An efficient synthesis, characterization, and antimicrobial screening of tetrahydropyrimidine derivatives

Article abstract of DOI:10.1007/s00044-013-0840-9

Several new substituted 1,2,3,4-tetrahydropyrimidine derivatives 2a-h have been synthesized by the modified Biginelli and Hantzsch reactions and compounds were characterized by spectral techniques. All compounds were evaluated for their in vitro antimicrobial activity against different strains of Gram-negative (E. coli and P. aeruginosa) and Gram-positive (S. aureus and S. pyogenus) bacteria and selected fungi C. albicans, A. niger, and A. clavatus using serial Broth dilution method (Mueller-Hinton broth dilution method). Compound 2e was found active against both Gram-negative and -positive bacterial strains used for present study, while compound 2b was good active against Gram-negative and S. pyogenus bacterial strains. Compounds 2a-h were not that much significant against (C. albicans, A. niger, and A. clavatus) selected fungal strains. Springer Science+Business Media 2013.

Full text of DOI:10.1007/s00044-013-0840-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:2417–2425
DOI 10.1007/s00044-013-0840-9
ORIGINAL RESEARCH
An efficient synthesis, characterization, and antimicrobial
screening of tetrahydropyrimidine derivatives
•
Dinesh R. Godhani Purvesh B. Dobariya
•
•
Anand A. Jogel Anil M. Sanghani
•
Jignasu P. Mehta
Received: 2 July 2013 / Accepted: 9 October 2013 / Published online: 20 October 2013
Ó Springer Science+Business Media New York 2013
Abstract Several new substituted 1,2,3,4-tetrahydropyr-
imidine derivatives 2a–h have been synthesized by the
modified Biginelli and Hantzsch reactions and compounds
were characterized by spectral techniques. All compounds
were evaluated for their in vitro antimicrobial activity
against different strains of Gram-negative (E. coli and P.
aeruginosa) and Gram-positive (S. aureus and S. pyogenus)
bacteria and selected fungi C. albicans, A. niger, and A.
clavatus using serial Broth dilution method (Mueller–
Hinton broth dilution method). Compound 2e was found
active against both Gram-negative and -positive bacterial
strains used for present study, while compound 2b was
good active against Gram-negative and S. pyogenus bac-
terial strains. Compounds 2a–h were not that much sig-
nificant against (C. albicans, A. niger, and A. clavatus)
selected fungal strains.
therapeutic and pharmacological properties (Kappe, 1993).
These tetrahydropyrimidines have emerged as antihyper-
tensive agents, mitotic kinesis inhibitors (Kappe et al.,
2000; Kappe, 2002), a1a-adrenergic receptor antagonists
(Barrow et al., 2000) and hepatitis B virus replication
inhibitors (Deres et al., 2003).
These compounds are known to possess wide spectrum
of biological and therapeutic properties such as antibacte-
rial, antiviral, antitumor, and anti-inflammatory activities,
which represents the important of tetrahydropyrimidine
compounds as the class of multi-channel blockers (Fabian
and Semones, 1997; Eisner and Kuthan, 1972; Stout and
Meyers, 1982). Tetrahydropyrimidines (THPMs) with
other types of bioactivity, cerebrocrast (Klusa, 1995) has
been introduced as a neuroprotectant and cognition
enhancer. Recently, Desai et al. (2011) reported potent
in vitro anti-cancer activity of THPMs.
Keywords Tetrahydropyrimidine Á Biginelli reaction Á
Hantzsch reaction Á Antibacterial activity Á
Antifungal activity
The biological as well as medicinal importance of
1,2,3,4-tetrahydropyrimidine prompted us for further
modification in the heterocyclic frame work for synthesis
of new THPMs. The product one-pot synthesis that pre-
cipitated as pyrimidine compounds on cooling of the
reaction mixture was identified correctly by Biginelli as
3,4-dihydropyrimidin-2(1H)-one (Biginelli, 1893). This
has led to the development of multi-step approaches that
produce overall higher yield, but need the simplicity of
Biginelli synthesis.
Introduction
The pyrimidine has attracted increasing interest in the
synthetic organic chemistry because of their diverse
During the past decades, the scope of the original cy-
clocondensation reaction is gradually extended by variation
of all three building blocks, allowing access to a large
number of structurally diverse multi-functionalized dihy-
dropyridines (DHPs) and dihydropyrimidines (DHPMs).
These heterocyclic compounds exist in a variety of natural
and synthetic organic compounds. These developments led
to the preparation and pharmacological evaluation of
D. R. Godhani (&) Á P. B. Dobariya Á A. A. Jogel Á J. P. Mehta
Department of Chemistry (DST-FIST Sponsored), Maharaja
Krishnakumarsinhji Bhavnagar University, Mahatma Gandhi
Campus, Bhavnagar 364022, Gujarat, India
e-mail: drgodhani@yahoo.com
A. M. Sanghani
Chemistry Department, Sir P. P. Institute of Science,
Bhavnagar 364002, Gujarat, India
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Med Chem Res (2014) 23:2417–2425
Fig. 1 Reaction scheme of
4-aryl-N-(2,4-dimethylphenyl)-
6-methyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-
carboxamide
1,4-dihydropyridines (DHPs) and 3,4-dihydropyrimidines
(DHPMs) of biological interest (Loev et al., 1972; Meyer
et al., 1981, 1992; Kappe et al., 2000; Dallinger et al.,
2004). However, in spite of their potential utility many of
these reported one-pot protocols suffer from drawbacks
such as the use of expensive reagents, strong acidic con-
ditions, and long reaction times. THPMs, a variety of dif-
ferent combinatorial protocols based on the classical
Biginelli microwave reaction have been reported. Synthe-
ses of THPMs by using over silica sulfuric acid as a
reusable catalyst under solvent-free conditions are reported
(Armstrong et al., 1996; Salehi et al., 2003).
benzene: ethyl acetate (8:2 V/V) as mobile phase and
visualized under ultraviolet (UV) light or iodine vapor.
A Perkin-Elmer 2400 CHN analyzer is used for elemental
analysis of carbon, hydrogen, and nitrogen. IR spectra of
compounds were recorded on Thermo-Nicolet FT-IR-200
1
spectrophotometer in KBr disk (cm-1). H NMR and 13C
NMR spectra were recorded on Bruker (200 MHz) spec-
trometer using CDCl3 as a solvent and TMS as an internal
standard. Chemical shifts are reported in parts per million
(dppm). Mass spectral study of synthesized compounds (2a–
h) was carried out using the Shimadzu GC–MS (Shimadzu
2010 plus) direct probe method. Compounds 2a–h were
synthesized using random synthesizer Syrris IKA-RCA
with safety control under similar experimental conditions
for each batch of synthesis.
In extension of our efforts to understand the effect of
functional groups on the conformational and features in the
‘‘Biginelli’’ class of compounds, we present herein a sys-
tematic study of THPMs-containing thiol group (Nayak
et al., 2010) moiety as depicted in reaction scheme (Fig. 1).
General procedure
General procedure for synthesis N-(2,4-dimethylphenyl)-3-
Experimental
oxobutanamide (1)
Materials and methods
A brief summary of method adopted for preparation of
compound 1 is described here (Banik et al., 2007; Desai
et al., 2012). A mixture of substituted aniline (0.01 mol)
and ethyl acetoacetate (0.012 mol) in 40 mL toluene was
refluxed for 12 h in the presence of few drops of NaOH in
water. The reaction was monitored with the help of TLC.
Melting points are determined on an electro-thermal
melting point apparatus and are uncorrected. Completion of
reaction and purity of all compounds were checked on
aluminum-coated TLC plates 60 F245 (E. Merck) using
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Med Chem Res (2014) 23:2417–2425
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Table 1 Analytical data of the prepared compounds 2a–h
Sr. no.
Ar
Molecular formula
% Yield
M.P. °C
Elemental analysis (%)
C
H
N
Req.
Obs.
Req.
Obs.
Req.
Obs.
2a
2b
2c
4-Cl–C6H4
C20H20ClN3Os
C20H21N3O2s
C20H20N4O3s
C20H21N3O2s
C21H23N3O2s
C20H20N4O3s
C20H20ClN3Os
C20H20N4O3s
60
60
61
69
63
55
58
67
175
140
166
152
165
172
130
170
62.25
65.37
60.59
65.37
66.12
60.59
62.25
60.59
62.23
65.36
60.57
65.36
66.11
60.58
62.24
60.57
5.22
5.76
5.08
5.76
6.08
5.08
5.22
5.08
5.21
5.75
5.07
5.75
6.06
5.06
5.20
5.07
10.89
11.44
14.13
11.44
11.01
14.13
10.89
14.13
10.89
11.44
14.12
11.42
11.00
14.13
10.88
14.12
2-OH–C6H4
3-NO2–C6H4
3-OH–C6H4
4-OCH3–C6H4
2-NO2–C6H4
3-Cl–C6H4
2d
2e
2f
2 g
2 h
4-NO2–C6H4
The excess of toluene was distilled off and the reaction
mixture was taken in hexane with continuous mechanical
starring at constant RPM. The product was isolated in
hexane and filtered off. The crude product was dried. The
dried product was dissolved in aqueous NaOH solution so
that, impurity of raw material can be removed using fil-
tration. The crystallization of compound 1 was carried out
in ethanol.
was poured onto crushed ice. The solid obtained was filtered
and dried. The crystallization of final compounds was carried
out in ethanol as described elsewhere. (Desai et al., 2011)
and their analytical data are summarized in Table 1.
Elemental and characterization data of 4-(4-
chlorophenyl)-N-(2,4-dimethylphenyl)-6-methyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxamide (2a)
Elemental and characterization data of N-(2,4-
White crystalline powder; m.p.: 175 °C yield 60 % IR
(KBr): cm-1: 2920 (C–H-stretching asymmetric), 2855 (C–
H-stretching symmetric), 1630 (C=O-stretching of amide),
1383 (–CH3-bending vibration), 1514 (C=C-stretching ring
skeletal), 1089 (C–H inplane bending), 1270 (C–N stretch-
ing), 3251 (N–H stretching), 1013 (C–O stretching), 1488
(–CH def asymmetric), 750 (C–H o.o.p. bending 1,4-disub-
tituted benzene ring), 811 (C–H o.o.p. bending 1,2,4-tri-
subtituted benzene ring). 1H-NMR (200 MHz, CDCl3 dppm):
2.23 (s, 6H, attached phenyl ring, –CH3), 1.72 (s, 1H, tetra-
hydropyrimidine ring, –SH), 1.81 (s, 3H, Ar–CH3), 5.41 (d,
1H, tetrahydropyrimidine ring, –CH), 6.63–7.50 (m, 7H, Ar–
H) 7.86 and 8.23 (s, 2H, tetrahydropyrimidine ring, –NH and
C–NH). 13C NMR (200 MHz, CDCl3 dppm): 114.6–159.3
(Ar–C, 12C, C-8-13, C-16-20, C-5), 16.3, 21.5, and 17.3
(Ar–CH3, 3C, C-14-15, C-6), 163.2 (–C=O, 1C, C-7), 174.2
(–C–S, 1C, tetrahydropyrimidine ring, C-3), 57.4 (Ar–C, 1C,
C-4), 106.2 and 159.4 (C=C, 2C, tetrahydropyrimidine ring,
C-1-2). GC–MS: m/z [M?1]? 369. Anal. Calcd. for
C20H20ClN3OS: C, 62.25; H, 5.22; N, 10.89; Found C, 62.23;
H, 5.21; N, 10.89 (Table 1).
dimethylphenyl)-3-oxobutanamide (1)
White crystallized powder; m.p.: 225 °C yield 78 % IR
(KBr): cm-1: 2921 (C–H-stretching asymmetric), 2866 (C–
H-stretching asymmetric), 1644 (C=O-stretching of amide),
1382 (–CH3-bending vibration), 3058 (C–H-stretching aro-
matic), 1511 (C=C-stretching ring skeletal), 1091 (C–H in-
plane bending), 1267 (C–N stretching), 3245 (N–H
stretching), 1010 (C–O stretching). 1H NMR (200 MHz
CDCl3 dppm) 2.24 (s, 6H, dimethylphenyl ring –CH3), 1.74
(s, 3H, –CH3), 6.45–7.21 (m, 3H, Ar–H), 7.25 (s, 1H, –C–
NH, amide), 7.32 and 7.21 (s, 2H, Ar–H). 13C NMR
(200 MHz, CDCl3 dppm): 114.4–143.0 (Ar–C, 6C, C-1-6),
21.5 and 17.3 (Ar–CH3, 2C, C-7, C-8), 165.6 and 201.6
(–C=O, 2C, C-9, C-11), 51.4 (C–C, 1C, C-10, attached of
oxobutamide), 27.9 (O=C–CH3, 1C, C-12). GC–MS: m/z
[M?1]? 205.15. Anal. Calcd. for C12H15NO2: C, 70.22; H,
7.37; N, 6.82; Found C, 70.21; H, 7.36; N, 6.82 %.
General procedure for synthesis of 4-(aryl)-N-(2,4-
dimethylphenyl)-6-methyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (2a–h)
Elemental and characterization data of N-(2,4-
dimethylphenyl)-4-(2-hydroxyphenyl)-6-methyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxamide (2b)
A mixture of compound (1) (0.01 mol), 4-chlorobenzalde-
hyde (0.01 mol), substituted diketone (0.012 mol), and
thiourea (0.015 mol) in methanol was placed in the round
bottom flask and was refluxed about for 12 h in the presence
of catalytic amount concentrated HCl. The reaction mixture
White crystal powder; m.p.: 140 °C yield 60 % IR (KBr):
cm-1: 2918 (C–H-stretching asymmetric), 2855 (C–H-
stretching symmetric), 1643 (C=O-stretching of amide),
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Med Chem Res (2014) 23:2417–2425
1380 (–CH3-bending vibration), 3057 (C–H stretching),
1504 (C=C-stretching ring skeletal), 1089 (C–H inplane
bending), 1261(C–N stretching), 3242 (N–H stretching),
1009 (C–O stretching), 3315 (N–H stretching), 1458 (–CH
def asymmetric), 750 (C–H o.o.p. bending 1,4-disubtituted
(C=C-stretching ring skeletal), 1088 (C–H inplane bending),
1263 (C–N stretching), 3237 (N–H stretching), 1017 (C–O
stretching), 3318 (N–H stretching), 1455 (–CH def asym-
metric), 753 (C–H o.o.p. bending 1,4-disubtituted benzene
ring), 817 (C–H o.o.p. bending 1,2,4-trisub). 1H NMR
(200 MHz, CDCl3 dppm): 2.17 (s, 6H, attached phenyl ring,
–CH3), 1.73 (s, 1H, tetrahydropyrimidine ring, –SH), 1.87 (s,
3H, Ar–CH3), 5.44 (s, 1H, tetrahydropyrimidine ring,–CH),
6.54–7.54 (m, 7H, Ar–H) 7.87 and 8.26 (s, 2H, amide tetra-
1
benzene ring), 815 (C–H o.o.p. bending 1,2,4-trisub). H
NMR (200 MHz, CDCl3 dppm): 2.19 (s, 6H, attached phenyl
ring, –CH3), 1.74 (s, 1H, tetrahydropyrimidine ring, –SH),
1.86 (s, 3H, Ar–CH3), 5.43 (s, 1H, tetrahydropyrimidine
ring, –CH), 6.74–7.52 (m, 7H, Ar–H) 7.83 and 8.22 (s, 2H,
–NH). 3.51 (s, 1H, phenol Ar–OH). 13C NMR (200 MHz,
CDCl3 dppm): 112.3–156.5 (Ar–C, 12C, C-8-13, C-16-20,
C-5), 17.7, 21.5, and 17.6 (Ar–CH3, 3C, C-14-15, C-6),
163.8 (–C=O, 1C, C-7), 174.4 (–C–S, 1C, tetrahydropyr-
imidine ring, C-3), 59.5 (Ar–C, 1C, C-4), 108.7 and 159.7
(C=C, 2C, tetrahydropyrimidine ring, C-1-2). GC–MS: m/z
[M?1]? 338. Anal. Calcd. for C20H21N3O2S: C, 65.37; H,
5.76; N, 11.44; Found C, 65.36; H, 5.75; N, 11.44.
hydropyrimidinering, –NH). 3.53(s, 1H, phenolAr–OH). 13
C
NMR (200 MHz, CDCl3 dppm): 112.6–156.7 (Ar–C, 12C,
C-8-13, C-16-20, C-5), 17.8, 21.6, and 17.5 (Ar–CH3, 3C,
C-14-15, C-6), 163.4 (–C=O, 1C, C-7), 174.2 (–C–S, 1C,
tetrahydropyrimidine ring, C-3), 59.2 (Ar–C, 1C, C-4), 108.4
and 159.3 (C=C, 2C, tetrahydropyrimidine ring, C-1-2). GC–
MS: m/z [M?1]? 342. Anal. Calcd. for C20H21N3O2S: C,
65.37; H, 5.76; N, 11.44; Found C, 65.36; H, 5.75; N, 11.42.
Elemental and characterization data of N-(2,4-
dimethylphenyl)-4-(4-methoxyphenyl)-6-methyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxamide (2e)
Elemental and characterization data of N-(2,4-
dimethylphenyl)-6-methyl-4-(3-nitrophenyl)-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxamide (2c)
White powder; m.p.: 165 °C yield 63 % IR (KBr): cm-1
:
Yellow amorphous powder; m.p.: 166 °C yield 61 % IR
(KBr): cm-1: 2929 (C–H-stretching asymmetric), 2850 (C–
H-stretching symmetric), 1640 (C=O-stretching of amide),
1378 (–CH3-bending vibration), 3052 (C–H stretching),
1506 (C=C-stretching ring skeletal), 1088 (C–H inplane
bending), 1243 (C–N stretching), 3233 (N–H stretching),
1008 (C–O stretching), 1447 (–CH def asymmetric), 750 (C–
H o.o.p. bending 1,4-disubtituted benzene ring), 813 (C–H
o.o.p. bending 1,2,4-trisubstituted), 1218 (N–O stretching).
1H NMR (200 MHz, CDCl3 dppm): 2.21 (s, 6H, attached with
phenyl ring, –CH3), 1.76 (s, 1H, tetrahydropyrimidine ring,
–SH), 1.88 (s, 3H, Ar–CH3), 5.46 (s, 1H, tetrahydropyrimi-
dine ring, Ar–H), 6.83–7.54 (m, 7H, Ar–H) 7.85 and 8.24 (s,
2H, amide in tetrahydropyrimidine ring, –NH). 13C NMR
(200 MHz, CDCl3 dppm): 114.7–159.1 (Ar–C, 12C, C-8-13,
C-16-20, C-5), 17.9, 21.6, and 17.6 (Ar–CH3, 3C, C-14-15,
C-6), 163.1 (–C=O, 1C, C-7), 174.1 (–C–S, 1C, tetrahydro-
pyrimidine ring, C-3), 57.9 (Ar–C, 1C, C-4), 106.4 and 159.1
(C=C, 2C, tetrahydropyrimidine ring, C-1-2). GC–MS: m/z
[M?1]? 380. Anal. Calcd. for C20H20N4O3S: C, 60.59; H,
5.08; N, 14.13; Found C, 60.57; H, 5.07; N, 14.12.
2918 (C–H-stretching asymmetric), 2870 (C–H-stretching.
symmetric), 1643 (C=O-stretching of amide), 1388 (–CH3-
bending vibration), 3058 (C–H stretching), 1517 (C=C-
stretching ring skeletal), 1083 (C–H-inplane bending), 1258
(C–N stretching), 3245 (N–H stretching), 1010 (C–O
stretching), 3311 (N–H stretching) 1455 (–CH def. asym-
metric), 756 (C–H o.o.p. bending 1,4-disubtituted benzene
ring), 811 (C–H o.o.p. bending 1,2,4-trisubtituted benzene
ring). 1H NMR (200 MHz, CDCl3 dppm): 2.24 (s, 6H, attached
phenyl ring, –CH3), 1.76 (s, 1H, tetrahydropyrimidine ring,
–SH), 1.82(s, 3H, Ar–CH3),5.44(s, 1H, tetrahydropyrimidine
ring,–CH), 6.73–7.57 (m, 7H, Ar–H) 7.86 and 8.27 (s, 2H,
amide tetrahydropyrimidine ring, –NH), 2.34 (s, 3H, attached
phenyl ring, Ar–OCH3). 13C NMR (200 MHz, CDCl3 dppm):
114.3–157.4 (Ar–C, 12C, C-8-13, C-16-20, C-5), 17.8, 21.8,
and17.6(Ar–CH3, 3C, C-14-15,C-6), 163.4(–C=O,1C, C-7),
174.4 (–C–S, 1C, tetrahydropyrimidine ring, C-3), 57.7 (Ar–
C, 1C, C-4), 106.3 and 158.3 (C=C, 2C, tetrahydropyrimidine
ring, C-1-2), 56.2 (Ar–OCH3, 1C, C-21). GC–MS: m/z
[M?1]? 364. Anal. Calcd. for C21H23N3O2S: C, 66.12; H,
6.08; N, 11.01; Found C, 66.11; H, 6.06; N, 11.00.
Elemental and characterization data of N-(2,4-
dimethylphenyl)-4-(3-hydroxyphenyl)-6-methyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxamide (2d)
Elemental and characterization data of N-(2,4-
dimethylphenyl)-6-methyl-4-(2-nitrophenyl)-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxamide (2f)
White crystalline powder; m.p.: 152 °C yield 69 % IR (KBr):
cm-1: 2918 (C–H-stretching asymmetric), 2856 (C–H-
stretching symmetric), 1638 (C=O-stretching of amide), 1378
(–CH3-bending vibration), 3048 (C–H stretching), 1515
Yellow crystalline powder; m.p.: 172 °C yield 55 % IR
(KBr): cm-1: 2922 (C–H-stretching asymmetric), 2856 (C–
H-stretching symmetric), 1646 (C=O-stretching of amide),
1383 (–CH3-bending vibration), 3051 (C–H stretching),
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Med Chem Res (2014) 23:2417–2425
2421
1515 (C=C-stretching ring skeletal), 1081 (C–H-inplane
bending), 1266 (C–N stretching), 3245 (N–H stretching),
1010 (C–O stretching), 1456 (–CH def asymmetric), 753 (C–
H o.o.p. bending 1,4-disubtituted benzene ring), 814 (C–H
o.o.p. bending 1,2,4-trisub), 1221 (N–O stretching). 1H
NMR (200 MHz, CDCl3 dppm): 2.23 (s, 6H, dimethylphenyl
attached phenyl ring, –CH3), 1.77 (s, 1H, tetrahydropyrim-
idine ring, –SH), 1.89 (s, 3H, Ar–CH3),5.42 (s, 1H, tetra-
hydropyrimidine ring, –CH), 6.78–7.54 (m, 7H, Ar–H) 7.85
1276 (C–N stretching), 3245 (N–H stretching), 1015 (C–O
stretching), 1455 (–CH def asymmetric), 753 (C–H o.o.p.
bending 1,4-disubtituted benzene ring), 817 (C–H o.o.p.
bending 1,2,4-trisub), 1223 (N–O stretching). 1H NMR
(200 MHz, CDCl3 dppm): 2.26 (s, 6H, attached with phenyl
ring, –CH3), 1.75 (s, 1H, tetrahydropyrimidine ring, –SH),
1.84 (s, 3H, Ar–CH3), 5.41 (s, 1H, tetrahydropyrimidine ring,
–CH), 6.71–7.56 (m, 7H, Ar–H) 7.84 and 8.23 (s, 2H amide
tetrahydropyrimidine ring, –NH). 13C NMR (200 MHz,
CDCl3 dppm): 114.3–159.1 (Ar–C, 12C, C-8-13, C-16-20,
C-5), 17.7, 21.6, and 17.4 (Ar–CH3, 3C, C-14-15, C-6), 163.3
(–C=O, 1C, C-7), 174.5 (–C–S, 1C, tetrahydropyrimidine
ring, C-3), 57.4 (Ar–C, 1C, C-4), 106.4 and 159.4 (C=C, 2C,
tetrahydropyrimidine ring, C-1-2). GC–MS: m/z [M?1]?
360. Anal. Calcd. for C20H20N4O3S: C, 60.59; H, 5.08; N,
14.13; Found C, 60.57; H, 5.07; N, 14.12.
and 8.24 (s, 2H, amide tetrahydropyrimidine ring, –NH). 13
C
NMR (200 MHz, CDCl3 dppm): 114.4–159.1 (Ar–C, 12C,
C-8-13, C-16-20, C-5), 17.8, 21.7, and 17.8 (Ar-CH3, 3C,
C-14-15, C-6), 163.4 (–C=O, 1C, C-7), 174.2 (–C–S, 1C,
tetrahydropyrimidine ring, C-3), 57.8 (Ar–C, 1C, C-4), 106.3
and 159.3 (C=C, 2C, tetrahydropyrimidine ring, C-1-2). GC–
MS: m/z [M?1]? 348. Anal. Calcd. for C20H20N4O3S: C,
60.59; H, 5.08; N, 14.13; Found C, 60.58; H, 5.06; N, 14.13.
Statistical analysis
Elemental and characterization data of 4-(3-
chlorophenyl)-N-(2,4-dimethylphenyl)-6-methyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxamide (2g)
Standard deviation value is expressed in terms of SD.
Based on calculated value by using one-way ANOVA
method followed by independent two-sample Turkey test;
it has been observed that differences below 0.001 levels are
considered as statistically significant.
White crystalline powder; m.p.: 130 °C yield 58 % IR
(KBr): cm-1: 2923 (C–H-stretching asymmetric), 2855 (C–
H-stretching symmetric), 1644 (C=O-stretching of amide),
1384 (–CH3-bending vibration), 3066 (C–H stretching),
1517 (C=C-stretching ring skeletal), 1089 (C–H-inplane
bending), 1263 (C–N stretching), 3243 (N–H stretching),
1011 (C–O stretching), 1453 (–CH def. asymmetric), 755
(C–H o.o.p. bending 1,4-disubtituted benzene ring), 815 (C–
H o.o.p. bending 1,2,4-trisubtituted benzene ring). 1H NMR
(200 MHz, CDCl3 dppm): 2.27 (s, 6H, attached phenyl ring–
CH3), 1.74 (s, 1H, tetrahydropyrimidine ring, –SH), 1.88 (s,
3H, Ar–CH3), 5.44 (s, 1H, tetrahydropyrimidine ring,–CH),
6.89–7.57 (m, 7H, Ar–H) 7.84 and 8.21 (s, 2H, amide tet-
rahydropyrimidine ring, –NH). 13C NMR (200 MHz, CDCl3
Result and discussion
Methodology adopted for preparation
of tetrahydropyrimidines
Several methodologies are taking on for the preparation of
tetrahydropyrimidines. Summary of adopted approaches
are given here.
Messer et al., (1992) and Dunbar et al., (1993) used
pyrimidine ring as initiator and reduced in the presence of Pd
catalyst to yield the target compound of tetrahydropyrimi-
dine. Sawant and Sarode (2011) prepared tetra-
hydropyrimidine-using AlCl3 and con. HCl. Both methods
are not efficient enough to get significant yield of final tar-
geted compounds. Therefore, we have modified Biginelli
reaction in which we used diketone, aromatic aldehyde and
thiourea in methanol medium. This modified reaction helps
us to get significant yield of our targeted moieties.
dppm): 114.2–159.2 (Ar–C, 12C, C-8-13, C-16-20, C-5),
17.4, 19.6 and 17.4 (Ar–CH3, 3C, C-14-15, C-6), 163.3
(–C=O, 1C, C-7), 174.3 (–C–S, 1C, tetrahydropyrimidine
ring, C-3), 57.8 (Ar–C, 1C, C-4), 106.3 and 159.4 (C=C, 2C,
tetrahydropyrimidine ring, C-1-2). GC–MS: m/z [M?1]?
235. Anal. Calcd. for C20H20ClN3OS: C, 62.25; H, 5.22; N,
10.89; Found C, 62.24; H, 5.21; N, 10.87.
Elemental and characterization data of N-(2,4-
dimethylphenyl)-6-methyl-4-(4-nitrophenyl)-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxamide (2h)
Chemistry of 4-(4-chlorophenyl)-N-(2,4-
dimethylphenyl)-6-methyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (2a)
White–yellowcrystallinepowder;m.p.:170 °Cyield67 %IR
(KBr): cm-1: 2911 (C–H-stretching asymmetric), 2871 (C–
H-stretching symmetric), 1665 (C=O-stretching of amide),
1384 (–CH3-bending vibration), 3060 (C–H stretching), 1511
(C=C-stretching ring skeletal), 1088 (C–H-inplane bending),
IR spectra
The IR spectrum of the compound 2a (molecular formula
C20H20ClN3O2 m.w. 369) clearly shows a C=O has given
123

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Med Chem Res (2014) 23:2417–2425
absorption peak appear in the range of 1,630 cm-1 is due
to stretching vibration, while ring skeletal C=C shows
medium intensity absorption stretching at 1,514 cm-1
,
respectively. C–N-stretching band at 1,270 cm-1 and high
frequency region of IR spectra of this compound contains
vibration N–H-stretching vibration band indicate at
3,251 cm-1 indicates ring closure of the pyrimidine ring
corresponding secondary amine. The presence of 1,2,4-
trisubstituted benzene ring is confirmed due to absorption
bands at 750 and 811 cm-1, respectively. The presence of
substituted (C–Cl) benzene ring is also confirmed with
weak intensity absorption peak corresponding to low fre-
quency region at 650 cm-1 of synthesized compound 2a.
1H NMR spectra
Fig. 2 Compound 2a showed the presence of tetrahydropyrimidine
moiety in its structure
Proton-NMR spectra of compound 2a suggests that C-10,
C-12, C-13, C-16, C-17, C-19, and C-20, seven protons are
attached to chemically equivalent environment in com-
pound 2a, which is appeared at d = 6.63–7.50 ppm. Sec-
ondary amide (attached to C-8) proton which is appeared as
singlet at d = 8.23 ppm. Proton of –SH group in pyrimi-
dine ring appeared as a singlet at d = 1.72 ppm. Protons of
methyl group (C-14 and C-15) appeared as singlet at
d = 2.23 ppm, while methyl group (C-6), which is
appeared as singlet at d = 1.81 ppm. C–NH of pyrimidine
ring is appeared as a singlet at d = 7.86 ppm and protons
of C-4 of pyrimidine ring is appeared as a doublet at
d = 5.41 ppm.
Antimicrobial activity
The newly synthesized compounds 2a–h were screened for
their antimicrobial activity against Gram-positive bacteria
S. aureus (MTCC-96), S. pyogenus (MTCC-442) and
Gram-negative (E. coli (MTCC-443), P. aeruginosa
(MTCC-1688), and fugal pathogens C. albicans (MTCC
227) A. niger (MTCC 282) and A. clavatus (MTCC 1323)
All MTCC were collected from Institute of Microbial
Technology, Chandigarh. The activity of compounds was
determined at Microcare Laboratory, Surat using standard
protocol of Mueller–Hinton Broth (Becton–Dickinson,
USA). The compounds were dissolved in dimethyl sulf-
oxide (DMSO) for the disk-diffusion test, and minimal
inhibitory concentrations (MIC) were determined by
making a series of dilutions in an appropriate substrate
ranging from 16.23 to 811.75 lM/L. The final concentra-
tion of DMSO did not exceed 2 %. The sensitivity of
microorganisms to the investigated species was tested by
measuring the zone of inhibition of a given concentration
of the extract by the disk-diffusion method and by deter-
mining the minimal inhibitory concentration (MIC) (Clinical
and Laboratory Standards Institute, 2008a, b). Bacterial
inoculum were obtained from bacterial cultures incubated
for 24 h at 37 °C on Mueller–Hinton agar substrate and
diluted to approximately 108 CFU/mL, according to the 0.5
McFarland standard. Suspensions of fungal spores were
prepared from fresh mature (3–7 days old) cultures that
grew at 30 °C on a PDA substrate (Gein, 2012). Spores
were rinsed with sterile distilled water, in order to deter-
mine turbidity spectrophotometrically at 530 nm. The set
was further diluted to approximately 106 CFU/mL
according to the procedure recommended by the NCCLS
(CLSI; formerly NCCLS). The standard disk-diffusion
method was used to study antimicrobial activity. The
13C NMR spectra
Looking at, chemical shifts of final compound 2a vary from
d = 16.3–174.2 ppm. Carbon nuclei under the influence of
a strong electronegative environment appeared in the
downfield, e.g., carbon of tetrahydropyrimidine ring
C-7 has chemical shift value of d = 163.2 ppm due to
the influence of oxygen atom. Similarly, C-3 in tetrahy-
dropyrimidine (–C–S) ring has chemical shift at
d = 174.2 ppm due to influence of sulfur atom, while C-1
of same ring is appeared at d = 106.5 ppm due to influence
of nitrogen atom presence in the tetrahydropyrimidine ring.
Carbons are chemically equivalent in nature for C-5, C-8-
13, and C-16-20 of compound 2a show chemical shift at
around d = 114.6–159.3 ppm, which suggest the presence
of phenyl rings in the structure, while chemical shifts of
C-6, C-14, and C-15 are appeared at d = 17.3–21.5 ppm
indicates the presence of three methyl groups. GC–MS data
suggest that molar mass of compound 2a is m/z [M?1]?
369.
1
Based on H NMR and 13C NMR and mass spectrum
data, carbon enumeration of compound 2a is described in
Fig. 2.
123

Med Chem Res (2014) 23:2417–2425
2423
appropriate inoculums were seeded in Mu¨eller–Hinton agar
(for bacteria) or SD agar (for fungi) in Petri dishes. Paper
disks (7 mm in diameter) were laid on the inoculated
substrate after being soaked with 15 lL of different con-
centration of 16.23–811.75 lM/L. Antimicrobial activity
was determined by measuring the diameter of the zone of
inhibition around the disk. As a positive control of growth
inhibition, ampicillin and chloramphenicol were used in
the case of bacteria, nystatin in the case of fungi. A DMSO
solution was used as a negative control for the influence of
solvents and all experiments were performed in duplicate.
The minimal inhibitory concentration (MIC) was deter-
mined by the broth tube dilution method. A series of
dilutions with concentrations ranging from 16.23 to
811.75 lM/L was used in the experiment with each extract
for every microorganism tested. The starting solutions of
extracts with a concentration of 16.23 lM/L were obtained
by measuring of a certain quantity of extract and dissolving
it in DMSO. Twofold dilutions of extracts were prepared in
Mueller–Hinton bullion for bacterial cultures and SD bul-
lion for fungal cultures in test tubes. The boundary dilution
without any visible growth was defined as the minimal
inhibitory concentration (MIC) for the tested microorgan-
ism at the given compounds concentration. All experiments
were performed in duplicate.
Antimicrobial assay
Compounds (2a–h) were screened against Gram-positive,
-negative bacteria and fungi strains. The compounds 2b,
2g, and 2h showed good activity against E. coli bacteria
(MIC = 324.7–405.7 lM/L), while compound 2e showed
excellent activity against E. coli (MIC = 202.93 lM/L).
Similarly, compounds 2b and 2e showed good activity
against P. aeruginosa. Compounds 2a, 2b, 2c, 2d, 2f, 2g,
and 2h showed moderate activity against S. aureus with
MIC = ranging from 649.4 to 811.75 lM/L. Compounds
2b, 2e, and 2f showed good activity against S. pyogenus
(MIC = 324.7–405.87 lM/L). Results also suggest that
compound 2e was more effective against all bacterial
strains studied in this work (Table 2). Results of antifungal
activity were of suggestive that the synthesized compounds
were not equally effective against three fungal pathogens.
The data revealed that electron-releasing groups as nitro,
hydroxyl, and chloro, present on phenyl ring were
increased the antibacterial property. These results clearly
demonstrated that when compounds 1 converted into cor-
responding final compounds 2a–h, exhibited significant
antibacterial activity. Based on MIC values, it is our
observation that the presence of 4-OCH3, 4-NO2, 2-OH,
and 4-Cl group at the position of tetrahydropyrimidines led
to a significant variation in the antimicrobial activity. A
series of compounds when substituted by electron-releasing
123

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Med Chem Res (2014) 23:2417–2425
Biginelli P (1893) The condensation reaction described by Biginelli.
Gazz Chim Ital 23:360–416
groups like nitro, hydroxyl, methoxy, and chloro present on
aromatic ring enhanced the antimicrobial activity.
Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS)
(2008a) National Committee for Clinical Laboratory Standards,
Performance standards for antimicrobial susceptibility testing;
16th Informational supplement. CLSI document M7-A7, 940
West Valley Road, Wayne, Pennsylvania, USA, 19087-1898
Clinical and Laboratory Standards Institute (CLSI) (2008b) Reference
method for broth dilution antifungal susceptibility testing of
yeast; approved standard-3rd edition. CLSI document M27-A3
(ISBN 1-56238-666-2) Clinical and Laboratory Standards Insti-
tute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania,
USA, 28:19087-1898
Dallinger D, Stadler A, Kappe CO (2004) Solid and solution phase
synthesis of bioactive tetrahydropyrimidines. Pure Appl Chem
76:1017–1024. doi:10.1351/pac200476051017
Deres K, Schroder CH, Paessens A, Goldmann S, Hecker HJ (2003)
Inhibition of hepatitis B virus replication by drug-induced
depletion of nucleocapsids. Science 299:893–896. doi:10.1126/
science.1077215
Desai NC, Chhabaria MT, Dodiya A, Bhavsar AM, Baldaniya BB (2011)
Synthesis, characterization, anticancer activity and QSAR-studies
of some new tetrahydropyrimidines. Med Chem Res 20:1331–1339
Desai NC, Pandya DD, Satodiya HM, Rajpara KM, Joshi VV,
Vaghani HV (2012) An efficient, solvent-free microwave-
assisted synthesis and antimicrobial screening of 1,6-tetrahydro-
pyrimidine analogues. Med Chem Res 21:4412–4421
Dunbar PG, Durant GJ, Fang Z, Rho T, Abuh YF, El-Assadi AA,
Ngur D, Periyasamy S, Hoss W, Messer WS Jr, Design (1993)
synthesis, and neurochemical evaluation of 5-(3-alkyl-1,2,4-
oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidines as M1 receptor
agonists. J Med Chem 36:842–847
Conclusion
In this work, we have disclosed the Biginelli synthesis for
tetrahydropyrimidines (THPMs). Using readily available
ethyl acetoacetate as reaction mediator and Toluene as sol-
vent, the yields in the one-pot Biginelli protocol can be
increased on average by 40–55 % as compared with the
traditional methods. This one-pot synthesis of tetrahydro-
pyrimidines is therefore, superior in simplicity and yield
compared to alternative multistep strategies that have been
reported. The use of ethyl acetoacetate in related cyclization
reactions is currently underway in our laboratories and will
be reported in due course. A series of 4-aryl-N-(2,4-
dimethylphenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimid-
ine-5-carboxamide were synthesized successfully. The
Antimicrobial activity profile of the title compounds were
evaluated against Gram-positive bacteria, -negative bacteria,
and three fungal pathogens. Looking to the structure–activity
relationship, remarkable inhibition was observed in com-
pounds bearing Ar = 4-chlorophenyl, 2-hydroxyphenyl,
3-nitrophenyl, 3-hydroxyphenyl, 4-methoxyphenyl, and
4-nitrophenyl substituents. Compound 2e, which contains
4-methoxyphenyl group, was found very efficient against
both Gram-negative and -positive bacteria. This is can be
attributed to the fact that 4-methoxyphenyl derivative may
interrupt the growth of all bacterial strains at one or more
stages, studied in this study. This study also suggests less
lipophilic, less bulky, and electron-withdrawing substituent
may increase the potency, and also reveals that further in-
depth study of 4-methoxyphenyl derivative will be extracted
more relative information about the structure–activity rela-
tionship with antimicrobial screening.
Eisner U, Kuthan J (1972) The chemistry of dihydropyridines. Chem
Rev 72:1–42. doi:10.1021/cr60275a001
Fabian WMF, Semones MA (1997) Conformational analysis of
4-aryl-tetrahydropyrimidine calcium channel modulators.
A
comparison of Ab Initio. Semiempirical and X-ray crystallo-
graphic studies. Tetrahedron. 53:2803–2816
Gein VL, Kholkin IV, Zamaraeva TM, Voronina EV, Vakhrin MI
(2012) Synthesis and antimicrobial activity of N,6-diaryl-4-
methyl-2-thioxo-1,2,3,6-tetrahydropyrimidine-5-carboxamides.
Pharma Chem J 46:2
Kappe CO (1993) 100 Years of the Biginelli dihydropyridine
synthesis. Tetrahedron 49:6937–6963
Kappe CO (2002) Recent advances in the Biginelli dihydropyrimidine
synthesis. New tricks from an old dog. Acc Chem Res
33:879–888. doi:10.1021/ar000048h
Kappe CO, Uray G, Verdino P, Shishkin OV (2000) X-ray structure,
conformational analysis, enantioseparation, and determination of
absolute configuration of the mitotic kinesin Eg5 inhibitor
monastrol. Tetrahedron 56:1859–1862. doi:10.1016/S0040-
4020(00)00116-2
Acknowledgments Authors are thankful to Microcare Laboratory,
Surat for antimicrobial study and are appreciate thanks to CSMCRI,
Bhavnagar for providing facility of H NMR and 13C NMR.
1
References
Klusa V (1995) Cerebrocrast, neuroprotectant, cognition enhancer.
Drugs Fut 20:135–138. doi:10.1021/jo951706s
Armstrong RW, Tempest AP, Brown PA, Keating SD (1996)
Multiple-component condensation strategies for combinatorial
library synthesis. Acc Chem Res 29:123–131
Loev B, Ehrreich SJ, Tedeschi RE (1972) Dihydropyridines with
potent hypotensive activity prepared by the Hantzsch reaction.
J Pharm Pharmacol 24:917–1008
Banik BK, Reddy AT, Datta A, Mukhopadhyay C (2007) Microwave-
induced bismuth nitrate-catalyzed synthesis of dihydropyrimi-
dones via Biginelli condensation under solventless conditions.
Tetrahedron Lett 48:7392–7394
Mager PP, Coburn RA, Solo AJ, Triggle DJ, Rothe H (1992) QSAR,
diagnostic statistics and molecular modelling of 1,4-dihydropyri-
dine calcium antagonists: a difficult road ahead. Drug Des Discov
8:273–289
Barrow JC, Nantermet PG, Slenick HG (2000) In vitro and in vivo
evaluation of dihydropyrimidinone c-5 amides as potent and
selective a1a receptor antagonists for the treatment of benign
prostatic hyperplasia. J Med Chem 43:2703–2718. doi:10.1021/
jm990612y
Messer WS Jr, Dunbar PG, Rho T, Periyasamy S, Ngur D, Ellerbrock
BR, Bohnett M, Ryan K, Durant GJ, Hoss W (1992) Synthesis,
biochemical activity and behavioral effects of aseries of 1,4,5,6-
tetrahydropyrimidines as novel ligands for M1 receptors. Bioorg
Med Chem Lett 2:781–786
123

Med Chem Res (2014) 23:2417–2425
2425
Meyer H, Bossert F, Wehinger E, Stoepel K, Vater W (1981)
Synthesis and comparative pharmacological studies of 1,4-
dihydro-2,6-dimethyl-4-(3-nitrophenyl) pyridine-3,5-dicarbonsa
uereestern with non identical ester functions. Arzneim Forsch
31:407–409
Nayak SK, Venugopala KN, Chopra DV, Guru Row TN (2010) Effect
of substitution on molecular conformation and packing features
in a series of aryl substituted ethyl-6-methyl-4-phenyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylates. Cryst Eng Com-
mun 12:1205–1216
Salehi P, Dabiri M, Zolfigol MA, Bodaghi Fard MA (2003) Efficient
synthesis of 3,4-dihydropyrimidin-2(1H)-ones over silica sulfu-
ric acid as a reusable catalyst under solvent-free conditions.
Heterocycles 60:2435–2440
Sawant R, Sarode V (2011) Synthesis, spectral characterization and
analgesic activity of 2-Methylthio-1,4-Dihydropyrimidines. Ira J
Pharm Res 10(4):733–739
Stout DM, Meyers AI (1982) Recent advances in the chemistry of
dihydropyridines. Chem Rev 82:223–243. doi:10.1021/cr00048a
004
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