Synthesis, antimicrobial, antioxidant activities of novel 6-aryl-5-cyano thiouracil derivatives

Article abstract of DOI:10.1016/j.ejmech.2013.08.032

A series of 6-aryl-5-cyano thiouracil derivatives (2a-c to 11a-c) was synthesized from 6-aryl-4-hydrazino-2-thioxo-1,2-dihydropyrimidine-5- carbonitriles (1a-c).The products were characterized by analytical and spectral data (IR, ¹H NMR, ¹³C NMR and mass spectra). All compounds were screened for their in-vitro antibacterial and antifungal activities. Compounds 7a, 7g and 9a-c showed pronounced antimicrobial activity than standards. Some of the newly synthesized compounds were evaluated for antioxidant activity. Compounds 1c, 5c and 8c displayed promising free radical scavenging activity and found to be more potent than standard, ascorbic acid (vitamin C).

Full text of DOI:10.1016/j.ejmech.2013.08.032

Accepted Manuscript
Synthesis, antimicrobial, antioxidant activities of novel 6-aryl- 5-cyano thiouracil
derivatives
M.S. Mohamed, M.M. Youns, N.M. Ahmed
PII:
S0223-5234(13)00549-7
10.1016/j.ejmech.2013.08.032
EJMECH 6379
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 March 2013
Revised Date: 17 August 2013
Accepted Date: 21 August 2013
Please cite this article as: M.S. Mohamed, M.M. Youns, N.M. Ahmed, Synthesis, antimicrobial,
antioxidant activities of novel 6-aryl- 5-cyano thiouracil derivatives, European Journal of Medicinal
Chemistry (2013), doi: 10.1016/j.ejmech.2013.08.032.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
►A series of novel 6-aryl-5-cyano thiouracils (2a-c to11a-c) was synthesized from
6-aryl-4-hydrazino-2-thioxo-1, 2-dihydropyrimidine-5-carbonitriles (1a-c).
►Antimicrobial and antioxidant studies were performed. ► Potent activities were
observed.
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Synthesis, antimicrobial, antioxidant activities of novel 6-aryl- 5-cyano thiouracil
derivatives
M.S.Mohamed a*, M. M.Youns b and N.M.Ahmed a
a Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Helwan
University, Ein Helwan, Cairo, Egypt
b Biochemistry Department, Faculty of Pharmacy, Helwan University, Ein Helwan,
Cairo, Egypt
* Correspondence; e-mail: doctor-mosaad@hotmail.com
ABSTRACT
A series of 6-aryl-5-cyano thiouracil derivatives (2a-c to11a-c) was synthesized from 6-
aryl-4-hydrazino-2-thioxo-1,2-dihydropyrimidine-5-carbonitriles (1a-c) .The products
were characterized by analytical and spectral data (IR,1HNMR,13CNMR and mass spec-
tra).All compounds were screened for their in-vitro antibacterial and antifungal
activities. Compounds 7a,7g and 9a-c showed pronounced antimicrobial activity than
standards. Some of the newly synthesized compounds were evaluated for antioxidant
activity. Compounds 1c,5c and 8c displayed promising free radical scavenging activity
and found to be more potent than standard, ascorbic acid (vitamin C).
Keywords: Synthesis, 6-aryl -5-cyano thiouracils, antibacterial, antifungal, antioxidant
activity, radical scavenging.
Graphical abstract
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1. Introduction
Pyrimidine constitutes an important component of nucleic acid and it is used as building
blocks in pharmaceutics for the synthesis of antiviral, anticancer,antioxidant,antiinflam-
matory, antibacterial and antifungal agents[1-7].Similarly, the related thiouracil deriv-
atives are potential therapeutics as antiviral, antioxidant, anticancer and antimicrobial
agents [8-11]. Moreover, a literature survey revealed that the thiouracil carbonitrile ring
system has occupied a marked position in the design and synthesis of novel chemothera-
peutic agents with remarkable antitumor I, HCV inhibitorsII and antimicrobial activities
III(Fig.1)[12-14].Reports from our laboratory[15] and from others [16] revealed the
synthesis of 4-hydrazinothiopyrimidine-5-carbonitriles from 4-chloro derivatives. These
hydrazino derivatives exerted promising antibacterial, antifungal and anticancer
activities [17-19]. In addition, the reactions of hydrazino pyrimidines with formic acid,
trietylorthoformate(TEOF)and CS2(one carbon donor moieties) afforded the corres-
ponding triazolopyrimidines[20-25],which are known to exhibit interesting Pharma-
ceutical activities [26, 27]. In the light of the aforementioned facts, and in continuation
for our interest in the synthesis of biologically active heterocyclic compounds, we report
herein the synthesis and biological evaluation of novel 6-aryl-5-cyano thiouracil
derivatives as antimicrobial and antioxidant agents.
Fig.1. Structures of some potent 5-cyano -2-thiouracils.
2. Results and discussion
Chemistry
The synthetic pathways adopted for the preparation of the desired new compounds are
illustrated in charts 1, 2.
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Synthesis of hydrazino pyrimidines 1a-c was achieved through two synthetic pathways
as previously reported from our laboratory [15], the first of which was the reaction of 6-
aryl-5-cyano-2-thiouracils with PoCl3/PCl5 to obtain the chloro derivatives. Reaction of
the latter compounds with hydrazine hydrate (99%) in methanol afforded 1a-c. Hydraz-
ino derivatives1a-c were used as key compounds for this study and for synthesis of other
fused heterocycles. In this investigation ,and in continuation for our previous work [28-
31] in the synthesis of different fused triazolo pyrimidines, refluxing of compounds 1a-c
with acetic anhydride ,formic or acetic acid ,ethyl chloroformate (ECF) and carbon
disulfide ; gave 7-aryl-3-methyl-5-thioxo-5,6-dihydro-[1,2,4]triazolo [4,3-c] pyrimidine-
8-carbonitriles 2a-c ,7-aryl-5-thioxo-5,6-dihydro[1,2,4]triazolo[1,5-c]-pyrimidine-8-car-
bonitriles 3a-c,7-aryl-2-methyl-5-thioxo-5,6-dihydro [1,2,4]triazolo [1,5-c]-pyrimidine-
8-carbonitriles 4a-c,7-aryl-3-oxo-5-thioxo-2,3,5,6-tetrahydro[1,2,4]-triazolo[4,3-c]pyri-
midine-8-carbonitriles 5a-c and 7-aryl-3,5-dithioxo-2,3,5,6-tetrahydro[1,2,4]triazolo [4,
3-c]pyrimidine-8-carbonitriles6a-c,respectively[16,25,32,33]as revealed in scheme1.
Furthermore, reaction of 4-hydrazinopyrimidines with different aromatic aldehydes in
ethanol afforded the corresponding 4-[(2E)-2-substituted (benzylidene) hydrazino]-6-
(aryl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitriles7a-i[34,35],while cyclocon-
densation of compounds1a-c with diethyloxalate,triethylorthoformate and acetyl acetone
afforded carbonitriles(8-10)a-c,respectively[12,25,19].Finally,the reaction of compou-
nds 1a-c with aqueous solution of sodium nitrite in HCl at 0-5˚C afforded triazolo[4,3-
c]pyrimidine-8-carbonitriles11a-c[36].The structures of new compounds were confirm-
ed by MS,IR,1H NMR,13CNMR as well as elemental analysis. IR spectra of compounds
1a-c showed CN bands at 2200-2230 cm-1and two absorbance bands at 3490 -3200 cm-1
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corresponding NHNH2 as reported [16] .1HNMR spectra showed three singlet of
1
NHNH2 around 5.1 and 5.7 ppm. The HNMR spectra of compounds 2a-c showed a
singlet signal at 2.3-2.5 ppm corresponding to CH3 of triazole ring. The 13C NMR
spectra displayed signals for CH3 aliphatic carbon and for C=N carbon of triazole ring.
The1HNMR spectra for 3a-c revealed singlet signals for CH=N and one NH instead of
three signals like in 1a-c ,for 4a-c signals for CH3 and one NH. The 13CNMR spectra of
products were compatible with the proposed structure. Also,the structures of 5a-c were
1
established on the basis of IR, which showed the presence of C=O while H NMR
13
spectra revealed two signal for NH proton. The CNMR spectra displayed signals for
1
C=O carbon. The HNMR spectra of 6a-c showed two singlet signals for NH proton.
Moreover, the13CNMR spectra showed the presence of two C=S signals corresponding
to C=S of thiouracil and triazole ring. Compounds7a-i and 8a-c were confirmed by
spectral data and the mass spectra studies of these compounds gave additional evidence
for the proposed structures.1HNMR for 9a-c showed singlet signals for CH=N and one
NH .The13CNMR spectra displayed signals for C=N carbon. Also, MS spectra gave their
1
molecular ion peaks.The HNMR of 10a-c showed two singlet signals at 2-2.53 ppm
corresponding to two CH3 group and only one singlet corresponding to NH. The IR and
13CNMR spectra of products 10a-c were compatible with the proposed structure. The IR
spectra of 11a-c showed the absence of NHNH2 as well as the presence of C=N and 1H
NMR showed a singlet signal for NH proton.
Scheme 1
Scheme 1. Synthetic Pathway for the preparation of compounds 1 ~ 6 (a - c).
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Scheme 2
Scheme 2. Synthetic Pathway for the preparation of compounds 7 (a-i) ~11 (a - c).
Biological results and discussion
Antimicrobial studies
Antimicrobial studies (Table I) showed that compounds 7a-i and 9a-c possess a pron-
ounced antimicrobial activity against Staphylococcus aureus ,Bacillus cereus, Escheric-
hia coli,Candida albicans and Aspergillus flavus compared to the reference drugs, while
compounds 1a-c,10a-c and 11a-c showed marked antibacterial activity against SA,BC
and EC but didn’t show any antifungal activity except 1a showed moderate activity
against Aspergillus flavus. Compounds 6a-c exhibited high activities also towards BC
,EC and CA,while inactive against SA and AF, compounds 8a-c towards Staphylo-
coccus aureus and Aspergillus flavus only. Compounds 5a-c possess high activities
towards Staphylococcus aureus only.Compounds2a-c,3a-c and 4a-c were either inactive
or weakly active against the tested microorganisms.
The present study revealed that conversion of 4-hydrazino pyrimidines to hydrazones
(7a-i) or triazolo[4,3-c] pyrimidines (9a-c) caused a pronounced inhibition effect against
Gram-positive (Staphylococcus aureus, Bacillus cereus), Gram-negative (Escherichia
coli) bacteria and fungi (Candida albicans and Aspergillus flavus).On the other hand,
increased antibacterial activity was achieved by cyclization to triazolo[4,3-c]pyrimidines
, pyazolopyrimidines or tetrazolo pyrimidines as in 6,10 and 11a-c ,while cyclization
to triazolo[4,3-c] pyrimidines or pyrimidotriazines as in 5, 8a-c increased antibacterial
activity only against Staphylococcus aureus or S.aureus and Asp.flavus, respectively.
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However, cyclization to triazolo [1,5-c]pyrimidines or triazolo[4,3-c] pyrimidines as
in 3,4a-c or 2a-c either decreased or diminished antimicrobial activity , respectively
(see Inline Supplementary,Figs.S1-S10).
The structure activity relationship suggested that conversion of thiouracils to hydrazones
or triazolo [4, 3-c] pyrimidines showed higher antibacterial and antifungal activities than
other derivatives.
Table 1 and Figure 2,3.
Table I. Antimicrobial activity results of newly synthesized compounds, expressed as
inhibition zone diameter (mm) and MIC (µg /ml) with the standard drugs.
Fig.2: Antibacterial activity (Gram +ve,-ve) of synthesized compounds.
Fig.3: Antifungal activity of synthesized compounds.
Antioxidant evaluation
The DPPH radical scavenging ability of some synthesized compounds was evaluated
and compared to those of the well-known antioxidant ascorbic acid (table II).
Our compounds were able to reduce DPPH in a concentration-dependent manner. The
tested samples were statistically different (P < 0.05, Kruskal-Wallis test) over the dose
range used. The maximum scavenging activity of DPPH was produced with compounds
1c,5c and 8c with IC50 of 6.21,9.34,8.55 µg/mL, respectively (Fig.4,5).On critical
overview of synthesized compounds, it has been found that compounds with electron
donating group (-OCH3) on phenyl ring exhibited potent antioxidant activity.
Table II and Figure 4, 5.
Table II. IC50 values (in µg/mL) for DPPH scavenging ability of the compounds
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Fig.4. Screening of antioxidant activity by the DPPH assay shows that 1c, 5c and 8c
have highest activity (more potent than the reference drug (RF). Each value represents a
mean ± SEM (n= 3).
Fig.5. DPPH radical scavenging activity of compound 1c. Each value represents a mean
± SEM (n = 3).
Conclusions
Evaluation of the new compounds established that the most promising antimicrobial
compounds are 4- [(2E)-2-benzylidene- hydrazino]-6-(4-fluoro-phenyl)-2-thioxo-1, 2, 3,
4-tetrahydropyrimidine-5-carbonitrile (7a), [(2E)-2-(4-methoxybenzylidene)-hydrazino]
-6-(4-fluoro-phenyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile(7g),7-(4-fluo-
oro-phenyl)-5-thioxo-5,6-dihydro[1,2,4] triazolo[4,3-c]- pyrimidine-8-carbonitrile(9a),
7-(4-Bromo-phenyl)-5-thioxo-5,6-dihydro[1,2,4]triazolo[4,3-c]-pyrimidine-8-carbonitr-
ile (9b) and 5-thioxo-7-(3,4,5-trimethoxy-phenyl)-5,6-dihydro- [1,2,4] triazolo[4,3-c]-
pyrimidine-8-carbonitrile(9c).Compounds 1c,5c and 8c were found to possess promising
antioxidant activity when compared with standard ascorbic acid (vitamin C).
3. Experimental
3.1. Chemistry
All melting points are uncorrected and measured using Electro- thermal IA 9100
apparatus (Shimadzu, Japan). IR spectra were recorded as potassium bromide pellets on
a Perkin-Elmer 1650 spectrophotometer (USA), Faculty of Science, Cairo University,
Cairo, Egypt.1HNMR and 13C-NMR spectra (see Supplementary data) were determined
on a Varian Mercury (300 MHz) spectrometer (Varian UK) and chemical shifts were
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expressed as ppm against TMS as internal reference (Faculty of Science, Cairo
University, Cairo, Egypt). Mass spectra were recorded on 70 eV EI Ms-QP 1000 EX
(Shimadzu, Japan), Faculty of Science, Cairo University, and Cairo, Egypt.
Microanalyses were operated using Vario, Elementar apparatus (Shimadzu, Japan),
Organic Microanalysis Unit, Faculty of Science, Cairo University, Cairo, Egypt and the
results were within the accepted range (0.40) of the calculated values. Column
Chromatography was performed on (Merck) Silica gel 60 (particle size 0.06-0.20 mm).
3.2.General method for preparation of I a - c
Synthesis of compounds was performed according to the literature [16].A mixture of the
appropriate thiouracil a (2.65 g,0.01 mol), b (2.90 g,0.01 mol) , c (3.26 g,0.01 mol) and
hydrazine hydrate (20 mL, 99%) was refluxed in methanol (30 mL) for 20m,cooled ,
stirred for 24 h and poured onto ice - water. The solid obtained was filtered, dried and
recrystallized from DMF/ water to yield compounds 1a- c, respectively.
3.2.1.6-(4-Fluoro-phenyl)-4-hydrazino-2-thioxo-1,2-dihydropyrimidine-5-carbonitrile
1a.Yield 65%,m.p.200-202˚C.IR (max /cm-1): 3460-3200( NH2,2NH ),2200 (CN) ,1270
(C=S).1HNMR(300MHz,DMSO-d6):δ5.1-5.7(s,3H,NH,NH2,D2Oexchangeable),7.3-7.8
(m, 4H,Ar-H),10.3(s,1H,NH,D2O exchangeable).13CNMR(300MHz,DMSO-d6):δ90.42,
114.17,115.38,125.27,131.26,157.96,159.57,161.82,175.71.MS (EI): m/z:261[M+](15.4
%).Anal. Calcd for C11H8FN5S(261.27):C,50.57;H,3.09; N,26.80;S,12.27; Found: C,50.
42; H,2.90; N,26.93;S,12.43.
3.2.2.6-(4-Bromo-phenyl)-4-hydrazino-2-thioxo-1,2-dihydropyrimidine-5-carbonitrile
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1b. Yield 66%, m.p.190-192˚C.IR (max/cm-1):3490-3264 (NH2, 2NH), 2225(CN),1275
(C=S).1H NMR (300MHz,DMSO-d6):δ 5.2-5.7(s,3H,NH,NH2, D2O exchangeable), 7.3-
7.8(m,4H,Ar-H),10.5(s,1H,NH,D2O exchangeable).13CNMR(300MHz,DMSO-d6):δ87.9
,117,122.3,128.4,131.7,133.9,164,169,182.MS(EI):m/z:321[M+](24.8%),323[M+2]
(24.7%).Anal.Calcd for C11H8BrN5S(320.97): C,41.01; H, 2.50; N,21.74; S,9.95; Found:
C,41.18;H,2.64;N,21.62;S,10.02.
3.2.3.4-hydrazino-2-thioxo-6-(3,4,5-trimethoxy-phenyl)-1,2-dihydropyrimidine-5-carbo-
nitrile 1c.Yield 68%,m.p.215-217˚C.IR (max/cm-1):3490-3300 (NH2, 2NH),2230 (CN),
1268(C=S).1HNMR (300 MHz, DMSO-d6):δ 3.1- 3.7(s,9H,3OCH3),5.1-5.7 (s, 3H,NH,
NH2,D2Oexchangeable),6.6,6.8(s,2H,Ar-H),10.2(s,1H,NH,D2O exchangeable).13CNMR
(300MHz,DMSO-d6):δ55.4,55.7,85.8,115,117,129.2,132.4,160,163,167.5,183.MS (EI):
m/z:333[M+](15.3%).Anal.Calcd for C14H15N5O3S (333.36):C,50.44; H, 4.54;N,21.01;
S,9.62;Found:C,50.58;H,4.42;N,21.10;S,9.77.
3.3.General method for preparation of 2 a - c
A mixture of the appropriate thiouracil derivative 1a - c (0.01 mol) and acetic anhydride
(30 mL) was heated under reflux for 2h.The solid obtained was filtered, dried and
recrystallized from acetic acid to yield compounds 2a- c, respectively.
3.3.1.7-(4-Fluoro-phenyl)-Aryl-3-methyl-5-thioxo-5, 6-dihydro-[1, 2, 4] triazolo [4, 3-c]
pyrimidine-8-carbonitrile 2a.Yield55%, m.p.170-172˚C.IR (max/cm-1):3262(NH), 2216
(CN),1270(C=S),1598(C=N).1H NMR (300 MHz,DMSO-d6):δ2.3(s,3H,CH3),6.9-7.9
(m,4H,Ar-H),9.1(s,1H,NH,D2Oexchangeable).13CNMR(300MHz,DMSO-d6):δ20.5,
86.5,113.3,115.2,127.2,130.4,150.3,152.4,159.5,163.2,177.2.MS (EI):m/z:285[M+]
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(40 %). Anal. Calcd for C13H8FN5S (285.29):C,54.73;H,2.83;N,24.55;S,11.24;
Found:C,54.80; H,2.90; N,24.70;S,11.34.
3.3.2.7-(4-Bromo-phenyl)-3-methyl-5-thioxo-5,6-dihydro-[1,2,4]triazolo[4,3-c]pyrimid-
ine-8-carbonitrile 2b.Yield 53%,m.p.208-210˚C.IR (max/cm-1):3250(NH),2220(CN),
1275(C=S),1595(C=N).1HNMR(300MHz,DMSO-d6):δ2.5(s,3H,CH3),7-7.9(m,4H,Ar-
H),9.4(s,1H,NH,D2Oexchangeable).13CNMR(300MHz,DMSO-d6):δ20.3,92.6,114.05,
115.4,128.8,133.4,150.3,151.4,160.5,162,175.2.MS (EI): m/z:345[M +] (55.1%),347
[M+2](55.3%).Anal. Calcd for C13H8BrN5S (344.97):C,45.10;H,2.33;N,20.23;S,9.26
;Found:C,45.30;H,2.50;N,20.40;S,9.34.
3.3.3.3-Methyl-5-thioxo-7-(3,4,5-trimethoxy-phenyl)-5, 6-dihydro-[1,2,4] triazolo [4, 3-
c]pyrimidine-8-carbonitrile2c.Yield 50%,m.p.223-225˚C.IR(max/cm-1):3277(NH),2222
(CN),1277(C=S),1590(C=N).1HNMR(300 MHz,DMSO-d6):δ2.52(s,3H,CH3),3.4-3.86
(s,9H,3OCH3),6.82,7(s,2H,Ar-H),10.14(s,1H,NH,D2O exchangeable.13CNMR(300MHz
,DMSO-d6):δ20.4,55.4,55.7,88.7,114,115.3,130.4,151.7,152,153.9, 159.3,168,178.MS
(EI):m/z:357[M+](60%).Anal. Calcd for C16H15N5O3S(357.38):C,53.77;H,4.23;N,19.60
;S,8.97;Found:C,53.80; H,4.30;N,19.77;S,9.04.
3.4. General method for preparation of 3a-c and 4a-c
A mixture of appropriate thiouracil derivative 1a- c (0.01 mol) and formic or acetic acid
(30 mL) was heated under reflux for 8h ,then cooled and poured onto ice - water. The
solid obtained was crystallized from acetic acid to yield compounds 3,4a-c, respectively.
3.4.1.7-(4-Fluoro-phenyl)-5-thioxo-5,6-dihydro[1,2,4]triazolo[1,5-c]-pyrimidine-8-car-
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bonitril 3a.Yield 45%,m.p.228-230˚C.IR(max/cm-1):3225(NH),2216(CN),1270 (C=S),
1613(C=N).1HNMR(300MHz,DMSO-d6):δ8.60(s,1H,C3-H),7-7.7(m,4H,Ar-H),9.2(s,
1H,NH,D2O exchangeable ).13CNMR(300MHz,DMSO-d6):82.3,115,117.2,127.4,130.1
,149.7,151.2,161.5,166,176.2.MS(EI):m/z:271[M+](56%).Anal.Calcd for C12H6FN5S
(271.27):C,53.13;H,2.23;N,25.82;S,11.82;Found:C,53.33; H,2.41;N,25.95; S, 11.94.
3.4.2.7-(4-Bromo-phenyl)-5-thioxo-5,6-dihydro[1,2,4]triazolo[1,5-c]-pyrimidine-8-car-
Bonitrile 3b.Yield47%,m.p.218-220˚C.IR(max/cm-1):3322(NH),2220(CN),1270(C=S),
1612(C=N).1HNMR(300MHz,DMSO-d6):δ8.5(s,1H,C3-H),7.2-ٌ7.6(m,4H,Ar-H),9.5(s,
1H,NH,D2Oexchangeable).13CNMR(300MHz,DMSO-d6):δ85.1,117.2,122.3,128.4,131.
7,148.5,153.1,163,165.1,175.7.MS(EI):m/z:331[M+](59.2%),333[M+2] (59.4%).Anal.
Calcd for C12H6BrN5S (330.95):C,43.39;H,1.82;N,21.08;S,9.65;Found: C,43.51; H,1.94;
N,21.26;S,9.78.
3.4.3.5-thioxo-7-(3,4,5-trimethoxy-phenyl)-5,6-dihydro[1,2,4]triazolo[1,5-c]-pyrimidine
-8-carbonitrile 3c.Yield 45%, m.p. 208-210˚C.IR(max/cm-1):3230(NH),2222(CN),1270
(C=S),1609(C=N).1HNMR(300MHz,DMSO-d6):δ3-3.8(s,9H,3OCH3),8.63(s,1H,C3-H),
7,7. 5(s,2H,Ar-H),9.3(s,1H,NH,D2O exchangeable ).13CNMR(300 MHz,DMSO-d6):δ
55.4,55.7, 80.9,115.1,117.2,129.2,132.4,148.5,152.2,161.8,165.3,176.1.MS(EI):m/z:343
[M+](42%).Anal.Calcd for C15H13N5O3S (343.36):C,52.47;H,3.82;N,20.40; S,9.34;
Found: C,52.58; H,3.97; N,20.61; S,9.44.
3.4.4.7-(4-Fluoro-phenyl-2-methyl-5-thioxo-5,6-dihydro[1,2,4]triazolo[1,5-c]-pyrimid-
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ine-8-carbonitrile 4a.Yield52%,m.p.233-235˚C. IR (max/cm-1):3225(NH),2216(CN),
1270(C=S),1607(C=N).1HNMR(300MHz,DMSO-d6):δ2.2(s,3H,CH3),7.1-7.8(m,4H,
Ar-H),9.8(s,1H,NH,D2Oexchangeable).13CNMR(300MHz,DMSO-d6):δ20.2, 84.3,113,
117.2,124.4,127.1,143.7,150.3,162.5,165,182.MS (EI): m/z: 285[M+](60%).Anal. Calcd
for C13H8FN5S (285.29):C,54.73;H,2.83;N,24.55;S,11.24;Found:C,54. 88;H,2.94;N, 24.
68; S,11.35.
3.4.5.7-(4-Bromo-phenyl-2-methyl-5-thioxo-5,6-dihydro[1,2,4]triazolo[1,5-c]-pyrimid-
ine-8-carbonitrile 4b.Yield 54%, m.p.222-224˚C.IR (max/cm-1):3333(NH),2216(CN),
1270(C=S),1602(C=N).1H NMR(300MHz,DMSO-d6):δ 2.3(s,3H,CH3),7.2-7ٌ .7(m,4H,
Ar-H),9.3(s,1H,NH,D2O exchangeable).13CNMR(300 MHz,DMSO-d6):δ20.2,82.5,
117.2,122.3,128.4,131.7,140.2,151.2,160.3,166.9,182.MS EI): m/z:345[M+] (62.1%),
347[M+2](62.3%).Anal.Calcd for C13H8BrN5S (344.97): C,45.10;H, 2.33; N,20.23; S,
9.26; Found: C,45.22; H,2.55; N,20.36; S,9.39.
3.4.6.2-Methyl-5-thioxo-7-(3,4,5-trimethoxy-phenyl)-5,6-dihydro[1,2,4]triazolo[1,5-c]-
Pyrimidine-8-carbonitrile 4c.Yield 55%,m.p.228-230˚C.IR(max/cm-1):3240(NH), 2216
(CN),1270(C=S),1605(C=N).1HNMR(300MHz,DMSO-d6):δ2.2(s,3H,CH3),3-3.7(s,9H
,3OCH3),7,7.4(s,2H,Ar-H),9.2 (s,1H,NH,D2O exchangeable).13CNMR(300 MHz,DMSO
-d6):δ20.3,55.4,55.6,81.1,115.1,117,129.2,132.4,148.5,151.9,162.3,166.8,182.MS(EI):
m/z:357 [M+](71%).Anal.Calcd for C16H15N5O3S (357.38):C,53.77; H,4.23; N,19.60;
S, 8.97; Found: C,52.89;H,4.35;N,19.72;S,9.10.
3.5. General method for preparation of 5a - c
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ACCEPTED MANUSCRIPT
A mixture of appropriate thiouracil derivative 1a - c (0.01 mol) and ethylchloroformate
(0.02 mol) in pyridine (30 mL) was heated under reflux for 12h and then poured on
HCl. The solid obtained was crystallized from dimethyl formamide to yield compounds
5a- c, respectively.
3.5.1.7-(4-Fluoro-phenyl)-3-oxo-5-thioxo-2,3,5,6-tetrahydro[1,2,4]-triazolo [4,3-c]pyr-
imidine-8-carbonitrile 5a.Yield 51%,m.p.178-180˚C. IR(max/cm-1):3430-3100(NH),
2215(CN),1661(C=O),1625(C=N).1HNMR(300MHz,DMSO-d6):δ7.1-7.5(m,4H,Ar-H)
,8.1,9.2(s,2H,NH,D2Oexchangeable).13CNMR(300MHz,DMSO-d6): δ77,113,117,124.4,
143.7,147.8,155,165,170,178.MS (EI):m/z:287[ M+](35%).Anal.Calcd for C12H6FN5OS
(287.27):C,50.17; H, 2.11;N,24.38;S,11.16; Found:C,50.29;H,2.25;N,24.49;S,11.27.
3.5.2.7-(4-Bromo-phenyl)-3-oxo-5-thioxo-2,3,5,6-tetrahydro[1,2,4]-triazolo[4,3-c]pyr-
imidine-8-carbonitrile 5b.Yield 50%,m.p.173-175˚C.IR (max/cm-1):3450-3190(NH),
2225(CN),1673(C=O),1635(C=N).1HNMR(300MHz,DMSO-d6):δ7.3-7.8(m,4H,Ar-H),
8.3,9(s,2H,NH,D2Oexchangeable).13CNMR(300MHz,DMSO-d6):δ75, 117,122.1,128.4,
133.2,147.8,158,168,170,177.MS(EI):m/z:347[M+](43.3%),349 [M+2] (43.5%).Anal.
Calcd for C12H6BrN5OS (346.95): C,41.40; H,1.74; N,20.11; S, 9.21; Found: C,41.55;
H,1.83;N,20.23;S,9.35.
3.5.3.3-Oxo-5-thioxo-7-(3, 4, 5-trimethoxy-phenyl)-2, 3, 5, 6-tetrahydro [1, 2,4]-triazolo
[4, 3-c] pyrimidine-8-carbonitrile 5c.Yield 53%, m.p.185-187˚C.IR(max/cm-1):3400-
- 14 -

ACCEPTED MANUSCRIPT
3180(NH),2225(CN),1677(C=O),1635(C=N).1HNMR(300MHz,DMSO-d6):δ3-3.7(s,9H
,3OCH3),7.1,7.6(s,2H,Ar-H),8.1,9.2(s,2H,NH,D2Oexchangeable).13CNMR(300MHz,
DMSO-d6):δ55.55,55.75,78.04,104.82,114.05,122,130.45,146.79,152,157.98,159.79,
178.04. MS (EI):m/z:359 [M+](40%).Anal.Calcd for C15H13N5O4S(359.35):C,50.13;H,
3.65; N,19.49; S,8.92; Found: C,50.26; H,3.77; N,19.55; S,9.10.
3.6. General method for preparation of 6a - c
To an ice cold solution of appropriate thiouracil derivative 1a-c (0.01 mol) and KOH
(0.01 mol) in ethanol (20 mL) was added dropwise with stirring CS2 (10mL), then the
reaction mixture was refluxed on a water-bath for 5h. The reaction mixture cooled,
poured onto ice -water, neutralized with HCl, filtered, crystallized from ethanol to yield
compounds 6 a- c, respectively.
3.6.1.7-(4-Fluoro-phenyl)-3,5-dithioxo-2,3,5,6-tetrahydro[1,2,4]triazolo[4,3-c]pyrimid-
ine-8-carbonitrile 6a.Yield 54 %, m.p.240-242˚C.IR (max/cm-1):3333-3160 (NH),2227
(CN),1650(C=S),1580(C=N).1HNMR(300MHz,DMSO-d6):δ6.6-7.6(m,4H,Ar-H),9.1,10
(s,2H,NH,D2O exchangeable).13CNMR(300MHz,DMSO-d6):δ78.7,113.8,117.2,124.4,
127.1,143.7,166,173,178,185.MS (EI) :m/z: 303[M+](32%).Anal.Calcd For C12H6FN5S2
(303.33):C,47.51;H,1.99;N,23.09;S,21.14;Found:C,47.73;H,2.12; N, 23.28; S,21.27.
3.6.2.7-(4-Bromo-phenyl)-3,5-dithioxo-2,3,5,6-tetrahydro[1,2,4]triazolo[4,3-c]pyrimid-
ine-8-carbonitrile 6b.Yield50 %,m.p.230-232˚C. IR (max/cm-1):3335-3180 (NH),2225
(CN),1649(C=S),1585(C=N).1H NMR (300MHz, DMSO-d6): δ6.8-7.9(m, 4H,Ar-H),
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ACCEPTED MANUSCRIPT
,9.3,10.1(s,2H,NH,D2O exchangeable).13CNMR(300MHz,DMSO-d6): 86.78,110.27,
115.28,128.51,130.04,147.47,164.04,172.04,175.72,186.01. MS(EI): m/z:363[M+]
(27.2%),365[M+2](27.3%).Anal.Calcd for C12H6BrN5S2(362.92):C,39.57;H,1.66;N,
19.23;S,17.61;Found:C,39.77;H,1.84;N,19.44;S,17.72.
3.6.3.3,5-Dithioxo-7-(3,4,5-trimethoxy-phenyl)-2,3,5,6-tetrahydro[1,2,4]triazolo[4,3-c
]pyrimidine-8-carbonitrile 6c.Yield 55%, m.p.243-245˚C.IR (max/cm-1):3345-3166
(NH),2200(CN),1652(C=S),1580(C=N).1HNMR(300MHz,DMSO-d6):δ3.1-3.7(s,9H,
3OCH3),7,7.4(s,2H,Ar-H),9.1,10.2(s,2H,NH,D2Oexchangeable).13CNMR(300 MHz,
DMSO-d6):δ55.4,55.7,79.5,115.1,117.4,129.2,132.4,148.5,167,174,179.2,186.MS (EI):
m/z: 375[M+] (37%).Anal.Calcd for C15H13N5O3S2 (375.42): C,47.99; H,3.49; N,18.65;
S,17.08; Found: C,48.21; H,3.60; N,18.78;S,17.15.
3.7.General method for preparation of 7a- i
A mixture of appropriate thiouracil derivative 1a - c(0.01 mol) and appropriate aldehyde
(0.01 mol) in ethanol (30 mL) was heated under reflux for 4h. The solid obtained was
crystallized from benzene to yield compounds 7a- i, respectively.
3.7.1.4-[(2E)-2-benzylidene-hydrazino]-6-(4-fluoro-phenyl)-2-thioxo-1,2,3,4-tetrahydr-
opyrimidine-5-carbonitrile 7a.Yield 70%, m.p.148-150 ˚C. IR(max/cm-1): 3375-3180
(NH),2213(CN),1608(C=N).1HNMR(300MHz,DMSO-d6):δ2.3(s,1H,NH,D2O exchang-
eable),6.9-7.8(m,8H,Ar-H),8.6(s,1H,N=CH),9.9(s,1H,NH,D2Oexchangeable).13CNMR
(300MHz,DMSO-d6):δ88,115.5,116.2,127.4,127.8,129.4,130.5,132.5,151.1,154.1,161.3
- 16 -

ACCEPTED MANUSCRIPT
,164,168.2,180.MS(EI):m/z:349[M+](25%).Anal.Calcd for C18H12FN5S(349.38): C,61.
88;H,3.46; N,20.04;S,9.18; Found:C,61.95;H,3.66;N,20.12;S,9.26.
3.7.2.4- [(2E)-2-benzylidene-hydrazino]-6-(4-bromo-phenyl)-2-thioxo-1,2,3,4-tetrahydr-
opyrimidine-5-carbonitrile 7b.Yield 72%,m.p.158-160˚C.IR(max/cm-1):3380-3180(NH)
,2220(CN),1606(C=N).1HNMR(300MHz,DMSO-d6):δ2.2(s,1H,NH,D2O exchangeable)
,7-7.8(m,8H,Ar-H),8.5(s,1H, N=CH),9.7(s,1H, NH,D2O exchangeable).13C NMR(300
MHz, DMSO-d6): 86,115,116.2,124.4,127.1,127.4,129.4,133.3,143.7,151.1,155.2,164,
167.3,180.MS(EI):m/z:409[M+] (22.3%),411[M+2] (22.1%).Anal.Calcd for C18H12Br
N5S (409):C,52.69; H,2.95; N,17.07;S,7.82;Found: C,52.79; ,3.04; N,17.16; S,7.95.
3.7.3.4- [(2E)-2-benzylidene-hydrazino]-6-(3,4,5-trimethoxy-phenyl)-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carbonitrile 7c.Yield 75%,m.p.123-125˚C.IR (max/cm-1): 3369-
3190(NH),2215CN),1605(C=N).1HNMR(300MHz,DMSO-d6):δ2.1(s,1H,NH,D2Oexch-
angeable),3-3.7(s,9H,3OCH3),7-7.9(m,6H,Ar-H),8.6(s,1H,N=CH),9.6(s,1H,NH,D2O
exchangeable).13CNMR(300MHz,DMSO-d6):δ55.5,55.7,85,115,117,122.3,127.4, 128.
4,129.4,131.7,133.9,151.1,156.2,162,166.2,180.MS(EI):m/z:421[M+](20%).Anal. Calcd
for C21H19N5O3S(421.47):C,59.84;H,4.54;N,16.62;S,7.61;Found:C,59.98;H,4.66; N,16.
75; S,7.68.
3.7.4.4- [(2E)-2-(4-fluorobenzylidene)-hydrazino]-6-(4-fluoro-phenyl)-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carbonitrile 7d.Yield 72%, m.p.157-159˚C.IR(max/cm-1): 3460-
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ACCEPTED MANUSCRIPT
3200(NH),2225(CN),1610(C=N).1HNMR(300MHz,DMSO-d6):δ2.4(s,1H,NH,D2O
exchangeable),6.8-7.8(m,8H,Ar-H),8.4(s,1H,N=CH),9.6(s,1H,NH,D2Oexchangeable).
MS(EI):m/z:367[M+](12%).Anal.CalcdforC18H11F2N5S(367.37):C,58.85;H,3.02;N,
19.06;S,8.73;Found:C,58.98;H,3.14; N,19.20;S,8.88.
3.7.5.4- [(2E)-2-(4-fluorobenzylidene)- hydrazino]-6-(4-bromo-phenyl)-2-thioxo-1,2, 3,
4-tetrahydropyrimidine-5-carbonitrile 7e.Yield73%,m.p.166-168˚C.IR(max/cm-1): 3490
-32640(NH),2222(CN),1611(C=N).1HNMR(300MHz,DMSO-d6):δ2.3(s,1H,NH,D2O
exchangeable),7-7.8(m,8H,Ar-H),8.5(s,1H,N=CH),9.5(s,1H,NH,D2O exchangeable).MS
(EI):m/z:427[M+](10%).429[M+2](10.3).Anal.Calcd for C18H11BrFN5S(426.99):C,50.
48;H,2.59;N,16.35;S,7.49; Found:C,50.57;H,2.66;N,16.55;S,7.54 .
3.7.6.4- [(2E)-2-(4-fluorobenzylidene)-hydrazino]-6-(3,4,5-trimethoxy-phenyl)-2-thioxo
-1,2,3,4-tetrahydropyrimidine-5-carbonitrile 7f.Yield 70%,m.p.128-130˚C.IR (max /
cm-1):3490-3300(NH),2220(CN),1609(C=N).1HNMR(300MHz, DMSO-d6): δ2.2 (s,1H,
NH, D2O exchangeable),3-3.7(s,9H,3OCH3) ,6.7-7.8 (m,6H,Ar-H),8.5 (s,1H,N=CH),9.7
(s,1H,NH,D2O exchangeable).MS(EI):m/z:439[M+](13%).Anal.Calcd for C21H18FN5
O3S(439.46):C,57.39;H,4.13; N,15.94; S,7.30; Found: C,57.49; H,4.24; N,16.04; S.7.42.
3.7.7.4- [(2E)-2-(4-methoxybenzylidene)-hydrazino]-6-(4-fluoro-phenyl)-2-thioxo-1,2,3,
4-tetrahydropyrimidine-5-carbonitrile 7g.Yield 72%,m.p.155-157˚C.IR(max/cm-1):
3450-3200(NH), 2215(CN),1608 (C=N).1H NMR (300 MHz, DMSO-d6):δ 2.49 (s,1H,
NH,D2O exchangeable),3.37(s,3H,OCH3),7.16-8.39(m,8H,Ar-H)8.59(s,1H,N=CH),9.5
- 18 -

ACCEPTED MANUSCRIPT
(s,1H,NH,D2O exchangeable).MS(EI):m/z:379[M+](14%).Anal.Calcd for C19H14FN5OS
(379.41):C,60.15;H,3.72;N,18.46;S,8.45; Found: C,60.26;H,3.78;N,18.59; S,8.51.
3.7.8.4- [(2E)-2-(4-methoxybenzylidene)-hydrazino]-6-(4-bromo-phenyl)-2-thioxo-1,2,3,
4-tetrahydropyrimidine-5-carbonitrile 7h.Yield 69%, m.p.152-154˚C.IR(max/cm-1) :
3500-3200(NH),2217(CN),1607(C=N).1H NMR(300 MHz, DMSO-d6):δ2.3 (s,1H,NH,
D2O exchangeable),3.5(s,3H,OCH3),7-7.8 (m,8H,Ar-H),8.5(s,1H,N=CH),9.5(s,1H,NH,
D2O exchangeable).MS (EI):m/z:439[M+](11.2%),441[M+2](11.4%).Anal. Calcd for
C19H14BrN5OS(439.01):C,51.83;H,3.20;N,15.91;S,7.28;Found:C,51.96; H,3.38;N,16.
08;S,7.34.
3.7.9.4- [(2E)-2-(4-methoxybenzylidene)-hydrazino]-6-(3,4,5-trimethoxy-phenyl)-2-thiox
o-1,2,3,4-tetrahydropyrimidine-5-carbonitrile 7i.Yield 68%, m.p. 134-136˚C.IR (max /
cm-1):3550-3300(NH),2225(CN),1609(C=N).1HNMR(300 MHz,DMSO-d6):δ2.2(1s,1H,
NH,D2O exchangeable),3-3.81 (s,12H,4OCH3),7-7.9(6H,m,Ar-H),8.5(s,1H,N=CH),9.7
(s,1H,NH,D2O exchangeable).MS(EI):m/z:451[M+](15%).Anal.Calcd for C22H21N5O4S
(451.49):C,58.52;H,4.69;N,15.51;S,7.10;Found:C,58.70;H,4.78;N,15.80;S,7.15.
3.8. General method for preparation of 8a- c
A solution of appropriate thiouracil derivative 1a - c (0.01 mol) and diethyloxalate (0.01
mol) in absolute ethanol (40 mL) was heated under reflux for 10 h. The solid obtained
was crystallized from benzene to yield compounds 8a- c,respectively.
3.8.1.8-(4-Fluoro-phenyl)-3,4-dioxo-6-thioxo-3,4,6,7-tetrahydro-2H-pyrimido[6,1-c]-
[1,2, 4]triazine-9-carbonitrile 8a.Yield 65%, m.p.228-230˚C. IR (max/cm-1):3400-3220
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ACCEPTED MANUSCRIPT
(NH),2230(CN),1664(C=O),1275(C=S).1HNMR(300MHz,DMSO-d6):δ6.59-7.07(m,4H
,Ar-H),10,11.7(s,2H,NH,D2O exchangeable).13CNMR(300MHz,DMSO-d6): δ82.5,113,
117.3,124.4,127.1,143.7,155.2,161.7,165.9,176.7,178.5.MS(EI):m/z:315[M+](8%).Anal
.Calcd for C13H6FN5O2S(315.28):C,49.52;H,1.92;N,22.21;S,10.17; Found:C,49.66;H,2.
02;N,22.45;S,10.26.
3.8.2.8-(4-Bromo-phenyl)-3,4-dioxo-6-thioxo-3,4,6,7-tetrahydro-2H-pyrimido[6,1-c]-
[1,2,4]triazine-9-carbonitrile 8b.Yield 66%,m.p.220-222˚C.IR (max/cm-1):3455-3200
(NH),2213(CN),1671(C=O),1270(C=S).1HNMR(300MHz,DMSO-d6):δ7-7.5(m, 4H,Ar-
H),8.2,9.3(s,2H,NH,D2O exchangeable).13CNMR(300MHz,DMSO-d6):δ85.6,117.1,122.
3,128.4,131.7,133.9,157.3,163,166.2,175.7,178.6.MS(EI):m/z:375[M+] (10.2%),377[M
+2](10.4%).Anal.Calcd for C13H6BrN5O2S(374.94):C,41.51;H,1.61;N,18.62;S, 8.52;
Found:C,41.75; H,1.77;N,18.79;S,8.66.
3.8.3.3,4-Dioxo-6-thioxo-8(3,4,5-trimethoxy-phenyl)-3,4,6,7-tetrahydro-2H-pyrimido
[6,1-c]- [1,2, 4]triazine-9-carbonitrile 8c.Yield 69%, m.p.250-252˚C. IR (max/cm-1):
3500-3210(NH),2215(CN),1677(C=O),1275(C=S).1HNMR(300MHz,DMSO-d6):δ3-
3.74(s,9H,3OCH3,7,7.4(s,2H,Ar-H),8.5,9(s,2H,NH,D2Oexchangeable).13CNMR(300
MHz,DMSO-d6):δ,55.4,55.7,80.7,115.1,117.2,129.2,132.4,148.5,155.3,161.3, 165.9,
175.2,176.1.MS(EI):m/z:387[M+](7%).Anal.Calcd for C16H13N5O5S(387.36): C,49.61;
H,3.38; N,18.08;S,8.28;Found: C,49.80; H,3.45; N,18.28;S,8.34.
3.9. General method for preparation of 9a- c
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ACCEPTED MANUSCRIPT
A mixture of appropriate thiouracil derivative 1a - c (0.01mol) and triethyl orthoformate
(30 mL) was heated under reflux for 2 h. The solid obtained was crystallized from acetic
acid to yield compounds 9a- c, respectively.
3.9.1.7-(4-Fluoro-phenyl)-5-thioxo-5,6-dihydro[1,2,4] triazolo[4,3-c]-pyrimidine-8-car-
bonitrile 9a.Yield 61%, m.p.165-167˚C.IR (max/cm-1):3251(NH),2218(CN),1580(C=N)
.1H NMR (300MHz,DMSO-d6): δ7.5-7.55 (m, 4H, Ar-H),8.1(s,1H,C5-H),10.47 (s,1H,
NH,D2O exchangeable).13C NMR (300 MHz, DMSO-d6): 85.4, 112,117.2,122.3,131.7,
142.2,148,155,168.3,184.MS (EI):m/z:271[M+](40%).Anal.Calcd for C12H6FN5S (271.
27):C,53.13;H,2.23;N,25.82;S,11.82; Found:C,53.25;H,2.33;N,25.95; S,11.90.
3.9.2.7-(4-Bromo-phenyl)-5-thioxo-5,6-dihydro[1,2,4]triazolo[4,3-c]-pyrimidine-8-car-
bonitrile 9b.Yield 62%,m.p.180-182˚C.IR(max/cm-1):3218(NH),2217(CN),1585(C=N).
1HNMR(300MHz,DMSO-d6):δ7-7.6 (m,4H, Ar-H),8.60 (s,1H,C5-H),9.5(s,1H, NH,D2O
exchangeable).13CNMR(300MHz,DMSO-d6):85.5,112,117.9, 129.2,131.3,148.5,155.6,
160.2,167.6,182. MS (EI): m/z: 331 [M+] (30.3%).333 [M+2] (30.4%). Anal.Calcd for
C12H6BrN5S(330.95):C,43.39; H,1.82; N,21.08; S,9.65; Found: C, 43.48; H, 1.96; N,
21.19; S, 9.76.
3.9.3.5-Thioxo-7-(3, 4,5-trimethoxy-phenyl)-5,6-dihydr [1,2,4] triazolo [4,3-c]-pyrimid-
ine-8-carbonitrile 9c.Yield 65%,m.p.188-190˚C.IR (max/cm-1):3230(NH), 2219(CN),
1586(C=N).1H NMR(300 MHz, DMSO-d6):δ3-3.7(s,9H,3OCH3),7.2,7.7(s,2H,Ar-H),
8.62(s,1H,C5-H ),9.3(s,1H,NH ,D2O exchangeable ).13CNMR (300MHz,DMSO-d6):
55.4,55.6,90,115,117.9,127.8,130.5,148.3,155,158,166,178. MS(EI): m/z: 343 [M+]
(51%). Anal.Calcd for C15H13N5O3S (343.36): C,52.47; H,3.82; N,20.40; S,9.34;
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ACCEPTED MANUSCRIPT
Found: C,52.55;H,3.99;N,20.65;S,9.44 .
3.10. General method for preparation of 10 a- c
A mixture of appropriate thiouracil derivative 1a- c (0.01 mol) and acetyl acetone (0.1
mL, 0.001 mol) in acetic acid (15 mL) was refluxed for 15h. The reaction mixture was
cooled and poured onto ice - water. The solid obtained was crystallized from ethanol to
yield compounds 10a- c,respectively.
3.10.1.4-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(4-fluoro-phenyl)-2-thioxo-1,2-dihydropyrim-
idine-5-carbonitrile 10a.Yield 50%,m.p.245-247˚C.IR(max/cm-1):3325(NH),2220 (CN),
1606 (C=N).1H NMR (300 MHz, DMSO-d6):δ2,2.5(s, 6H, 2CH3),6.7(s,1H ,C4-H),7- 7.5
(m,4H,Ar-H),9.1(s,1H,NH,D2Oexchangeable).13CNMR(300MHz,DMSO-d6):11.78, 14.
04,86.78,111.64,113.89,115.28,126.89,127.92,130.04,147.32,152.47,158.51,159.66,175.
72.MS(EI):m/z:325[M+](23%),Anal.Calcd for C16H12FN5S(325.36):C,59.06;H,3.72 ;
N,21.52; S,9.86; Found: C,59.18; H,3.90; N, 21.77; S,9.92.
3.10.2.4-(3, 5-dimethyl-1H-pyrazol-1-yl)-6-(4-bromo-phenyl)-2-thioxo-1,2-dihydro
pyrimidine-5-carbonitrile 10b.Yield 52%,m.p.238-240˚C.IR(max/cm-1):3345(NH),2225
(CN),1608(C=N).1HNMR(300MHz,DMSO-d6):δ2.25,2.4(s,6H,2CH3),6.8(s,1H,C4-H),
7.2-7.7(m,4H,Ar-H),9.3(s,1H,NH,D2Oexchangeable).13CNMR(300MHz,DMSO-d6):12,
15.3,90,110,115,117,129.2,132.4,148,149,152,162,169,179.MS(EI):m/z:385[M+](30.1
%).387 [M+2] (30.3%). Anal. Calcd for C16H12BrFN5S (385):C,49.75; H,3.13; N,18.13;
S,8.30; Found: C,49.95; H,3.41; N,18.29;S,8.39.
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3.10.3.4-(3, 5-Dimethyl-1H-pyrazol-1-yl)-2-thioxo-6(3,4,5-trimethoxy-phenyl)-1,2-di-
hydropyrimidine-5-carbonitrile 10c.Yield 53%, m.p. 230-232˚C. IR (max/cm-1):3365:
(NH),2222(CN),1609(C=N).1HNMR(300MHz,DMSO-d6):δ2.4,2.53(s,6H,2CH3),3-3.7
(s,9H,3OCH3),6.73(s,1H,C4-H ),7.2,7.3(s,2H, Ar-H),9.3(s,1H,NH,D2O exchangeable).
13CNMR(300 MHz,DMSO-d6):11,19.3,55.4,55.7,92,111,114,117, 127.2 ,134.4,146,
147,155,163,165,177.MS(EI):m/z:397[M+](32%).Anal.Calcd for C19H19N5O3S (397.
45):C,57.42;H,4.82;N,17.62; S,8.07;Found:C,57.66;H,4.95;N, 7.77;S,8.27.
3.11.General method for preparation of 11a- c
A cold solution (0 - 5˚C) of sodium nitrite (1g, 0.144mol ) in H2O (15 mL) was added
gradually within 15 min to a cold stirred solution of appropriate thiouracil derivative 1
a- c (0.01 mol) in 2N HCl (15mL ).After addition, the reaction mixture was then
further stirred for 4 h at the same temperature ,then it was diluted with cold H2O. The
solid obtained was crystallized from ethanol to yield compounds 11a- c, respectively.
3.11.1.7-(4-Fluoro-phenyl)-5-thioxo-5,6-dihydrotetrazolo[1,5-c]pyrimidine-8-carbonit-
rile 11a.Yield 50%, m.p.218-220˚C.IR (max/cm-1):3325(NH),2220 (CN),1510 (C=N).
1HNMR(300MHz,DMSO-d6):δ7-7.6(m,4H,Ar-H),9(s,1H,NH,D2O exchangeable).MS
(EI):m/z:272[M+](45%).Anal.Calcd for C11H5FN6S(272.26):C,48.53;H,1.85; N,30.87
;S,11.78;Found:C,48.67;H,1.98;N,30.95;S,11.88.
3.11.2.7-(4-Bromo-phenyl)-5-thioxo-5,6-dihydrotetrazolo[1,5-c]pyrimidine-8-carbonit-
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rile 11b.Yield 54%, m.p. 208-210˚C.IR (max/cm-1):3330(NH),2223 (CN),1521 (C=N).
1H NMR (300MHz,DMSO-d6):δ7.1-7.8 (m,4H,Ar-H),9.4(s,1H,NH, D2O exchangeable).
MS(EI):m/z:332[M+](60.1%).334 [M+2](60.3%).Anal. Calcd for C11H5BrN6S (331.95):
C,39.66; H,1.51;N,25.22;S,9.62; Found:C,39.79;H,1.72;N,25.45; S,9.80.
5-Thioxo-7-(3,4,5-trimethoxy-phenyl)-5,6-dihydrotetrazolo[1,5-c]pyrimidine-8-carboni-
trile 11c.Yield 55 %,m.p.200-202˚C.IR (max/cm-1):3322(NH),2222 (CN), 1520 (C=N).
1H NMR (300 MHz, DMSO-d6):δ3.4-3.8(s,9H,3OCH3),6.6,6.8(s,2H,Ar-H),10.14(s,1H
,NH,D2O exchangeable).MS(EI):m/z:344[M+](56%).Anal.Calcd for C14H12N6O3S (344.
34): C,48.83; H,3.51; N,24.41; S,9.31; Found:C,48.96; H,3.76;N, 24.66; S,9.42.
Biological assay
Antimicrobial activity
Materials and methods
Antibacterial activity was examined by the disc-diffusion method and the MIC method
under standard conditions of the National Committee for Clinical Laboratory Standards
[37]. The in-vitro antimicrobial activity of the synthesized compounds was investigated
against several pathogenic representative Gram-positive bacteria (S.aureus ATCC12600
, B.cereus ATCC14579), Gram-negative bacteria (Escherichia coli ATCC11775), and
(Candida albicans ATCC26555,Aspergillus flavus ATCC 11495) as a representative for
fungi. All microorganisms used were obtained from culture collection of the Department
of Microbiology, Micro Analytical Centre, Faculty of Science, Cairo University, Cairo,
Egypt. Media for disc sensitivity tests were the nutrient agar and Muller-Hinton agar
(MHA) purchased from Difco (USA). Non-sterile powder of the tested compound was
dissolved in sterile DMSO to yield: 10 mg mL–1, and passed through a 0.2µm membrane
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filter (Millipore Corp, USA). The filtrates were dispensed as 2 mL samples into sterile,
small screw-capped vials, frozen and kept stored at _15 °C. The vials were refrozen after
thawing. Disc diffusion sensitivity test was done in the manner identical to that of Bauer
et al. [38]. DMSO showed no inhibition zones.
Penicillin (Bioanalyse, Turkey) and Fluconazole (Sigma- Aldrich, USA) were used as
reference substances. Inhibition zones were measured in millimeters at the end of an
incubation period of 48 h at 28 °C (see Inline Supplementary,Figs.S1-S10).
MIC (minimum inhibitory concentration) for each tested compound was determined on
Mueller_Hinton agar (MHA), by micro dilution technique according to NCCLS
guidelines 1997 [39].All bacterial isolates were sub cultured in MHA plates and
incubated overnight at 37 °C and all Candida isolates were sub cultured in SDA plates at
35 °C for 24-48 h. The microorganisms were passage at least twice to ensure purity and
viability. The solution of the newly synthesized compounds and standard drugs were
prepared at 1024,512,256,128,64,32 and 16 µg/mL concentrations using serial two folds
dilutions in DMSO (dimethyl sulphoxide), each concentration was mixed with sterile
nutrient agar (Sigma - Aldrich) in sterile plate, bacteria inoculums were added to each
well of the micro dilution trays. The trays were incubated at 37°C in a humid chamber
and MIC end points were read after 24 h of incubation.
Antioxidant activity
Determination of radical scavenging activity using DPPH assay:
Thiouracil derivatives (1, 2, 5a, c),7a,7g and 8a, c were evaluated for their antioxidative
potential through invitro DPPH radical scavenging model. The DPPH (2, 2-diphenyl-1-
picrylhydrazyl) radical scavenging effect was carried out according to reported methods
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[40,41]. Compounds of different concentrations were prepared in DMSO, 1 mL of each
compound solutions having different concentrations (10, 25,50,100,200 and 300 µM)
were taken in different test tubes, 4 mL of 0.1 mM DMSO solution of DPPH was added
and shaken vigorously. The tubes were then incubated in the dark room at room
temperature for 20 minutes. A DPPH blank was prepared without compound, and
DMSO was used for the baseline correction. Changes (decrease) in the absorbance at
517 nm were measured using a UV-visible spectrophotometer (Shimadzu 160A).
Scavenging of DPPH free radicals was calculated from the following equation:
DPPH scavenging activity (%) = [(Ac-At)/Ac] × 100
Where; Ac is absorbance of control, At is absorbance of compound.
Results
The relation between DPPH scavenging percentage against compound concentrations is
plotted to get compound concentration providing a 50% decrease in absorbance of
DPPH radical (IC50%). The radical scavenging activities were expressed as IC50.
Vitamin C served as reference compound. The results have been given in (Table II
, Fig.4, 5).
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