Cytotoxic and antimicrobial activities of some novel heterocycles employing 6-(1,3-diphenyl-1H-pyrazol-4-yl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile

Article abstract of DOI:10.1515/hc-2019-0008

A pyrimidinethione derivative namely, 6-(1,3-diphenyl-1H-pyrazol-4-yl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile, was readily synthesized and reacted with carbon electrophiles in an attempt to synthesize selected fused heterocycles. The r

Full text of DOI:10.1515/hc-2019-0008

Heterocycl. Commun. 2019; 25: 107–115
Research Article
Open Access
SCayyetdoK.tRoamxaidcana*,nEmdanaAn. Et. Eilm-HeilwcranodbHainaaln Aa. cSatlliavmities of some
novel heterocycles employing 6-(1,3-diphenyl-
1H-pyrazol-4-yl)-4-oxo-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carbonitrile
https://doi.org/10.1515/hc-2019-0008
several new pharmacophores containing pyrimidine,
Received May 13, 2019; accepted July 12, 2019.
pyrimidinethione, and thiazolopyrimidine moieties incor-
Abstract:
A
pyrimidinethione derivative namely, porated into the pyrazole nucleus, in an effort to obtain
6-(1,3-diphenyl-1H-pyrazol-4-yl)-4-oxo-2-thioxo-1,2,3,4- compounds with enhanced potency.
tetrahydropyrimidine-5-carbonitrile, was readily synthe-
sized and reacted with carbon electrophiles in an attempt
to synthesize selected fused heterocycles. The reactivity Results and discussion
of 6-(1,3-diphenyl-1H-pyrazol-4-yl)-4-oxo-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carbonitrile was investigated A one-pot cyclo-condensation reaction of 1,3-diphenyl-
towards selected nitrogen nucleophiles. Thiation and hyd- 1H-pyrazole-4-carboxaldehyde (1) with ethyl cyanoacetate
rolysis reactions of the tetrahydropyrimidine derivative and thiourea yielded 6-(1,3-diphenyl-1H-pyrazol-4-yl)-
were investigated. Antitumor and antimicrobial activity 4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile
evaluation of some of the synthesized products exhibited (2) (Scheme 1) [7]. There are two possible tautomeric struc-
promising results.
tures for pyrimidine 2 as depicted in Scheme 1.
The reactions of pyrimidine 2 with selected carbon
Keywords: Antitumor and antimicrobial activity, Carbon electrophiles were studied (see schemes 2 and 3) in an
electrophiles, Tetrahydropyrimidine, Thiation, Thiazolo- attempt to design and synthesize N-heterocycles with
pyrimidine.
pharmacological activity. The reactions of the thioamide
and iminothiol tautomers of pyrimidinethione 2, based on
their thermodynamic and kinetic properties under expe-
rimental conditions are discussed below. The conjugate
base of the iminothiol tautomer has been found to be ther-
Introduction
The chemistry of the pyrazole heterocycles has received modynamically more stable than that of the thioamide
great attention during the last few decades because of tautomer in the presence of a base (basicity is thermody-
their outstanding pharmacological activities e.g. antivi- namically controlled). This is due to back donation invol-
ral, antitumor and antimicrobial [1-7]. Considering all of ving the vacant d-orbital of the sulfur atom. Further, the
these benefits and in continuation of our work to syn- sulfur anion is a stronger nucleophile than the nitrogen
thesize new heterocycles, starting from 1,3-diphenyl-1H- anion (nucleophilicity is kinetically controlled), making
pyrazole-4-carboxaldehyde [1-3], we introduce herein the iminothiol tautomer kinetically more reactive than
the thioamide tautomer. Thus, under the experimental
conditions used, the iminothiol tautomer is more thermo-
dynamically and kinetically favored than the thioamide
* Corresponding author: Sayed K. Ramadan, Chemistry
tautomer.
Department, Faculty of Science, Ain Shams University, Abassia,
Chloroacetyl chloride was reacted with pyrimidi-
Cairo 11566, Egypt, e-mail: sayed.karam2008@sci.asu.edu.eg
nethione 2 in refluxing sodium ethoxide to yield thiazolo-
Eman A. E. El-Helw and Hanan A. Sallam, Chemistry Department,
pyrimidine derivative 3 as pale-yellow crystals (Scheme 2).
The structure of compound 3 was deduced from its
Faculty of Science, Ain Shams University, Abassia, Cairo 11566,
Egypt
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Sayed K. Ramadan et al.: Cytotoxic and antimicrobial activities of some novel heterocycles employing
Scheme 1ꢂSynthesis of pyrazolylpyrimidinethione 2.
analytical and spectral data. The IR spectrum exhibited
the stretching absorption bands of C=O groups of thiazoli-
dinone and pyrimidinone at ν 1731 and 1690 cm–1, respec-
1
tively. The singlet in the H-NMR spectrum at δ 3.58 ppm
Scheme 2ꢂCondensation of pyrimidinethione 2 with carbon
electrophiles.
integrated to two protons, corresponding to methylene
CH2 protons. It is possible the reaction takes place via the
2
tetrahedral mechanism (rather than the SN mechanism),
by attack of the more nucleophilic sulfur atom on the car-
bonyl group of acid chloride followed by cyclization. In
the tetrahedral mechanism, the S-C bond is formed before
the C-Cl bond is broken and a lot of energy is accumula-
ted in the reaction medium which offsets the activation
2
energy and reactivity increase compared with the SN
mechanism.
Chloroacetonitrile reacted with 2 to yield amino-
thiazolopyrimidine derivative 4 (Scheme 2). The spectral
data of compound 4 agreed with the assigned structure.
The S-alkylated product 5 was obtained from condensa-
tion of 2 with 1,2-dichloroethane in refluxing dioxane and
anhydrous sodium acetate (Scheme 2). The IR spectrum
showed the characteristic stretching absorption bands for
1
NH, C≡N and C=O groups. The H-NMR spectrum showed
a singlet signal at δ 11.71 ppm, integrated to one exchan-
geable proton corresponding to a NH proton, as well as
two triplet signals of CH2-CH2 protons (cf. Experimental).
Ethyl cyanoacetate was treated with pyrimidinethione 2 in
refluxing dioxane to yield pyrimidothiazine derivative 6
as yellow crystals (Scheme 2). The reaction could proceed
via nucleophilic attack of the thiol group on the ester car-
Scheme 3ꢂReaction of pyrimidinethione 2 with carbon electrophiles.
bonyl group to eliminate ethanol molecule, followed by derivative 7 (Scheme 3). The spectral data of compound
1,6-exo-dig cyclization.
7 was in accord with the proposed structure. The IR spec-
Treatment of pyrimidine 2 with maleic anhydride trum displayed the characteristic stretching absorption
in refluxing dry toluene produced thiazolopyrimidine bands for OH, C≡N and C=O groups. The formation of
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compound 7 could be interpreted via the ring opening 11 was produced. The structures of compounds 10 and 11
of maleic anhydride due to nucleophilic attack by the were substantiated from their analytical and spectral data.
NH of pyrimidine on the C=O of anhydride, followed by
Thiation of compound 2 was undertaken using phos-
1,5-exo-trig cyclization via intra thia-Michael addition, phorus pentasulfide in refluxing toluene to produce
then finally dehydrogenation. The driving force for dehy- dithioxopyrimidine 12, which was reacted with ethyl chlo-
drogenation of the desired product is to be more thermo- roacetate to yield the di-S-alkylated product 13 (Scheme 5).
dynamically stable via conjugation with the carboxylic The IR spectrum of compound 12 showed the absence
carbonyl group.
of the stretching absorption band of the carbonyl group
Pyrimidinethione 2 was treated with 3’-nitro-w- and displayed the corresponding absorption band of C=S
bromoacetophenone in N,N-dimethylformamide and group. The IR spectrum of compound 13 lacked the NH
anhydrous potassium carbonate and the N-alkylated absorption band and showed the absorption bands cha-
product 8 was obtained (Scheme 3). The IR spectrum racteristic for the ester carbonyl group. The 1H-NMR spec-
displayed two absorption stretching bands for carbonyl trum of compound 13 showed the absence of NH singlets
groups at ν 1735 and 1702 cm–1, as well as the appearance (cf. Experimental). Compelling evidence for structure 13
of a CH2 singlet in its 1H-NMR spectrum. The higher values was derived from the fact that, when the reaction was
of ν C=O may be attributed to mutual induction between carried out in an equimolar ratio, the yield was 43%,
the two carbonyl groups.
however, by utilizing a double molar ratio of ethyl chlo-
Thiazolopyrimidine 9 was obtained when pyrimidi- roacetate, the yield was increased to 75%.
nethione 2 reacted with dimethyl acetylenedicarboxylate
Hydrolysis of 5-cyanopyrimidine 2 in 70% sulfuric acid
in refluxing methanol (Scheme 3). The IR spectrum exhibi- yielded pyrimidine-5-carboxylic acid 14 (Scheme 5). The
ted three absorption bands for C=O groups at ν 1736, 1719 IR spectrum revealed the disappearance of the absorption
and 1695 cm–1. It was assumed that the higher values of ν band of the cyano group and the appearance of the sub-
C=O of the two ester groups may be due to mutual induc- maxima for ν OH of the carboxylic group at 3408 cm–1, as
tion between the two carbonyl groups. The formation of well as the stretching absorption band of carboxylic car-
1
compound 9 could be explained via thia-Michael addi- bonyl group at ν 1709 cm–1. The H-NMR spectrum was in
tion on the C≡C followed by aza-Michael addition to give accord with the assigned structure.
1,5-endo-trig cyclization, then finally dehydrogenation.
This work was extended to investigate the reactivity of
pyrimidinethione 2 towards 1,2-diaminoethane and thiou- Pharmacology
rea (Scheme 4). The, pyrimidine dimer 10 was obtained
from the reaction of compound 2 with 1,2-diaminoethane Antimicrobial activity evaluation
in refluxing ethanol. It seemed that the lactim form is
more stable than the lactam form, due to its aromaticity. Six compounds were screened for their antimicrobial
When thiourea was treated with pyrimidinethione 2 in activity against Gram-positive bacteria (Staphylococcus
refluxing ethanol/acetic acid, the pyrimidine derivative aureus), Gram-negative bacteria (Escherichia coli), and
Scheme 4ꢂReaction of pyrimidine 2 with 1,2-diaminoethane and thiourea.
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Sayed K. Ramadan et al.: Cytotoxic and antimicrobial activities of some novel heterocycles employing
yeast-like pathogenic fungi (Aspergillus Niger and Candida Structure-Activity Relationship for Antimicrobial Activity
albicans). The antimicrobial screening was carried out
using a standard well agar diffusion assay according to Analysis of the previous results concerning the structu-
Cheesbrough [8]. The broad-spectrum antibiotic Amoxi- ral modifications that occurred only at positions-2,3 of
cillin and the antifungal Fluconazole were used at a con- the parent pyrimidine scaffold, revealed that Thiazolo-
centration of 100 μg/ml as positive controls. The results pyrimidine derivative 4, with a free amino group showed
obtained (Table 1 and Figure 1) revealed that of the com- strong inhibitory activity against all tested strains except
pounds tested 4, 8, 12 and 14 exhibited the highest antimi- Aspergillus Niger. Incorporation of 3-nitroacetophenone
crobial activity. The rest of the tested compounds showed moiety at position-3, as in compound 8, produced excel-
moderate to weak antimicrobial activity.
lent activity. Replacement of an oxygen atom with sulfur
as in compound 12 increased the activity [6]. Hydrolysis
of the cyano group as in compound 14 achieved good
activity.
In vitro anticancer activity
Six compounds were investigated for their in vitro cyto-
toxic activity against two human carcinoma cell lines;
namely, colon cancer HCT-116 and mammary gland breast
cancer MCF-7 cell lines, using doxorubicin as a standard
anticancer drug. The anticancer activity was expressed as
IC50 values (the concentration of the test compound requi-
red to kill 50% of the cell population). The results depic-
ted in Table 2 and Figure 2 revealed that the most potent
compounds are 8 and 12 against the tested cell lines. The
rest of compounds exhibited moderate activity.
Structure-activity relationship for anticancer activity
The inhibitory activity of the tested derivatives could be
correlated to structure variation and modifications. By
investigating the variation in selectivity of the highly
potent compounds, it was revealed that the existence of
Scheme 5ꢂThiation and hydrolysis of compound 2.
Table 1ꢀAntibacterial and antifungal activity (as inhibition zone in mm diameter).
Compound
Antibacterial Activity
Antifungal Activity
Staphylococcus aureus (G+)
Escherichia coli (G–)
Candida albicans
Aspergillus Niger
Amoxicillin
28
—
25
—
—
—
22
0
Fluconazole
20
11
17
12
17
16
17
3
4
6
8
12
14
6
8
24
14
20
23
15
22
11
21
19
13
0
9
0
0
0
G: Gram reactionꢁ0.0: no activity (inhibition zone less than 7 mm).
7-10: weak activityꢁ11-15: moderate activity.ꢁMore than 15: strong activity.
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Antimicrobial Activity
30
25
20
15
10
5
0
Staphylococcus
aureus (G+)
Escherichia
Compounds
Candida
albicans
Aspergillus
Amoxicillin
Fluconazole
3
4
6
8
12
14
Figure 1ꢂAntimicrobial activity of the tested compounds.
Table 2ꢀIn vitro cytotoxic activity of the tested compounds against
6-(1,3-diphenyl-1H-pyrazol-4-yl)-4-oxo-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carbonitrile with carbon electro-
philes and nitrogen nucleophiles. Thiation and hydrolysis
of 6-(1,3-diphenyl-1H-pyrazol-4-yl)-4-oxo-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carbonitrile were also studied.
Some of the synthesized compounds exhibited promising
antimicrobial and antitumor activities.
different human cancer cell lines.
Compound
In vitro cytotoxicity IC50 (μM)
HCT-116
MCF-7
doxorubicin
3
4
6
8
12
14
5.23 0.3
30.48 2.4
39.55 2.8
35.07 2.7
13.72 1.3
17.79 1.6
43.06 3.2
4.17 0.2
28.67 2.7
41.28 3.6
30.59 2.8
15.53 1.4
19.58 1.8
49.71 3.9
Experimental
Chemistry
IC50 (μM): 1-10 (very strong); 11-20 (strong); 21-50 (moderate);
51-100 (weak), and > 100 (non-cytotoxic).
All melting points were measured on a Gallenkamp elec-
tric melting point apparatus and are uncorrected. The
the free thioamide group of the parent pyrimidine scaf- FTIR spectra (ν, cm–1) were recorded using potassium
fold led to enhanced activity. Also, the presence of the bromide disks on a Fourier Transform Infrared Thermo
NO2 group in compound 8 as a strong electron attracting Electron Nicolet iS10 Spectrometer (Thermo Fisher Scien-
group rendered the molecule positively charged, forming tific Inc., Waltham, MA, USA) at the Central Laboratory
an electrostatic attraction with the DNA nucleobases [1].
1
of Faculty of Science, Ain Shams University. The H-NMR
spectra (δH, ppm) were run at 400 MHz on a BRUKER
400 BB NMR Spectrometer (BRUKER, Manufacturing &
Engineering Inc., Anaheim, CA, USA) using tetramethyl
silane (TMS) as an internal standard in deuterated dime-
Conclusion
Selected fused heterocycles bearing
a
pyrazole thylsulfoxide (DMSO-d6) at the Faculty of Pharmacy, Ain
scaffold were synthesized via the reaction of Shams University. The mass spectra (MS) were recorded
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Sayed K. Ramadan et al.: Cytotoxic and antimicrobial activities of some novel heterocycles employing
Cytotoxic Activity
80
70
60
50
40
30
20
10
0
HCT-116
MCF-7
14
Compounds
doxorubicin
3
4
6
8
12
Figure 2ꢂCytotoxic activity of the tested compounds on HCT-116 and MCF-7 cell lines.
on a Shimadzu GC-MS-QP-1000 EX mass spectrometer Synthesis of 7-(1,3-diphenyl-1H-pyrazol-4-yl)-2,5-
(Shimadzu Scientific Instruments, Inc., USA) operating at dioxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-
70 eV at The Regional Center for Mycology and Biotech- carbonitrile (3)
nology of Al-Azhar University, Nasr City, Cairo, Egypt.
Elemental analyses were carried out at the Microana- A solution of pyrimidinethione 2 (0.74 g, 2 mmol), chlo-
lytical Unit, Faculty of Science, Ain Shams University, roacetyl chloride (0.22 g, 2 mmol) and sodium ethoxide
using a Perkin-Elmer 2400 CHN elemental analyzer and (0.05 g/10 ml ethanol) in ethanol (20 ml) was heated
satisfactory analytical data ( 0.4) were obtained for all under reflux for 10 h. The reaction mixture was poured
compounds. The reactions were monitored by thin layer onto ice/HCl (10%). The precipitated solid was filtered
chromatography (TLC) using Merck Kiesel gel 60 F254 ana- off, washed with water, dried, and then recrystallized
lytical sheets obtained from Fluka. The pharmacologi- from dioxane to yield thiazolopyrimidine derivative 3
cal activity assays were carried out at the Pharmacology as pale-yellow crystals, mp. 270-272oC, yield 68%. FTIR
Department, Faculty of Pharmacy, Mansoura University, (KBr, ν, cm–1): 2216 (C≡N), 1731 (C=O thiazolidine), 1690
Mansoura, Egypt.
(C=O pyrimidine). 1H-NMR (400 MHz, DMSO-d6) δH, ppm:
9.12 (s, 1H, C5-H pyrazole), 7.98-7.43 (m, 10H, Ar-H), 3.58
(s, 2H, CH2). MS (m/z, %): 411 (M.+, 15). Anal. Calcd. for
C22H13N5O2S (411.08): C, 64.22; H, 3.18; N, 17.02. Found: C,
64.01; H, 3.03; N, 17.05.
6-(1,3-Diphenyl-1H-pyrazol-4-yl)-5-cyano-4-oxo-1,2,3,4-
tetrahydropyrimidine-2-thione (2)
A mixture of pyrazole aldehyde 1 (2.48 g, 0.01 mol), ethyl
cyanoacetate (1.13 g, 0.01 mol), thiourea (0.76 g, 0.01 mol) Synthesis of 3-amino-7-(1,3-diphenyl-1H-pyrazol-4-yl)-
and potassium carbonate (4.14 g, 0.03 mol) in absolute 5-oxo-5H-thiazolo[3,2-a]pyrimidine-6-carbonitrile (4)
ethanol (60 ml) was heated under reflux for 12 h, cooled,
and neutralized with glacial acetic acid. The product was A solution of compound 2 (0.74 g, 2 mmol), chloroace-
crystallized from methanol to give the pyrimidine 2 as yel- tonitrile (0.15 g, 2 mmol) and freshly prepared anhyd-
lowish orange, mp. 196-198oC (Lit. [7] 195-197oC).
rous sodium acetate (0.16 g, 2 mmol) in dioxane (10 ml)
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was heated under reflux for 8 h. After concentration the Synthesis of 2-(6-cyano-7-(1,3-diphenyl-1H-pyrazol-4-
reaction mixture was then cooled and the solid product yl)-3,5-dioxo-5H-thiazolo[3,2-a]pyrimidin-2(3H)-ylidene)
that formed was filtered off and recrystallized from acetic acid (7)
an ethanol/dioxane mixture (2:1) to give compound 4
as brown crystals, mp >360°C, yield 62%. FTIR (KBr, ν, A mixture of 2 (0.74 g, 2 mmol) and maleic anhydride
1
cm–1): 3425, 3351 (NH2), 2216 (C≡N), 1668 (C=O). H-NMR (0.19 g, 2 mmol) in dry toluene (20 ml) was heated under
(400 MHz, DMSO-d6) δH, ppm: 9.10 (s, 1H, C5-H pyra- reflux for 6 h. The precipitated solid was cooled, collec-
zole), 7.99-7.42 (m, 11H, Ar-H + CH), 6.55 (s, 2H, NH2, D2O- ted and recrystallized from ethanol to give thiazolopyrimi-
exchangeable). MS (m/z, %): 410 (M.+, 12). Anal. Calcd. dine derivative 7 as yellow crystals, mp. 252-254oC, yield
for C22H14N6OS (410.09): C, 64.38; H, 3.44; N, 20.48. Found: 69%. FTIR (KBr, ν, cm–1): 3424 (br. OH), 2227 (C≡N), 1670
1
C, 64.09; H, 3.15; N, 20.46.
(C=O). H-NMR (400 MHz, DMSO-d6) δH, ppm: 13.19 (br.s,
1H, COOH, D2O-exchangeable), 9.21 (s, 1H, C5-H pyrazole),
8.07-7.43 (m, 10H, Phenyl), 6.66 (s, 1H, CH=). MS (m/z, %):
467 (M.+, 11). Anal. Calcd. for C24H13N5O4S (467.07): C, 61.67;
H, 2.80; N, 14.98. Found: C, 61.54; H, 2.11; N, 14.91.
Synthesis of 2-((2-chloroethyl)thio)-4-(1,3-diphenyl-
1H-pyrazol-4-yl)-6-oxo-1,6-dihydro-pyrimidine-5-
carbonitrile (5)
A mixture of compound 2 (0.74 g, 2 mmol), 1,2-dichloro- Synthesis of 6-(1,3-diphenyl-1H-pyrazol-4-yl)-3-(2-
ethane (0.20 g, 2 mmol) and freshly prepared sodium (3-nitrophenyl)-2-oxoethyl)-4-oxo-2-thioxo-1,2,3,4-
acetate (0.16 g, 2 mmol) in dioxane (20 ml) was heated tetrahydropyrimidine-5-carbonitrile (8)
under reflux for 10 h. The excess solvent was evaporated
under vacuum. The residue was triturated with water. The A solution of pyrimidinethione 2 (0.74 g, 2 mmol) and
solid was filtered off and recrystallized from benzene to 3’-nitro-w-bromoacetophenone (0.48 g, 2 mmol) in N,N-
give compound 5 as yellow crystals, mp. 150-152oC, yield dimethylformamide (20 ml) containing anhydrous potas-
54%. FTIR (KBr, ν, cm–1): 3435 (NH), 2211 (C≡N), 1660 sium carbonate (0.27 g, 2 mmol) was stirred at room
(C=O). 1H-NMR (400 MHz, DMSO-d6) δH, ppm: 11.71 (s, 1H, temperature for 8 h. The reaction mixture was poured
NH, D2O-exchangeable), 9.07 (s, 1H, C5-H pyrazole), 7.9 8 - onto ice and acidified with dilute HCl (10%). The sepa-
7.30 (m, 10H, Phenyl), 3.25-3.20 (t, 2H, CH2Cl, J= 7.2 Hz), rated solid was collected and recrystallized from an
3.07-3.01 (t, 2H, SCH2, J= 7.2 Hz). Anal. Calcd. for C22H16Cl- ethanol/dioxane mixture (1:1) to give compound 8 as
N5OS (433.08): C, 60.90; H, 3.72; N, 16.14. Found: C, 60.72; beige crystals, mp. 264-266oC, yield 68%. FTIR (KBr, ν,
H, 3.57; N, 16.17.
cm–1): 3143 (NH), 2224 (C≡N), 1735 (C=O acetophenone),
1702 (C=O pyrimidine), 1531, 1354 (NO2), 1234 (C=S).
1H-NMR (400 MHz, DMSO-d6) δH, ppm: 13.77 (s, 1H, NH,
D2O-exchangeable), 9.19 (s, 1H, C5-H pyrazole), 8.09-
7.36 (m, 14H, Ar-H), 4.50 (s, 2H, CH2). Anal. Calcd. for
C28H18N6O4S (534.11): C, 62.91; H, 3.39; N, 15.72. Found: C,
62.70; H, 3.11; N, 15.75.
Synthesis of 8-(1,3-diphenyl-1H-pyrazol-4-yl)-4-imino-
2,6-dioxo-3,4-dihydro-2H,6H-pyrimido[2,1-b][1,3]
thiazine-7-carbonitrile (6)
To a solution of compound 2 (0.74 g, 2 mmol) in dioxane
(20 ml), ethyl cyanoacetate (0.22 g, 2 mmol) was added
and the reaction mixture was heated under reflux for 6 h. Synthesis of dimethyl 6-cyano-7-(1,3-diphenyl-1H-
The reaction mixture was concentrated and then cooled pyrazol-4-yl)-5-oxo-5H-thiazolo[3,2-a]pyrimidine-2,3-
to room temperature. The precipitated solid was collec- dicarboxylate (9)
ted and recrystallized from ethanol to give compound 6
as yellow crystals, mp. 274-276oC, yield 66%. FTIR (KBr, An equimolar mixture of pyrimidine 2 (0.74 g, 2 mmol)
ν, cm–1): 3448 (NH), 2220 (C≡N), 1700 (C=O thiazine), 1670 and dimethyl acetylenedicarboxylate (0.28 g, 2 mmol) in
(C=O pyrimidine). 1H-NMR (400 MHz, DMSO-d6) δH, ppm: methanol (20 ml) was heated under reflux for 4 h. The
13.15 (s, 1H, NH, D2O-exchangeable), 9.20 (s, 1H, C5-H precipitated solid was filtered off, while hot, and rec-
pyrazole), 8.10-7.47 (m, 10H, Phenyl), 3.46 (s, 2H, CH2). rystallized from methanol to give compound 9 as yellow
MS (m/z, %): 438 (M.+, 28). Anal. Calcd. for C23H14N6O2S crystals, mp. 284-286oC, yield 83%. FTIR (KBr, ν, cm–1):
(438.09): C, 63.00; H, 3.22; N, 19.17. Found: C, 62.87; H, 3.11; 2239 (C≡N), 1736, 1719 (C=O two ester), 1695 (C=O pyrimi-
1
N, 19.20.
dine). H-NMR (400 MHz, DMSO-d6) δH, ppm: 9.19 (s, 1H,
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C5-H pyrazole), 8.09-7.37 (m, 10H, Ar-H), 3.73 (s, 6H, 2 CH3). cm–1): 3418 (NH), 2211 (C≡N), 1222 (C=S). MS (m/z, %): 387
Anal. Calcd. for C26H17N5O5S (511.10): C, 61.05; H, 3.35; N, (M.+, 24). Anal. Calcd. for C20H13N5S2 (387.06): C, 62.00; H,
13.69. Found: C, 60.42; H, 3.09; N, 13.76.
3.38; N, 18.07. Found: C, 61.76; H, 3.02; N, 18.00.
Synthesis of 2,2’-(ethane-1,2-diylbis(azanediyl))bis(4-
(1,3-diphenyl-1H-pyrazol-4-yl)-6-hydroxypyrimidine-5-
carbonitrile) (10)
Synthesis of diethyl 2,2’-((5-cyano-6-(1,3-diphenyl-
1H-pyrazol-4-yl)pyrimidine-2,4-diyl)bis(sulfanediyl))
diacetate (13)
To a solution of pyrimidine 2 (0.74 g, 2 mmol) in absolute A solution of dithioxopyrimidine derivative 12 (0.77 g,
ethanol (20 ml), 1,2-diaminoethane (0.12 g, 2 mmol) was 2 mmol), ethyl chloroacetate (0.24 g, 2 mmol or 0.49 g,
added and the reaction mixture was heated under reflux 4 mmol) and anhydrous sodium acetate (0.16 g, 2 mmol
for 4 h. The obtained precipitate was collected, while hot, or 0.33 g, 4 mmol) in absolute ethanol (20 ml) was heated
and recrystallized from dioxane to give compound 10 as under reflux for 2 h. The reaction mixture was cooled to
white crystals, mp. >360oC, yield 61%. FTIR (KBr, ν, cm–1): room temperature. The precipitated solid was collected
1
3422 (br. OH, NH), 2211 (C≡N). H-NMR (400 MHz, DMSO- and recrystallized from ethanol to give compound 13 as
d6) δH, ppm: 11.30 (s, 2H, OH, D2O-exchangeable), 9.04 (s, brown crystals, mp. 102-104oC, yield 43% [1:1 mol], 75% [1:2
1
2H, C5-H pyrazole), 8.41 (s, 2H, NH, D2O-exchangeable), mol]. FTIR (KBr, ν, cm–1): 2210 (C≡N), 1733 (C=O). H-NMR
7.9 1-7.44 (m, 20H, Ar-H), 3.11 (s, 4H, CH2). Anal. Calcd. for (400 MHz, DMSO-d6) δH, ppm: 9.10 (s, 1H, C5-H pyrazole),
C42H30N12O2 (734.26): C, 68.65; H, 4.12; N, 22.88. Found: C, 7.9 8 -7.41 (m, 10H, Ar-H), 4.40 (s, 4H, 2 S-CH2), 4.21-3.92 (q,
68.32; H, 3.87; N, 22.84.
4H, 2 CH2CH3, J= 6.8 Hz), 1.21-1.42 (t, 6H, 2 CH2CH3, J= 6.8
Hz). Anal. Calcd. for C24H19N5O2S2 (473.10): C, 60.87; H,
4.04; N, 14.79. Found: C, 60.45; H, 3.72; N, 14.76.
Synthesis of 1-(5-Cyano-4-(1,3-diphenyl-1H-pyrazol-4-
yl)-6-oxo-1,6-dihydropyrimidin-2-yl)thiourea (11)
Synthesis of 6-(1,3-diphenyl-1H-pyrazol-4-yl)-4-oxo-
A solution of compound 2 (0.74 g, 2 mmol) and thiourea 2-thioxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylic
(0.15 g, 2 mmol) in absolute ethanol (20 ml) was heated acid (14)
under reflux for 5 h in the presence of 3-drops of glacial
acetic acid. The reaction mixture was concentrated and A solution of pyrimidine 2 (0.74 g, 2 mmol) in sulfuric acid
the separated solid was filtered off, after cooling and rec- 70% (10 ml) was heated under reflux for 3 h. The reaction
rystallized from a benzene/ethanol mixture (1:1) to give mixture was allowed to cool and poured onto ice, then
compound 11 as yellow crystals, mp. 258-260oC, yield neutralized by NH4OH. The precipitate was then filtered
49%. FTIR (KBr, ν, cm–1): 3473, 3422 (NH, NH2), 2214 (C≡N), off, dried and recrystallized from ethanol to give the cor-
1
1696 (C=O), 1233 (C=S). H-NMR (400 MHz, DMSO-d6) δH, responding acid derivative 14 as grey powder, mp. >360oC,
ppm: 11.01 (s, 2H, 2 NH, D2O-exchangeable), 9.14 (s, 1H, yield 57%. FTIR (KBr, ν, cm–1): 3408 (br. OH), 1709 (C=O
1
C5-H pyrazole), 7.08 (s, 2H, NH2, D2O-exchangeable), 8.01- acid), 1655 (C=O pyrimidine), 1225 (C=S). H-NMR (400
7.46 (m, 10H, Ar-H). MS (m/z, %): 413 (M.+, 17). Anal. Calcd. MHz, DMSO-d6) δH, ppm: 13.02 (s, 2H, COOH and NHCS,
for C21H15N7OS (413.11): C, 61.01; H, 3.66; N, 23.71. Found: C, D2O-exchangeable), 11.20 (s, 1H, NHCO, D2O-exchange-
60.74; H, 3.19; N, 23.76.
able), 9.11 (s, 1H, C5-H pyrazole), 7.99-7.42 (m, 10H, Ar-H).
MS (m/z, %): 392 (M.+, 32). Anal. Calcd. for C20H16N4O3S
(392.09): C, 61.21; H, 4.11; N, 14.28. Found: C, 60.92; H, 3.89;
N, 14.31.
Synthesis of 6-(1,3-diphenyl-1H-pyrazol-4-yl)-2,4-
dithioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (12)
A suspension of compound 2 (0.74 g, 2 mmol) and phos- Biological evaluation
phorus pentasulfide (0.22 g, 1 mmol) in dry toluene (20
ml) was heated under reflux for 2 h. The precipitated solid Antimicrobial assay
was collected while hot and recrystallized from ethanol to
yield the corresponding dithioxopyrimidine derivative 12 The antimicrobial activities of the synthesized com-
as brown crystals, mp. 160-162oC, yield 74%. FTIR (KBr, ν, pounds were examined against two bacterial strains,
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Sayed K. Ramadan et al.: Cytotoxic and antimicrobial activities of some novel heterocycles employingꢁ
ꢁ115
Staphylococcus aureus and Escherichia coli, and two References
fungal strains, Aspergillus Niger and Candida albicans,
using a standard well agar diffusion assay [8]. Plates con-
taining nutrient agar medium and sabouraud dextrose
agar medium (for bacteria and fungi, respectively) were
surface inoculated with 106 CFU/ml of freshly prepared
microorganisms. Using a 6-mm sterile cork borer wells
were punched in the agar and filled separately with 100 μl
of the tested compounds (100 μg/ml in DMSO). The plates
were left in a refrigerator for 2 h to allow diffusion of the
tested compounds. After that, the plates were incubated
for 24 h at 37°C for bacteria and for 72 h at 28°C for fungi
Following incubation, the inhibition zones surround-
ing the wells were measured in millimeters. Amoxicillin
and Fluconazole were used as the standard against bac-
teria and fungi, respectively, at the same concentration
(100 μg/ml).
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Cytotoxicity Assay
Materials and methods
Two cell lines were utilized for this work: colorectal
carcinoma (colon) HCT-116 and mammary gland breast
cancer MCF-7. The cell lines were obtained from ATCC
via a holding company for biological products and vac-
cines (VACSERA), Cairo, Egypt. Doxorubicin was used
as a standard anticancer drug for comparison, accor-
ding to a previously reported MTT method [9]. Chemical
reagents: The reagents RPMI-1640 medium, MTT, and
DMSO (Sigma Co., St. Louis, USA), fetal bovine serum
(GIBCO, UK).
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