Synthesis, antitumor and antimicrobial testing of some new thiopyrimidine analogues

Article abstract of DOI:10.1248/cpb.c12-00557

The synthesis of some new 4-chloro-pyrimidine-5-carbonitriles (3b-d), 4-substituted-amino-pyrimidine-5-carbonitriles (4a-g), trioxo and dioxo-thiazolo[3,2-a]pyrimidine-6-carbonitriles (5a-c and 6a-h) have been described. The obtained compounds were evaluated for their in-vitro antitumor activity. A single dose (10 μM) of the test compounds was used in the National Cancer Institute (NCI) 60 cell lines panel assay. Compounds 3c and 4f showed high inhibitory activity against leukemia, whereas, compounds 3b and 4d, g displayed moderate activity. On the other hand, all compounds were screened for their in-vitro antibacterial and antifungal activities. Compounds 3d and 4b exhibited significant antibacterial activity against Staphylococcus aureus. Compound 4e showed two folds inhibitory activity against Entrobacter aerogener compared with the reference drug Tobramycin.

Full text of DOI:10.1248/cpb.c12-00557

October 2012
Regular Article
Chem. Pharm. Bull. 60(10) 1305–1313 (2012)
1305
Synthesis, Antitumor and Antimicrobial Testing of Some New
Thiopyrimidine Analogues
,a
Azza Taher Taher* and Amira Atef Helwab
a Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Cairo University; Cairo 11562, Egypt: and
b Department of Organic Chemistry, Faculty of Pharmacy, Misr University for Science and Technology; Al-
Motamayez District, 6th of October City, Cairo 11562, Egypt.
Received June 23, 2011; accepted July 19, 2012; advance publication released online August 3, 2012
The synthesis of some new 4-chloro-pyrimidine-5-carbonitriles (3b–d), 4-substituted-amino-pyrim-
idine-5-carbonitriles (4a–g), trioxo and dioxo-thiazolo[3,2-a]pyrimidine-6-carbonitriles (5a–c and 6a–h)
have been described. The obtained compounds were evaluated for their in-vitro antitumor activity. A single
dose (10µM) of the test compounds was used in the National Cancer Institute (NCI) 60 cell lines panel assay.
Compounds 3c and 4f showed high inhibitory activity against leukemia, whereas, compounds 3b and 4d, g
displayed moderate activity. On the other hand, all compounds were screened for their in-vitro antibacterial
and antifungal activities. Compounds 3d and 4b exhibited significant antibacterial activity against Staphylo-
coccus aureus. Compound 4e showed two folds inhibitory activity against Entrobacter aerogener compared
with the reference drug Tobramycin.
Key words thiopyrimidine derivative; synthesis; antitumor; antimicrobial activity
Thiouracil, 5-Flurouracil1) (A) and other pyrimidine com-
Results and Discussion
pounds are important synthons in anticancer, antibacterial and
Chemistry The 6-chloro-pyrimidine derivatives 3a–d is
antifungal chemotherapy.2–5) S-Alkylation (B) and N-alkyla- used as a key precursor in the present study. They were syn-
tion (C) products of thiouracil analogues have been recently thesized using the reported method for compound 3a.14) (Chart
reported as novel antibacterial, and cytotoxic agents.6,7) 1). Thus compounds 1a–d was treated with alkyl halides to
Thiazolopyrimidines (D), the bioisostere analogues of the give the corresponding S-alkylated derivatives 2a–d followed
anticancer purines such as Thioguanine8,9) (Lanvis®) (E), are by phosphoryl chloride treatment to afford 3a–d. The struc-
potentially bioactive molecules reported to display significant ture of the newly prepared compounds 3b–d was confirmed
1
anticancer and antimicrobial activities10–13) (Fig. 1).
by elemental analyses and spectral data (IR, H-NMR and
The development of potent and effective novel antineo- MS). The IR spectra of these compounds showed the disap-
1
plastic drugs is one of the most intensely persuaded goals of pearance of both NH and C=O bands. H-NMR spectra of
contemporary medicinal chemistry. In view of the biological compounds 3b–d showed the absence of NH signal. Moreover
significance of thiouracils and in continuation of our previous mass spectrum of compound 3c showed molecular ion peaks
efforts in the synthesis of new thiouracils and uracil-related at m/z 307.05 (M+) (33.17%) and 309.05 (M+2) (13.09%) in
heterocycles,7) we would like to report herein the synthesis of ratio 3:1 indicating the presence of chlorine atom.
new thioxo-chloro-pyrimidines (3b–d), substituted-aminopy-
2-Alkylthio-6-aryl-4-substituted-aminopyrimidine-5-car-
rimidines (4a–g) and thiazolopyrimidines (5a–c, 6a–h) and bonitriles (4a–g) were prepared through direct reaction of
their antitumor, antimicrobial testing.
2-alkylthio-4-aryl-6-chloropyrimidine-5-carbonitrile with var-
ious amines, in the presence of potassium carbonate in boiling
Fig. 1. Some Literature Cited Antitumor Thiopyrimidine Analogues
The authors declare no conflict of interest.
*To whom correspondence should be addressed. e-mail: azzataher2005@yahoo.com
© 2012 The Pharmaceutical Society of Japan

1306
Vol. 60, No. 10
dry benzene.15) Supporting evidence for structures of newly
prepared compounds 4a–g was provided by microanalyses and
1
1
spectral data (IR, H-NMR and MS). The H-NMR spectra of
compounds 4a–c showed signals of piperidyl protons around
1
δ 1.39, 1.70 and 3.87ppm. While H-NMR spectrum of 4d
pointed to triplet signal at δ 3.27 and δ 3.72ppm of morphinyl
protons.
On the other hand, in Chart 2 the thiazolopyrimidinetriones
5a–c were prepared in 50–55% yield, by treatment of 1a–c
with oxalyl chloride under dry condition. Compounds 5a–c
may exist in one of two forms F and G. Based on the litera-
ture reports16,17) the chemical shift of the pyrimidinone carbo-
nyl is markedly affected by the nature of the adjacent nitro-
gen. To distinguish between the two forms F and G, 13C-NMR
of compounds 5b were recorded. The data showed signals for
the carbonyl carbon resonance at δ=175.99ppm. This chem-
ical shift suggests that N(3) near to C=O is sp2-hybridized
(pyridine type as in compound I) appears at 170–175ppm and
different from the sp3-hybridized nitrogen (pyrrole type), the
carbonyl carbon C-4, which appears at 160–164ppm (com-
pound H) (Fig. 2).16,17) Based on the above finding, we con-
clude that the isolated products are found in each case in one
form namely, F rather than G.
Fig. 2. 13C-NMR Chemical Shift of Reported Thiouracil
1
IR, H-NMR, and mass spectra were consistent with the
proposed structures.
Moreover, reaction of 6-aryl-4-oxo-2-thioxo-1,2,3,4-te-
trahydropyrimidine-5-carbonitriles (1a–h) with chloroacetyl
chloride in dry benzene18) afforded thiazolopyrimidinediones
6a–h. Confirmatory evidence for structures of the novel syn-
thesized compounds 6a–h was given by elemental analyses
1
and spectral data (IR, H-NMR and MS). The IR spectra
showed the disappearance of NH band and appearance of
two absorption bands at 1740, 1650cm−1 of two carbonyl
Fig. 3. Three Dimension of Compounds 6a–h
1
group. H-NMR showed that CH2 of thiazolidinone appeared
as two singlet peaks around at δ 3.47 and 4.45ppm. Based
Antimicrobial Activity The testing of the antimicrobial
on literature survey19,20) and three dimension data (Fig. 3) of activity of all novel derivatives was carried out at Department
compounds 6a–h, it revealed that one proton of (CH2) was of Microbiology, Faculty of Pharmacy, Misr University for
incorporated within the shielding cone of the aromatic ring in Science and Technology using the disc diffusion technique
the 6-position so appeared in ranging 3.47–3.78ppm, while the according to Wiart.25) The tested strains included Staphylococ-
other proton was affected by deshielding effect of the adjacent cus aureus ATCC 25923, Escherichia coli ATCC 25922, En-
carbonyl group so appeared in ranging 4.00–4.45ppm. This terobacter aerogener ATCC 23048, Klebsiella ATCC 23495,
supports the hypothesis that the cyclization may occur at N(1) Salmonella, and Candida albicans. The diameters of the
rather than N(3).
measured zones showing complete inhibition were recorded to
Biological Evaluation The antitumor screening of novel the nearest millimeter. The mean of the inhibition zone was
synthesized compounds was carried-out at the National tabulated in Table 2.
Cancer Institute (NCI), Bethesda, MD, U.S.A. However, the
Determination of the Minimum Inhibitory Concen-
antimicrobial testing was carried-out at Department of Micro- tration (MIC) MIC values of the synthesized compounds
biology, Faculty of Pharmacy, Misr University for Science and (3b–d, 4b,f, 5a,b and 6d,f,g) were determined with S. aureus
Technology, Cairo, Egypt.
ATCC 25923 using Tobramycin as reference drug according to
Preliminary in-Vitro Antitumor Screening The synthe- broth dilution method.26) The results were represented in Table
sized compounds (3b,c, 4a–g, 5a–c, and 6a–h) were subjected 3 and Fig. 4.
to the NCI’s disease-oriented human cell lines screening assay
Results of in-Vitro Antitumor Screening The obtained
to be evaluated for their in-vitro antitumor activity. A single results of tested compounds showed that compounds 3b,c and
dose (10µm) of the tested compounds was used in the full NCI 4f possessed an excellent activity against some leukemia cell
60 cell lines panel assay which includes nine tumor subpanels line with GI values >70%. However, other compounds exhib-
namely; leukemia, non-small cell lung, colon, CNS, mela- ited moderate to weak activity against most of the cell lines.
noma, ovarian, renal, prostate, and breast cancer cells.21–24)
Regarding the activity toward individual cell lines: the
The data reported as mean graph of the percent growth of the 4-chlorothiopyrimidine derivative 3b showed remarkable ac-
treated cells, and presented as percentage growth inhibition tivity against CCRF-CEM, K-562 and MOLT-4 leukemia cell
(GI%). The obtained results of the tested thioxopyrimidinones lines with GI values 93, 84 & 93, respectively. In addition, it
and their analogues are shown in Table 1.
exerted moderate activity against HCT-116 colon cancer and

October 2012
1307
Table 1. Percentage Growth Inhibition (GI%) of in Vitro Sensitive Tumor Cell Lines at 10µm Concentration of Tested Compounds
Compds. No.
Cell line
CCRF-CEM
GI%
Compds. No.
Cell line
OVCAR- 5
GI%
3b
Leukemia
93
84
93
22
4e
4f
Ovarian cancer
Leukemia
13
93
29
15
K-562
CCRF-CEM
RPMI-8226
NCI-H522
MOLT-4
RPMI-8226
Non-small cell lung
cancer
Non-small cell lung
cancer
NCI-H522
14
Colon cancer
HCT-116
34
Colon cancer
HCT-116
HCT-15
SW-620
LOX IMVI
UO-31
38
13
28
30
14
36
17
25
72
13
10
HCT-115
SW-620
U251
12
30
12
38
12
14
22
22
27
21
23
CNS cancer
Melanoma
Melanoma
Renal cancer
Leukemia
LOX IMVI
M14
3c
4a
CCRF-CEM
K-562
Renal cancer
Leukemia
UO-31
4g
CCRF-CEM
K-562
MOLT-4
SR
MOLT-4
RPMI-8226
NCI-H226
Leukemia
CCRF-CEM
MOLT-4
Non-small cell lung
cancer
Non-small cell lung
cancer
NCI-H522
18
NCI-322m
26
Melanoma
UACC-62
UACC-62
OVAR-5
786-0
16
17
11
17
17
19
28
18
CNS cancer
SF-295
18
18
19
20
21
19
15
22
4c
Melanoma
SF-539
Ovarian cancer
Renal cancer
U251
Prostate cancer
Breast cancer
PC-3
A-498
MCF7
4d
Leukemia
CCRF-CEM
MOLT-4
RPMI-8226
T-47D
MDA-MB-231/ATCC
Hop-92
5a
Non-small cell lung
cancer
SR
19
12
5b
5c
Renal cancer
Renal cancer
UO-31
A498
22
30
Non-small cell lung
cancer
EKVX
Hop-92
21
24
16
Leukemia
Melanoma
RPMI-8226
UACC-62
Hop-92
23
20
12
NCI-H522
HCT-15
Colon cancer
6a
Non-small cell lung
cancer
HT29
18
18
18
15
Prostate cancer
Leukemia
PC-3
SR
16
15
27
11
CNS cancer
Melanoma
SF-295
6b
6d
6g
SNB-75
SK-MEL-5
Renal cancer
UO-31
EKVX
Non-small cell lung
cancer
UACC-62
OVCAR- 4
786-0
23
16
20
29
17
34
12
17
13
21
11
Hop-92
14
Ovarian cancer
Renal cancer
A498
RXF 393
UO-31
Prostate cancer
Breast cancer
PC-3
MCF-7
MDA-MB-231/ATCC
T-47D
MDA-MB-468
LOX-IMVI melanoma with GI values 38 & 30, respectively. lines.
Meanwhile, compound 3c registered distinctive potential
As an exception of compound 4f which proved to be having
selectivity against SR leukemia panel and moderate activity an excellent selective activity to CCRF-CEM with GI value
against CCRF-CEM leukemia cell line with GI values 72 & 93% and moderate activity against HCT-116, SW-620 colon
36%, respectively. The 4-substituted aminothiopyrimidines cancer and LOX-IMVI melanoma with GI values 34, 30 &
4a–g recorded low activity against most of the tumor cell 38%, respectively. Meanwhile, compound 4d showed moderate

1308
Vol. 60, No. 10
Table 2. The Preliminary Antimicrobial and Antifungal Screening Test for the Prepared Compounds Using Tobramycin and Fluconazole as References,
DMSO as Control
Compds. No.
Staph. aureus
Salmoenella
E. coli
Klebsiella
Enterobacter
Candida albicans
3b
18mm
19mm
22mm
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
3c
3d
—
—
—
4a
—
—
—
4b
22mm
—
—
—
—
4c
—
—
—
4d
—
—
—
—
4e
—
14mm
—
34 mm
—
7mm
—
4f
15mm
20mm
13mm
9mm
—
4g
—
—
—
5a
—
—
—
5b
—
—
—
5c
—
—
—
6a
—
—
—
—
6b
—
—
—
9mm
—
6c
—
—
—
6d
7mm
—
—
—
—
6e
6f
—
—
—
7mm
10mm
—
—
—
7mm
—
6g
—
—
6h
—
—
7mm
—
Tobramycin
Fluconazole
Sulphadiazine
Sulphamethoxazole
DMSO
29mm
23mm
20mm
28
21
20
—
—
—
—
16
18
—
—
—
—
—
—
—
—
—
—
Table 3. Minumum Inhibitory Concentration Values of the Compound with S. aureus
Sulphamethox-
Compds. No.
3c
3d
4b
4f
5a
5b
6d
128
Sulphadiazine
8
Tobramycin
1
azole
MIC value (µg/mL)
16
8
8
32
64
64
16
zone 18, 19 and 15mm compared with 29mm of the reference
Tobramycin drug. On the other hand, compounds 3d and 4b,g
registered promising activity with an inhibition zone 22, 22
and 20mm, respectively. While compound 4e proved to be
most active member against E. aerogener ATCC 23048 with
an inhibition zone doubling the reference drug. Compounds 4e
and 6b,f,h displayed poor activity against C. albicans.
Structure Activity Correlation 4-Chlorothiopyrimidine
compounds 3c and 3b have higher activity than that of 4a and
4b respectively, and this could be assigned to the presence of
the active chlorine atom which favors the potency than that
piperidine moiety.27)
Compound 4a (4-flouroisopropyl derivative) showed selec-
tive activity toward CCRF-CEM, K-562 leukemia cell lines
and H522 non-small cell lung cancer compared to 4-bromo
isopropyl analogy 4c. This could be assigned to the higher
electronegativity of the fluorine atom at the aromatic ring
Fig. 4. Minumum Inhibitory Concentration Values of Compounds
3b–d, 4f, 5a,b & 6d,f,g
activity against UO-31 renal cancer with GI value 34%.
Considering the fused thiazolopyrimidinones 5a–c and manipulated the selectivity of the compound against specific
6a–h demonstrated mild activity against the tested cell lines. cell lines.28,29) In addition that, compound 4c (4-bromoisopro-
The highest activity within these classes of compounds was pyl piperidino derivative) showed moderate selective activity
observed with compound 5c against A498 renal cancer with against UACC-62 melanoma, OVACAR-5 ovarian cancer
GI value 30%.
and 786-0, A 498 renal cancer compared to compound 4b
Results of Antimicrobial Activity Compounds 3b,c and (3-bromoethylpiperidino derivative). This is may be referred
4f showed moderate activity against S. aureus with inhibition to the combined factors of the difference in the position of

October 2012
1309
Chart 1
the halogen atom and the lipophilic character of isopropyl the pyrimidinone nucleus afforded the hybrid series 5a–c and
thioether substituent which enhances the potency than that of 6a–h, with diminished the potency of antitumor activity.
the methyl or ethyl derivatives. The hydrophobicity–activity
relationship of alkyl moieties was found in various bioactive an inhibitory activity against S. aureus. It may be that the
compounds.30–33)
effect of chlorine atom at position-4 favors the antibacterial
On the other hand, all chloro derivatives 3b–d possessed
Compound 4d (morpholino derivative) displayed good activity. Meanwhile, compounds 4b and 4g showed decent
activity against all leukemia panels, EKVX, HOP-92, antibacterial activity, this may be attributed to the presence
NCI-H522 non small cell lung cancer, HCT-15, HT29 colon of piperidino and sulphamethoxazole moieties which enhances
cancer, SF-295, SNB-75 CNS cancer, SK-MEL-5 melanoma, the antimicrobial activity.39,40) Additionally, compounds 3d, 4b
OVCAR- 4 ovarian cancer, RXF 393, UO-31 renal cancer, and 4g exhibited comparable activity to the sulphadiazine and
PC-3 prostate cancer and MCF-7, MDA-MB-231/ATCC, sulphamethoxazole reference drugs against S. aureus, while
T-47D, MDA-MB-468 breast cancer compared to piperidino compound 4e displayed equipotent activity toward E. coli
analogy 4c. It seems that morpholino derivative is more in comparison with sulphadiazine standard drug. Moreover,
preferred than piperidino congener.34–37) Compound 4f dis- compound 4e showed an excellent inhibition activity against
played an excellent inhibition activity against CCRF-CEM, E. aerogener ATCC 23048, this is due to the presence of sul-
RPMI-8226 leukemia cell lines, HCT-116, SW-620 colon phadiazine moiety.41)
cancer, LOX-IMVI melanoma and good inhibitory activity
against NCI-H522 non-small cell lung cancer, HCT-15 colon
cancer, U251 CNS cancer, M14 melanoma and UO-31 renal
Conclusion
According to the results of bioactivities, it is noted that
cancer compared to 4e (sulphadiazine derivative). It is pre- compounds 3b,c and 4f showed promising antitumor activity
sumably due to sulphamethoxazole moiety which enhances the against leukemic cell panel than the other pyrimidine deriv-
antitumor activity.38) On the other hand, compound 4e exerted atives. Compounds 4d,f,g possessed moderate to weak anti-
selective activity against OVCAR-5 ovarian cancer compared cancer activity against most of the cancer cell line used. On
to compound 4f. Compound 4g (sulphamethoxazole derivative) the other hand, compounds 3d and 4b exhibited significant
registered decent inhibition activity against most of leukemia inhibitory activity against S. aureus than the other tested
cell lines, in addition to NCI-H226, NCI-322m non-small cell compounds with MIC 8µg/mL. Compound 4e was the most
lung cancer, SF-295, Sf-539, U251 CNS cancer, PC-3 prostate active derivative against E. aerogener ATCC 23048 with an
cancer and MCF7, T-47D, MDA-MB-231/ATCC breast cancer inhibition zone 34mm about two folds more than that of the
compared to piperidino analogy 4b due to the presence of sul- reference Tobramycin antibiotic. Compounds 4e and 6b,f,h
phamethoxazole moiety.38) Moreover, in chart 2 Compound 5a displayed antifungal activity against C. albicans. In conclu-
showed superior inhibition activity against Hop-62 non-small sion, the study leads to the identification of novel cytotoxic
lung cell line more than 6a congener. Compound 5b dis- compounds 3b,c and 4f as potential anticancer activity against
played selective activity against HOP-62 non-small cell lung certain leukemia panels. Whereas, compounds 3d and 4b
cancer and UO-31 renal cancer compared to thiazolidindione exhibited significant antibacterial activity against S. aureus.
derivative 6b. In addition, compound 5c recorded an inhibi- Compound 4e showed twice an inhibitory activity in com-
tory activity toward CCRF-CEM, RPMI-8226 leukemia cell parison with that of the reference compound. These findings
lines, H226 non-small cell lung cancer, SNB-75 CNS cancer, demonstrated a new potential for 4-substitutedpyrimidin de-
LOXIMVI, UACC-257, UACC-62 melanoma, A498, CAKI-1 rivatives which could be useful templates for further deriva-
renal cancer and MCF7 breast cancer compared to thiazolidin- tives to obtain more potent antitumor agent(s).
dione analogy 6c. Generally, the fusion of thiazolone ring with

1310
Vol. 60, No. 10
Chart 2
3.23 (q, 2H, J=7.2Hz S–CH2–CH3), 7.51–8.06 (m, 4H,
Experimental
General Melting points were determined on Stuart appa- ArH); 13C-NMR (DMSO): δ 13.83, 25.56, 100.98, 114.60,
ratus and the values given are uncorrected. IR spectra were 121.90, 127.99, 130.94, 131.42, 134.75, 136.01, 162.10, 166.20,
recorded on Shimadzu IR 435 spectrophotometer and values 175.00; MS (EI) m/z: 353.00 (M+, 72.61%), 355.00 (M+2,
were represented in cm1. H- and 13C-NMR were carried out 100%), 357.00 (M+4, 27.52%); Anal. Calcd for C13H9BrClN3S
1
on Varian Gemini 300MHz spectrophotometer at The Micro- (354.65): C, 44.03; H, 2.56; N, 11.85. Found: C, 43.86; H, 2.28;
analytical Center, Cairo University, Cairo, Egypt, using TMS N, 11.92.
as an internal standard and chemical shifts were recorded in
6-(4-Bromophenyl)-4-chloro-2-(isopropylthio)pyrimidine-5-
ppm on δ scale. The electron impact (EI) mass spectra were carbonitrile (3d): Yellowish white micro crystals; yield 84%;
recorded on Hewlett Packard 5988 spectrometer at The Micro- mp: 88–90°C; IR (KBr, cm−1): 2970, 2866 (C–H aliphatic),
1
analytical Center, Cairo University, Cairo, Egypt, Analytical 2220 (CN); H-NMR (DMSO-d6): δ 1.41 (d, 6H, J=6.9Hz
thin layer chromatoghraphy (TLC) on silica gel olates contain- CH3–CH–CH3), 3.96 (sept, 1H, J=6.9Hz CH3–CH–CH3),
ing UV indicator was employed routinely to follow the course 7.63–7.91(m, 4H, ArH); 13C-NMR (DMSO): δ 22.13, 22.42,
of reactions and to check the purity of products. Elemental 36.81, 101.26, 114.60, 126.29, 130.52, 130.59, 131.64, 131.91,
microanalyses were performed at The Microanalytical Center, 133.39, 162.38, 167.25, 174.62; Anal. Calcd for C14H11BrClN3S
Cairo University, Cairo, Egypt, and were within 0.4%.
(368.68): C, 45.42; H, 3.01; N, 11.40; S, 8.70. Found: C, 45.57;
Compounds 1a–h, 2a–d and 4-chloro-6-(4-chlorophenyl)-2- H, 2.98;N, 11.29; S, 8.3.
(methylthio)pyrimidine-5-carbonitrile (3a) were prepared fol-
2-Alkylthio-6-aryl-4-substitutedaminopyrimidine-5-car-
bonitrile (4a–g) A suspension of 3a–d (0.0015mol), anhy-
lowing reported procedures.7,14)
2-Alkylthio-6-aryl-4-chloropyrimidine-5-carbonitrile drous potassium carbonate (0.28g, 0.002mol) and substituted
(3a–d) A suspension of 2a–d (0.0017mol) and phosphorus amine (0.0015mol) in dry benzene (10mL) was heated under
oxychloride (6mL) was heated under reflux for 5h. Excess reflux for 8h. The reaction mixture was filtered while hot. The
phosphorus oxychloride was evaporated under reduced pres- formed precipitate was filtered, washed with water, dried and
sure to half its volume. The reaction mixture was poured onto crystallized from methanol.
ice cold water (15mL) with continuous stirring. The formed
6-(4-Fluorophenyl)-2-(isopropylthio)-4-(piperidin-1-yl)py-
precipitate was filtered, dried and crystallized from acetone.
rimidine-5-carbonitrile (4a): White micro crystals; yield 62%;
4-Chloro-6-(4-fluorophenyl)-2-(isopropylthio)pyrimidine-5- mp: 108–110°C; IR (KBr, cm−1): 2927,2862 (C–H aliphatic),
1
carbonitrile (3b): White micro crystals; yield 79%; mp: 2202 (CN); H-NMR (DMSO-d6): δ 1.37 (d, 6H, CH3–CH–
86-87°C; IR (KBr, cm−1): 2981, 2877 (C–H aliphatic), 2225 CH3), 1.39 (br s, 2H, CH2 piperidine), 1.66 (br, 4H, 2CH2
1
(CN); H-NMR (DMSO-d6): δ 1.39 (d, 6H, J=6.9Hz, CH3– piperidine), 3.80 (sept, 1H, CH3– CH–CH3), 3.87 (br, 4H,
CH–CH3), 4.03 (sept, 1H, J=6.9Hz CH3–CH–CH3), 7.38–7.44 2CH2 piperidine), 7.34–7.39 (dd, 2H, J=8.7Hz, C2,C6 ArH),
(dd, 2H, J=8.7, C2,C6 ArH), 7.99–8.04 (dd, 2H, J=8.7Hz, 7.88–7.92 (dd, 2H, J=8.7, C3,C5 ArH); 13C-NMR (DMSO): δ
J=5.4Hz, C3,C5 ArH); 13C-NMR (DMSO): δ 22.06 (2C), 22.45(2C), 23.66, 25.05(2C), 35.66, 47.83(2C), 82.92, 115.16,
36.69, 101.04, 114.65, 115.78, 116.08, 130.65, 131.68, 131.75, 115.46, 117.92, 131.60, 131.72, 132.68, 161.21, 165.36, 168.76,
162.45, 165.97, 167.04, 174.43; MS (EI) m/z: 307.05 (M+, 172.43; MS (EI) m/z: 356.15 (M+, 74.12%), 313.10 (100%);
33.17%), 309.05 (M+2, 13.09%), 274.05 (100%); Anal. Calcd Anal. Calcd for C19H21FN4S (356.46): C, 64.02; H, 5.94; N,
for C14H11ClFN3S (307.77): C, 54.63; H, 3.60; N, 13.65; S, 15.72. Found: C, 63.83; H, 5.96; N, 15.60.
10.42. Found: C, 54.78; H, 3.63; N, 13.53; S, 10.21.
6-(3-Bromophenyl)-2-(ethylthio)-4-(piperidin-1-yl)pyrimi-
6-(3-Bromophenyl)-4-chloro-2-(ethylthio)pyrimidine-5- dine-5-carbonitrile (4b): Buff micro crystals; yield 65%; mp:
carbonitrile (3c): White micro crystals; yield 69%; mp: 220–221°C; IR (KBr, cm−1): 2922,2852 (C–H aliphatic), 2200
1
180–182°C; IR (KBr, cm−1): 2966, (C–H aliphatic), 2229 (CN); (CN); H-NMR (DMSO-d6): δ1.42 (t, 3H, J=7.2Hz, S–CH2–
1H-NMR (DMSO-d6): δ 1.33 (t, 3H, J=7.2Hz S–CH2–CH3), CH3), 1.58 (br s, 2H, CH2 piperidine), 1.75 (br, 4H, 2CH2

October 2012
1311
piperidine), 3.15 (q, 2H, J=7.2Hz, S–CH2–CH3), 3.93 (br, 4H, 3.35; N, 14.71; S, 11.22. Found: C, 48.01; H, 3.15; N, 14.40; S,
2CH2 piperidine), 7.32–7.99 (m, 4H, ArH); 13C-NMR (DMSO): 10.96.
δ 14.38, 23.65, 24.86, 25.52 (2C), 47.81(2C), 83.39, 117.71,
5-Aryl-2,3,7-trioxo-2,3-dihydro-7H-thiazolo[3,2-a]-py-
121.42, 128.16, 130.47, 131.58, 133.62, 138.47, 160.93, 168.40, rimidine-6-carbonitrile (5a–c) A mixture of 1c, 1d or 1g
172.52; Anal. Calcd for C18H19BrN4S (403.34): C, 53.60; H, (0.001mol), anhydrous potassium carbonate (0.28g, 0.002mol)
4.75; N, 13.89; S, 7.95. Found: C, 53.57; H, 4.77; N, 13.89; S, and oxalyl chloride (0.13mL, 0.0015mol) in dry benzene
7.88.
(15mL) was heated under reflux for 8h. The reaction mixture
6-(4-Bromophenyl)-2-(isopropylthio)-4-(piperidin-1-yl)py- was filtered while hot and the filtrate was left to cool. The
rimidine-5-carbonitrile (4c): Faint yellow micro crystals; formed precipitate was filtered, washed with water, dried and
yield 59%; mp: 128–130°C; IR (KBr, cm−1): 2931, 2850 (C–H crystallized from methanol.
1
aliphatic), 2206 (CN); H-NMR (DMSO-d6): δ 1.37 (d, 6H,
5-(4-Chlorophenyl)-2,3,7-trioxo-2,3-dihydro-7H-thiazolo-
J=6.9Hz, CH3–CH–CH3), 1.40 (s, 2H, CH2 piperidine), 1.66 [3,2-a]pyrimidine-6-carbonitrile (5a): Dark yellow micro crys-
(br, 4H, 2CH2 piperidine), 3.77 (sept, 1H, J=6.9Hz, CH3– tals; yield 53%; mp: 244–246°C; IR (KBr, cm−1): 2229 (CN)
1
CH–CH3), 3.87 (br, 4H, 2CH2 piperidine), 7.70–7.81 (m, 4H, 1720 (N–C=O), 1702 (S–C=O), 1680 (CH–CO–N); H-NMR
ArH); 13C-NMR (DMSO-d6) δ 22.50(2C), 23.62, 25.48(2C), (DMSO-d6): δ 7.63–7.73 (m, 4H, ArH); MS (EI) m/z: 316.0
35.71, 47.83(2C), 83.05, 117.73, 124.73, 131.10(2C), 131.33(2C), (M-1, 0.42%), 138.00 (100%); Anal. Calcd for C13H4ClN3O3S
135.48, 161.22, 169.90, 172.55; MS (EI) m/z: 416.15 (M+, (317.71): C, 49.15; H, 1.27; N, 13.23. Found: C, 49.40; H, 1.33;
60.38%), 418.15 (M+2, 64.18), 375.10 (100%); Anal. Calcd for N, 13.41.
C19H21BrN4S (417.37): C, 54.68; H, 5.07; N, 13.42. Found: C,
54.71; H, 5.40; N, 13.67.
5-(3-Bromophenyl)-2,3,7-trioxo-2,3-dihydro-7H-thiazolo-
[3,2-a]pyrimidine-6-carbonitrile (5b): Yellow micro crystals;
6-(4-Bromophenyl)-2-(isopropylthio)-4-morpholinopyrimi- yield 58%; mp: 170–172°C; IR (KBr, cm−1): 2229 (CN) 1720
dine-5-carbonitrile (4d): White micro crystals; yield 55%; mp: (N–C=O), 1701 (S–C=O), 1674 (CH–CO–N); 1H-NMR
140–141°C; IR (KBr, cm−1): 2966, 2866 (C–H aliphatic), 2202 (DMSO-d6): δ 7.50–7.89 (m, 4H, ArH); 13C-NMR (DMSO-d6)
1
(CN); H-NMR (DMSO-d6): δ 1.38 (d, 6H, CH3–CH–CH3), δ 91.19, 114.31, 120.70, 121.28, 127.79, 130.55, 131.26, 131.33,
3.27 (t, 4H, 2CH2–N morpholine), 3.73 (sep, 1H, CH3–CH– 134.50, 134.65, 158.20, 159.24, 175.99; MS (EI) m/z: 316 (M-1,
CH3), 3.88 (t, 4H, 2CH2–O morpholine), 7.45–7.77(m, 4H, 0.42%), 138 (100%); Anal. Calcd for C13H4BrN3O3S (362.16):
ArH)); 13C-NMR (DMSO-d6) δ 22.41 (2C), 35.72, 46.96 C, 43.11; H, 1.11; N, 11.60; Found: C, 43.00; H, 1.19; N, 11.78.
(2C), 65.75 (2C), 83.53, 117.63, 124.89, 130.88, 131.06, 131.18,
5-(4-Fluorophenyl)-2,3,7-trioxo-2,3-dihydro-7H-thiazolo-
131.38, 135.19, 161.44, 168.79, 172.56; MS (EI) m/z: 418.00 [3,2-a]pyrimidine-6-carbonitrile (5c): Brownish red micro
(M+, 41.79%), 420.00 (M+2, 40.83%), 385.00 (100%); Anal. crystals; yield 51%; mp: 122–123°C; IR (KBr, cm−1): 2225
Calcd for C18H19BrN4OS (419.34): C, 51.56; H, 4.57; N, 13.36. (CN) 1720 (N–C=O), 1700 (S–C=O), 1689 (CH–CO–N);
Found: C, 51.29; H, 4.32; N, 13.20.
4-((6-(4-Chlorophenyl)-5-cyano-2-(methylthio)pyrimid- m/z: 301.10 (M+, 3.06%), 77.00 (100%); Anal. Calcd for
in-4-yl)amino)-N-(pyrimidin-2-yl)benzenesulfonamide (4e): C13H4FN3O3S (301.25): C, 51.83; H, 1.34; N, 13.95. Found: C,
1H-NMR (DMSO-d6): δ 7.03–7.87 (m, 4H, ArH); MS (EI)
Brownish yellow micro crystals; yield 62%; mp: 158–159°C; 51.91; H, 1.70; N, 13.58.
IR (KBr, cm−1): 3425, 3356 (2NH), 2931, 2870 (C–H ali-
5-Aryl-2,7-dioxo-2,3-dihydro-7H-thiazolo[3,2-a]pyrimi-
phatic), 2222 (CN); 1H-NMR (DMSO-d6): δ 2.61 (s, 3H, dine-6-carbonitrile (6a–h) A mixture of 1a–h (0.001mol),
S–CH3), 7.65–8.47 (m, 11H, ArH), 8.41, 8.50 (2s, 2H, NH ex- anhydrous potassium carbonate (0.28g, 0.002mol) and chloro-
changeable by D2O); Anal. Calcd for C22H16ClN7O2S2 (509.99): acetyl chloride (0.12mL, 0.0015mol) in dry benzene (15mL)
C, 51.81; H, 3.16; N, 19.23. Found: C, 51.94; H, 3.76; N, 19.33. was heated under reflux for 10h. The reaction mixture was
4-((6-(4-Chlorophenyl)-5-cyano-2-(methylthio)pyrimid- filtered while hot. The formed precipitate was filtered, washed
in-4-yl)amino)-N-(5-methylisoxazol-3-yl)benzenesulfonamide with water, dried and crystallized from methanol.
(4f): Buff micro crystals; yield 57%; mp: 126-128°C; IR (KBr,
5-(4-Chlorophenyl)-2,7-dioxo-2,3-dihydro-7H-thiazolo[3,2-
cm−1): 3400, 3300 (2NH), 2900, 2850 (C–H aliphatic), 2220 a]pyrimidine-6-carbonitrile (6a): Dark yellow micro crystals;
1
(CN); H-NMR (DMSO-d6): δ 2.30 (s, 3H, CH3 of oxazole), yield 90%; mp: >360°C; IR (KBr, cm−1): 2927 (C–H ali-
1
2.64 (s, 3H, S–CH3), 6.16 (s, 1H, CH of oxazole), 7.63–8.03 phatic), 2214 (CN), 1735 (S–C=O), 1685 (N–C=O); H-NMR
(m, 8H, ArH), 10.23, 11.41 (2s, 2H, NH exchangeable by D2O); (DMSO-d6) δ 3.52, 4.35 (2s, 2H, N–CH2–CO), 7.39–7.90 (m,
Anal. Calcd for C22H17ClN6O3S2 (512.99): C, 51.51; H, 3.34; N, 4H, ArH); 13C-NMR (DMSO): δ 57.09, 91.10, 114.63, 128.72,
16.38. Found: C, 51.90; H, 3.39; N, 16.72.
128.76, 130.72, 130.83, 133.85, 137.17, 158.46, 165.92, 169.29,
4-((6-(3-Bromophenyl)-5-cyano-2-(ethylthio)pyrimidin-4- 176.21; MS (EI) m/z: 302.20 (M-1, 6.02%), 303.20 (M+,
yl)amino)-N-(5-methylisoxazol-3-yl)benzenesulfonamide (4g): 7.33%), 304.20 (M+1, 17.90%), 305.20 (M+2, 8.29%), 203.15
Buff micro crystals; yield 60%; mp: 234–235°C; IR (KBr, (100%); Anal. Calcd for C13H6ClN3O2S (303.72): C, 51.41; H,
cm−1): 3429, 3302 (2NH), 2966, 2927 (C–H aliphatic), 2214 1.99; N, 13.83. Found: C, 51.52; H, 2.11; N, 13.94.
1
(CN); H-NMR (DMSO-d6): δ 1.20 (t, 3H, J=7.2Hz S–CH2–
5-(3-Bromophenyl)-2,7-dioxo-2,3-dihydro-7H-thiazolo[3,2-
CH3), 2.15 (s, 3H, CH3 of oxazole), 3.00 (q, 2H, J=7.2Hz a]pyrimidine-6-carbonitrile (6b): Dark yellow micro crys-
S–CH2–CH3), 5.87 (s, 1H, CH of oxazole), 7.54–7.99 (m, tals; yield 64%; mp: 220–221°C; IR (KBr, cm−1): 2927, 2854
4H, ArH), 7.83, 10.20 (2s, 2H, NH exchangeable by D2O); (C–H aliphatic), 2218 (CN), 1700 (S–C=O), 1681 (N–C=
1
13C-NMR (DMSO): δ 13.31, 14.21, 29.25, 82.10, 101.34, 114.71, O); H-NMR (DMSO-d6) δ 3.78, 3.90 (2s, 2H, N–CH2–CO),
115.68, 120.52, 121.12, 122.85, 128.67, 128.95, 130.50, 130.89, 7.40–7.91 (m, 4H, ArH); Anal. Calcd for C13H6BrN3O2S
133.62, 133.98, 136.61, 137.29, 160.67, 162.35, 165.77, 166.88, (348.17): C, 44.85; H, 1.74; N, 12.07. Found: C, 45.16; H, 1.82;
175.26; Anal. Calcd for C23H19BrN6O3S2 (571.47): C, 48.34; H, N, 12.19.

1312
Vol. 60, No. 10
5-(4-Fluorophenyl)-2,7-dioxo-2,3-dihydro-7H-thiazolo[3,2- microtiter tray in duplicate. Cell culture (1.8mL) containing a
a]pyrimidine-6-carbonitrile (6c): Dark buff micro crystals; cell population of 6×104 cells/mL was pippeted into each well.
yield 69%; mp: 270–271°C; IR (KBr, cm−1): 2930 (C–H ali- Controls, containing only phosphate buffer saline and DMSO
1
phatic), 2220 (CN), 1700–(S–C=O), 1685 (N–C=O); H-NMR at identical dilutions, were also prepared in the same manner.
(DMSO-d6): δ 3.76, 4.40 (2s, 2H, N–CH2–CO), 7.24–7.28 (m, These cultures were incubated in a humidified incubator at
1H, C2ArH), 7.29–7.38 (m, 1H, C6 ArH), 7.81–7.84 (m, 1H, C3 37°C. The incubator was supplied with 5% CO2 atmosphere.
ArH), 7.85–7.96 (m, 1H, C5 ArH); MS (EI) m/z: 287.00 (M+, After 48h, cells in each well were diluted 10 times with saline
50.7%), 75.00 (100%); Anal. Calcd for C13H6FN3O2S (287.27): and counted by using a coulter counter. The counts were cor-
C, 54.35; H, 2.11; N, 14.63; Found: C, 54.72; H, 2.29; N, 14.88. rected for the dilution.19–22)
5-(4-Bromophenyl)-2,7-dioxo-2,3-dihydro-7H-thiazolo[3,2-
Antimicrobial Screening Antimicrobial activity was car-
a]pyrimidine-6-carbonitrile (6d): Brownish yellow micro ried out using the disc diffusion technique according to Wiart.
crystals; yield 62%; mp: 210–212°C; IR (KBr, cm−1): 2935, The newly synthesized compounds were diluted in DMSO
2846 (C–H aliphatic), 2225 (CN), 1735-(S–C=O), 1654 (N– with concentration 10mg/mL, 6mm filter paper Whatman no1
1
C=O); H-NMR (DMSO-d6): δ 3.72, 4.07 (2s., 2H, N–CH2– was soaked in 50µL of each diluted compound. Both positive
CO), 7.62–7.88 (m, 4H, ArH); Anal. Calcd for C13H6BrN3O2S and negative control disc were applied using Tobramycin
(348.17): C, 44.85; H, 1.74; N, 12.07. Found: C, 44.85; H, 1.72; (10µg/mL) and DMSO, respectively. The tested strains in-
N, 12.07.
cludes (S. aureus ATCC 25923, E. coli ATCC 25922, En-
2,7-Dioxo-5-phenyl-2,3-dihydro-7H-thiazolo[3,2-a]pyrimi- terobacter aerogener ATCC 23048, Klebsiella ATCC 23495,
dine-6-carbonitrile (6e): Faint brown micro crystals; yield Salmonella, Candida albican).
60%; mp: 258–260°C; IR (KBr, cm−1): 2927 (C–H aliphatic),
Muller–Hinton agar was inoculated with microorganisms
2229 (CN), 1700 (S–C=O), 1678 (N–C=O); 1H-NMR suspension that adjusted in its density to that of 0.5 McFar-
(DMSO-d6): δ 3.62, 4.00 (2s, 2H, N–CH2–CO), 7.46–7.82 (m, land standard. Within 15min from preparation the adjusted
5H, ArH); Anal. Calcd for C13H7N3O2S (269.28): C, 57.98; H, suspension a sterile non toxic cotton swap on a wooden
2.62; N, 15.60. Found: C, 58.13; H, 2.78; N, 15.94.
applicator was dipped into the standardized inoculums and
5-(2-Fluorophenyl)-2,7-dioxo-2,3-dihydro-7H-thiazolo[3,2- rotates the soaked swab firmly against the upper inside wall
a]pyrimidine-6-carbonitrile (6f): Brownish red micro crystals; of the tube to express excess fluid. Entire agar surface of the
yield 62%; mp: 230–231°C; IR (KBr, cm−1): 2900 (C–H ali- plate was streaked with the swab three times, turning the
1
phatic), 2210 (CN), 1700 (S–C=O), 1650 (N–C=O); H-NMR plate 60° angle between each streaking. Then the inoculated
(DMSO-d6): δ 3.47, 3.51 (2s, 2H, N–CH2–CO), 7.23–7.79 (m, plate was allowed to dry before applying discs for five to
4H, ArH); Anal. Calcd for C13H6FN3O2S (287.27): C, 54.35; H, fifteen minutes with lid in place. Discs containing the tested
2.11; N, 14.63; Found: C, 54.70; H, 2.26; N, 14.80; S, 8.10.
synthesized products were applied using aseptic technique on
5-(2-Bromophenyl)-2,7-dioxo-2,3-dihydro-7H-thiazolo[3,2- the surface of agar and plates were incubated immediately at
a]pyrimidine-6-carbonitrile (6g): Brownish yellow micro 37°C for 14–19h or later if necessary. The diameters of the
crystals; yield 60%; mp: 130–132°C; IR (KBr, cm−1): 2970, measured zones showing complete inhibition were recorded to
2881 (C–H aliphatic), 2229 (CN), 1743 (S–C=O), 1647 (N– the nearest millimeter. The mean of the zone of inhibition was
1
C=O); H-NMR (DMSO-d6): δ 3.79, 4.10 (2s, 2H, N–CH2– tabulated in Table 2.
CO), 7.33–7.80 (m, 4H, ArH); MS (EI) m/z: 348.15 (M+1,
Determination of the Minimum Inhibitory Concentra-
0.19%), 349.05 (M+2, 3.69%), 270.15 (100%); Anal. Calcd for tion of the Synthesized Product MIC values of the synthe-
C13H6BrN3O2S (348.17): C, 44.85; H, 1.74; N, 12.07; Found: C, sized compounds with Staphylococcus aureus ATCC 25923
45.18; H, 1.88; N, 12.32.
were determined using broth dilution method. The tested
5-(4-(Dimethylamino)phenyl)-2,7-dioxo-2,3-dihydro-7H- compounds were dissolved in DMSO and further dilutions in
thiazolo[3,2-a]pyrimidine-6-carbonitrile (6h): Yellow micro Muller–Hinton broth were prepared to make 256, 128, 64, 32,
crystals; yield 72%; mp: 300–301°C; IR (KBr, cm−1): 16, 8, 4, 2, 1µg/mL . Both negative and positive control were
2935,2819 (C–H aliphatic), 2202 (CN), 1700 (S–C=O), 1651 prepared to ensure the sterility of the medium and viability of
(N–C=O); 1H-NMR (DMSO-d6): δ 2.94 (br, 6H, CH3–N– the tested strain respectively, also, a control test was carried
CH3), 3.60, 4.36 (2s, 2H, N–CH2–CO), 6.77–7.72 (m., 4H, out using inoculated broth with DMSO to test the solvent ef-
ArH); 13C-NMR (DMSO): δ 38.07 (2C), 59.12, 80.11, 110.96, fect which was found to be inactive in culture medium. The
120.59 (2C), 124.84 (2C), 129.22 (2C), 151.24, 162.03, 172.11, results were represented in Table 3 and Fig. 4.
174.09; MS (EI) m/z: 312.00 (M+, 32.25%), 148.40 (100%);
Anal. Calcd for C15H12N4O2S (312.35): C, 57.68; H, 3.87; N,
Acknowledgements Thanks are due to the NCI, Bethesda,
17.94. Found: C, 58.08; H, 3.97; N, 18.21.
MD, U.S.A. for performing the antitumor testing of the syn-
Antitumor Screening Under a sterile condition, cell thesized compounds. The authors would like to express their
lines were grown in RPMI 1640 media (Gibco, NY, U.S.A.) sincere thanks to Dr. Mahmoud Abdel-Ati Fouaad, Depart-
supplemented with 10% fetal bovine serum (Biocell, CA, ment of Microbiology, Faculty of Pharmacy, Misr University
U.S.A.), 5×105 cell/mL was used to test the growth inhibition for Science and Technology for carrying out the antimicrobial
activity of the synthesized compounds. The concentrations screening.
of the compounds ranging from 0.01−100µm were prepared
in phosphate buffer saline. Each compound was initially sol-
ubilized in dimethyl sulfoxide (DMSO), however, each final
dilution contained less than 1% DMSO. Solutions of different
concentrations (0.2mL) were pipetted into separate well of a
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