Synthesis of novel thiazol-5-ylpyrimidine derivatives and their antimicrobial evaluation

Article abstract of DOI:10.1002/jhet.4116

Novel thiazol-5-ylpyrimidine derivatives were designed and synthesized. The chemical structures of all new synthesized compounds were assigned by studying their elemental analyses and spectral data (FT-IR, ¹HNMR, ¹³C NMR, and MS). Th

Full text of DOI:10.1002/jhet.4116

Synthesis of Novel Thiazol-5-ylpyrimidine Derivatives and Their
Antimicrobial Evaluation
1Sanjay U. Deshmukh,1 Raghunath B. Toche,2* Sushama J. Takate,3 Supriya P. Salve,3
Ram W. Sabnis4
1Department of Chemistry, KRT Arts, BH Commerce & AM Science College, Nashik 422002,
India
2†Dadasheb Bidkar Arts, Science & Commerce College, Peth, Nashik422208, India
†Affiliated to SPP University, Pune, India
3Department of Chemistry, New Art, Commerce and Science College, Ahmednagar 414001
India
4Georgia-Pacific LLC, 133 Peachtree Street NE, Atlanta, GA 30303, USA
Correspondence
Raghunath B. Toche, Dadasheb Bidkar Arts, Science & Commerce College, Peth,
Nashik422208, India, Email: raghunath_toche@rediffmail.com
ABSTRACT
Novel thiazol-5-ylpyrimidine derivatives were designed and synthesized. The chemical
structures of all new synthesized compounds were assigned by studying their elemental analyses
and spectral data (FT-IR, 1HNMR, 13C NMR and MS). The target compounds, 8 and 9a-9d were
evaluated for their antimicrobial activity in vitro against Gram-positive bacteria, Bacillus subtilis
and Staphylococcus aureus, Gram-negative bacteria, Salmonella abony and Escherichia Coli and
fungi, Aspergillus flavus and Fusarium oxysporum. In particular, compounds 9a-9c exhibited
moderate to good activity against Gram-positive bacteria, Staphylococcus aureus, Gram-negative
bacteria, Salmonella abony and fungus, Fusarium oxysporum in comparison with reference
drugs.
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Keywords: Thiazolylpyrimidines, Thiazoles, Pyrimidines, Oxopropanenitrile, Antimicrobial,
Antibacterial, Antifungal activity.
1 INTRODUCTION
The sulfur containing heterocyclic compounds, thiazoles exhibited key role in medicinal
chemistry and subsequently materialised as a pharmacophore. The thiazoles moiety have
attracted a great interest due to their ready accessibility, varied chemical reactivity, and wide
range of biological activities like anticancer agents,[1] Antidiabetic agents,[2] antitubercular
agents,[2] anti-Alzheimer’s agents,[3] antifungal agents,[4] antiviral agents,[4] and antimicrobial
agents.[4]
Pyrimidines are being building blocks of nucleic acids such as DNA and RNA, which
make life possible. DNA damaged by exogenous physical and chemical agents is the most
common cause of cancer. Conversely, some of the commonly anticancer drugs can kill malignant
cell by damaging their DNA. Number of substituted pyrimidine derivatives are reported in
literature having anticancer activity. Broprimine,[5] the 2-amino-5-bromo-6-phenylpyrimidin-
4(3H)-one, is a well-known antiviral and antineoplastic agent.
Recently, thiazolylpyrimidines were extensively studied because of their magnificent
pharmacological and therapeutic properties, such as cyclin dependent kinase (CDK)
inhibitors,[6,10] Jak2 inhibitors,[7] spleen tyrosine kinase (SYK) inhibitors,[8] and their promising
antibacterial,[9] antitumor,[10] and antihyperglycemic activities.[11] Further, cancer cells often have
a high demand for antiapoptic proteins in order to resist programmed cell death. CDK9 inhibition
selectively targets survival proteins and reinstates apoptosis in cancer cells. Thiazol-5-
ylpyrimidine derivative I inhibits CDK9 with IC50 = 7 nM and shows over 80-fold selectivity for
CDK9 over CDK2. X-ray crystal structure of compound I bound to CDK9 and CDK2 provide
2
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insights into the binding modes. Compound I demonstrates potent anticancer activity against
primary chronic lymphocytic leukemia cells with a therapeutic window 31- and 107-fold over
those of normal B- and T-cells.[10]
I
Inspired by the above-mentioned potential biological activities of thiazolylpyrimidines,
and our ongoing work aims for the synthesis of new molecules of pharmacological interest.[12-16]
In this paper, we disclosed an efficient method towards the synthesis of new series of
thiazolylpyrimidine derivatives and were screened for their antimicrobial activity.
2 RESULTS AND DISCUSSION
2.1 Chemistry
The synthetic method has been described in two reaction schemes to synthesize the key
intermediate compound 6 (Scheme 1) and final thiazolylpyrimidines 9a-9d (Scheme 2). The
entire reaction sequence for synthesizing key intermediate compound 6 from compound 1 is
provided in Scheme 1. The first starting compound, ethyl 2-amino-4-methylthiazole-5-
carboxylate[17] 2 was prepared in excellent yield (84%) via heating 2-chloro ethylacetoacetate 1
with thiourea in ethanol. The structure of compound 2 was established by studying elemental
1
analysis, FTIR, H NMR and mass spectral analyses. IR spectrum showed absorption bands at
3373.24 cm-1 and 1673.46 cm-1 due to NH2 group and CO group, respectively. 1H NMR spectrum
3
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exhibited signals at δ 7.70 ppm and δ 2.37 ppm for NH2 and CH3 protons of thiazole 2,
respectively`.
Ethyl 2-iodo-4-methylthiazole-5-carboxylate[18] 3 was synthesized from ethyl 2-amino-4-
methylthiazole-5-carboxylate 2 via Sandmeyer reaction, by performing diazotization of ethyl 2-
amino-4-methylthiazole-5-carboxylate with sodium nitrite/acid at 0°C and coupled with
potassium iodide at 0°C in water followed by continuous stirring for 24 h at room temperature.
Structure of compound 3 was elucidated using elemental analysis data, FTIR, 1H NMR and mass
spectral analyses. The IR spectrum showed stretching frequency at 1711 cm-1 because of CO
group and showed absence of band for NH2 group. 1H NMR spectrum exhibited singlet at δ 2.62
ppm due to CH3 protons and disappearance of singlet, which corresponds to NH2 of thiazole 2.
Ethyl 2-(4-chlorophenyl)-4-methylthiazole-5-carboxylate[19] 5 was prepared from ethyl 2-
iodo-4-methylthiazole-5-carboxylate
3
by Suzuki coupling reaction. Ethyl-2-iodo-4-
methylthiazole-5-carboxylate 3 and (4-chlorophenyl)boronic acid 4 and Pd(PPh3)4 in THF under
nitrogen atmosphere afforded ethyl 2-(4-chlorophenyl)-4-methylthiazole-5-carboxylate 5.
1
Structure 5 was elucidated by analysing elemental analysis, FT-IR, H NMR and mass spectral
analyses.
The key intermediate, 3-(2-(4-chlorophenyl)-4-methylthiazol-5-yl)-3-oxopropanenitrile 6
was synthesized by condensing 2-(4-chlorophenyl)-4-methylthiazole-5-carboxylate 5 with
acetonitrile in THF using n-BuLi under nitrogen atmosphere below -10°C. Structure 6 was
1
illustrated using elemental analysis data, FT-IR, H NMR and mass spectral analyses. The IR
spectrum showed stretching band at 2263 cm-1 and 1676 cm-1 for CN and CO. The ester carbonyl
stretching frequency of compound 5 at 1709 cm-1 has now shifted in compound 6 at 1676 cm-1.
1
Further, there is prominent stretching frequency at 2263 cm-1due to nitrile group. H NMR
4
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spectrum exhibited distinct singlet at δ 4.68 ppm corresponding to two CH2 protons of compound
6.
Scheme 1: Synthesis compound 6 as key intermediate.
The entire reaction sequence for synthesizing compounds 9a-9d from key intermediate
compound 6 is provided in Scheme 2. Compound, 2-(2-(4-chlorophenyl)-4-methylthiazole-5-
carbonyl)-3-(dimethylamino) acrylonitrile 7 was prepared in excellent yield (92%) via heating 3-
(2-(4-chlorophenyl)-4-methylthiazol-5-yl)-3-oxopropanenitrile 6 with dimethylformamide-
1
dimethylacetal (DMF-DMA). Structure 7 was established by elemental analysis data, FT-IR, H
13
NMR, C NMR and mass spectral analyses. IR spectrum showed stretching at 1633 cm-1 for
1
C=C, enamine group. H NMR spectrum exhibited singlet at δ 3.528 ppm and δ 3.558 ppm
corresponding to six protons of 2N-CH3 and no singlet corresponding to CH2CN of thiazole 7.
Compound,
2-amino-4-(2-(4-chlorophenyl)-4-methylthiazol-5-yl)pyrimidine-5-
carbonitrile 8 was synthesized in high yield (87%) via heating 2-(2-(4-chlorophenyl)-4-
methylthiazole-5-carbonyl)-3-(dimethylamino) acrylonitrile 7 in ethanol with guanidine and
5
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anhydrous potassium carbonate. The Structure of the compound 8 was elucidated by elemental
1
analysis data, FT-IR, H NMR, 13C NMR and Mass spectral analyses. IR spectrum showed
stretching at 3402 cm-1 and 3177 cm-1 for the presence of NH2 group and absence of stretching
1
due to C=C, enamine group. H NMR spectrum exhibited singlet at δ 8.782 ppm and δ 7.970
ppm corresponding to pyrimidine-H and NH2 protons, respectively and no proton signals
corresponding to six 2N-CH3 protons in compound 8.
The compounds, amide analogs of 2-amino-4-(2-(4-chlorophenyl)-4-methylthiazol-5-
yl)pyrimidine-5-carbonitrile 9a-9d were prepared in high yields (70-77%) via heating 2-amino-
4-(2-(4-chlorophenyl)-4-methylthiazol-5-yl)pyrimidine-5-carbonitrile 8 in THF, triethylamine
and 4-dimethylaminopyridine with corresponding acid chlorides. Structures 9a-9d were
illustrated by elemental analysis as well as FT-IR, 1H NMR and mass spectral analyses.
9a:R = 3-F-Ph
9b: R = 4-F-Ph
9c: R = cyclohexyl
6
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9d: R = isobutyl
Scheme 2: General synthetic route of the target compounds 9a-9d
2.2 Biology
2.2.1 Antimicrobial activity
The newly synthesized compounds, 8 and 9a-9d were screened to determine their antimicrobial
activity in vitro against two pathogenic Gram-positive bacteria viz. Bacillus subtilis and
Staphylococcus aureus, two pathogenic Gram-negative bacteria viz Salmonella abony and
Escherichia Coli and fungal culture of Aspergillus flavus and Fusarium oxysporum. The drugs
used as reference were Ciprofloxacin for antibacterial and Luliconazole for antifungal tests,
correspondingly.
2.2.2 Antibacterial assay
The antibacterial activity of the synthesized compounds was studied against two Gram-positive
strains Bacillus subtilis and Staphylococcus aureus, two Gram-negative strains Salmonella abony
and Escherichia Coliusing Ciprofloxacin as standard by employing the Agar diffusion method.
The media prepared using solid culture such as 10 g peptone, 10 g sodium chloride, 5 g yeast
extract and 20 g agar were dissolved in 1000 ml of distilled water. The stock solution of culture
was inoculated in broth media and grown at 37°C for 18 hours and was revived. This stock
solution was added in the petridishes and bores were made in the plate. For 18 hours old cultures
were (100 μL 10−4 cfu) inoculated for each plate and spread consistently on the plates. The bores
were filled after 20 minutes containing different concentrations of samples and antibiotic. After
that at 37°C all the dishes were incubated and zone of inhibition in diameter (mm) noted after 24
hours.[20,21]
7
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2.2.3 Antifungal assay
The antifungal activity of the synthesized compounds was studied against two fungal strains
Aspergillus flavus and Fusarium oxysporumusing Luliconazole by using Agar diffusion method.
The media Czapek-Dox Agar was prepared using solid culture composition of 30 g sucrose, 2 g
sodium nitrite, 1 g K2HPO4, 0.5 g MgSO4.7H2O, 0.5 g KCl, 0.01 g FeSO4, and 20 g agar
dissolved in 1000 mL of distilled water. This stock culture was inoculated in broth media and
grown at 27°C for 48 hours and revived. This stock solution was poured in the petri plate and
wells were made in each petri plate. For 48 hours old cultures (100 μL 104 CFU) inoculated for
each plate and spread consistently on the plate. The wells were filled after 20 minutes containing
different concentrations of samples and antibiotic. After that at 27°C all the plates incubated and
zone of inhibition noted in diameter (mm) after 96 hours.[20,21]
It was observed that the compound 8 exhibited moderate antibacterial activity against
Gram-negative S. abony of inhibition zone 13 mm/mg vs 40 mm/mg as compared to reference
drug Ciprofloxacin. The antifungal activity of compound 8 against fungi F. oxysporum was
comparable inhibition zone 12 mm/mg when compared to that of the reference drug
Luliconazole 17 mm/mg.
Further, the compound 9a exhibited good to moderate antibacterial activity against Gram-
positive S. aureus and Gram-negative S. abony of inhibition zones 14, 11 mm/mg vs 23, 40
mm/mg as compared to reference drug Ciprofloxacin, while its antifungal activity against fungi
F. oxysporum was good producing inhibition zone 13 mm/mg when compared to that of the
reference drug Luliconazole 17 mm/mg.
Also, the compound 9b exhibited good to moderate antibacterial activity against Gram-
positive S. aureus and Gram-negative S. abony of inhibition zones 15, 10 mm/mg vs 23, 40
8
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mm/mg of the reference drug Ciprofloxacin, while its antifungal activity against fungi F.
oxysporum was potent producing inhibition zone 11 mm/mg when compared to that of the
reference drug Luliconazole 17 mm/mg.
Further, the compound 9c exhibited good to moderate antibacterial activity against Gram-
positive S. aureus and Gram-negative S. abony of inhibition zones 14, 12 mm/mg vs 23, 40
mm/mg of the reference drug Ciprofloxacin, while its antifungal activity against fungi F.
oxysporum was potent producing inhibition zone 11 mm/mg when compared to that of the
reference drug Luliconazole 17 mm/mg.
Furthermore, the compound 9d exhibited good antibacterial activity against Gram-
positive S. aureus of inhibition zone 14 mm/mg vs 23 mm/mg of the reference drug
Ciprofloxacin, while it represented weak to complete loss of antifungal potency. Additional
modification and optimization are needed to get new candidates of more significant antimicrobial
activity against various types of bacteria and fungi.
TABLE 1 Antimicrobial activity of the samples against Gram-positive bacteria, Gram-negative
bacteria, and Fungi
Antibacterial activity
Antifungal activity
Sample
B. subtilis S. aureus S. abony E. coli A. flavus F. oxysporum
-
-
14
13
11
10
12
-
-
-
12
13
11
11
-
8
-
-
-
9a
-
-
15
-
-
-
-
9b
9c
14
-
14
-
-
9d
Ciprofloxacin
Luliconazole
30
N/A
23
40
N/A
26
N/A
N/A
23
N/A
17
N/A
9
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3 MATERIALS AND METHODS
The chemicals and solvents used were dried and purified by using standard literature procedures
and moisture was removed from the glass apparatus using CaCl2 drying tubes. The melting
points were determined in open capillary tubes with Gallenkamp melting point apparatus and are
uncorrected. FT-IR spectra were recorded on Bruker FTIR-TENSOR II spectrophotometer using
1
13
Platinum ATR discs. H NMR spectra and C NMR spectra were recorded on Varian Mercury
300 NMR spectrophotometer at 300 MHz frequency and Bruker 400 NMR spectrophotometer at
400 MHz and 100 MHz frequency in CDCl3 or dimethyl sulfoxide (DMSO-d6) using
tetramethylsilane (TMS) as internal standard. Chemical shifts were recorded in δ ppm and
multiplicities are given as s (singlet), bs (broad singlet), d (doublet), t (triplet), q (quartet), or m
(multiplet). Mass spectra were recorded on a Shimadzu LC-MS QP 2020A mass spectrometer
with an ionization potential of 70 eV. Elemental analyses were performed on Thermo Quest
Flash 1112 Series EA analyzer. Reactions were monitored by thin layer chromatography, carried
out on 0.2 mm silica gel 60 F254 (Merck) plates using UV light (254 and 366 nm) for detection.
Regular reagent grade chemicals were commercially available and used without further
purification or prepared by standard literature procedures. All the compounds were prepared by
conventional methods.
4. EXPERIMENTAL
4.1 Synthesis of ethyl 2-amino-4-methylthiazole-5-carboxylate (2)
A mixture of 2-chloro ethylacetoacetate (20g, 0.125mol), thiourea (11.09g, 0.145mol) in EtOH
(100mL) was reflux for 24 h (monitored by TLC). The solvent was evaporated under reduced
pressure and the residue was stirred in ice-water mixture. The resultant precipitate was subjected
10
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to filtration, dried and recrystallized from ethanol as white solid, yield 84%; mp 172-175°C. IR
(KBr, cm-1): ῡ = 3373.24 (NH2), 1673.46 (C=O, ester); 1H NMR (300 MHz, DMSO-d6, δ, ppm):
7.70 (bs, 2H, NH2), 4.17-4.10 (q, J = 7.1Hz, 2H, CH2), 2.37 (s, 3H, CH3), 1.24-1.19 (t, J = 7.1Hz,
3H, CH3); MS m/z (%): 185.43 (M-1), 186.05 (100.0), 187.05 (8.6), 188.04 (4.5); Anal. Calcd.
(%) for C7H10N2O2S: C, 45.15; H, 5.41; N, 15.04. Found: C, 45.37; H, 5.48; N, 15.12.
4.2 Synthesis of ethyl 2-iodo-4-methylthiazole-5-carboxylate (3)
To a solution of ethyl 2-amino-4-methylthiazole-5-carboxylate (10 g, 0.05 mol) in acetonitrile
(100 mL) was added p-toluenesulfonic acid monohydrate (20.4 g, 0.107 mol) and the reaction
mixture was stirred at room temperature for 4 h. The mixture was cooled to 0°C, followed by
addition of aqueous solution of NaNO2 (5.5 g, 0.080 mol) and KI (13.36 g, 0.080 mol) in water
(25 mL), maintaining temperature below 0°C. The reaction mixture was further stirred for 24 h at
room temperature followed by addition of aqueous sodium meta bisulphate and was extracted
with ethyl acetate (5 x 100 mL). The combined organic layers were dried over anhydrous
Na2SO4, the solvent evaporated in vacuum, dried and recrystallized from ethanol as pale yellow
1
solid, yield 72%; mp 175-177°C. IR (KBr, cm-1): ῡ =1711 (C=O); H NMR (300 MHz, DMSO-
d6, δ, ppm): 4.29-4.22 (q, J = 7.1 Hz, 2H, CH2) 2.62 (s, 3H, CH3), 1.29-1.24 (t, J = 7.1 Hz, 3H,
CH3); MS m/z (%): 297 (M+), 296.93 (100.0), 297.94 (7.7), 298.93 (4.6), 297.93 (1.2); Anal.
Calcd. (%) for C7H8INO2S: C, 28.30; H, 2.71; N, 4.71. Found: C, 28.53; H, 2.77; N, 4.79.
4.3 Synthesis of ethyl 2-(4-chlorophenyl)-4-methylthiazole-5-carboxylate (5)
To a solution of ethyl-2-iodo-4-methylthiazole-5-carboxylate(9.3g, 0.03 mol) and (4-
chlorophenyl)boronic acid(5.87 g, 0.037 mol) in dry THF (50 mL), was added Pd(PPh3)4 (903
mg, 25 mol%) under nitrogen atmosphere and the mixture was stirred at room temperature for
30 min. Aqueous Na2CO3 (10%) solution was added to the mixture and was heated under reflux
11
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for 2 days. The THF was evaporated under reduced pressure, the residue was stirred in ice-water
mixture and was extracted with ethyl acetate (5 x 100 mL). The combined organic layers were
dried over anhydrous Na2SO4 and the solvent evaporated in vacuum. The crude compound was
purified by silica gel column chromatography, using 2% ethyl acetate-petroleum ether mixture as
eluent afforded as white solid, yield 70%; mp 165-167°C. IR (KBr, cm-1): ῡ = 2988(CH),
1709(C=O), 1522(Ar-C=C); 1H NMR (300 MHz, DMSO-d6, δ, ppm): 8.02-8.00 (d, J = 9 Hz, 2H,
Ar-H), 7.60-7.57 (d, J = 9 Hz, 2H, Ar-H), 4.31-4.29 (q, J = 6.9 Hz, 2H, CH2), 2.69(s, 3H, CH3),
1.33-1.30 (t, J = 6.9 Hz, 3H, CH3); MS m/z (%): 282.53 (M+H)+, 281.03 (100.0), 283.02 (36.5),
282.03 (15.1), 284.03 (5.2), 283.03 (1.5), 285.02 (1.5); Anal. Calcd. (%) for C13H12ClNO2S: C,
55.42; H, 4.29; N, 4.97. Found: C, 55.47; H, 4.31; N, 4.98.
4.4 Synthesis of 3-(2-(4-chlorophenyl)-4-methylthiazol-5-yl)-3-oxopropanenitrile (6)
A solution of ethyl 2-(4-chlorophenyl)-4-methylthiazole-5-carboxylate(4.6 g, 0.014 mol) and
acetonitrile (1.10 mL, 0.021 mol) in dry THF (25 mL) was stirred under nitrogen atmosphere
below -10°C. To this solution, n-BuLi (9.65 mL, 1.6 molar solution in hexane, 0.0154 mol) was
slowly added maintaining the temperature below -5°C. The reaction mixture was stirred for 30
min followed by addition of 2N HCl (10 mL) below 0°C and was extracted with ethyl acetate (2
x 200 mL). The combined organic layers were dried over anhydrous Na2SO4, the solvent
evaporated in vacuum, dried and recrystallized from methanol as pale yellow solid, yield 84%;
1
mp 151-153°C. IR (KBr, cm-1): ῡ = 3444 (CH), 2263 (CN), 1676 (C=O); H NMR (300 MHz,
DMSO-d6, δ, ppm): 8.04-8.02 (d, J = 9 Hz, 2H, Ar-H ), 7.63-7.60 (d, J = 9 Hz, 2H, Ar-H), 4.68
13
(s, 2H, CH2CN), 2.71 (s, 3H, CH3); C NMR (DMSO-d6, δ, ppm): 182.93, 168.89, 160.29,
136.90, 131.10, 130.00, 128.84, 115.57, 33.38, 18.71; MS m/z (%): 277.41 (M+1)+, 276.01
(100.0), 278.01 (36.6), 277.02 (14.2), 279.01 (5.7), 280.01 (1.7), 277.01 (1.5), 278.02 (1.3);
12
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Anal. Calcd. (%) for C13H9ClN2OS: C, 56.42; H, 3.28; N, 10.12. Found: C, 56.67; H, 3.12; N,
10.22.
4.5 Synthesis of 2-(2-(4-chlorophenyl)-4-methylthiazole-5-carbonyl)-3-
(dimethylamino)acrylonitrile(7)
A mixture of 3-(2-(4-chlorophenyl)-4-methylthiazol-5-yl)-3-oxopropanenitrile (1g, 0.0036 mol)
and dimethylformamide-dimethylacetal (DMF-DMA) (5mL) was heated under reflux for 2 h
(monitored by TLC). The DMF-DMA was evaporated under reduced pressure and the crude
product was washed with n-pentane gave as yellow solid, yield 92%; mp 125-127°C. IR (KBr,
1
cm-1): ῡ = 2191 (CN), 1698 (C=O, ketone), 1633 (C=C, enamine), 1592, 1571 (Ar-C=C); H
NMR (400 MHz, CDCl3, δ, ppm): 7.98 (s, 1H, olefin-H), 7.94-7.92 (d, J = 8.8 Hz, 2H, p-Cl-Ph),
7.44-7.42 (d, J = 8.8 Hz, 2H, p-Cl-Ph), 3.52 (s, 3H, N-CH3), 3.55 (s, 3H, N-CH3), 2.72 (s, 3H,
CH3); MS m/z: 332.40 (M+1)+; Anal. Calcd. (%) for C16H14ClN3OS: C, 57.91; H, 4.25; N, 12.66.
Found: C, 57.75; H, 4.18; N, 12.61.
4.6 Synthesis of 2-amino-4-(2-(4-chlorophenyl)-4-methylthiazol-5-yl)pyrimidine-5-
carbonitrile (8)
To
a
solution of 2-(2-(4-chlorophenyl)-4-methylthiazole-5-carbonyl)-3-(dimethylamino)
acrylonitrile (1g, 0.003 mol) in ethanol (10 mL), was added guanidine (0.214 g, 0.0036 mol) and
anhy. K2CO3 (1.2 g, 0.009 mol) and was heated under reflux for 6 h (monitored by TLC). The
ethanol was evaporated under reduced pressure, the residue was stirred in ice-water mixture and
acidified with 2N dil. HCl. The resultant precipitate was subjected to filtration and recrystallized
from ethanol as brown solid, yield 87%; mp 253-255°C. IR (KBr, cm-1): ῡ = 3402, 3177 (NH2,
amine), 2215 (CN), 1658, 1579 (Ar-C=C); 1H NMR (400 MHz, DMSO-d6, δ, ppm): 8.78 (s, 1H,
pyrimidine-H), 8.02-8.00 (d, 2H, J = 8.8 Hz, p-Cl-Ph), 7.97 (bs, 2H, NH2), 7.62-7.60 (d, J = 8.8
13
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13
Hz, 2H, p-Cl-Ph), 2.58 (s, 3H, CH3); C NMR (DMSO-d6, δ, ppm): 166.57, 164.257, 163.392,
161.657, 155.058, 136.101, 131.518, 129.929, 128.542, 128.165, 117.762, 94.545; MS m/z:
328.28 (M+1)+; Anal. Calcd. (%) for C15H10ClN5S: C, 54.96; H, 3.07; N, 21.37. Found: C, 54.91;
H, 3.09; N, 21.32.
4.7 General Procedure for synthesis of amide analogs of 2-amino-4-(2-(4-chlorophenyl)-4-
methylthiazol-5-yl) pyrimidine-5-carbonitrile (9a-9d)
To a solution of 2-amino-4-(2-(4-chlorophenyl)-4-methylthiazol-5-yl)pyrimidine-5-carbonitrile
(0.1 g, 0.0003 mol) in THF (5 mL), was added triethylamine (0.3 mL), catalytic amount of 4-
dimethylaminopyridine (0.036 g, 0.00003 mmol) and acid chloride(0.057 g, 00036 mol) and was
heated under reflux for 6 h (monitored by TLC). The reaction mixture was stirred in ice-water
mixture and was extracted with ethyl acetate (2 x 10 mL). The combined organic layers were
dried over anhydrous Na2SO4 and the solvent evaporated in vacuum. The crude compound was
washed by diethyl ether and n-pentane gave pure solids, yields 70-77%.
4.7.1 N-(4-(2-(4-chlorophenyl)-4-methylthiazol-5-yl)-5-cyanopyrimidin-2-yl)-3-fluoro-
benzamide (9a)
Yellow solid, yield 70%; mp 209-211°C. IR (KBr, cm-1): ῡ = 3494 (NH, amide), 2227 (CN),
1
1716 (C=O, amide), 1602 (Ar-C=C); H NMR (400 MHz, DMSO-d6, δ, ppm): 11.74 (s, 1H,
CONH), 9.20 (s, 1H, pyrimidine-H), 8.06-8.04 (m, 4H, p-F-Ph), 7.81-7.79 (d, J = 8 Hz, 2H, p-
Cl-Ph), 7.78-7.49 (d, J = 8 Hz, 2H, p-Cl-Ph), 2.76 (s, 3H, CH3); MS m/z: 450.41(M+1)+; Anal.
Calcd. (%) for C22H13ClFN5OS: C, 58.73; H, 2.91; N, 15.57. Found: C, 58.76; H, 2.95; N, 15.56.
4.7.2 N-(4-(2-(4-chlorophenyl)-4-methylthiazol-5-yl)-5-cyanopyrimidin-2-yl)-4-fluoro-
benzamide (9b)
14
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Off white solid, yield 77%; mp 201-203°C. IR (KBr, cm-1): ῡ = 3494 (NH, amide), 2227 (CN),
1
1716 (C=O, amide), 1602 (Ar-C=C); H NMR (400 MHz, DMSO-d6, δ, ppm): 9.30 (s, 1H,
pyrimidine-H), 7.99-7.93 (m, 4H, p-F-Ph), 7.63-7.61 (d, J = 8.8 Hz, 2H, p-Cl-Ph), 7.41-7.36 (d, J
13
= 8 Hz, 2H, p-Cl-Ph), 2.39 (s, 3H, CH3); MS m/z: 448.33(M-1); C NMR (DMSO-d6, δ, ppm):
171.101, 168.266, 166.952, 165.235, 164.434, 161.591, 160.184, 158.478, 136.638, 132.939,
132.842, 131.089, 130.032, 128.697, 126.091, 115.831, 103.841, 18.547; Anal. Calcd. (%) for
C22H13ClFN5OS: C, 58.73; H, 2.91; N, 15.57. Found: C, 58.76; H, 2.95; N, 15.56.
4.7.3 N-(4-(2-(4-chlorophenyl)-4-methylthiazol-5-yl)-5-cyanopyrimidin-2-yl)cyclohexane
carboxamide (9c)
Pale yellow solid, yield 72%; mp 219-221°C. IR (KBr, cm-1): ῡ = 3422 (NH, amide), 2220 (CN),
1
1677 (C=O, amide), 1578, 1500 (Ar-C=C); H NMR (400 MHz, DMSO-d6, δ, ppm): 11.12 (s,
1H, CONH), 9.18 (s, 1H, pyrimidine-H), 8.05-8.03 (d, J = 8.8 Hz, 2H, p-Cl-Ph), 7.63-7.61 (d, J =
8.8 Hz, 2H, p-Cl-Ph), 2.73 (s, 3H, CH3), 2.67-2.61 (m, 1H, CH), 1.85-1.39 (m, 10H, cyclohexyl);
MS m/z: 438.20 (M+1)+; Anal. Calcd. (%) forC22H20ClN5OS: C, 60.34; H, 4.60; N, 15.99.
Found: C, 60.41; H, 4.61; N, 15.96.
4.7.4 N-(4-(2-(4-chlorophenyl)-4-methylthiazol-5-yl)-5-cyanopyrimidin-2-yl)isobutyramide
(9d)
Off white solid, yield 70%; mp 237-239°C. IR (KBr, cm-1): ῡ = 3422 (NH, amide), 2220 (CN),
1
1677 (C=O, amide), 1578, 1500 (Ar-C=C); H NMR (400 MHz, DMSO-d6, δ, ppm): 11.18 (s,
1H, CONH), 9.18 (s, 1H, pyrimidine-H), 8.05-8.03 (d, J = 8.8 Hz, 2H, p-Cl-Ph), 7.63-7.61 (d, J =
8.8 Hz, 2H, p-Cl-Ph), 2.73 (s, 3H, CH3), 2.90-2.85 (m, 1H, CH), 1.11-1.10 (m, 6H, 2CH3); MS
m/z: 396.56 (M-1); Anal. Calcd. (%) for C19H16ClN5OS: C, 57.35; H, 4.05; N, 17.60. Found: C,
57.39; H, 4.06; N, 17.63.
15
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5 CONCLUSIONS
We have designed and synthesized a novel series of thiazolylpyrimidine derivatives. All new
synthesized compounds, 8 and 9a-9d were screened for antimicrobial activity in vitro against
two pathogenic Gram-positive bacteria viz. Bacillus subtilis and Staphylococcus aureus, two
pathogenic Gram-negative bacteria viz. Salmonella abony and Escherichia Coli and fungal
culture of Aspergillus flavus and Fusarium oxysporum. Our results showed that compounds 9a-
9c exhibited moderate to good activity against Gram-positive bacteria, Staphylococcus aureus,
Gram-negative bacteria, Salmonella abony and fungus, Fusarium oxysporum. Therefore, it was
concluded that thiazolylpyrimidne derivatives could be developed as novel and promising
antimicrobial agents.
ACKNOWLEDGEMENT
Authors thank to SPPU Pune for financial support and for spectral analysis and KTHM College
Nashik for all types of support to do research work.
ORCID
Raghunath B. Toche: https://orcid.org/0000-0001-9555-9277
Ram W. Sabnis: https://orcid.org/0000-0001-7289-0581
REFERENCES AND NOTES
[1]
P. C. Sharma, K. K. Bansal, A. Sharma, D. Sharma,A. Deep Eur. J. Med. Chem.2020,
188, 112016.
16
This article is protected by copyright. All rights reserved.

[2]
R. Mishra, P. K. Sharma, P. K. Verma, I. Tomer, G. Mathur, P. K. Dhakad, J. Heterocycl.
Chem.2017, 54, 2103.
[3]
[4]
C. B. Mishra, S. Kumari, M. Tiwari, Eur. J. Med. Chem. 2015, 92, 1.
S. J. Kashyap, V. K. Garg, P. K. Sharma, N. Kumar, R. Dudhe, J. K. Gupta, Med. Chem.
Res.2012, 21, 2123.
[5]
[6]
H. I. Skulnick, S. D. Weed, E. E. Eidson, H. E. Renis, D. A. Stringfellow, W. Wierenga,
J. Med. Chem.1985, 28, 1864.
N. A. McIntyre, C. McInnes, G. Griffiths, A. L. Barnett, G. Kontopidis, A. M. Z. Slawin,
W. Jackson, M. Thomas, D. I. Zheleva, S. Wang, D. G. Blake, N. J. Westwood, P. M.
Fischer, J. Med. Chem. 2010, 53, 2136.
[7]
[8]
[9]
H. Guan, M. L. Lamb, B. Peng, S. Huang, N. DeGrace, J. Read, S. Hussain, J. Wu, C.
Rivard, M. Alimzhanov, G. Bebernitz, K. Bell, M. Ye, M. Zinda, S. Ioannidis, Bioorg.
Med. Chem. Lett. 2013, 23, 3105.
L. J. Farmer, G. Bemis, S. D. Britt, J. Cochran, M. Connors, E. M. Harrington, T. Hoock,
W. Markland, S. Nanthakumar, P. Taslimi, E. T. Haar, J. Wang, D. Zhaveri, F. G.
Salituro, Bioorg. Med. Chem. Lett.2008, 18, 6231.
R. Butta, S. Donthamsetty, P. Adivireddy, P. Venkatapuram, J. Heterocycl. Chem. 2017,
54, 524.
[10] H. Shao, S. Shi, S. Huang, A. J. Hole, A. Y. Abbas, S. Baumli, X. Liu, F. Lam, D. W.
Foley, P. M. Fischer, M. Noble, J. A. Endicott, C. Pepper, S. Wang, J. Med. Chem. 2013,
56, 640.
[11] M. R. Bhosle, A. R. Deshmukh, S. Pal, A. K. Srivastava, R. A. Mane, Bioorg. Med.
Chem. Lett. 2015, 25, 2442.
17
This article is protected by copyright. All rights reserved.

[12] R. Watpade, A. Bholay, R. B. Toche, J. Heterocycl. Chem. 2017, 54, 3434.
[13] R. B. Toche, P. Nikam, Chem. Biol. Interface2015, 5, 246.
[14] S. K. Kanawade, R. B. Toche, D. P. Rajani, Eur. J. Med. Chem. 2013, 64, 314.
[15] P. S. Patil, R. B. Toche, Front. Drug, Chem. Clin. Res. 2018, 1, 1.
[16] S. K. Kanawade, R. B. Toche, S. P. Patil, A. E. Desai, S. S. Bhamare, Eur. J. Med. Chem.
2011, 46, 4682.
[17] B. S. Kuarm, J. V. Madhav, B. Rajitha, Lett. Org. Chem. 2011, 8, 549.
[18] L. A. Adams, P. Sharma, B. Mohanty, O. V. Ilyichova, M. D. Mulcair, M. L. Williams,
E. C. Gleeson, M. Totsika, B. C. Doak, S. Caria, K. Rimmer, J. Horne, S. R. Shouldice,
M. Vazirani, S. J. Headey, B. R. Plumb, J. L. Martin, B. Heras, J. S. Simpson, M. J.
Scanlon, Angew. Chem. Int. Ed. 2015, 54, 2179.
[19] M. L. Sierra, V. Beneton, A. Boullay, T. Boyer, A. G. Brewster, F. Donche, M. Forest,
M. Fouchet, F. J. Gellibert, D. A. Grillot, M. H. Lambert, A. Laroze, C. Le Grumelec, J.
M. Linget, V. G. Montana, V. Nguyen, E. Nicodeme, V. Patel, A. Penfornis, O. Pineau,
D. Pohin, F. Potvain, G. Poulain, C. B. Ruault, M. Saunders, J. Toum, H. E. Xu, R. X.
Xu, P. M. Pianetti, J. Med. Chem. 2007, 50, 685.
[20] E. J. Threlafall, I. S. T. Fisher, L. R. Ward, H. Tschape, P. Gerner-Smidt, Microb. Drug
Resist. 1999, 5, 195.
[21] J. F. Prescott, J. D. Baggot, R. D. Walker, Antimicrobial Therapy in Veterinary Medicine,
3rd ed., Iowa State University Press, Ames, 2000, 12.
SUPPORTING INFORMATION
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