Synthesis, antiviral, antituberculostic, and antibacterial activities of some novel, 4-(4-substituted phenyl)-6-(4-nitrophenyl)-2-(substituted imino)pyrimidines

Article abstract of DOI:10.1002/ardp.200600121

A variety of novel 4-(4-substituted phenyl)-6-(4-nitrophenyl)-2-substituted imino) pyrimidines were synthesized by reacting 4-(4-substituted phenyl)-6-(4-nitrophenyl)-2-amino pyrimidines with different substituted aromatic aldehydes, coumarin chloroisatin

Full text of DOI:10.1002/ardp.200600121

Arch. Pharm. Chem. Life Sci. 2007, 340, 95–102
A. A. Siddiqui et al.
95
Full Paper
Synthesis, Antiviral, Antituberculostic, and Antibacterial
Activities of Some Novel, 4-(4-substituted phenyl)-6-
(4-nitrophenyl)-2-(substituted imino)pyrimidines
Anees A. Siddiqui1, Ramadoss Rajesh1, Mojahid-Ul-Islam1, Veerachamy Alagarsamy2, and
Erick De Clercq3
1 Department of Pharmaceutical Chemistry, Hamdard University, New Delhi, India
2 Department of Pharmaceutical Chemistry, Arulmigu Kalasalingam College of Pharmacy, Krishnan Koil, India
3 Rega Institute for Medical Research, Katholieke University, Leuven, Belgium
A variety of novel 4-(4-substituted phenyl)-6-(4-nitrophenyl)-2-substituted imino) pyrimidines
were synthesized by reacting 4-(4-substituted phenyl)-6-(4-nitrophenyl)-2-amino pyrimidines with
different substituted aromatic aldehydes, coumarin chloroisatin. The 4-(4-substituted phenyl)-6-
(4-nitrophenyl)-2-amino pyrimidines were synthesized by reacting 3-(49-substituted phenyl)-1-(4-
nitrophenyl)-2-propen-1-ones with guanine hydrochloride. 3-(4-Substituted phenyl)-1-(4-nitrophe-
nyl)-2-propen-1-ones were synthesized by reacting 4-nitroacetophenone with different para-sub-
stituted aromatic aldehydes. Spectral data (IR, NMR, and mass spectra) confirmed the structures
of the synthesized compounds. The synthesized compounds were investigated for their antiviral,
antituberculostic, and antibacterial activities. The results of antiviral, antituberculostic, and
antibacterial activities indicated that the synthesized compounds exhibited mild to potent activ-
ities compared to the respective reference standards.
Keywords: Antibacterial / Antituberculostic / Antiviral / Pyrimidine /
Received: September 9, 2006; accepted: November 21, 2006
DOI 10.1002/ardp.200600121
been able to completely inhibit the replication of the
virus. Since the efficiency of currently used antiviral com-
pounds are doubtful, there is a need to envisage new
classes of antiviral agents from both synthetic and nat-
ural sources. Pyrimidine and its derivatives are note-
worthy for their physiological and biological impor-
tance. They attracted the attention of medicinal chemists
due to their wide range of biological activities like antic-
ancer [1–4], antiviral [5–11], antituberculostic [12, 13],
and antibacterial [14–17] activities. In view of the above
mentioned facts and inspired by the research envisaged
on pyrimidine and its derivatives, particularly in relation
to microbial infections [1–17], in the present study, we
aimed to synthesize some novel 4-(4-substituted phenyl)-
6-(4-nitrophenyl)-2-(substituted imino) pyrimidines for
their antiviral, antituberculostic, and antibacterial activ-
ities. The title compounds were synthesized by reacting
4-(4-substituted phenyl)-6-(4-nitrophenyl)-2-amino pyrimi-
dines with different substituted aromatic aldehydes,
chloroisatin and coumarin. The 4-(4-substituted phenyl)-
Introduction
Many human illnesses are caused by infections with
microbes like viruses or bacteria or fungi. Amongst those
various illnesses to human beings certain viral, bacterial,
tubercular, and fungal infections are more common
because of their tendency to develop new strains under
any circumstance and to develop resistance against the
available drugs. This stimulated scientists to engage in
the development of several compounds with novel enti-
ties to combat the illnesses caused by them. Infectious
microbial diseases remain a pressing problem world-
wide, because microbes have resisted prophylaxis or ther-
apy longer than any other form of life. For viral diseases,
till today, no antiviral candidate substance tested has
Correspondence: Ramadoss Rajesh, Huclin Research Ltd., 4th Floor, Ti-
cel Bio Park, CSIR Road, Taramani, Chennai-600 113, Tamilnadu, India
E-mail: rrajeshmpharm@yahoo.com
Fax: +91 44 22540-211
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A. A. Siddiqui et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 95–102
6-(4-nitrophenyl)-2-amino pyrimidines were synthesized concentration of test drug required to kill 50% of exposed
by reacting 3-(4-substituted phenyl)-1-(4-nitrophenyl)-2- cell population), nontoxic concentrations of all the
propen-1-ones with guanine hydrochloride. The starting synthesized compounds were subjected for antiviral
material 3-(4-substituted phenyl)-1-(4-nitrophenyl)-2-pro- activity against different viral strains. The results of the
pen-1-ones was synthesized by reacting 4-nitroacetophe- antiviral activity testing (Table 2) indicate that pyrimi-
none with different para-substituted aromatic aldehydes dines with chlorophenyl, dimethylaminophenyl 4, and
(Scheme 1). Spectral data (IR, NMR, and mass spectra) con- pyrimidines with methoxyphenyl, furfuryl 25 substitu-
firmed the structures of the synthesized compounds; the tions were excellent in their action. Next in the order,
purity of these compounds was ascertained by microana- pyrimidines with chlorophenyl, methoxyphenyl 5, and
lysis (Table 1).
trimethoxyphenyl 6 substitutions, pyrimidines with
dimethylaminophenyl, methoxyphenyl, and trimethoxy-
phenyl 13–15 substitutions, pyrimidines with methoxy-
phenyl and p-chlorophenyl 21, p-N-dimethylaminophe-
nyl 22, methoxyphenyl 23, 3,4,6-trimethoxyphenyl 24
Results and discussion
The results of the cytotoxicity assay indicate that all the substitutions have exhibited comparable activity with
compounds showed varying degrees of cytotoxicity, i.e. the reference drugs brivudin and ribavirin. The rest of
from 80 to 400 lg/mL. Based on the CTC50 (the minimum the synthesized compounds exhibited mild activity. The
Table 1. Physical data of 4-(49-substituted phenyl)-6-(49-nitrophenyl)-2-(substituted imino)pyrimidines.
Comp.
R
R1
Molecular
formulaa)
Molecular
weight
Mp
(8C)
Yield
%
3
–Cl
C23H14N4O2Cl2
C25H20N5O2Cl
C24H17N4O3Cl
449
458
445
82
102
127
68
70
72
4
5
–Cl
–Cl
6
–Cl
C26H21N4O5Cl
503
92–94
65
7
8
–Cl
–Cl
C21H13N4O3Cl
C25H15N4O3Cl
405
453
172–-175
90–92
70
68
9
–Cl
C24H13N5O3Cl2
C25H20N5O2Cl
490
457
110
138
71
69
12
–N(CH3)2
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Arch. Pharm. Chem. Life Sci. 2007, 340, 95–102
Novel Pyrimidines
97
Table 1. Physical data of 4-(49-substituted phenyl)-6-(49-nitrophenyl)-2-(substituted imino)pyrimidines.
Comp.
R
R1
Molecular
formulaa)
Molecular
weight
Mp
(8C)
Yield
%
13
–N(CH3)2
C27H26N6O2
442
83
73
14
15
–N(CH3)2
–N(CH3)2
C26H23N5O3
453
98
69
C27H27N5O5
501
123
75
16
17
–N(CH3)2
–N(CH3)2
C23H19N5O3
C27H20N5O3
413
462
152
131
72
75
18
21
22
–N(CH3)2
–OCH3
–OCH3
C26H19N6O3Cl
C24H17N4O3Cl
C26H23N5O3
498
444
454
107
186
135
68
62
70
23
24
–OCH3
–OCH3
C25H20N4O4
C27H24N4O6
440
500
105
76
78
65
25
26
27
–OCH3
–OCH3
–OCH3
C22H16N4O4
C26H17N4O4
C25H16N5O4Cl
400
449
485
152
110
75
85–87
70
78
a)
All compounds gave satisfactory elemental analysis (l 0.4% of theoretical values).
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A. A. Siddiqui et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 95–102
Table 2. Cytotoxicity and antiviral activity of 4-(49-substituted phenyl)-6-(49-nitrophenyl)-2-(substituted imino) pyrimidines in E6SM cell
cultures.
Compound
Minimum cytotoxic
Minimum inhibitory concentration (l/mL)a)
Herpes simplex virus-1 Herpes simplex Vaccinia virus Vesicular stomatitis Herpes simplex virus-1
concentration (lg/mL)b)
(KOS)
virus-2 (G)
virus
TK KOS ACV
3
4
5
6
7
8
9
12
13
14
15
16
17
18
21
22
F16
16
80
A16
A16
A3.2
A16
A16
A400
A400
A400
A16
A16
A16
A16
A80
A80
A400
A16
A16
A16
A16
A3.2
A80
A80
240
240
A16
A3.2
A16
A16
80
240
A400
A16
A16
A16
A16
A80
A80
A400
A16
16
A16
A16
A3.2
A80
A80
1.92
48
A16
A16
A3.2
A16
A16
240
240
A400
A16
A16
A16
A16
A80
A80
A400
A16
A16
A16
A16
A3.2
A80
A80
80
A3.2
A3.2
A16
A16
A400
A400
A400
A16
A16
A16
A16
A80
A80
A400
A16
A16
A16
A16
A16
A16
A400
A400
A400
A16
A16
A16
A16
A80
A80
A400
A16
A16
A16
A16
80
A400
A400
F400
F16
80
80
80
F80
F80
F400
80
80
80
80
16
400
400
A400
A400
23
24
25
26
A3.2
A80
A80
0.0768
240
A3.2
A80
A80
A400
240
27
Brivudin
Ribavirin
240
a)
Required to cause a microscopically detectable alteration of normal cell morphology.
Required to reduce virus- induced cytopathogenicity by 50%.
b)
Table 3. In vitro antitubercular activity of 4-(49-substituted phe-
nyl)-6-(49-nitrophenyl)-2-(substituted imino) pyrimidines.
showed equipotent activity in comparison with the refer-
ence drugs. The rest of compounds showed comparable
activity. In antibacterial screening (Table 4), the screened
compounds exhibited mild to moderate activity against
Klebsiella pneumoniae. Against other bacterial strains,
only few compounds showed activity comparable to that
of the standard drugs and especially against Staphylococ-
cus aureus; only few compounds were shown to have mild
to moderate activity.
In summary, the synthesis of a new series of 4-(4-substi-
tuted phenyl)-6-(4-nitrophenyl)-2-substituted imino) pyri-
midines derivatives is described here. These derivatives
exhibit moderate antiviral, antituberculostic, and anti-
bacterial activity. However, QSAR studies should be car-
ried out to predict the structure-activity relationship for
all the compounds.
Compound
Concentration Percentage
(lg/mL)a)
Inhibition
3
4
5
6
7
9
15
26
6.25
6.25
6.25
6.25
6.25
6.25
6.25
6.25
6.25
0.25
0.031
10.0
1.17
2.31
98
97
100
100
82
80
78
65
70
98
95
99
99
27
Rifampicin
Isoniazid
Tobramycin
Ethionamide
PAS
99
a)
The concentration represents their MIC's.
The authors are grateful to Rega Institute for Medical Re-
search, Katholieke University, Leuven, Belgium for screening
antiviral activity and to National Institute of Allergy and In-
fectious Diseases, Tuberculosis antimicrobial Acquisition and
Coordinating Facility (TAACF), Southern Research Institute,
Birmingham (USA) for screening antituberculostic activity.
The authors are also thankful to Hamdard University, New Del-
hi for providing facilities to carry out the research work.
result of antituberculostic activity (Table 3) indicates
that the pyrimidines with chloro and p-methoxyphenyl
5, trimethoxyphenyl 6 showed more potent activity than
the reference drugs. Pyrimidines with chloro and chloro-
phenyl 3, and p-N-dimethylaminophenyl 4 substitutions
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Arch. Pharm. Chem. Life Sci. 2007, 340, 95–102
Novel Pyrimidines
99
Table 4. Antibacterial activity of 4-(49-substituted phenyl)-6-(49-
nitrophenyl)-2-(substituted imino) pyrimidines.
Compound
Concentra-
tion
(lg/mL)
Zone of Inhibition (in mm)
Gram-positive Gram-negative
Staph.
aureus
M. luteus E. coli
K. pneumo-
niae
5
6
100
200
500
100
200
500
100
200
500
100
200
500
100
200
500
100
200
500
100
200
500
100
200
500
100
200
500
100
200
500
50
–
–
–
16
19
21
–
–
–
–
–
–
–
18
–
–
–
–
–
–
–
–
–
–
16
19
–
–
–
–
–
–
–
–
–
–
–
–
–
18
20
–
–
–
15
18
20
–
–
–
–
–
17
–
–
–
–
–
–
16
19
21
15
18
20
17
20
22
21
20
7
8
–
17
20
–
–
–
–
–
–
–
9
12
17
18
24
26
–
–
18
20
–
–
–
15
18
20
–
–
–
–
–
–
23
22
15
19
–
–
–
–
–
–
–
–
–
–
–
–
25
23
15
18
20
17
19
20
–
Scheme 1. Synthesis of 4-(49-substituted phenyl)-6-(49-nitrophe-
nyl)-2-(substituted imino) pyrimidines.
–
–
15
17
22
21
25
Ampicillin
Chloram-
phenicol
Experimental
50
Chemistry
Melting points were determined in open capillaries on a Veego
VMP-1 melting point apparatus (Veego Instruments, Mumbai,
India) and are uncorrected. IR spectra were recorded in KBr on a
Perkin Elmer IR spectrophotometer (values in cm– 1; Perkin
Elmer), NMR spectra (in DMSO) on a JEOL Fx-100 FT-NMR spectro-
photometer using TMS as an internal standard and Mass spectra
on a JEOL Bx 102/DA-6000 spectrometer (JEOL, Tokyo, Japan).
The preparation of 4-(4-substitutedphenyl)-6-(4-nitrophenyl)-2-
(substituted imino) pyrimidines involves three steps.
(–) Denotes no activity
CO=CH); MS (m/z) 288 [M+ +1]; Anal. Calcd. for C15H10NO3Cl: C,
62.62; H, 3.50; N, 4.86. Found: C, 62.65; H, 3.48; N, 4.89. Adopting
this procedure, starting materials 10 and 19 were prepared by
reported procedure [18].
Preparation of 4-(4-chlorophenyl)-6-(4-
nitrophenyl)pyrimidin-2-amine (2)
Preparation of 3-(4-chlorophenyl)-1-(4-nitrophenyl)-2-
propen-1-one (1)
A mixture of 3-(49-chlorophenyl)-1-(49-nitrophenyl)-2-propen-1-
one 1 (0.01 mol) and guanidine hydrochloride (0.015 mol) was
added to NaOH (0.045 mol), water (2 mL), and ethanol (50 mL).
The reaction mixture was refluxed for 10 h and poured onto
crushed ice. The resultant solid was washed with dilute ethanol,
dried, and recrystallized from ethanol-chloroform mixture to
yield a yellow crystalline solid. Yield: 78.5%, mp 1258C; IR (KBr)
cm– 1: 3418 (NH2), 1595.07 (C=N), 1412 (NO2), 817 (C-Cl); 1H-NMR
(DMSO-d6) d: 4.80 (s, 2H, NH2), 6.63–8.21 (m, 8H, Ar-H), 8.45 (s, 1H,
Pyr-C-5); MS (m/z) 326 [M+]; Anal. Calcd. for C16H10N4O2Cl: C, 58.99;
H, 3.09; N, 17.20. Found: C, 58.95; H, 3.12; N, 17.16. Adopting this
An aqueous solution of sodium hydroxide (10% w/v, 10 mL) was
added to a solution of 4-chlorobenzaldehyde (0.02 mol) and 4-
nitroacetophenone (0.02 mol) in ethanol (6 mL). The reaction
mixture was stirred overnight at room temperature and then
poured into water (100 mL). After neutralization with HCl (10%
w/v), a pale yellow solid, which separated, was recrystallized
from ethanol to obtain a pale-yellow crystalline compound.
Yield: 85%, mp 1018C; IR (KBr) cm– 1: 2888 (CH), 1681.05 (C=O),
1
1606 (C=C), 1343 (NO2); H-NMR (DMSO-d6) d: 6.82 (d, 1H, J = 8.7
HZ, CH-Ar), 7.51–8.02 (m, 8H, Ar-H), 8.12 (d, 1H, J = 8.7 Hz,
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procedure, intermediates 11 and 20 were prepared by reported
procedure [19].
5-Chloro-3-{[4-(4-chlorophenyl)-6-(4-nitrophenyl)-
pyrimidin-2-ylimino]-methyl}-1,3-dihydro-indol-2-one (9)
IR (KBr) cm– 1: 3354 (NH), 1600 (CO), 1522 (C = N), 1370 (NO2), 776
1
(Ar-Cl); H-NMR (DMSO-d6) d: 3.91 (s, 1H, CH), 6.36 (s, 1H, CH),
Preparation of (4-chlorobenzylidene)-[4-(4-chlorophenyl)-
6-(4-nitrophenyl)pyrimidin-2-yl]amine (3)
6.55–7.78 (m, 12H, Ar-H), 8.25 (brs, 1H, NH); MS (m/z) 491 [M+ +1];
Anal. Calcd. for C24H13N5O3Cl2 : C, 58.79; H, 2.67; N, 14.28. Found:
C, 58.52; H, 2.55; N, 14.13.
Equimolar quantities of 4-(4-cholorophenyl)-6-(4-nitrophenyl)-2-
aminopyrimidine 2 (0.002 mol) and 3,4,5-trimethoxybenzalde-
hyde (0.002 mol) were dissolved in warm ethanol (75 mL) con-
taining glacial acetic acid (1 mL). The reaction mixture was
refluxed for 11 h and poured onto crushed ice. The resultant
pale-yellow solid was dried and recrystallized from ethanol to
get a pale-yellow crystalline solid. Yield: 65%, mp 92–948C; IR
(4-Chloro-benzylidene)-[4-(4-dimethylamino-phenyl)-6-
(4-nitro-phenyl)-pyrimidin-2-yl]-amine (12)
(KBr) cm– 1: 2920 (CH), 1590 (C=N), 815 (Ar-Cl); 1H-NMR (DMSO-d6)
d: 3.02 (s, 6H, N(CH3)2), 6.62 (s, 1H, CH), 7.41-8.45 (m, 13H, Ar-H);
MS (m/z) 458 [M+ +1]; Anal. Calcd. for C25H20N5O2Cl: C, 65.57; H,
4.40; N, 15.29. Found: C, 65.61; H, 4.43; N, 15.27.
1
(KBr) cm– 1: 2972 (CH), 1582 (C=N), 1390 (NO2), 845.3 (C-Cl); H-
NMR (DMSO-d6): d: 6.38 (s, 1H, CH), 6.52–8.69 (m, 13H, Ar-H); 13C-
NMR (CDCl3): d 115.4, 121.1 (2C), 127.2 (2C), 128.2 (2C), 129.4
(2C), 129.7 (2C), 129.9, 130.6 (2C), 132.8, 135.4, 139.6, 143.7,
149.4, 165.6, 166.8 (2C), 182.4; MS (m/z) 450 [M+ +1]; Anal. Calcd.
for C23H14N4O2Cl2: C, 61.48; H, 3.14; N, 12.47. Found: C, 61.40; H,
3.00; N, 12.13. Adopting this procedure, compounds 3–9, 12–
18, and 21–27 were prepared.
(4-Dimethylaminobenzylidene)-[4-(4-
dimethylaminophenyl)-6-(4-nitrophenyl)pyrimidin-2-
yl]amine (13)
IR (KBr) cm– 1: 2924 (CH), 1596 (C=N);1H-NMR (DMSO-d6) d: 3.00 (s,
12H, 2-N(CH3)2, 6.63 (s, 1H, CH), 7.67–8.41 (m, 13H, Ar-H); MS
(m/z) 443 [M+ +1]; Anal. Calcd. for C27H26N6O2: C, 69.51; H, 5.61; N,
18.01. Found: C, 69.55; H, 5.63; N, 18.00.
[4-(4-Chlorophenyl)-6-(4-nitrophenyl)pyrimidin-2-yl]-(4-
dimethylamino-benzylidene)amine (4)
IR (KBr) cm– 1: 2970 (CH), 1585 (C = N), 1388 (NO2), 845.7 (C-Cl); 1H-
NMR (DMSO-d6): d: 3.11 (s, 6H, N(CH3)2), 6.34 (s, 1H, CH), 6.52–8.72
(m, 13H, Ar-H); MS (m/z) 459 [M+ +1]; Anal. Calcd. for C25H20N5O2Cl
: C, 65.57; H, 4.40; N, 15.29. Found: C, 65.52; H, 4.35; N, 15.13.
[4-(4-Dimethylaminophenyl)-6-(4-nitrophenyl)pyrimidin-2-
yl]-(4-methoxy-benzylidene)amine (14)
1
IR (KBr) cm– 1: 2972 (CH), 1582 (C=N); H-NMR (DMSO-d6): d: 3.08
(s, 6H, N(CH3)2) 3.89 (s, 3H, OCH3), 6.39 (s, 1H, CH), 7.52–8.62 (m,
13H, Ar-H); MS (m/z) 454 [M+ +1]. Anal. Calcd. for C26H23N5O3: C,
68.86; H, 5.11; N, 15.44. Found: C, 68.52; H, 5.25; N, 15.13.
[4-(4-Chlorophenyl)-6-(4-nitrophenyl)pyrimidin-2-yl]-(4-
methoxy-benzylidene)amine (5)
IR (KBr) cm– 1: 2972 (CH), 1582 (C=N), 1390 (NO2), 845.3 (C-Cl); 1H-
NMR (DMSO-d6): d: 3.86 (s, 3H, OCH3), 6.35 (s, 1H, CH), 6.49-8.67
(m, 13H, Ar-H); MS (m/z) 445 [M+]; Anal. Calcd. for C24H17N4O3Cl: C,
64.80; H, 3.85; N, 12.59. Found: C, 64.62; H, 3.95; N, 12.23.
[4-(4-Dimethylaminophenyl)-6-(4-nitrophenyl)pyrimidin-2-
yl]-(3,4,5-trimethoxy-benzylidene)amine (15)
IR (KBr) cm– 1: 2968 (CH), 1589 (C=N);1H-NMR (DMSO-d6): d: 3.04 (s,
6H, N(CH3)2), 3.84 (s, 9H, OCH3), 6.34 (s, 1H, CH), 7.47–8.59 (m,
11H, Ar-H); 13C-NMR (CDCl3): d 39.4 (2C), 54.7 (3C), 104.3 (2C),
127.6 (2C), 128.9 (2C), 130.4 (2C), 130.6 (2C), 131.2 (2C), 134.9,
139.2, 142.7, 143.1, 148.9 (2C), 164.6, 165.9 (2C), 182.4; MS (m/z)
502 [M+ +1]; Anal. Calcd. for C27H27N5O5: C, 64.66; H, 5.43; N,
13.96. Found: C, 64.52; H, 5.25; N, 14.00.
[4-(4-Chlorophenyl)-6-(4-nitrophenyl)pyrimidin-2-yl]-
(3,4,5-trimethoxy-benzylidene)amine (6)
IR (KBr) cm– 1: 2968 (CH), 1589 (C=N), 1398 (NO2), 850 (C-Cl); 1H-
NMR (DMSO-d6): d: 3.86 (s, 9H, OCH3), 6.38 (s, 1H, CH), 6.47–8.59
(m, 11H, Ar-H); MS (m/z) 504 [M+ +1]; Anal. Calcd. for C26H21N4O5Cl:
C, 61.84; H, 4.19; N, 11.09. Found: C, 61.80; H, 4.16; N, 11.13.
[4-(4-Dimethylaminophenyl)-6-(4-nitrophenyl)pyrimidin-2-
[4-(4-Chlorophenyl)-6-(4-nitrophenyl-pyrimidin-2-yl)furan-
yl]furan-2-ylmethylene-amine (16)
2-ylmethylene-amine (7)
IR (KBr) cm– 1: 2979 (CH), 1598 (C = N); 1H-NMR (DMSO-d6): d 3.10
(s, 6H, N(CH3)2), 6.42 (s, 1H, CH), 7.27–8.39 (m, 12H, Ar-H); MS (m/
z) 414 [M+ +1]; Anal. Calcd. for C23H19N5O3: C, 66.82; H, 4.63; N,
16.94. Found: C, 66.62; H, 4.35; N, 16.90.
1
IR (KBr) cm– 1: 2976 (CH), 1594 (C=N), 1388 (NO2), 850 (C-Cl); H-
NMR (DMSO-d6): d: 6.38 (s, 1H, CH), 6.27–8.39 (m, 12H, Ar-H); MS
(m/z) 406 [M+ + ]; Anal. Calcd. for C21H13N4O3Cl: C, 62.31; H, 3.24;
N, 13.84. Found: C, 62.52; H, 3.35; N, 13.63.
[4-(4-Chlorophenyl)-6-(4-nitrophenyl)-pyrimidin-2-yl]-
Chromen-2-ylidene-[4-(4-dimethylaminophenyl)-6-(4-
chromen-2-ylidene-amine (8)
nitrophenyl)pyrimidin-2-yl]amine (17)
IR (KBr) cm– 1: 3028 (CH), 1700 (Cyclic C-O-C), 1617 (C = N), 1375
(NO2), 828.6 (C-Cl); 1H-NMR (DMSO-d6): d: 6.48 (d, 1H, J = 10.2 Hz,
CH), 6.72 (d, 1H, J = 10.2 Hz, CH), 6.92-8.48 (m, 13H, Ar-H); MS (m/z)
454 [M+ +1]; Anal. Calcd. for C25H15N4O3Cl: C, 66.01; H, 3.32; N,
12.31. Found: C, 66.05; H, 3.31; N, 12.35.
IR (KBr) cm– 1: 2920 (CH), 1700 (Cyclic C-O-C), 1596 (C=N), 1370.5
1
(NO2); H-NMR (DMSO-d6) d 3.02 (s, 6H, N(CH3)2), 6.48 (d, 1H, J =
10.4 Hz, CH), 6.64 (d, 1H, J = 10.4 Hz, CH), 7.35–8.42 (m, 13H, Ar-
H); MS (m/z) 463 [M+ +1]; Anal. Calcd. for C27H21N5O3: C, 69.96; H,
4.56; N, 15.11. Found: C, 69.93; H, 4.60; N, 15.13.
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Arch. Pharm. Chem. Life Sci. 2007, 340, 95–102
Novel Pyrimidines
101
5-Chloro-3-{[4-(4-dimethylaminophenyl)-6-(4-
5-Chloro-3-{[4-(4-methoxyphenyl)-6-(4-
nitrophenyl)pyrimidin-2-ylimino]-methyl}-1,3-dihydro-
nitrophenyl)pyrimidin-2-ylimino]methyl}-1,3-dihydro-
indol-2-one (18)
indol-2-one (27)
IR (KBr) cm– 1: 3354 (NH), 1600 (CO), 1522 (C=N), 1370 (NO2), 776
(Ar-Cl); 1H-NMR (DMSO-d6) d 3.03 (s, 6H, N(CH3)2), 6.55–7.78 (m,
12H, Ar-H), 8.45 (s, 1H, NH); MS (m/z) 498 [M+]; Anal. Calcd. for
C26H19N6O3Cl: C, 62.59; H, 3.83; N, 16.84. Found: C, 62.58; H, 3.87;
N, 16.82
IR (KBr) cm– 1: 3355 (NH), 2928 (CH), 1661 (CO), 1602 (C =N), 1368
(NO2), 823.4 (Ar-Cl); 1H-NMR (DMSO-d6) d 3.51 (s, 3H, OCH3), 6.65–
8.45 (m, 12H, Ar-H); 13C-NMR (CDCl3): d 35.9, 54.7, 113.9(2C),
115.9, 127.2 (2C), 127.9 (2C), 128.2 (2C), 129.3 (2C), 129.9 (2C),
131.1, 139.7, 142.8, 147.6, 161.2, 163.2, 165.4 (2C), 167.7, 179.8;
MS (m/z) 484 [M+ –1]; Anal. Calcd. for C25H16N5O4Cl: C, 61.79; H,
3.31; N, 14.41. Found: C, 61.78; H, 3.35; N, 14.45.
(4-Chlorobenzylidene)-[4-(4-methoxyphenyl)-6-(4-
Biological evaluation
The synthesized compounds were evaluated for their antiviral,
antituberculostic, and antibacterial activities.
nitrophenyl)pyrimidin-2-yl]amine (21)
IR (KBr) cm– 1: 2924 (CH), 1603 (C=N), 1366 (NO2), 824 (Ar-Cl); 1H-
NMR (DMSO-d6) d 3.81 (s, 3H, OCH3), 6.33 (s, 1H, CH), 6.66–8.73
(m, 13H, Ar-H); MS (m/z) 445 [M+ +1]; Anal. Calcd. for C24H17N4O3Cl:
C, 64.79; H, 3.85; N, 12.59. Found: C, 64.70; H, 3.84; N, 12.63.
Cytotoxicity assay
Each synthesized compound was separately dissolved in 1 mL of
distilled dimethyl sulphoxide (DMSO) and the volume was
brought up to 10 mL with maintenance medium to obtain a
stock solution of 1 mg/mL concentration. It was sterilized by fil-
tration and further dilutions were made from the stock. The
cytotoxicity assay was carried out using 0.1 mL of the cell sus-
pension, containing 10000 cells seeded in each well of a 96-well
microtitre plate (Tarsons India Pvt. Ltd., Kolkata, India). Fresh
medium containing different concentrations of the test sample
was added 24 h after the seeding. Control cells were incubated
without the test sample and with DMSO. The little percentage of
DMSO present in the wells (maximal 0.2%) was found not to
affect the experiment. The microtitre plates were incubated at
378C for a period of 72 h. 16 wells were used for each concentra-
tion of the test sample. The morphology of the cells was
inspected daily and observed for microscopically detectable
alterations, i. e., loss of monolayer, granulation, and vacuoliza-
tion in the cytoplasm. The CTC50 (the minimum concentration of
test drug required to kill 50% of exposed cell population) of each
test drug were determined by the standard MTT assay [20].
(4-Dimethylaminobenzylidene)-[4-(4-methoxyphenyl)-6-
(4-nitrophenyl)pyrimidin-2-yl]amine (22)
IR (KBr) cm– 1: 2924 (CH), 1596 (C = N), 1391 (NO2);1H-NMR (DMSO-
d6) d 3.06 (s, 6H, N(CH3)2), 3.84 (s, 3H, OCH3), 6.65 (s, 1H, CH),
7.67–8.41 (m, 13H, Ar-H); MS (m/z) 455 [M+ +1]; Anal. Calcd. for
C26H23N5O3: C, 68.86; H, 5.11; N, 15.44. Found: C, 68.52; H, 5.25;
N, 15.13.
3.1.19 (4-Methoxybenzylidene)-[4-(4-methoxyphenyl)-6-
(4-nitrophenyl)pyrimidin-2-yl]amine (23)
IR (KBr) cm-1: 2924 (CH), 1596 (C=N), 1391 (NO2); 1H-NMR (DMSO-
d6) d 3.89 (s, 6H, 2-OCH3), 6.67 (s, 1H, CH), 7.67-8.41 (m, 13H, Ar-H);
MS (m/z) 441 [M+ +1]; Anal. Calcd. for C25H20N4O4: C, 68.18; H,
4.58; N, 12.72. Found: C, 68.22; H, 4.25; N, 12.53.
[4-(4-Methoxyphenyl)-6-(4-nitrophenyl)pyrimidin-2-yl]-
Antiviral assay (MIC)
(3,4,5-trimethoxy-benzylidene)amine (24)
Confluent cell cultures in 96-well microtiter plates were inocu-
lated with 100 CCID50 of virus, (1 CCID50 being the virus dose
required to infect 50% of the cell cultures). After a 1–2 h period
of virus adsorption, residual virus was removed, and the cell cul-
tures were incubated at 378C in the presence of varying concen-
trations of the test compounds (dilutions were made based upon
CTC50). Viral cytopathogenicity was recorded microscopically as
soon as it reached completion in the control virus-infected cell
cultures that were not treated with the test compounds after 7–
8 days of post infection. The antiviral activity of the compounds
was expressed as the effective concentration required for inhi-
biting the viral cytopathic effect by 50% (MIC or EC50). The CTC50
and MIC of the test compounds were compared with the stan-
dard drugs brivudin (BVDU) and ribavirin under similar condi-
tions. By adopting the above procedure, the MIC or EC50 for all
the synthesized compounds were determined [21, 22].
IR (KBr) cm– 1: 2940 (CH), 1586 (C=N), 1391 (NO2), 1015, 823; H-
1
NMR (DMSO-d6) d 3.86 (s, 12H, 4-OCH3), 6.32 (s, 1H, CH), 6.66–8.23
(m, 11H, Ar-H); MS (m/z) 500 [M+]; Anal. Calcd. for C27H24N4O6: C,
64.79; H, 4.83; N, 11.19. Found: C, 64.71; H, 4.80; N, 11.17.
Furan-2-ylmethylene-[4-(4-methoxyphenyl)-6-(4-
nitrophenyl)pyrimidin-2-yl]amine (25)
IR (KBr) cm– 1: 2928 (CH), 1604 (C = N), 1370 (NO2), 845; 1H-NMR
(DMSO-d6): d 3.81 (s, 3H, OCH3), 6.32 (s, 1H, CH), 6.61-8.47 (m, 12H,
Ar-H); MS (m/z) 400 [M+]; Anal. Calcd. for C22H16N4O4: C, 65.99; H,
4.02; N, 13.99. Found: C, 65.96; H, 4.07; N, 13.98.
Chromen-2-ylidene-[4-(4-methoxyphenyl)-6-(4-
nitrophenyl)pyrimidin-2-yl]amine (26)
IR (KBr) cm– 1: 2920 (CH), 1700 (Cyclic C-O-C), 1596 (C=N), 1370.5
(NO2); 1H-NMR (DMSO-d6) d 3.82 (s, 3H, OCH3), 6.38 (d, 1H, J = 10.2
Hz, CH), 6.61 (d, 1H, J = 10.4 Hz, CH), 6.72–8.47 (m, 13H, Ar-H);
MS (m/z) 449 [M+]; Anal. Calcd. for C26H18N4O4: C, 69.32; H, 4.02; N,
12.43. Found: C, 69.32; H, 4.04; N, 12.40.
Antitubercular activity (Alamar blue susceptibility test
(MABA))
Antimicrobial susceptibility testing was performed in black,
clear-bottomed, 96-well microplates (black view plates; Packard
Instrument Company, Meriden, CT, USA) in order to minimize
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102
A. A. Siddiqui et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 95–102
background fluorescence. Outer perimeter wells were filled
with sterile water to prevent dehydration in experimental wells.
Initial drug dilutions were prepared in either dimethyl sulfox-
ide or distilled de-ionized water, and subsequent twofold dilu-
tions were performed in 0.1 mL of 7H9GC (no Tween 80) in the
microplates. BACTEC 12B-passaged inocula were initially diluted
1:2 in 7H9GC, and 0.1 mL was added to wells. Subsequent deter-
mination of bacterial titers yielded 16106 CFU/mL in plate wells
for M. tuberculosis H37Rv. Frozen inocula were initially diluted
1:20 in BACTEC 12B medium followed by a 1:50 dilution in
7H9GC. Addition of 1/10 mL to wells resulted in final bacterial
titers of 2.06105 CFU/mL for M. tuberculosis H37Rv. Wells contain-
ing drug only were used to detect autofluorescence of the com-
pounds. Additional control wells consisted of bacteria only (B)
and medium only (M). Plates were incubated at 378C. Starting at
day 4 of the incubation, 20 lL of 106Alamar blue solution (Ala-
mar Biosciences/Accumed, Westlake, OH, USA) and 12.5 mL of
20% Tween 80 were added to one B well and one M well, and
plates were re-incubated at 378C. Wells were observed at 12 and
24 h for a color change from blue to pink and for a reading of
50 000 fluorescence units (FU). Fluorescence was measured in a
Cytofluor II microplate fluorometer (PerSeptive Biosystems, Fra-
mingham, MA, USA.) in bottom-reading mode with excitation at
530 nm and emission at 590 nm. If the B wells became pink by
24 h, reagent was added to the entire plate. If the well remained
blue or 50000 FU were measured, additional M and B wells were
tested daily until a color change occurred, at which time
reagents were added to all remaining wells. Plates were then
incubated at 378C, and results were recorded at 24 h post-
reagent addition. Visual minimum inhibitory concentration
(MIC) was defined as the lowest concentration of drug that pre-
vented a color change. For fluorometric MICs, a background sub-
traction was performed on all wells with a mean of triplicate M
wells. Percent inhibition was defined as 1-(test well FU/mean FU
of triplicate B wells)6100. The lowest drug concentration effect-
ing an inhibition of A90% was considered the MIC. The percen-
tage inhibitions of bacterial growth at 6.25 lg/mL of all the
selected synthesized compounds are depicted in Table 3 along
with the MIC's of standard drugs [23, 24]. The compounds which
were shown 90 and above of percentage inhibition have been
taken up for second phase of screening to determine their actual
MIC's by TAACF, Birmingham, USA.
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microbial strains viz., Staphylococcus aureus, Micrococcus luteus,
Escherichia coli, and Klebsiella pneumoniae were procured from
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was compared with that of the reference standard antibiotics
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i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com