Synthesis of novel thiopyrimidines: An investigation of anti-tubercular and antimicrobial activity

Article abstract of DOI:10.1080/17415993.2010.523894

A variety of novel sulfur-containing tricyclic pyrimidine derivatives have been synthesized via the reaction of 2-amino-4-oxo-4H-chromene-3-carbonitriles 3(a-f) with different reagents and characterized by IR, 1H NMR, 13C NMR, mass spectrometry and elemen

Full text of DOI:10.1080/17415993.2010.523894

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Synthesis of novel thiopyrimidines: an
investigation of anti-tubercular and
antimicrobial activity
Dhaval D. Haveliwala a , Nimesh R. Kamdar a , Prashant T. Mistry a
& Saurabh K. Patel a
a Department of Chemistry, Veer Narmad South Gujarat University,
Surat, 395007, Gujarat, India
Version of record first published: 17 Aug 2011.
To cite this article: Dhaval D. Haveliwala, Nimesh R. Kamdar, Prashant T. Mistry & Saurabh K. Patel
(2011): Synthesis of novel thiopyrimidines: an investigation of anti-tubercular and antimicrobial
activity, Journal of Sulfur Chemistry, 32:5, 451-462
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Journal of Sulfur Chemistry
Vol. 32, No. 5, October 2011, 451–462
Synthesis of novel thiopyrimidines: an investigation of
anti-tubercular and antimicrobial activity
Dhaval D. Haveliwala, Nimesh R. Kamdar, Prashant T. Mistry and Saurabh K. Patel*
Department of Chemistry, Veer Narmad South Gujarat University, Surat 395007, Gujarat, India
(Received 17 June 2010; final version received 12 September 2010)
A variety of novel sulfur-containing tricyclic pyrimidine derivatives have been synthesized via the reaction
of 2-amino-4-oxo-4H-chromene-3-carbonitriles 3(a–f) with different reagents and characterized by IR,
1H NMR, 13C NMR, mass spectrometry and elemental analysis. The anti-tubercular and antimicrobial
activities of the synthesized compounds were investigated.
R1
O
NH
R1
O
S
R1
O
NHCSNH2
N
R2
R3
R2
R3
R2
R3
N
NH
O
O
N
H
S
O
N
H
S
N
H
S
R4
R4
R4
4(a–f)
6(a–f)
5(a–f)
Keywords: thiopyrimidines; dithiopyrimidines; chromone; anti-tubercular activity; antimicrobial activity
1. Introduction
Condensed heterocyclic systems, especially those linked to a thiopyrimidine ring, play an impor-
tant role as potential medicinal agents in cancer and virus research (1, 2). Recent studies reported
the synthesis of some new fused thiopyrimidine candidates which showed antimicrobial (3),
anticonvulsant (4, 5), antiarrhythmic (6), anti-inflammatory (7) and anti-tumor (8–10) activities.
On the other hand, a wide range of biological activities have been attributed to compounds
containing chromone ring systems (11–17). If the chromone is fused with the thiopyrimidine
ring, the linked tricyclic compounds are expected to display interesting biological activities. So
our interest in the tricyclic compounds containing thiopyrimidine moieties leads us to search for
novel bio-active scaffold based on the use of aspirin as the starting material.
*Corresponding author. Emails: saurabh@vnsgu.ac.in; saurabh.silvina@gmail.com
ISSN 1741-5993 print/ISSN 1741-6000 online
© 2011 Taylor & Francis
http://dx.doi.org/10.1080/17415993.2010.523894
http://www.tandfonline.com

452 D.D. Haveliwala et al.
2. Results and discussion
2.1. Chemistry
An o-aminonitrile is one of the routes for the synthesis of the fused systems including thiopyrim-
idine (18–21). With the aim of obtaining thiopyrimidine-fused chromone derivatives, cyclization
was carried out by the reaction of substituted o-aminonitrile of chromones 3(a–f) and each of
carbon disulphide, ammonium thiocyanate and phenylisothiocyanate, respectively. Aspirins 1(a–
f) were used as the starting material to synthesize functionalized o-aminonitrile of chromones
3(a–f) followed by thionylation.
Reaction of acid chloride of aspirins 2(a–f) with malononitrile in the presence of diluted 10%
NaOH solution at 0–5 ◦C followed by treatment with 50% KOH solution and subsequent acidifi-
cation afforded substituted 2-amino-4-oxo-4H-chromene-3-carbonitriles 3(a–f) (22) (Scheme 1).
The IR spectra of 3(a–f) exhibited a sharp peak in the region of 3230–3295 cm−1 due to NH2
−
stretching vibrations and a sharp peak in the range of 2220–2240 cm−1 and 1660–1670 cm−1
,
R1
O
R2
R3
1–6 R1
R2
H
Br
NO2
Cl
R3
H
H
H
H
CH3
H
R4
H
Br
H
H
H
OH
OCOCH3
a
b
c
d
e
f
H
H
H
H
H
H
R4
H
H
1(a–f)
CH3
SOCl2
O
R1
R2
R3
R1
O
O
S
Cl
OCOCH3
R2
R3
CS2/Pyridine
Reflux; 6 h
NH
R4
N
H
S
2(a–f)
R4
4(a–f)
1. CH2CN2/dil. NaOH
2. 50% KOH
3. H+
R1
O
O
R1
O
NH
R2
CN
R2
R3
PhN=C=S
N
DMF/TEA; 10 h
R3
NH2
O
N
H
S
R4
R4
3(a–f)
5(a–f)
R1
O
NHCSNH2
N
R2
R3
NH4SCN
Glc. HAc; 8 h
O
N
H
S
R4
6(a–f)
Scheme 1. Synthetic route of compounds 4, 5, 6(a–f).

Journal of Sulfur Chemistry 453
1
− ≡
=
suggesting the presence of C N and >C O groups. H NMR spectra exhibited a broad signal
13
−
− ≡
=
in the region 8.95–9.30 δ ppm due to the NH2 proton. In C NMR, C N and >C O carbon
signals appeared in the region of 119.8–124.8 and 173.4–178.8 δ ppm, respectively.
The target ring system 4(a–f) was synthesized by the reaction of 3(a–f) with carbon disulfide
throughseveralintermediateswhosesubsequentrearrangementleadstofusedpyrimidinedithiones
4(a–f) (Scheme 1). The IR spectra of the products 4(a–f) confirmed the disappearance of the
1
−
− ≡
primary amine ( NH2) and nitrile ( C N) absorption bands. The H NMR spectrum of the
products 4(a–f) showed in each case the disappearance of the broad amine signal. Cyclization
reaction was further confirmed by the 13C NMR spectra of compounds 4(a–f). The reaction
products 5(a–f) were obtained in a one-step reaction through prolonged heating of 3(a–f) with
phenylisothiocyanate in DMF containing a catalytic amount of TEA (Scheme 1). Meanwhile,
compounds 3(a–f) in glacial acetic acid were treated with ammonium thiocyanate to give
1-(5-oxo-2-thioxo-1,5-dihydro-2H-chromeno[2,3-d]pyrimidin-4-yl)thiourea derivatives 6(a–f)
under refluxed temperature (Scheme 1). The structures of the reaction products were established
based on its elemental analysis and spectral data displayed in Section 5.
3. Biological activity
3.1. Anti-tubercular activity
Anti-tubercular activity was carried out against Mycobacterium tuberculosis H37Rv [MTCC-200]
at the concentration of 62.5 μg/ml, and the values are shown in Table 1. Rifampicin was used
as the standard drug. All the compounds were tested for their anti-tubercular activity by the agar
micro-dilution technique (23). The halogen group containing substituted derivatives, 6b and 6d,
displayed a relatively higher inhibitory activity in general. However, substitution at chromone
with a methyl group reduces the anti-tubercular activity. The presence of a dibromo group on
chromone caused a remarkable improvement in the anti-tubercular activity (Table 1).
3.2. Antibacterial activity
All the compounds were tested for their in vitro antibacterial activity against Staphylococcus
aureus [MTCC-96] and Streptococcus pyogenes [MTCC-442] as Gram-positive and Escherichia
coli [MTCC-443] and Pseudomonas aeruginosa [MTCC-1688] as Gram-negative bacterial
species. Ampicillin and chloramphenicol were used as standard drugs. Minimum inhibitory con-
centration (MIC, μg/ml) values are displayed in Table 2. All the compounds were tested for
their antibacterial activity by the two-fold serial dilution method (24). It is evident from the
results that, in general, the generation of thiopyrimidines ring system enhances the activity against
Table 1. The in vitro anti-tubercular activity of the synthesized compounds against M.
tuberculosis H37Rv.
Compound
% Inhibition
Compound
% Inhibition
Compound
% Inhibition
4a
4b
4c
4d
4e
4f
5
23
6
13
15
11
5a
5b
5c
5d
5e
5f
6
29
21
8
14
9
6a
6b
6c
6d
6e
6f
41
99
57
99
24
20
Note: Rifampicin: 99% inhibition at 40 μg/ml.

454 D.D. Haveliwala et al.
Table 2. The in vitro antimicrobial activity (MIC, μg/ml) of the synthesized compounds.
Minimum inhibition concentration (μg/ml)
Gram negative
E. coli P. aeruginosa
Gram positive
Fungal species
Compound
S. aureus
S. pyogenes
C. albicans
A. niger
A. clavatus
3a
3b
3c
3d
3e
3f
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
5f
6a
6b
6c
6d
6e
6f
250
250
500
250
250
500
100
100
100
200
150
250
200
200
250
500
200
500
125
200
250
500
150
250
125
200
100
500
100
100
500
250
250
125
250
250
100
50
250
200
250
250
150
200
250
200
250
250
200
100
100
150
200
100
200
200
125
200
250
250
500
250
250
250
200
250
500
250
200
200
250
250
200
250
250
200
200
250
200
200
250
100
50
1000
500
>1000
500
500
500
>1000
150
500
250
250
500
1000
500
1000
500
250
500
1000
>1000
1000
1000
>1000
>1000
>1000
500
>1000
>1000
1000
1000
500
1000
1000
>1000
>1000
1000
>1000
>1000
500
>1000
>1000
>1000
>1000
500
62.5
125
100
250
125
100
500
200
125
50
200
250
100
50
1000
500
1000
1000
1000
1000
>1000
1000
1000
>1000
1000
>1000
>1000
–
>1000
>1000
>1000
>1000
>1000
500
1000
500
>1000
500
62.5
150
150
500
62.5
250
500
–
–
>1000
>1000
–
200
250
50
–
A
B
C
–
100
–
100
–
–
–
100
D
–
–
–
–
500
100
100
Note: Ampicillin (A) and chloramphenicol (B) were used as standard drugs for bacterial strains while nystatin (C) and greseofulvin (D)
were used as standard drugs for fungal strains.
Gram-negative as well as Gram-positive species except S. pyogenes.Among the synthesized com-
pounds, 5a and 6d display significant antibacterial activity compared to chloramphenicol against
E. coli in the range of 62.5 and 50 μg/ml, respectively. Dithiopyrimidine 4a has a similar potency
comparedtoampicillinbutreplacementofthethiogroupatthefourthpositionbyathiourealinkage
decreased the antibacterial potency against E. coli. The imino compounds bearing 7-nitro as well
as 8 and 9-methyl derivatives show relatively higher potency than ampicillin against P. aeruginosa.
Similarly, dithiopyrimidine and 4-imino N-phenyl thiopyrimidine showed exceptional potency
while 7,9-dibromo and 8-methyl derivatives are comparable to chloramphenicol against S. aureus.
The activity results reveal that on E. coli species the activity is exceptionally enhanced than their
precursors, while on other species, the results are random (Table 2).
3.3. Antifungal activity
The antifungal activity of all the compounds was studied against the fungal strains viz. Aspergillus
niger [MTCC-282], Candida albicans [MTCC-227] and Aspergillus clavatus [MTCC-1323] by
two-fold serial dilution. Nystatin and greseofulvin were used as standard drugs. MIC values in
μg/ml are displayed in Table 2. The antifungal activity results indicated that the synthesized
compounds showed more fungicidal potency against C. albicans rather than against A. niger and
A. clavatus when compared with the standard drug. None of the compounds shows similar or

Journal of Sulfur Chemistry 455
high fungicidal potency against A. niger as well as A. clavatus. However, the compounds show
varied activity against C. albicans when compared with the standard drug greseofulvin. Even,
compound 4b showed a high potency comparable to the standard drug nystatin. Compounds 4d,
4e, 5e and 6e possess remarkably higher fungicidal potency while compounds 3b, 3d–f, 4c, 4f, 5b,
5d, 5f, 6b, 6d and 6f have similar potency as greseofulvin against C. albicans. The unsubstituted
and nitro-substituted thiopyrimidines were inactive, whereas the 8-methyl thiopyrimidines were
found to be more potent than the 9-methyl thiopyrimidines. Among the halo-substituted thiopy-
rimidines, mono halo-substituted thiopyrimidines showed consistently higher potency while the
dithiopyrimidine-bearing dibromo substitution showed remarkable fungicidal potency (Table 2).
4. Conclusion
We report the successful synthesis and anti-tubercular and antimicrobial activities of new tricyclic
chromone-fused thiopyrimidines. The assumed structures are confirmed by the IR, 1H NMR, 13
C
NMR, mass and elemental analysis. The anti-tubercular studies revealed that 6b and 6d showed
promising anti-tubercular activity against M. tuberculosis H37Rv. From the biological activity of
the newly synthesized compounds, it is evident that the fused thiopyrimidines containing tricyclic
chromone derivatives showed, in general, good antibacterial potency which suggests that the
introduction of sulfur is valuable for its activity.
5. Experimental
Melting points were taken in open capillaries and are uncorrected. IR spectra were recorded on
1
a Hitachi 270-50 double-beam spectrophotometer, using KBr pallets. H NMR and 13C NMR
spectra were recorded on aVarian 400 MHz spectrometer using TMS as the internal standard with
DMSO-d6 or CDCl3 as the solvent. Electron impact MS spectra were obtained on a Jeol SX-102
at 70 eV. The progress of the reaction was monitored by silica gel 60 F254 (Merck, 0.25 mm
thick)-coated TLC plates [mobile phase: toluene/ethyl acetate (7.5:2.5)], and visualization was
accomplished by UV light or iodine vapor.All the chemicals were purchased from the commercial
sources and purified by either distillation or recrystallization.
5.1. General procedure for the synthesis of compound 3(a–f)
A mixture of acetylated acids 1(a–f) (10.0 mmol) and thionyl chloride (15.0 mmol) was heated
on a water bath for 2 h till no more hydrogen chloride is evolved. The reaction mixture was left
to cool to room temperature and the excess of thionyl chloride was distilled out under anhydrous
condition, and the gummy solid product was used as early as possible in the next step. A mixture
of acid chlorides and malononitrile (5.0 mmol) was stirred vigorously for 10 min in the presence
of ice (5 g) and of 20% (w/v) NaOH solution (1 ml). Second portion of malononitrile (5.0 mmol)
and 20% (w/v) NaOH solution (1 ml) was added; the mixture was stirred for another 10 min, and
then warmed to 40 ◦C with stirring. After completion of the reaction, 50% of KOH solution was
added in 5 ml increments till the solution becomes clear. The solution was cooled, acidified with
conc. HCl and the solid product was separated by filtration, washed with water and dried.
5.1.1. 2-Amino-4-oxo-4H-chromene-3-carbonitrile (3a)
Yield 74%; mp >300 ◦C; IR (KBr) υ (cm−1): 3264 ( NH2), 2225 ( C N), 1666 (>C O);
−
− ≡
=
1
−
−
H NMR (300 MHz, DMSO-d6) δ (ppm): 7.43–7.93 (m, 4H, Ar H), 8.94 (2H, br s, NH2);

456 D.D. Haveliwala et al.
13CNMR(300 MHz, CDCl3)δ (ppm):120.2, 130.2, 132.0, 134.4, 135.4, 136.2(aromaticcarbons),
−
− ≡
−
=
191.5 (C NH2), 120.8 ( C N), 90.8 (>C CN), 175.8 (>C O); m/z: 186.04 (100.0%), 187.05
(11.0%). Anal. Calcd for C10H6N2O2: C, 64.52; H, 3.25; N, 15.05. Found: C, 64.46; H, 3.23;
N, 14.96.
5.1.2. 2-Amino-6,8-dibromo-4-oxo-4H-chromene-3-carbonitrile (3b)
Yield 78%; mp >300 ◦C; IR (KBr) υ (cm−1): 3277 ( NH2), 2228 ( C N), 1666 (>C O);
−
− ≡
=
1
−
−
H NMR (300 MHz, DMSO-d6) δ (ppm): 7.78 and 8.75 (s, 2H, Ar H), 9.01 (2H, br s, NH2);
13C NMR (300 MHz, CDCl3) δ (ppm): 109, 115 (2C–Br), 128, 135, 140, 152 (aromatic car-
−
− ≡
−
=
bons), 190.5 (C NH2), 120.9 ( C N), 91.8 (>C CN), 175.1 (>C O); m/z: 343.86 (100.0%),
341.86 (51.4%), 345.86 (48.7%), 344.87 (11.0%), 346.86 (5.7%), 342.87 (5.6%). Anal. Calcd
for C10H4N2O2Br2: C, 34.92; H, 1.17; N, 8.14. Found: C, 34.86; H, 1.24; N, 8.26.
5.1.3. 2-Amino-6-nitro-4-oxo-4H-chromene-3-carbonitrile (3c)
Yield 71%; mp >300 ◦C; IR (KBr) υ (cm−1): 3295 ( NH2), 2225 ( C N), 1661 (>C O);
−
− ≡
=
1H NMR (300 MHz, DMSO-d6) δ (ppm): 8.64–8.78 (m, 3H, Ar-H), 9.27 (br s 2H, NH2);
−
13
− −
C NMR (300 MHz, CDCl3) δ (ppm): 142.5 ( C NO2), 114, 120, 125, 144, 152 (aromatic car-
−
− ≡
−
=
bons), 187.6 (C NH2), 124.8 ( C N), 90.8 (>C CN), 174.4 (>C O); m/z: 231.03 (100.0%),
232.03 (12.1%), 233.03 (1.5%). Anal. Calcd for C10H5N3O4: C, 51.96; H, 2.18; N, 18.18. Found:
C, 52.01; H, 2.24; N, 18.06.
5.1.4. 2-Amino-6-chloro-4-oxo-4H-chromene-3-carbonitrile (3d)
Yield 75%; mp >300 ◦C; IR (KBr) υ (cm−1): 3237 ( NH2), 2239 ( C N), 1667 (>C O);
−
− ≡
=
1
−
−
H NMR (300 MHz, DMSO-d6) δ (ppm): 7.49–7.89 (3H, m, Ar H), 9.03 (2H, br s, NH2);
13C NMR (300 MHz, CDCl3) δ (ppm): 129.5 (C–Cl ), 119, 122, 125, 140, 155 (aromatic carbons),
−
− ≡
−
=
191.5 (C NH2), 120.8 ( C N), 90.8(>C CN), 175.8 (>C O); m/z: 220.00 (100.0%), 222.00
(32.0%), 221.01 (10.9%), 223.00 (3.7%). Anal. Calcd for C10H5N2O2Cl: C, 54.44; H, 2.28;
N, 12.70. Found: C, 54.51; H, 2.34; N, 12.86.
5.1.5. 2-Amino-7-methyl-4-oxo-4H-chromene-3-carbonitrile (3e)
Yield 77%; mp >300 ◦C; IR (KBr) υ (cm−1): 3230 ( NH2), 2222 ( C N), 1662 (>C O);
−
− ≡
=
1
−
−
H NMR (300 MHz, DMSO-d6) δ (ppm): 2.36 (3H, s, Ar CH3), 7.51–7.93 (3H, m, Ar H), 9.12
13
−
−
(2H, br s, NH2); C NMR (300 MHz, CDCl3) δ (ppm): 31.5 (C CH3), 113, 122, 125, 129
−
− ≡
− −
=
130, 155 (aromatic carbons), 185.5 (C NH2), 119.8 ( C N), 90.8 ( C CN), 178.8 (>C O);
m/z: 200.06 (100.0%), 201.06 (12.8%). Anal. Calcd for C11H8N2O2: C, 66.00; H, 4.03; N, 13.99.
Found: C, 66.08; H, 3.97; N, 14.07.
5.1.6. 2-Amino-8-methyl-4-oxo-4H-chromene-3-carbonitrile (3f)
Yield 76%; mp >300 ◦C; IR (KBr) υ (cm−1): 3253 ( NH2), 2226 ( C N), 1667 (>C O);
−
− ≡
=
1
−
−
H NMR (300 MHz, DMSO-d6) δ (ppm): 2.38 (3H, s, Ar CH3), 7.73–8.12 (3H, m, Ar H),
13
−
−
9.08 (2H, br s, NH2); C NMR (300 MHz, CDCl3) δ (ppm): 32.5 (C CH3), 114, 120,
123, 129 135, 158 (aromatic carbons), 191.5 (C NH2), 122.8 ( C N), 92.8 ( C CN),
173.4 (>C O); m/z: 200.06 (100.0%), 201.06 (12.8%). Anal. Calcd for C11H8N2O2: C, 66.00;
−
− ≡
− −
=
H, 4.03; N, 13.99. Found: C, 66.11; H, 3.95; N, 14.05.

Journal of Sulfur Chemistry 457
5.2. General procedure for synthesis of compounds 4(a–f)
A mixture of compound 3(a–f) (5.0 mmol) and carbon disulphide (5.0 mmol) in dry pyridine
(20 ml) was refluxed on a water bath for 6 h (monitored by TLC).After completion of the reaction,
the reaction mixture was left to cool to room temperature, poured into ice-cold water (50 ml) and
neutralized with dil. HCl (1:1). The separated product was filtered off, washed with water and
recrystallized from acetic acid to give compounds 4(a–f).
5.2.1. 2,4-Dithioxo-1,2,3,4-tetrahydro-5H-chromeno[2,3-d]pyrimidine-5-one (4a)
Yield 65%; mp 293–295 ◦C; IR (KBr) υ (cm−1): 3139 ( NH), 1661 (>C O), 1325 (>C S);
−
=
=
1
−
−
H NMR (300 MHz, DMSO-d6) δ (ppm): 7.24–7.38 (4H, m, Ar H), 8.18 (1H, s, NH), 9.08
(1H, s, NH); 13C NMR (300 MHz, CDCl3) δ (ppm): 114, 120, 125, 135, 144, 152 (aro-
−
=
=
=
matic carbons), 170.5 (>C O), 190.3 (2 >C S), 78 and 172 (C C at pyrimidine ring);
m/z: 261.99 (100.0%), 262.99 (13.6%), 263.98 (9.1%), 263.99 (1.4%), 264.99 (1.2%). Anal.
Calcd for C11H6N2O2S2: C, 50.37; H, 2.31; N, 10.68; S, 24.45. Found: C, 50.45; H, 2.37; N,
10.76; S, 24.50.
5.2.2. 7,9-Dibromo-2,4-dithioxo-1,2,3,4-tetrahydro-5H-chromeno[2,3-d]pyrimidine-5-one
(4b)
Yield 63%; mp 282–284 ◦C, IR (KBr) υ (cm−1): 3072 ( NH), 1676 (>C O), 1318 (>C S);
−
=
=
1
−
−
H NMR (300 MHz, DMSO-d6) δ (ppm): 7.58–7.94 (2H, s, Ar H), 8.16 (1H, s, NH),
13
−
−
9.00 (1H, s, NH); C NMR (300 MHz, CDCl3) δ (ppm): 110.5 and 116 (2 C Br), 119, 122,
=
=
=
125, 155 (aromatic carbons), 169.2 (>C O), 195.4 (2 >C S), 79 and 176 (C C at pyrim-
idine ring); m/z: 419.81 (100.0%), 421.80 (57.4%), 417.81 (51.0%), 420.81 (14.1%), 418.81
(7.3%), 422.81 (7.0%), 423.80 (4.7%), 419.80 (4.6%), 421.81 (1.4%), 422.80 (1.3%).Anal. Calcd
for C11H4N2O2Br2S2: C, 31.45; H, 0.96; N, 6.67; S, 15.27. Found: C, 31.41; H, 1.05; N, 6.61; S,
15.30.
5.2.3. 7-Nitro-2,4-dithioxo-1,2,3,4-tetrahydro-5H-chromeno[2,3-d]pyrimidine-5-one (4c)
Yield 57%; mp 298–300 ◦C; IR (KBr) υ (cm−1): 3152 ( NH), 1667 (>C O), 1325 (>C S);
−
=
=
1
−
−
H NMR (300 MHz, DMSO-d6) δ (ppm): 7.92–8.75 (3H, m, Ar H), 8.12 (1H, s, NH), 9.05
(1H, s, NH); 13C NMR (300 MHz, CDCl3) δ (ppm): 142.5 (C–NO2), 114, 120, 125, 144, 152
−
=
=
=
(aromatic carbons), 171.4 (>C O), 199.8 (2 >C S), 71 and 172 (C C at pyrimidine ring); m/z:
306.97 (100.0%), 307.98 (12.1%), 308.97 (9.4%), 307.97 (2.7%), 308.98 (1.5%), 309.97 (1.2%).
Anal. Calcd for C11H5N3O4S2: C, 42.99; H, 1.64; N, 13.67; S, 20.87. Found: C, 43.06; H, 1.57;
N, 13.71; S, 20.81.
5.2.4. 7-Chloro-2,4-dithioxo-1,2,3,4-tetrahydro-5H-chromeno[2,3-d]pyrimidine-5-one (4d)
Yield 66%; mp >300 ◦C; IR (KBr) υ (cm−1): 3169 ( NH), 1672 (>C O), 1333 (>C S);
−
=
=
1
−
−
H NMR (300 MHz, DMSO-d6) δ (ppm): 7.12–7.40 (3H, m, Ar H), 8.15 (1H, s, NH), 9.06
13
−
−
(1H, s, NH); C NMR (300 MHz, CDCl3) δ (ppm): 125.9 (C Cl), 111, 120, 125, 144,
=
=
=
152 (aromatic carbons), 176.9 (>C O), 198.8 (2 >C S), 71 and 172 (C C at pyrimidine
ring); m/z: 295.95 (100.0%), 297.95 (33.3%), 296.95 (14.4%), 297.94 (9.1%), 298.95 (5.0%),
299.94 (3.1%). Anal. Calcd for C11H5N2O2ClS2: C, 44.52; H, 1.70; N, 9.44; S, 21.61. Found:
C, 44.48; H, 1.73; N, 9.42; S, 21.57.

458 D.D. Haveliwala et al.
5.2.5. 8-Methyl-2,4-dithioxo-1,2,3,4-tetrahydro-5H-chromeno[2,3-d]pyrimidine-5-one (4e)
Yield 64%; mp >300 ◦C; IR (KBr) υ (cm−1): 3143 ( NH), 1678 (>C O), 1323 (>C S);
−
=
=
1
−
−
H NMR (300 MHz, DMSO-d6) δ (ppm): 2.36 (3H, s, CH3), 7.21–7.46 (3H, m, Ar H), 8.15
13
−
−
−
(1H, s, NH), 9.05 (1H, s, NH); C NMR (300 MHz, CDCl3) δ (ppm): 31.5 (C CH3), 113,
=
=
=
122, 125, 129 130, 155 (aromatic carbons), 175.1 (>C O), 196.2 (2 >C S), 77 and 179 (C C at
pyrimidine ring); m/z: 276.00 (100.0%), 277.01 (13.1%), 278.00 (9.2%), 277.00 (2.3%), 278.01
(1.4%), 279.00 (1.3%). Anal. Calcd for C12H8N2O2S2: C, 52.16; H, 2.92; N, 10.14; S, 23.21.
Found: C, 52.17; H, 2.89; N, 10.17; S, 23.23.
5.2.6. 9-Methyl-2,4-dithioxo-1,2,3,4-tetrahydro-5H-chromeno[2,3-d]pyrimidine-5-one (4f)
Yield 63%; mp >300 ◦C; IR (KBr) υ (cm−1): 3168 ( NH), 1668 (>C O), 1334 (>C S);
−
=
=
1
−
−
H NMR (300 MHz, DMSO-d6) δ (ppm): 2.36 (3H, s, CH3), 7.25–7.66 (3H, m, Ar H), 8.11
13
−
−
−
(1H, s, NH), 9.00 (1H, s, NH); C NMR (300 MHz, CDCl3) δ (ppm): 35.6 (C CH3),
114, 120, 126, 128 130, 150 (aromatic carbons), 176.1 (>C O), 196.2 (2 >C S), 77 and
180 (C C at pyrimidine ring); m/z: 276.00 (100.0%), 277.01 (13.1%), 278.00 (9.2%), 277.00
(2.3%), 278.01 (1.4%), 279.00 (1.3%). Anal. Calcd for C12H8N2O2S2: C, 52.16; H, 2.92; N,
10.14; S, 23.21. Found: C, 52.18; H, 2.87; N, 10.16; S, 23.27.
=
=
=
5.3. General procedure for synthesis of compounds 5(a–f)
A mixture of 3(a–f) (5.0 mmol) and phenyl isothiocyanate (5.0 mmol) in dimethylformamide
(20 ml) containing a catalytic amount of triethylamine (3–4 drops) was refluxed on a sand bath
for 10 h (monitored by TLC). After completion of the reaction, the mixture was left to cool
to room temperature and poured into ice-cold water (50 ml) for complete precipitation. Product
was separated by filtration, washed with water, dried and recrystallized from methanol to give
compounds 5(a–f).
5.3.1. 4-Imino-3-phenyl-2-thioxo-1,2,3,4-tetrahydro-5H-chromeno[2,3-d]pyrimidine-5-one
(5a)
Yield 67%; mp 154–156 ◦C; IR (KBr) υ (cm−1): 3071 ( NH), 1666 (>C O), 1316 (>C S);
−
=
=
1
−
−
H NMR (300 MHz, DMSO-d6) δ (ppm): 7.18–8.15 (9H, m, Ar H), 8.21 (1H, s, NH), 9.02
(1H, s, NH); 13C NMR (300 MHz, CDCl3) δ (ppm): 114, 118, 120, 122, 125, 128, 130, 131,
136, 139, 144, 146 (aromatic carbons) 178.2 (>C O), 181.3 (>C S), 164.8 (>C NH), 65 and
167 (C C at pyrimidine ring); m/z: 321.06 (100.0%), 322.06 (19.4%), 323.05 (4.5%), 323.06
(2.4%), 322.05 (1.1%). Anal. Calcd for C17H11N3O2S: C, 63.54; H, 3.45; N, 13.08; S, 9.98.
Found: C, 63.61; H, 3.41; N, 13.15; S, 10.01.
−
=
=
=
=
5.3.2. 7,9-Dibromo-4-imino-3-phenyl-2-thioxo-1,2,3,4-tetrahydro-5H-chromeno[2,3-d]
pyrimidine-5-one (5b)
Yield 65%; mp 226–228 ◦C; IR (KBr) υ (cm−1): 3115 ( NH), 1661 (>C O), 1326 (>C S);
−
=
=
1
−
−
H NMR (300 MHz, DMSO-d6) δ (ppm): 7.12–8.25 (7H, m, Ar H), 8.27 (1H, s, NH), 9.12
(1H, s, NH); 13C NMR (300 MHz, CDCl3) δ (ppm): 109 and 112 (C–Br), 119, 122, 123,
125, 129, 130, 135, 136, 139, 144, 147 (aromatic carbons), 178.2 (>C O), 185.2 (>C S),
164.4 (>C NH), 67 and 169 (C C at pyrimidine ring); m/z: 478.88 (100.0%), 480.87 (52.6%),
476.88 (50.8%), 479.88 (19.6%), 477.88 (10.4%), 481.88 (9.9%), 480.88 (2.4%), 482.87 (2.3%),
−
=
=
=
=

Journal of Sulfur Chemistry 459
478.87 (2.3%), 482.88 (1.1%), 479.87 (1.1%). Anal. Calcd for C17H9N3O2Br2S: C, 42.61;
H, 1.89; N, 8.77; S, 6.69. Found: C, 42.66; H, 1.94; N, 8.81; S, 6.73.
5.3.3. 4-Imino-7-nitro-3-phenyl-2-thioxo-1,2,3,4-tetrahydro-5H-chromeno[2,3-d]pyrimidine-
5-one (5c)
Yield 61%; mp 258–260 ◦C; IR (KBr) υ (cm−1): 3163 ( NH), 1670 (>C O), 1338 (>C S);
−
=
=
1
−
−
H NMR (300 MHz, DMSO-d6) δ (ppm): 7.88–8.72 (8H, m, Ar H), 8.10 (1H, s, NH), 9.06
(1H, s, NH); 13C NMR (300 MHz, CDCl3) δ (ppm): 145.6 (C–NO2) 118, 120, 122, 125,
128, 130, 131, 136, 139, 144, 146 (aromatic carbons), 178.2 (>C O), 181.3 (>C S), 164.8
(>C NH), 65 and 167 (C C at pyrimidine ring); m/z: 366.04 (100.0%), 367.05 (18.7%),
368.04 (4.8%), 368.05 (2.6%), 367.04 (2.3%). Anal. Calcd for C17H10N4O4S: C, 55.73; H, 2.75;
N, 15.29; S, 8.75. Found: C, 55.76; H, 2.81; N, 15.33; S, 8.78.
−
=
=
=
=
5.3.4. 7-Chloro-4-imino-3-phenyl-2-thioxo-1,2,3,4-tetrahydro-5H-chromeno[2,3-d]
pyrimidine-5-one (5d)
Yield 67%; mp 294–296 ◦C; IR (KBr) υ (cm−1): 3216 ( NH), 1686 (>C O), 1321 (>C S);
−
=
=
1
−
−
H NMR (300 MHz, DMSO-d6) δ (ppm): 7.33–8.27 (8H, m, Ar H), 8.68 (1H, s, NH), 9.34 (H,
s, NH); 13C NMR (300 MHz, CDCl3) δ (ppm): 145.2 (C–NO2), 117, 120, 122, 125, 128, 130,
−
=
=
=
135, 136, 139, 144, 146 (aromatic carbons), 179.2 (>C O), 189.3 (>C S), 166.8 (>C NH),
=
60 and 165 (C C at pyrimidine ring); m/z: 355.02 (100.0%), 357.02 (34.3%), 356.02 (20.5%),
358.02 (6.8%), 357.01 (4.5%), 359.01 (1.5%). Anal. Calcd for C17H10N3O2ClS: C, 57.39;
H, 2.83; N, 11.81; S, 9.01. Found: C, 57.44; H, 2.85; N, 11.78; S, 9.08.
5.3.5. 4-Imino-8-methyl-3-phenyl-2-thioxo-1,2,3,4-tetrahydro-5H-chromeno[2,3-d]
pyrimidine-5-one (5e)
Yield 64%; mp 276–278 ◦C; IR (KBr) υ (cm−1): 3186 ( NH), 1667 (>C O), 1326 (>C S);
−
=
=
1
−
−
H NMR (300 MHz, DMSO-d6) δ (ppm): 2.33 (3H, s, CH3), 7.28–8.46 (8H, m, Ar H), 8.24
(1H, s, NH), 9.20 (1H, s, NH); 13C NMR (300 MHz, CDCl3) δ (ppm): 45.2 (C–CH3), 117,
120, 122, 125, 128, 130, 135, 136, 139, 144, 146 (aromatic carbons), 179.2 (>C O), 189.3
(>C S), 166.8 (>C NH), 60 and 165 (C C at pyrimidine ring); m/z: 335.07 (100.0%),
336.08 (19.7%), 337.07 (4.7%), 337.08 (2.4%), 336.07 (1.9%). Anal. Calcd for C18H13N3O2S:
C, 64.46; H, 3.91; N, 12.53; S, 9.56. Found: C, 64.44; H, 3.88; N, 12.50; S, 9.53.
−
−
=
=
=
=
5.3.6. 4-Imino-9-methyl-3-phenyl-2-thioxo-1,2,3,4-tetrahydro-5H-chromeno[2,3-d]
pyrimidine-5-one (5f)
Yield 60%; mp 263–265 ◦C; IR (KBr) υ (cm−1): 3142 ( NH), 1672 (>C O), 1333 (>C S);
−
=
=
1
−
−
H NMR (300 MHz, DMSO-d6) δ (ppm): 2.34 (3H, s, CH3), 7.12–8.33 (8H, m, Ar H), 8.43
13
−
−
−
(1H, s, NH), 9.10 (1H, s, NH); C NMR (300 MHz, CDCl3) δ (ppm): 40.9 (C CH3),
117, 121, 122, 126, 128, 130, 134, 136, 139, 141, 146 (aromatic carbons), 175.2 (>C O),
185.3 (>C S), 165.8 (>C NH), 68 and 167 (C C at pyrimidine ring); m/z: 335.07 (100.0%),
336.08 (19.7%), 337.07 (4.7%), 337.08 (2.4%), 336.07 (1.9%). Anal. Calcd for C18H13N3O2S:
C, 64.46; H, 3.91; N, 12.53; S, 9.56. Found: C, 64.49; H, 3.95; N, 12.58; S, 9.59.
=
=
=
=

460 D.D. Haveliwala et al.
5.4. General procedure for synthesis of compounds 6(a–f)
A solution of compound 3(a–f) (5.0 mmol) and ammonium thiocyanate (15.0 mmol) in glacial
acetic acid (20 ml) was refluxed on a sand bath for 8 h (monitored by TLC). After completion
of the reaction, the reaction mixture was left to cool to room temperature and then poured into
ice-cold water (50 ml) for complete precipitation. The product was separated by filtration and
washed with water, dried well and recrystallized from methanol to give compounds 6(a–f).
5.4.1. 1-(5-Oxo-2-thioxo-1,5-dihydro-2H-chromeno[2,3-d]pyrimidine-4-yl)thiourea (6a)
Yield 75%; mp 284–286 ◦C; IR (KBr) υ (cm−1): 3362 ( NH2), 3140 ( NH), 1666 (>C O),
−
−
=
1
=
−
1321 (>C S); H NMR (300 MHz, DMSO-d6) δ (ppm): 7.12–8.20 (4H, m, Ar H), 8.45 (2H, s,
2NH), 9.31 (2H, br s, NH2); 13C NMR (300 MHz, CDCl3) δ (ppm): 114, 120, 123, 125, 144,
152 (aromatic carbons), 180.2 (>C S), 171 (>C N at pyrimidine ring), 184 (>C S at thiourea
linkage), 178 (>C O), 65 and 167 (>C C at pyrimidine ring); m/z: 304.01 (100.0%), 305.01
(16.1%), 306.00 (9.0%), 307.01 (1.2%). Anal. Calcd for C12H8N4O2S2: C, 47.36, H, 2.65, N,
18.41, S, 21.07. Found: C 47.42, H, 2.61, N, 18.34, S, 21.13.
−
−
=
=
=
=
=
5.4.2. 1-(7,9-Dibromo-5-oxo-2-thioxo-1,5-dihydro-2H-chromeno[2,3-d]pyrimidine-4-yl)
thiourea (6b)
Yield 78%; mp 219–221 ◦C; IR (KBr) υ (cm−1): 3354 ( NH2), 3157 ( NH), 1661 (>C O),
−
−
=
1
=
−
1328 (>C S); H NMR (300 MHz, DMSO-d6) δ (ppm): 7.29–7.75 (2H, s, Ar H), 8.02 (2H, s,
13
−
−
−
2NH), 9.02 (2H, br s, NH2); C NMR (300 MHz, CDCl3) δ (ppm): 103 and 111 (C Br), 115,
=
=
=
118, 125, 150 (aromatic carbon), 179.3 (>C S), 169 (>C N at pyrimidine ring), 185 (>C S at
=
=
thiourea linkage), 177 (>C O), 67 and 170 (>C C at pyrimidine ring); m/z: 461.83 (100.0%),
459.83 (48.9%), 463.83 (47.9%), 462.83 (14.6%), 463.82 (8.8%), 464.83 (8.0%), 460.83 (7.9%),
465.82 (4.5%), 462.82 (1.5%). Anal. Calcd for C12H6N4O2Br2S2: C, 31.19; H, 1.31; N, 12.12; S,
13.88. Found: C, 31.26; H, 1.34; N, 12.14; S, 13.83.
5.4.3. 1-(7-Nitro-5-oxo-2-thioxo-1,5-dihydro-2H-chromeno[2,3-d]pyrimidine-4-yl)
thiourea (6c)
Yield 67%; mp >300 ◦C; IR (KBr) υ (cm−1): 3308 ( NH2), 3149 ( NH), 1673 (>C O)
−
−
=
1
=
−
1321 (>C S); H NMR (300 MHz, DMSO-d6) δ (ppm): 7.49–8.66 (3H, m, Ar H), 8.00 (2H,
13
−
−
−
s, 2NH), 8.98 (2H, br s, NH2); C NMR (300 MHz, CDCl3) δ (ppm): 142.5 (C NO2),
=
=
114, 120, 125, 144, 152 (aromatic carbons), 181 (>C S), 178 (>C N at pyrimidine ring),
=
=
=
184 (>C S at thiourea linkage), 175(C O), 65 and 168 (C C pyrimidine ring); m/z: 348.99
(100.0%), 350.00 (13.2%), 350.99 (9.3%), 349.99 (3.4%), 351.00 (1.8%), 351.99 (1.4%). Anal.
Calcd for C12H7N5O4S2: C, 41.26; H, 2.02; N, 20.05; S, 18.36. Found: C, 41.28; H, 2.07;
N, 20.00; S, 18.34.
5.4.4. 1-(7-Chloro-5-oxo-2-thioxo-1,5-dihydro-2H-chromeno[2,3-d]pyrimidine-4-yl)
thiourea (6d)
Yield 74%; mp 279–281 ◦C; IR (KBr) υ (cm−1): 3326 ( NH2), 3206 ( NH), 1662 (>C O)
−
−
=
1
=
−
1329 (>C S); H NMR (300 MHz, DMSO-d6) δ (ppm): 7.58–8.45 (3H, m, Ar H), 8.43 (2H, s,
–2NH), 9.45 (2H, br s, NH2); 13C NMR (300 MHz, CDCl3) δ (ppm): 126.9 (C–Cl), 114, 118,
−
=
=
=
130, 135, 155 (aromatic carbon), 185 (>C S), 180 (>C N at pyrimidine ring), 185 (>C S at

Journal of Sulfur Chemistry 461
=
=
thiourea linkage), 178 (>C O), 66 and 170 (>C C at pyrimidine ring); m/z: 337.97 (100.0%),
339.97 (41.8%), 338.97 (16.1%), 340.97 (6.0%), 341.96 (3.1%).Anal. Calcd for C12H7N4O2S2Cl:
C, 42.54; H, 2.08; N, 16.54; S, 18.93. Found: C, 42.48; H, 2.14; N, 16.61; S, 18.87.
5.4.5. 1-(8-Methyl-5-oxo-2-thioxo-1,5-dihydro-2H-chromeno[2,3-d]pyrimidine-4-yl)
thiourea (6e)
Yield 77%; mp 265–267 ◦C; IR (KBr) υ (cm−1): 3311 ( NH2), 3193 ( NH), 1668 (>C O)
−
−
=
1
=
−
1325 (>C S); H NMR (300 MHz, DMSO-d6) δ (ppm): 2.30 (3H, s, CH3), 7.66–7.87 (3H,
m, Ar H), 8.21 (2H, s, 2NH), 8.42 (2H, br s, NH2); 13C NMR (300 MHz, CDCl3) δ (ppm):
31.5 (C CH3), 113, 122, 125, 129 130, 155 (aromatic carbons), 179.3 (>C S), 169 (>C N
at pyrimidine ring), 185 (>C S at thiourea linkage), 177 (>C O), 67 and 170 (>C C at
pyrimidine ring); m/z: 318.02 (100.0%), 319.03 (14.3%), 320.02 (9.3%), 319.02 (3.1%), 320.03
(1.6%), 321.02 (1.5%). Anal. Calcd for C13H10N4O2S2: C, 49.04; H, 3.17; N, 17.60; S, 20.14.
Found: C, 48.96; H, 3.22; N, 17.56; S, 20.06.
−
−
−
−
=
=
=
=
=
5.4.6. 1-(9-methyl-5-oxo-2-thioxo-1,5-dihydro-2H-chromeno[2,3-d]pyrimidine-4-yl)
thiourea (6f)
Yield 71%; mp 247–249 ◦C; IR (KBr) υ (cm−1): 3336 ( NH2), 3207 ( NH), 1678 (>C O)
−
−
=
1
=
−
1337 (>C S); H NMR (300 MHz, DMSO-d6) δ (ppm): 2.33 (3H, s, CH3), 7.46–8.25 (3H, m,
Ar H), 8.65 (2H, s, 2NH), 8.96 (2H, br s, NH2); 13C NMR (300 MHz, CDCl3) δ (ppm): 32.5
(C CH3), 114, 120, 123, 129, 135, (aromatic carbons), 181 (>C S), 178 (>C N at pyrimidine
ring), 184 (>C S at thiourea linkage), 175 (>C O), 65 and 168 (C C pyrimidine ring); m/z:
318.02 (100.0%), 319.03 (14.3%), 320.02 (9.3%), 319.02 (3.1%), 320.03 (1.6%), 321.02 (1.5%).
Anal. Calcd for C13H10N4O2S2: C, 49.04; H, 3.17; N, 17.60; S, 20.14. Found: C, 49.10; H, 3.13;
N, 17.64; S, 20.17.
−
−
−
−
=
=
=
=
=
5.5. Agar micro-dilution method
TheinvitroactivityofthecompoundsagainstM. tuberculosis H37Rv [MTCC-200]wasdetermined
by the agar micro-dilution technique. Two-fold dilutions of each test compound were added to
7H10 agar and M. tuberculosis H37Rv was used as the test organism. MIC is the concentration of
the compound that completely inhibits the growth and colony-forming ability of M. tuberculosis.
In a 24-well plate, 3 ml of middle brook 7H11 agar medium with OADC supplement was dispensed
in each well. The test compound was added to the middle brook medium agar before in duplicate,
so that the final concentration of the test compound in well was 62.5 μg/ml. The known colony-
forming unit (CFU) of the H37Rv culture was dispensed on top of the agar in each well in a
negative-pressure bio-safety hood. The plates were then incubated at 37 ◦C in a CO2 incubator.
The concentration at which the complete inhibition of colonies was observed was taken as the
MIC of the test drug.
5.6. The MIC
The definition of the MIC is “the lowest concentration which resulted in maintenance or reduction
of inoculums viability”. The determination of the MIC involves a semi-quantitative test procedure
which gives an approximation to the least concentration of the antimicrobial agent needed to
prevent the microbial growth. The serial dilution technique was applied for the determination of
MIC of the tested compounds against four species of bacterial strains S. aureus [MTCC-96] and

462 D.D. Haveliwala et al.
S. pyogenes [MTCC-442]asGram-positive, E. coli [MTCC-443]andP. aeruginosa[MTCC-1688]
as Gram-negative and three species of fungal strain A. niger [MTCC-282], C. albicans [MTCC-
227] and A. Clavatus [MTCC-1323]. Dilution series was set up with 62.5, 100, 125, 150, 200,
250, 500 and 1000 μg/ml of the nutrient broth medium, and to each tube, 1000 μl of standardized
suspension of the test microbes (107 cell/ml) was added and incubated at 37 ◦C for 24 h.
Acknowledgements
The authors are thankful to the Department of Chemistry, V.N.S.G. University, Surat, for providing the laboratory facil-
ities, Bioscience Department, V.N.S.G. University and D. Rajani, Microcare Laboratory, Surat, for anti-tubercular and
antimicrobial activity. The authors also thank SAIF, Chandigarh and COE, Vapi for analytical facilities.
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