6):d 62.22 (C-50), 63.15 (C-40), 69.32 (C-30),
74.56 (C-20), 122–140 (CN, Ar-C),152 (C-10), 153, 170 (C-2,4), 166
(C¼O); Analysis calcd. for: C16H15 ClN6O7(438.78): C, 43.80; H,
3.45; N, 19.15 Found: C, 43.75; H, 3.40; N, 19.10%.
3.3. Synthesis of compounds 16 and 17
A mixture of compound 6 (0.01mol) and different ketones namely,
4-ami-noacetophenone and cyclohexanone in a mixture of ethanol and
glacialacetic acid was refluxed for 4–5 h. The formed precipitate
was washed withwater, filtered and crystallized to give compounds
16 and 17.
NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS 1003
-
3.3.1.
2-(2-(1-(4-Aminophenyl)ethylidene)hydrazinyl)-4-(4-chloro-3-nitrophenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
(16)
Brown solid; Yield (65%), mp 200–202 �C; 1H NMR (DMSO-d6,
300MHz):d 2.3 (s, 3H, CH3), 5.4 (s, 2H, NH2), 6.8 (d, 2H, Ar-H),
7.6 (d, 2H, Ar-H),7.74, 7.96 (2d, 2H, Ar-H), 8.05 (s, 1H, Ar-H),
8.7 (s, 1H, NH, D2Oexchangeable), 11.10 (s, 1H, NH, D2O
exchangeable);
13C NMR (75MHz):d 15.5 (CH3), 94 (C-5), 114–147 (CN, Ar-C, N¼C),
153, 170 (C-2,4), 166(C¼O); Analysis Calcd. for C19H14ClN7O3
(423.82) C, 53.85; H, 3.33; N,23.13; Found C, 53.83, H, 3.33, N,
23.12.
3.3.2.
4-(4-Chloro-3-nitrophenyl)-2-(2-cyclohexylidenehydrazinyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
(17)
Yellow solid; Yield (67%), mp 290–292 �C; 1H NMR (DMSO-d6,
300MHz):d 1.59 (m, 6H, 3CH2), 2.3 (m, 4H, 2CH2), 7.74, 7.96 (2d,
2H, Ar-H), 8.05(s, 1H, Ar-H), 10.7 (s, 1H, NH exchangeable), 11.10
(s, 1H, NH exchange-able); 13C NMR (75MHz): d 25–34 (5CH2), 94
(C-5), 120–147 (CN, Ar-C),153,160,170 (C-2,4, N¼C), 166 (C¼O);
Analysis Calcd. for C17H15ClN6O3(386.80) C, 52.79; H, 3.91; N,
21.73; Found C, 52.80, H, 3.93, N, 21.72.
3.3.3.
N0-(4-(4-Chloro-3-nitrophenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)benzohydrazide
(18)
A solution of compound 6 (0.01mol) and benzoylchloride (10mL)
wasrefluxed for 3 hr, the mixture was allowed to cool and poured
onto coldwater. The solid that formed was filtered off, dried and
crystallized fromethanol to form compound 18. Brown solid; Yield
(77%), mp 100–102 �C;1H NMR (DMSO-d6, 300MHz): d 7.49–7.63 (m, 4H,
Ar-H), 7.93–7.96 (m,4H, Ar-H), 8.2 (s, 1H, NH, D2O exchangeable
12.0 (s, 1H, NH, D2Oexchangeable), 13.10 (s, 1H, NH, D2O
exchangeable);
13C NMR (75MHz):d 94 (C-5), 120–147 (CN, Ar-C), 153,170 (C-2,4),
164, 166 (2C¼O);Analysis Calcd. for C19H14ClN7O3 (423.82) C, 53.85;
H, 3.33; N, 23.13;Found C, 53.90, H, 3.33, N, 23.02.
4. Biology
4.1. Material and methods
4.1.1. In vitro antimicrobial assay4.1.1.1. Pathogenic strains.
Bacillus cereus B-3711 and Asperagillus flavus3357 were provided by
the Northern Regional Research Laboratory Illinois,USA (NRRL).
Listeria monocytogenes 598 was provided by the Departmentof Food
Science, University of Massachusetts, USA. Escherichia coli0157:H7,
Salmonella typhmirum and Staphylococcus aureus were isolated
1004 I. F. NASSAR ET AL.
-
from serologically identified by Dairy microbiological Lab.,
NationalResearch Center. Yersinia enterocolitica was obtained from
HungarianNational Collection of Medical Bacteria, OKI, Gyaliut 2-6,
H-1966Budapest, Hungary.
4.1.1.2. Experimental. The stock solutions of the compounds
under study(4–18) were prepared at conc. (1mg/mL) for antibacterial
and antifungalassay. Sterile discs were impregnated with 10mL of
each for anti-bacterialand antifungal assay 10lg/disk) a loading
control was also prepared contain-ing 10mL of Dimethyl sulfoxide
(DMSO) for each inoculated spread plate.The discs of (4–18) were
placed on the surface of the agar plat using sterileforceps, gently
press down each disc to ensure complete contact with theagar
surface. Antimicrobial activity of the selected compounds (4–18)
wasconducted against a wide range of human pathogenic
microorganisms Grampositive bacteria (S. aureus, B. cereus and L.
monocytogenes), Gram negativebacteria (Y. enterocolitica, S.
typhmirum, E. coli) and fungal strain (A. flavusand A. niger).The
inverted plates were incubated at (37 �C, 18 h) for bacteriaand (25
�C, 48 h) for fungi within 15min. after the discs are applied.
Thediameter of zones of complete inhibition was measured to the
nearest wholemillimeter, including the diameter of the disc, using
sliding calipers, which isheld on the back of the inverted Petri
plate. Plates were examined for growthinhibition and the diameter
of the inhibition zone measured. The strength ofthe activity was
classified as high activity for the inhibition zone havingdiameters
of (10–15mm) and low activity for the diameter ranging from(7–10mm)
and no activity for one with diameter less than 7mm.
4.1.2. The DPPH assay for evaluation of antioxidant activityIn
vitro the antioxidant activity was evaluated by
2.2-diphenyl-1-picrylhydra-zyl free radical scavenging assay
according to the method of Brand-Williamset al.[41] Briefly, 100,
200 and 300lL of each compound (1mg/mL) weretaken in different test
tubes and 3.9 of 0.1mM ethanol solution of DPPH wasadded and shaken
vigorously. The tubes were then incubated at 37Co for30min. Changes
in the absorbance were measured at 517nm against a blank,i.e.,
without DPPH using UV-Vis Shimadzu (UV-1601, PC)
spectrophotom-eter. Ethanol was used to zero spectrophotometer.
Measurement was per-formed in triplicate and an average was used.
The radical scavenging activitywas expressed as percentage
inhibition of DPPH using the following formula:
%Inhibition ¼ ðAcontrol– Atreatment=AcontrolÞ½ � � 100where
Acontrol is the absorbance of the control; Atreatment is the
absorbanceof the treatments. Butylated hydroxyl anisol (BHA) and
tert-Butylatedhydroxyl qunione (TBHQ) were used as positive
controls.
NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS 1005
-
4.1.3. Anti-tumor activity4.1.3.1. Cell culture. Human
Hepatocellular carcinoma cells (Hep G2) andHuman Breast
adenocarcinoma (MCF-7) was purchased from ATCC, USA,were used to
evaluate the cytotoxic effect of the tested samples. Cells
wereroutinely cultured in Dulbecco’s Minimum Essential Media
(DMEM),the media were supplemented with 10% fetal bovine serum
(FBS), 2mML-glutamine, containing 100 units/mL penicillin G sodium,
100 units/mLstreptomycin sulfate, and 250 ng/mL amphotericin B.
Cells were maintainedat sub-confluency at 37 �C in humidified air
containing 5% CO2. Forsub-culturing, monolayer cells were harvested
after trypsin/EDTA treatmentat 37 �C. Cells were used when
confluence had reached 75%. Testedsamples were dissolved in
dimethyl sulphoxide (DMSO), and then dilutedin the assay to reach
the intended concentration. All cell culture materialwas obtained
from Cambrex BioScience (Copenhagen, Denmark).All chemicals were
from Sigma/Aldrich, USA, except mentioned. Allexperiments were
repeated three times, unless mentioned.
4.1.3.2. Anti-tumor activity assay. Cytotoxicity of tested
samples was meas-ured against Hep G2 and MCF-7 cells using the MTT
Cell Viability Assay.MTT
(3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide)
assayis based on the ability of active mitochondrial dehydrogenase
enzymeof living cells to cleave the tetrazolium rings of the yellow
MTT and forma dark blue insoluble formazan crystals which is
largely impermeable tocell membranes, resulting in its accumulation
within healthy cells.Solubilization of the cells results in the
liberation of crystals, which arethen solubilized. The number of
viable cells is directly proportional to thelevel of soluble
formazan dark blue color. The extent of the reduction ofMTT was
quantified by measuring the absorbance at 570 nm.[42]
4.1.3.3. Reagents preparationMTT solution: 5mg/mL of MTT in
0.9%NaCl and dimethylsulfox-ide (DMSO).
4.1.3.4. Procedure. Cells (0.5� 105 cells/well), in serum-free
media, wereplated in a flat bottom 96-well microplate, and treated
with 20 mL of differ-ent concentrations (100, 50, 25 and 12.5
mg/mL) of the tested samples for24 h at 37� C, in a humidified 5%
CO2 atmosphere. After incubation, mediawere removed and 40 mL MTT
solution/well were added and Incubatedfor an additional 4 h. MTT
crystals were solubilized by adding 180 mLof dimethyl
sulphoxide/well and plate was shacked at room temperature,followed
by photometric determination of the absorbance at 570 nm
usingmicroplate ELISA reader. Triplicate repeats were performed for
each
1006 I. F. NASSAR ET AL.
-
concentration and the average was calculated. Data were
expressed as thepercentage of relative viability compared with the
untreated cells comparedwith the vehicle control, with cytotoxicity
indicated by
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AbstractIntroductionResults and discussionIn vitro antimicrobial
activityIn vitro antioxidant activityThe DPPH assay for evaluation
of antioxidant activity
Anti-tumor activity
ExperimentalChemistry6-(4-Chloro-3-nitrophenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile
(4)Ethyl
2-((4-(4-chloro-3-nitrophenyl)-5-cyano-6-oxo-1,6-dihydro-pyrimidin-2-yl)thio)acetate
(5)4-(4-Chloro-3-nitrophenyl)-2-hydrazinyl-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
(6)4-(4-Chloro-3-nitrophenyl)-2-((((4-chlorophenyl)amino)methyl)thio)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
(7)1-Acetyl-6-(4-chloro-3-nitrophenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile
(8)7-(4-Chloro-3-nitrophenyl)-5-oxo-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carbonitrile
(9)7-(4-Chloro-3-nitrophenyl)-5-oxo-3-thioxo-3,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carbonitrile
(10)N′-(4-(4-Chloro-3-nitrophenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)acetohydrazide
(11)4-(4-Chloro-3-nitrophenyl)-2-(2-(4-cyanobenzylidene)hydrazinyl)-6-oxo-1,6-dihydro-
pyrimidine-5-carbonitrile (12)Ethyl
N-(4-(4-chloro-3-nitrophenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)formo
hydrazonate (13)
General procedure to prepare compounds 14 and
154-(4-Chloro-3-nitrophenyl)-6-oxo-2-(2-D-Galactopentitolylidenehydrazinyl)-1,6-dihydropyrimidine-5-carbonitrile
(14)4-(4-Chloro-3-nitrophenyl)-6-oxo-2-(2-D-Xylotetritolylidenehydrazinyl)-1,6-dihydropyrimidine-5-carbonitrile
(15)
Synthesis of compounds 16 and
172-(2-(1-(4-Aminophenyl)ethylidene)hydrazinyl)-4-(4-chloro-3-nitrophenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
(16)4-(4-Chloro-3-nitrophenyl)-2-(2-cyclohexylidenehydrazinyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
(17)N′-(4-(4-Chloro-3-nitrophenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)benzohydrazide
(18)
BiologyMaterial and methodsIn vitro antimicrobial
assayPathogenic strainsExperimental
The DPPH assay for evaluation of antioxidant activityAnti-tumor
activityCell cultureAnti-tumor activity assay
Reagents preparationProcedure
AcknowledgmentsReferences