Novel pyrimidinone derivatives: Synthesis, antitumor and antimicrobial evaluation

Article abstract of DOI:10.1248/cpb.60.521

Starting from 6-aryl-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5- carbonitrile (4a-d), a series of mono- and dialkyl derivatives 5a-j and 6a, b was synthesized. Hydrazinolysis of 4a, b, d and 5d afforded the hydrazino derivatives 7a-c which were cyclised to give the triazolopyrimidinones 8a-c and the pyrimidotriazinones 9a-c through the reaction with formic acid and chloroacetyl chloride, respectively. Most of the newly synthesized compounds were evaluated for their in-vitro antitumor activity. Compounds 6a and b displayed promising anticancer activity against leukaemia, non-small cell lung, melanoma, and renal cancer. On the other hand, all compounds prepared were screened for their in-vitro antibacterial and antifungal activities. Compounds 5h and j showed significant activity against Staphylococcus aureus, while compounds 5e, 7c and 8c displayed moderate inhibitory activity against Candida albicans.

Full text of DOI:10.1248/cpb.60.521

April 2012
Regular Article
Chem. Pharm. Bull. 60(4) 521–530 (2012)
521
Novel Pyrimidinone Derivatives: Synthesis, Antitumor and Antimicrobial
Evaluation
,a
Azza Taher Taher* and Amira Atef Helwab
a Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Cairo University; P.O. Box 11562, Cairo,
b
Egypt: and Department of Organic Chemistry, Faculty of Pharmacy, Misr University for Science and Technology;
6-October, Egypt. Received December 8, 2011; accepted January 6, 2012
Starting from 6-aryl-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (4a–d), a series of
mono- and dialkyl derivatives 5a–j and 6a, b was synthesized. Hydrazinolysis of 4a, b, d and 5d afforded
the hydrazino derivatives 7a–c which were cyclised to give the triazolopyrimidinones 8a–c and the pyrimi-
dotriazinones 9a–c through the reaction with formic acid and chloroacetyl chloride, respectively. Most of
the newly synthesized compounds were evaluated for their in-vitro antitumor activity. Compounds 6a and b
displayed promising anticancer activity against leukaemia, non-small cell lung, melanoma, and renal cancer.
On the other hand, all compounds prepared were screened for their in-vitro antibacterial and antifungal ac-
tivities. Compounds 5h and j showed significant activity against Staphylococcus aureus, while compounds 5e,
7c and 8c displayed moderate inhibitory activity against Candida albicans.
Key words pyrimidinone; synthesis; antitumor activity; antimicrobial activity
Nowadays, cancer is the highly striking disease worldwide. synthesized. The alkylated derivatives 5b–d, f–h and 6a, b
It represents the second leading cause of human mortality af- and the biheterocyclic compounds triazolopyrimidines 8a–c
ter cardiovascular diseases. During the search for developing and pyrimidotriazines 9a–c were tested for their antitumor
more potent anticancer agents, several nitrogen heterocycles and antimicrobial activities and their structure–activity rela-
were synthesized and evaluated for their anticancer activity.1–5) tionship (SAR) was also studied.
A variety of anticancer drugs are currently in clinical use.
Some of these drugs are used successfully for the treatment of
several neoplastic diseases such as leukemia or testicular can-
Results and Discussion
Chemistry The synthetic pathways adopted for the prepa-
cer. However, the effect of anticancer drugs on solid tumors ration of the desired new compounds are illustrated in Charts
has been found to be rather low. For the sake of improving 1, 2. The starting pyrimidine derivatives 4a–d were synthe-
the efficacy of response to the anticancer chemotherapy, the sized in a one-pot three component reaction as outlined in
search for new drugs is always demanded. Literature survey Chart 1, according to reported procedure.17)
revealed the presence a large number of pyrimidine-based
The 2-alkylthio derivatives 5a–j were prepared through
antimetabolites which are usually structurally related to the the alkylation of the mercapto function of 4a–d with the ap-
endogenous substrates that they antagonize. 5-Fluorouracil propriate alkyl iodide, such as methyl iodide, ethyl iodide
°
A (5-FU), one of the early prepared metabolites, became one and isopropyl iodide, at −5 C. The structure of these mono-
of the most widely used antineoplastic agents due to its in- alkylated compounds was confirmed by elemental analyses
hibition of DNA synthesis.6–9) Recently, it was reported that and spectral data. The IR spectra showed the presence of new
2-((1H-benzoimidazol-2-yl)methylthio)-4-hydroxy-6-phenyl- bands at 2855–2877cm−1 corresponding to alkyl groups. The
pyrimidine-5-carbonitrile B possess significant in vitro anti-
proliferative activity.10) Furthermore, pyridotriazolopyrimidine
derivatives C were reported to possess a potent antitumor
activity11) (Fig. 1).
In the light of the aforementioned facts, and in continuation
to our interest in the synthesis of biologically active heterocy-
clic compounds, we report herein the synthesis of some new
4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine derivatives by
introducing various substituents (such as alkyl and substituted
phenyl) at 2-, 3- and 6-positions. In addition, the fusion of the
pyrimidinone ring with a triazole and a triazine heterocycles
was accomplished to improve the anticancer profiles.
On the other hand, some tetrahydro and dihydropyrim-
idinones D and E (Fig. 2), were shown to possess promising
antibacterial and fungicidal activities,12–15) especially against
the Gram positive (Gr+) pathogens Staphylococcus aureus
through inhibition of DNA polymerase IIIC.16) Bearing this
in mind, novel bioactive pyrimidinones (thiouracil analogues)
could be interesting target compounds.
Searching for new lead candidates, a series of 6-aryl-4-oxo-
Fig. 1. Structures of Some Literature Active Antitumor Agents
2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile 4a–d was
*To whom correspondence should be addressed. e-mail: azzataher2005@yahoo.com © 2012 The Pharmaceutical Society of Japan

522
Vol. 60, No. 4
yielded compounds 9a–c which were separated as hydrochlo-
ride salts.24) IR spectra showed an additional absorption band
1
around 1720cm−1 corresponding to the new νC=O. H-NMR
spectra of compounds 9a–c showed singlet of two protons
around δ 5.13, 4.26ppm assigned to the CH2 protons of the
triazine ring.
Biological Evaluation The antitumor screening of novel
synthesized compounds was carried in National Institue
Center (NCI), Bethesda, MD, U.S.A. In addition to the
antimicrobial activity was carried out in Department of
Microbiology, Faculty of Pharmacy, Misr University for
Science and Technology, Cairo, Egypt.
Preliminary in-Vitro Antitumor Screening The newly
synthesized compounds (5b–d, f–h, 6a,b, 8a–c, 9a–c) were
’
subjected to the NCI s disease-oriented human cell lines
screening assay to be evaluated for their in-vitro antitumor
activity. A single dose (10μM) of the test compounds were
used in the full NCI 60 cell lines panel assay which includes
nine tumor subpanels namely; leukaemia, non-small cell lung,
colon, CNS, melanoma, ovarian, renal, prostate, and breast
cancer cells.25–28) The data reported as mean graph of the per-
cent growth of the treated cells, and presented as percentage
growth inhibition (GI %). The obtained results of the tested
thioxopyrimidinones and their analogues are shown in Table 1.
Antimicrobial Activity Antimicrobial activity of all nov-
el derivatives was carried out in Department of Microbiology,
Faculty of Pharmacy, Misr University for Science and
Fig. 2. Structures of Some Literature Active Antimicrobial Agents
1H-NMR spectra of compounds 5b,c showed a singlet of three Technology using the disc diffusion technique according to
protons corresponding to the methyl group at δ 2.61, 2.79ppm, Warit.29) The tested Strains includes S. aureus ATCC 25923,
respectively. The H-NMR spectra of 5d–f showed the usual E. coli ATCC 25922, Enterobacter aerogener ATCC 23048,
1
pattern of triplet and quartet of the ethyl group while those of Klebsiella ATCC 23495, Salmonella, and Candida albicans.
5g–j showed a doublet and a septet of the isopropyl group (cf. The diameters of the measured zones showing complete inhi-
Experimental).
bition were recorded to the nearest millimetre. The mean of
On the other hand, compounds 6a,b were successfully the zone of inhibition was tabulated in Table 2.
prepared by direct alkylation of 4c,d using two equivalents
Determination of the Minimum Inhibitory Concentration
of alkyl halide at room temperature.18–21) IR spectra showed (MIC) MIC values of the synthesized compounds (5b–j, 7a,
1
the absence of νNH band. H-NMR spectrum of 6a showed 9a,c) were determined with Staphylococcus aureus (ATCC
two singlet at δ 2.72 and δ 3.61ppm corresponding to S–CH3 25923) using Tobramycin as reference drug according to broth
1
and N–CH3 respectively. H-NMR spectrum of 6b showed a dilution method.30) The results were represented in Table 3 and
triplet (δ=1.37ppm) and a quartet (δ=3.16ppm) correspond- Fig. 3.
ing to S–CH2–CH3, while the other triplet (δ=1.27ppm) and
Structure–Activity Relationship The obtained results of
quartet (δ=4.01ppm) were assigned to the N–CH2–CH3 group. tested compounds, alkyl and cyclised pyrimidinone analogues
In Chart 2, the synthesis 6-aryl-2-hydrazinyl-4-oxo-1,4- 5b–d, f–h, 6a,b, 8a–c and 9a–c showed potential pattern
dihydropyrimidine-5-carbonitrile 7a–c has been already of selectivity as broad spectra antitumor Table 1. Regarding
described by the reaction of 4a,b,d with a large excess the activity towards individual cell lines, compound 5h
of hydrazine hydrate and the yields were reported around (4-chloroisopropyl derivative) showed promising activity
60%.22) Alternatively, another approach was achieved by al- against Hop-92 non small cell lung cancer compared with
kylation of compound 4a, followed by treatment of the formed 5g (4-fluoroisopropyl derivative). In addition, compound 5h
S-alkylated products with hydrazine hydrate to afford a 30% exhibited moderate cytotoxic activity against leukaemia CCR
yield of 7a according to reported method.23) The IR spectra F-CEM and CNS SF-295 with GI 15.4 and 14.1%, respec-
of 7a,c showed two absorption bands at 3320 and 3282cm−1 tiviley. Chlorine atom increases lipophilicity of the compound
corresponding νNHNH2. 1H-NMR spectra showed the two which enhanced its absorption to cancer cell lead to increas-
characteristic singlets of NH2 around 7.07 and 7.57ppm re- ing the activity.31) Also, compound 5f (4-bromoethyl deriva-
spectively, exchangeable with D2O (cf. Experimental).
tive) exerted selective activity against leukaemia (K-562 and
The reaction of the hydrazino derivatives 7a–c with for- RPMI-8226) and renal cancer (ACHN and UO-31) cell lines
mic acid at reflux temperature afforded 5-aryl-7-oxo-1,7- relative to compound 5d (4-flouroethyl derivative). The more
dihydro[1,2,4]triazolo[4,3-a]pyrimidine-6-carbonitrile deriva- lipophilic chloro or bromo substituent which may enhance the
tives (8a–c). 1H-NMR spectra of the latter compounds are absorption to cancer cell line leading to increase in activity.
characterized by a singlet corresponding to H-3 proton of the Compound 5b (3-bromomethyl derivative) displayed moderate
triazole ring at δ 9.33, 9.35 and 9.30ppm, respectively.23) On activity against MOLT-4 leukaemia tumor cell line and UO-31
the other hand, the reaction of 7a–c with chloroacetyl chloride renal cancer compared to activity of 4-bromo analogy 5c. It

April 2012
523
Chart 1
seems that 3-bromo derivative is more preferred than 4-bromo the most active members of this study towards different tumor
congener. Compound 6b showed selective potency against non subpanels.
small cell lung cancer-322M, melanoma UACC-62 and renal
Compound 8b showed slightly activity against Hop-92
cancer TK-10 with GI values of 27.9, 30.3 and 31.9%, respec- non small cell lung cancer with GI 15.7%. Compound 9a
tively. In addition, compound 6a showed selective against displayed moderate activity against UO-31 renal cancer with
leukaemia K-562, renal cancer UO-31 and breast cancer T-47D GI 19.9%. In addition compound 9b showed slightly inhibi-
with GI values 26.6, 30.2 and 27.5%, respectively. From the tory activity against renal cancer A 498, A CHN, CAK-1 in
results we can note by increasing lipophilicity (logP), the GI 12.6, 12.4, 11.8%, respectively compared with other anal-
activity was increased towards solid tumors, in contrast by in- ogy 4-flouro and 4-bromo derivatives 9a and c, respectively.
creasing hydrophilicity the activity decrease towards solid tu- Dioxopyrimidotriazine derivatives (9a–c) exhibited selectivity
mours. This hydrophobic alkyl moiety was found in a diverse toward renal cancer cell line in comparison with triazolopy-
group of bioactive compounds.32–35) Close examination of data rimidine analogues (8a–c).
represented in Table 1, revealed that compounds 6a and 6b are

524
Vol. 60, No. 4
Chart 2
apparatus and the values given are uncorrected. IR spectra
Conclusion
According to the results of bioactivities, it is noted that were recorded on Shimadzu IR 435 spectrophotometer and
the dialkyl derivatives 6a,b showed higher antitumor activity values were represented in cm−1. 1H- and 13C-NMR were
than the monoalkyl derivatives 5b–d, f–h. Compounds 6a,b carried out on Varian Gemini 300MHz spectrophotometer at
exerted anticancer activity against most of the cancer cell line The Microanalytical Center, Cairo University, Cairo, Egypt,
used. On the other hand, compounds 5a–j exhibited promising using TMS as an internal standard and chemical shifts were
inhibition activity against Staphylococcus aureus than the oth- recorded in ppm on δ scale. The electron impact (EI) mass
er tested compounds. Compound 5h showed the most active spectra were recorded on Hewlett Packard 5988 spectrom-
one against Staphylococcus aureus ATCC 25923, Salmonella, eter at The Microanalytical Center, Cairo University, Cairo,
and Entrobacter aerogener ATCC 23048 with MIC of 16μg/ Egypt, Analytical thin layer chromatoghraphy (TLC) on silica
mL. Only compounds 5g–i and 7a showed moderate activ- gel olates containing UV indicator was employed routinely
ity against Entrobacter aerogener ATCC 23048. Compounds to follow the course of reactions and to check the purity of
5e, 7c and 8c displayed antifungal activity against Candida products. Elemental microanalyses were performed at The
albicans. In conclusion, the study leads to the identification Microanalytical Center, Cairo University, Cairo, Egypt, and
of novel cytotoxic compounds 6a,b against of most cell lines. were within 0.4%.
Whereas, compounds 5a–j exhibited significant antibacterial
activity against Staphylococcus aureus. The findings demon- 5-carbonitrile
strated a new potential for 2-thioxopyrimidin-4-one as lead 6-oxo-1,6-dihydropyrimidine-5-carbonitrile
6-Aryl-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-
(4a–d), 4-(4-chlorophenyl)-2-(methylthio)-
(5a) and
compounds for further development as medicinal agents.
6-(4-chlorophenyl)-2-hydrazinyl-4-oxo-1,4-dihydropyrimidine-
5-carbonitrile (7b) were prepared according to reported pro-
cedures.17–21)
Experimental
Chemistry Melting points were determined on Stuart
2-Alkylthio-4-aryl-6-oxo-1,6-dihydropyrimidine-5-

April 2012
525

526
Vol. 60, No. 4

April 2012
527
Table 2. The Preliminary Antimicrobial and Antifungal Screening Test for the Prepared Compounds Using Tobramycin and Fluconazole as References,
DMSO as Control
m.o Compd. No.
Staph. aureus
Salmonella
E. coli
Klebsiella
Enterobacter
Candida albicans
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
23
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
5b
10mm
7mm
9mm
12mm
7mm
12mm
18mm
12mm
15mm
—
—
5c
—
—
—
5d
—
—
5e
7mm
—
—
—
5f
—
—
5g
9mm
9mm
7mm
—
—
5h
7mm
—
—
5i
—
5j
7mm
—
—
—
6a
—
—
—
—
6b
—
—
7a
8mm
—
13mm
—
—
7c
7mm
—
—
—
—
8a
—
—
—
—
8b
—
—
—
8c
9a
7mm
—
—
—
8mm
—
—
—
—
9b
—
—
—
9c
12mm
29
—
Tobramycin
Fluconazole
DMSO
20
28
—
—
—
—
—
—
Table 3. Minumum Inhibitory Concentration Values of the Compound with Staphylococcus aureus
Compd. No.
5b
5c
5d
5e
5f
5g
5h
5i
5j
7a
9a
9c
Tobramycin
1
MIC value
64
64
64
64
64
32
16
32
16
64
128
64
(μg/mL)
1
2222 (CN), 1666 (C=O); H-NMR (DMSO-d6) δ: 2.61 (s, 3H,
S–CH3), 7.55–8.09 (m, 4H, ArH); Anal. Calcd for C12H8BrN3OS
(322.18): C, 44.74; H, 2.50; N, 13.04. Found: C, 44.60; H,
2.16; N, 12.85.
4-(4-Bromophenyl)-2-(methylthio)-6-oxo-1,6-dihydro-
°
pyrimidine-5-carbonitrile (5c): Yield 43%; mp 290–291 C;
IR (KBr, cm−1): 3400 (NH), 2931, 2873 (C–H aliphatic),
2222 (CN), 1666 (C=O); 1H-NMR (DMSO-d6) δ: 2.79 (s,
3H, S–CH3), 7.68–7.78 (m, 4H, ArH); MS (EI) m/z: 321.00
(M+, 92.18%), 323.00 (M+2, 100%); Anal. Calcd for
C12H8BrN3OS (322.18): C, 44.74; H, 2.50; N, 13.04. Found: C,
44.93; H, 2.65; N, 13.22.
Fig. 3. MIC Values of the Synthesized Compounds (5b-j, 7a and 9a,c)
2-(Ethylthio)-4-(4-fluorophenyl)-6-oxo-1,6-dihydro-
°
pyrimidine-5-carbonitrile (5d): Yield 41%; mp 228–230 C;
IR (KBr, cm−1): 3400 (NH), 2931, 2819 (C–H aliphatic),
carbonitrile (5a–j) A solution of appropriate alkyl iodide 2225 (CN), 1647 (C=O); 1H-NMR (DMSO-d6) δ: 1.33 (t,
(0.001mol) in dimethyl formamide (5mL) was added drop 3H, J=7.2Hz, S–CH2–CH3), 3.25 (q, 2H, J=7.2Hz, S–
wise with stirring to a solution of 4a–d (0.001mol) and anhy- CH2–CH3), 7.29–7.38 (dd, 2H, J=8.8Hz, C2, C6 ArH), 7.93–
drous potassium carbonate (0.14g, 0.001mol) in dimethylfor- 8.00 (dd, 2H, J=8.8, 5.6Hz, C3, C5 ArH), 8.05 (s, 1H, NH,
mamide (20mL) maintained at 0–5 C. After 3h the reaction exchangeable by D2O); MS (EI) m/z: 275.10 (M+, 100%); Anal.
°
mixture was poured into water (20mL), filtered and neutral- Calcd for C13H10FN3OS (275.30): C, 56.72; H, 3.66; N, 15.26.
ized with acetic acid (2mL). The obtained precipitate was fil- Found: C, 56.42; H, 3.39; N, 15.46.
tered, washed with water twice (10mL), dried and crystallized
from acetone.
4-(3-Bromophenyl)-2-(ethylthio)-6-oxo-1,6-dihydro-
°
pyrimidine-5-carbonitrile (5e): Yield 47%; mp 216–218 C;
4-(3-Bromophenyl)-2-(methylthio)-6-oxo-1,6-dihydro- IR (KBr, cm−1): 3300 (NH), 2935, 2873 (C–H aliphatic),
1
°
pyrimidine-5-carbonitrile (5b): Yield 30%; mp 248–249 C; 2225 (CN), 1654 (C=O); H-NMR (DMSO-d6) δ: 1.34 (t, 3H,
IR (KBr, cm−1): 3350 (NH), 2924, 2854 (C–H aliphatic), J=7.4Hz, S–CH2–CH3), 3.24 (q, 2H, J=7.4Hz, S–CH2–CH3),

528
Vol. 60, No. 4
7.50–8.07 (m, 4H, ArH), 13.72 (s, 1H, NH, exchangeable by dihydropyrimidine-5-carbonitrile (6a): Yield 54%; mp
−1
D2O); MS (EI) m/z: 335.05 (M+, 99.19%), 337.05 (M+2, 100%); 160–161 C; IR (KBr, cm ): 2936 (C–H aliphatic), 2218 (CN),
°
1
Anal. Calcd for C13H10BrN3OS (336.21): C, 46.44; H, 3.00; 1666 (C=O); H-NMR (CDCl3) δ: 2.72 (s, 3H, S–CH3), 3.61 (s,
N, 12.50. Found: C, 46.57; H, 2.85; N, 12.82.
3H, N–CH3), 7.35–8.14 (m, 4H, ArH); MS (EI) m/z: 335 (M+,
4 - (4 -Bromophenyl) -2- (et hylt h io) - 6 - oxo -1,6 - d i- 97.92%), 337 (M+2, 100%); Anal. Calcd for C13H10BrN3OS
hydropyrimidine-5-carbonitrile (5f): Yield 65%; mp: 228– (336.21): C, 46.44; H, 3.00; N, 12.50; S, 9.54. Found: C, 46.55;
−1
°
230 C; IR (KBr, cm ): 3300 (NH), 2927, 2862 (C–H ali- H, 3.05; N, 12.30; S, 9.45.
1
phatic), 2222 (CN), 1651 (C=O); H-NMR (DMSO-d6) δ: 1.32
4-(4-Bromophenyl)-1-ethyl-2-(ethylthio)-6-oxo-1,6-
(t, 3H, J=7.5Hz, S–CH2–CH3), 3.22 (q, 2H, J=7.5Hz, S–CH2– dihydropyrimidine-5-carbonitrile (6b): Yield 35%; mp
−1
°
CH3), 7.76, 7.87 (2d, 4H, J=9.0, ArH), 13.60 (s, 1H, NH, ex- 130–132 C; IR (KBr, cm ): 2919 (C–H aliphatic), 2237 (CN),
changeable by D2O); 13C-NMR (DMSO): 14.21, 24.99, 93.07, 1666 (C=O); H-NMR (DMSO-d6) δ: 1.27 (t, 3H, J=7.2Hz,
1
115.62, 125.50(2C), 130.55(2C), 131.62, 134.39, 160.78, 166.04, S–CH2–CH3), 1.37 (t, 3H, J=7.2Hz, N–CH2–CH3), 3.16 (q,
166.29; Anal. Calcd for C13H10BrN3OS (336.21): C, 46.44; H, 2H, J=7.2Hz, S–CH2–CH3), 4.06 (q, 2H, J=7.2Hz, N–CH2–
3.00; N, 12.50. Found: C, 46.57; H, 3.10; N, 12.77.
CH3), 7.79–7.93 (m, 4H, ArH); Anal. Calcd for C15H14BrN3OS
4-(4-Fluorophenyl)-2-(isopropylthio)-6-oxo-1,6-di- (364.26): C, 49.46; H, 3.87; N, 11.54. Found: C, 49.56; H, 3.75;
hydropyrimidine-5-carbonitrile (5g): Yield 42%; mp 238– N, 11.61.
−1
°
239 C; IR (KBr, cm ): 3300 (NH), 2935, 2862 (C–H aliphatic),
6-Aryl-2-hydrazinyl-4-oxo-1,4-dihydropyrimidine-5-
1
2222 (CN), 1651 (C=O); H-NMR (DMSO-d6) δ: 1.39 (d, 6H, carbonitrile (7a–c) A mixture of 4a,b,d or 5d (0.01mol)
J=6.8Hz, CH3–CH–CH3), 4.02 (sept, 1H, J=6.8Hz, CH3– wsa heated under reflux with an excess of 99% hydrazine hy-
CH–CH3), 7.35–7.49 (dd, 2H, J=8.8Hz, C2, C6 ArH), 7.98– drate (15mL) until the odour of hydrogen sulphide or C2H5SH
8.05 (dd, 2H, J=8.8, 5.6Hz, C3, C5 ArH), 13.75 (s, 1H, NH, ceased. The formed precipitate was filtered, dried and crystal-
exchangeable by D2O); Anal. Calcd for C14H12FN3OS (289.33): lized from ethanol.
C, 58.12; H, 4.18; N, 14.52. Found: C, 58.35; H, 3.93; N, 14.54.
6 - (4 - F l u o r o p h e n y l ) -2 - h y d r a z i n y l - 4 - o xo -1, 4 -
4-(4-Chlorophenyl)-2-(isopropylthio)-6-oxo-1,6- dihydropyrimidine-5-carbonitrile (7a): Yield 58%; mp 248–
−1
°
dihydropyrimidine-5-carbonitrile (5h): Yield 49%; mp 250 C; IR (KBr, cm ): 3320, 3282 (NH, NH2), 2198 (CN),
242–243 C; IR (KBr, cm ): 3400 (NH), 2927, 2866 (C–H 1674 (N–C=O); 1H-NMR (DMSO-d6) δ: 7.07 (s, 2H, NH2,
aliphatic), 2220 (CN), 1654 (C=O); H-NMR (CDCl3) δ: 1.28 exchangeable by D2O), 7.26–7.33 (dd, 2H, J=8.7Hz, C2, C6
−1
°
1
(d, 6H, J=6.6Hz, CH3–CH–CH3), 3.92 (sept, 1H, J=6.6Hz, ArH), 7.80–7.85 (dd, 2H, J=8.7, 5.7Hz, C3, C5 ArH); Anal.
CH3–CH–CH3), 7.28,7.80 (2d, 4H, J=8.8Hz, ArH), 12.21(s, Calcd for C11H8FN5O.H2O (263.23): C, 50.19; H, 3.83; N,
1H, NH, exchangeable by D2O); Anal. Calcd for C14H12ClN3OS 26.61; Found: C, 50.32; H, 3.95; N, 26.93.
(305.78): C, 54.99; H, 3.96; N, 13.74. Found: C, 55.13; H, 3.92;
N, 13.98.
6 - (4 - B r o m o p h e n y l ) -2 - h y d r a z i n y l - 4 - o x o -1, 4 -
dihydropyrimidine-5-carbonitrile (7c): Yield 39%; mp
−1
°
4-(3-Bromophenyl)-2-(isopropylthio)-6 -oxo-1,6 - >350 C; IR (KBr, cm ): 3331, 3280 (NH, NH2), 2198 (CN),
dihydropyrimidine-5-carbonitrile (5i): Yield 25%; mp 150– 1685 (N–C=O); 1H-NMR (DMSO-d6) δ: 7.57 (s, 2H, NH2,
−1
°
151 C; IR (KBr, cm ): 3380 (NH), 2927, 2866 (C–H ali- exchangeable by D2O), 7.61–7.70 (m, 4H, ArH), 10.2 (s, 2H,
1
phatic), 2225 (CN), 1658 (C=O); H-NMR (DMSO-d6) δ: 1.39 2NH, exchangeable by D2O); MS (EI) m/z: 306 (M+1, 1.74%),
(d, 6H, J=6.8Hz, CH3–CH–CH3), 3.95 (sept, 1H, J=6.8Hz, 307 (M+2, 0.78%), 55 (100%); Anal. Calcd for C11H8BrN5O.
CH3–CH–CH3), 7.54–8.06 (m, 4H, ArH), 13.21 (s, 1H, H2O (324.12): C, 40.72; H, 3.09; N, 21.60. Found: C, 40.35; H,
NH, exchangeable by D2O); Anal. Calcd for C14H12BrN3OS 2.72; N, 21.28.
(350.23): C, 48.01; H, 3.45; N, 12.00; S, 9.16. Found: C, 47.83;
H, 3.35; N, 12.18; S, 9.00.
5-Aryl-7-oxo-1,7-dihydro[1,2,4]triazolo[4,3-a]pyrimi-
dine-6-carbonitrile (8a–c) A mixture of 7a–c (0.01mol)
4-(4-Bromophenyl)-2-(isopropylthio)-6 -oxo-1,6 - and formic acid (35mL) was heated under reflux for 10h. The
dihydropyrimidine-5-carbonitrile (5j): Yield 41%; mp 220– formed precipitate after pouring onto ice cold water was fil-
−1
°
222 C; IR (KBr, cm ): 3400 (NH), 2931, 2866 (C–H ali- tered, dried and crystallized from ethanol.
1
phatic), 2245 (CN), 1651 (C=O); H-NMR (DMSO-d6) δ: 1.37
5-(4-Fluorophenyl)-7-oxo-1,7-dihydro-[1,2,4]triazolo[4,3-a]-
°
(d, 6H, J=6.8Hz, CH3–CH–CH3), 3.99 (sept, 1H, J=6.8Hz, pyrimidine-6-carbonitrile (8a): Yield 36%; mp 240–241 C; IR
CH3–CH–CH3), 7.61–7.95 (m, 4H, ArH), 12.9 (s, 1H, NH, (KBr, cm−1): 3417 (NH), 2229 (CN), 1670 (N–C=O); H-NMR
1
exchangeable by D2O); MS (EI) m/z: 349.05 (M+, 35.23%), (DMSO-d6) δ: 7.36–7.44 (dd, 2H, J=8.7, 6.9Hz, C2, C6 ArH),
351.05 (M+2, 36.77%), 316.10 (100%); Anal. Calcd for 7.89–7.96 (dd, 2H, J=8.7Hz, C3, C5 ArH), 8.12 (s, 1H, NH, ex-
C14H12BrN3OS (350.23): C, 48.01; H, 3.45; N, 12.00. Found: C, changeable by D2O), 9.33 (s, 1H, N–CH); 13C-NMR (DMSO):
47.93; H, 3.42; N, 12.37.
1-Alkyl-2-(alkylthio)-4-aryl-6-oxo-1,6-dihydro- 161.98, 165.29, 168.44; Anal. Calcd for C12H6FN5O (255.21): C,
pyrimidine-5-carbonitrile (6a,b) mixture of 4c,d 56.48; H, 2.37; N, 27.44. Found: C, 56.29; H, 2.35; N, 27.37.
(0.5g, 0.0016mol), potassium carbonate (0.45g, 0.0032mol) 5-(4-Chlorophenyl)-7-oxo-1,7-dihydro-[1,2,4]triazolo[4,3-a]-
83.22, 115.52, 120.47, 131.10(2C), 132.40(2C), 150.13, 155.31,
A
°
and appropriate alkyl iodide such as methyl iodide and/or ethyl pyrimidine-6-carbonitrile (8b): Yield 77%; mp >300 C;
iodide in dimethyl formamide (20mL) was stirred 3h at room IR (KBr, cm−1): 3250 (NH), 2225 (CN), 1693 (N–C=O);
temperature and then diluted with water (10mL). The formed 1H-NMR (DMSO-d6) δ: 7.57, 7.88 (2d, 4H, J=8.4Hz, ArH),
precipitate was filtered. The filtrate was acidified with acetic 8.13 (s, 1H, NH, exchangeable by D2O), 9.35 (s, 1H, N–CH);
acid (2mL). The obtained precipitate was filtered, dried and MS (EI) m/z: 271.00 (M+, 68.86%), 273.00 (M+2, 22.92%),
crystallized from acetone.
57 (100%); Anal. Calcd for C12H6ClN5O (271.66): C, 53.05; H,
4-(3-Bromophenyl)-1-methyl-2-(methylthio)-6-oxo-1,6- 2.23; N, 25.78. Found: C, 52.72; H, 2.07; N, 25.51.

April 2012
529
°
5-(4-Bromophenyl)-7-oxo-1,7-dihydro-[1,2,4]triazolo[4,3-a]- fied incubator at 37 C. The incubator was supplied with 5%
°
pyrimidine-6-carbonitrile (8c): Yield 54%; mp 320–322 C; CO2 atmosphere. After 48h, cells in each well were diluted 10
IR (KBr, cm−1): 3525 (NH), 2222 (CN), 1689 (N–C=O); times with saline and counted by using a coulter counter. The
1H-NMR (DMSO-d6) δ: 7.74, 7.82 (2d, 4H, J=8.7Hz ArH), counts were corrected for the dilution.25–28)
8.12 (s, 1H, NH, exchangeable by D2O), 9.30 (s, 1H, N–CH);
Antimicrobial Screening Antimicrobial activity was
13C-NMR (DMSO): 83.25, 116.40, 124.68, 130.36, 131.33(2C), carried out using the disc diffusion technique according to
133.04(2C), 135.44, 150.07, 155.03, 168.34; MS (EI) m/z: Wiart.29) The newly synthesized compound were diluted in
315.00 (M+, 100%), 317.00 (M+2, 96.76%); Anal. Calcd for DMSO with at a concentration 10mg/mL, 6mm filter paper
C12H6BrN5O (316.11): C, 45.59; H, 1.91; N, 22.15. Found: C, Whatman No. 1 was soaked in 50μL of each diluted com-
45.30; H, 1.84; N, 22.30.
pound. Both positive and negative control discs were applied
6-Aryl-3,8-dioxo-1,3,4,8-tetrahydro-2H-pyrimido[2,1- using Tobramycin (10μg/mL) and DMSO, respectively. The
c][1,2,4]triazine-7-carbonitrile Hydrochloride Salt (9a–c) tested Strains includes S. aureus ATCC 25923, E. coli ATCC
A mixture of 7a–c (0.001mol) and chloroacetyl chloride 25922, Enterobacter aerogener ATCC 23048, Klebsiella
(0.12mL, 0.0015mol) in dry benzene (15mL) was heated un- ATCC 23495, Salmonella, and Candida albicans. Muller
der reflux for 7h. The formed precipitate was filtered, dried hinton agar were inoculated with microorganisms suspension
and crystallized from methanol.
6-(4-Fluorophenyl)-3,8-dioxo-1,3,4,8-tetrahydro-2H- Within 15min from preparation the Adjusted Suspension a
pyrimido[2,1-c][1,2,4]triazine-7-carbonitrile Hydrochloride sterile non toxic cotton swap on a wooden applicator was
that Adjusted in its density to that of 0.5 McFarland standard.
−1
°
Salt (9a): Yield 48%; mp 310–311 C; IR (KBr, cm ): 3444 dipped into the standardized inoculums and rotates the
(NH), 2924 (C–H aliphatic), 2214 (CN), 1720 (CH2–C=O), soaked swab firmly against the upper inside wall of the tube
1
1666 (N–C=O); H-NMR (DMSO-d6) δ: 5.13 (s, 2H, CH2– to express excess fluid. Entire agar surface of the plate was
°
C=O), 7.39–7.95 (m, 4H, ArH), 9.18 (s, 2H, 2NH, exchange- streaked with the swab three times, turning the plate 60
able by D2O); 13C-NMR (DMSO): 24.99, 83.00, 117.02, 128.34, angle between each streaking. Then the inoculated Plate was
129.95, 130.92(2C), 136.22(2C), 146.10, 154.09, 157.30, 161.13; allowed to dry before applying discs for five to fifteen min-
Anal. Calcd for C13H8FN5O2·2HCl (358.16): C, 43.60; H, 2.81; utes with lid in place. Discs containing the tested synthesized
N, 19.55. Found: C, 43.60; H, 2.79; N, 19.57.
6-(4-Chlorophenyl)-3,8-dioxo-1,3,4,8-tetrahydro-2H- of agar and plates Incubated immediately at 37 C for 14–19h
pyrimido[2,1-c][1,2,4]triazine-7-carbonitrile Hydrochloride or later if necessary. The diameters of the measured zones
products were applied using aseptic technique on the surface
°
−1
°
Salt (9b): Yield 28%; mp 230–231 C; IR (KBr, cm ): 3236 showing complete inhibition were record to the nearest mil-
(NH), 2974 (C–H aliphatic), 2222 (CN), 1724 (CH2–C=O), limeter. The mean of the zone of inhibition was tabulated in
1
1651 (N–C=O); H-NMR (DMSO-d6) δ: 4.26 (s, 2H, CH2– Table 2.
C=O), 7.61, 7.85 (2d, 4H, J=8.7Hz, ArH), 10.55, 10.59 (s,
Determination of the Minimum Inhibitory Concentration
2H, 2NH, exchangeable by D2O); MS (EI) m/z: 301.00 (M+, (MIC) MIC values of the synthesized compounds with
35.59%), 303.05 (M+2, 13.08%), 86.10 (100%); Anal. Calcd Staphylococcus aureus (ATCC 25923) were determined using
for C13H8ClN5O2·2HCl (374.61): C, 41.68; H, 2.69; N, 18.70. broth dilution method.30) The tested compounds were dis-
Found: C, 41.58; H, 2.30; N, 19.02.
6-(4-Bromophenyl)-3,8-dioxo-1,3,4,8-tetrahydro-2H- were prepared to make 256, 128, 64, 32, 16, 8, 4, 2, 1μg/mL.
pyrimido[2,1-c][1,2,4]triazine-7-carbonitrile Hydrochloride Both negative and positive control were prepared to ensure the
solved in DMSO and further dilutions in Muller Hinton broth
−1
°
Salt (9c): Yield 41%; mp >320 C; IR (KBr, cm ): 3414 (NH), sterility of the medium and viability of the tested strain re-
2947 (C–H aliphatic), 2222 (CN), 1797 (CH2–C=O), 1693 spectively, also, a control test was carried out using inoculated
1
(N–C=O); H-NMR (DMSO-d6) δ: 4.26 (s, 2H, CH2–C=O), broth with DMSO to test the solvent effect which was found
7.69–7.85 (m, 4H, ArH), 10.55 (s, 2H, 2NH, exchangeable to be inactive in culture medium Table 3.
by D2O); MS (EI) m/z: 345.00 (M+, 23.67%), 346.00 (M+1,
4.22%), 347.00 (M+2, 11.52%), 348.05 (M+4, 4.85%), 55.05
(100%); Anal. Calcd for C13H8BrN5O2·2HCl (419.06): C, 37.26; Bethesda, MD, U.S.A. for performing the antitumor testing
H, 2.41; N, 16.71. Found: C, 37.52; H, 2.01; N, 16.61. of the synthesized compounds. The authors would like to ex-
Acknowledgements Thanks are due to the NCI,
Biological Studies. Antitumor Screening Under a sterile press their sincere thanks to Dr. Mahmoud Abdel-Ati Fouaad,
condition, cell lines were grown in RPMI 1640 media (Gibco, Department of Microbiology, Faculty of Pharmacy, Misr
NY, U.S.A.) supplemented with 10% fetal bovine serum University for Science and Technology for carrying out the
(Biocell, CA, U.S.A.), 5×105 cell/mL was used to test the antimicrobial screening.
growth inhibition activity of the synthesized compounds. The
concentrations of the compounds ranging from 0.01–100μM
were prepared in phosphate buffer saline. Each compound was
initially solubilized 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 microtiter tray in duplicate. Cell culture (1.8mL)
containing a cell population of 6×104 cells/mL was pippeted
into each well. Controls, containing only phosphate buffer
saline and DMSO at identical dilutions, were also prepared in
the same manner. These cultures were incubated in a humidi-
References
1) Rashad A. E., Mahmoud A. E., Ali M. M., Eur. J. Med. Chem., 46,
1019–1026 (2011).
2) Hafez H. N., El-Gazzar A. R., Bioorg. Med. Chem. Lett., 19, 4143–
4147 (2009).
3) Large J. M., Torr J. E., Raynaud F. I., Clarke P. A., Hayes A.,
Stefano F., Urban F., Shuttleworth S. J., Saghir N., Sheldrake P.,
Workman P., McDonald E., Bioorg. Med. Chem., 19, 836–851
(2011).
4) Tangeda S. J., Garlapati A., Eur. J. Med. Chem., 45, 1453–1458
(2010).

530
5) Kraljević T. G., Krištafor S., Šuman L., Kralj M., Ametamey S.
Vol. 60, No. 4
21) Ram V. J., Vanden Berghe D. A., Vlietinck A. J., J. Heterocyclic
Chem., 21, 1307–1312 (1984).
M., Cetina M., Raić-Malić S., Bioorg. Med. Chem., 18, 2704–2712
(2010).
22) Attaby F. A., Eldin S. M., Naturforsch, 54b, 788–798 (1999).
23) Attaby F. A., Eldin S. M., Hanafi E. A., Arch. Pharm. Res., 20,
620–628 (1997).
24) Taher A., Raafat M., Bull. Fac. Pharm. Cairo Univ., 43, 43–55
(2005).
“
’
”
6) Callery P., Gannett P., Foye s Principles of Medicinal Chemistry,
5th ed. Lippincott Williams & Wilkins, Philadelphia, 2002, pp.
934–935.
7) Jain K. S., Chitre T. S., Miniyar P. B., Kathiravan M. K., Bendre
V. S., Veer V. S., Shahane S. R., Shishoo C. J., Current Sci., 90(6), 25) Grever M. R., Schepartz S. A., Chabner B. A., Semin. Oncol., 19,
793–803 (2006). 622–638 (1992).
8) Sawyer R. C., Stolfi R. L., Martin D. S., Balis M. E., Biochem. 26) Monks A., Scudiero D., Skehan P., Shoemaker R., Paull K.,
Pharmacol., 38, 2305–2311 (1989).
9) Ghoshal K., Jacob S. T., Biochem. Pharmacol., 53, 1569–1575
(1997).
Vistica D., Hose C., Langley J., Cronise P., Vaigro-Wolff A., Gray-
Goodrich M., Campbell H., Mayo J., Boyd M., J. Natl. Cancer Inst.,
83, 757–766 (1991).
10) Abdel-Mohsen H. T., Ragab F. A., Ramla M. M., El Diwani H. I.,
Eur. J. Med. Chem., 45, 2336–2344 (2010).
11) El-Nassan H. B., Eur. J. Med. Chem., 46, 2031–2036 (2011).
12) Prachayasittikul S., Worachartcheewan A., Nantasenamat C.,
27) Boyd M. R., Paull K. D., Drug Der. Res., 34, 91–109 (1995).
28) Skehan P., Storeng R., Scudiero D., Monks A., McMahon J., Vistica
D., Warren J. T., Bokesch H., Kenney S., Boyd M. R., J. Natl.
Cancer Inst., 82, 1107–1112 (1990).
Chinworrungsee M., Sornsongkhram N., Ruchirawat S., Pra- 29) Wiart C., Evid. Based Complement. Alternat. Med., 4, 299 (2007).
chayasittikul V., Eur. J. Med. Chem., 46, 738–742 (2011).
13) Amr A. E., World J. Chem, 4(2), 201–206 (2009).
30) Andrews J., J. Antimicrob. Chemother., 48 (Suppl. S1), 5–16 (2001).
“
’
31) Williams D. A., Lemke T. L., Foye s Principles of Medicinal
”
14) Habib N. S., Soliman R., El-Tombary A. A., El-Hawash S. A.,
Shaaban O. G., Arch. Pharm. Res., 30, 1511–1520 (2007).
15) Said M., Abouzid K. H., Mouneer A., Ahmedy A., Osman A. M.,
Arch. Pharm. Res., 27, 471–477 (2004).
16) Rose Y., Ciblat S., Reddy R., Belley A. C., Dietrich E., Lehoux
D., McKay G. A., Poirier H., Far A. R., Delorme D., Bioorg. Med.
Chem. Lett., 16, 891–896 (2006).
Chemistry, 5th ed., Lippincott Williams & Wilkins, 2002, p. 48.
32) Bello C., Dal Bello G., Cea M., Nahimana A., Aubry D., Garuti A.,
Motta G., Moran E., Fruscione F., Pronzato P., Grossi F., Patrone F.,
Ballestrero A., Dupuis M., Sordat B., Zimmermann K., Loretan J.,
Wartmann M., Duchosal M. A., Nencioni A., Vogel P., Bioorg. Med.
Chem., 19, 7720–7727 (2011).
33) Balzarini J., Orzeszko B., Maurin J. K., Orzeszko A., Eur. J. Med.
Chem., 42, 993–1003 (2007).
17) Kambe S., Saito K., Kishi H., Synthesis, 1979, 287–289 (1979).
18) Ram V. J., Goel A., Nath M., Srivastava P., Bioorg. Med. Chem. 34) Yeh V. S., Kurukulasuriya R., Madar D., Patel J. R., Fung S.,
Lett., 4, 2653–2656 (1994).
Monzon K., Chiou W., Wang J., Jacobson P., Sham H. L., Link J. T.,
Bioorg. Med. Chem. Lett., 16, 5408–5413 (2006).
35) Rho H. S., Baek H. S., Ahn S. M., Yoo J. W., Kim D. H., Kim H.
G., Bioorg. Med. Chem. Lett., 19, 1532–1533 (2009).
19) Ram V. J., J. Prakt. Chem., 331, 893–905 (1989).
20) Ram V. J., Vanden Berghe D. A., Vlietinck A. J., Liebigs Ann.
Chem., 1987, 797–801 (1987).