Synthesis and Antitubercular Activity of 2-(substituted phenyl/benzyl-amino)-6-(4-chlorophenyl)-5-(methoxycarbonyl)-4-methyl-3,6-dihydropyrimidin-1-ium Chlorides

Article abstract of DOI:10.1111/cbdd.12065

A series of 2-(substituted phenyl/benzyl-amino)-6-(4-chlorophenyl)-5-(methoxycarbonyl)-4-methyl-3,6-dihydropyrimidin-1-ium chlorides 7-13 and 15 was synthesized in their hydrochloride salt form. The title compounds were characterized by FT-IR, NMR (¹H and ¹³C) and elemental analysis. They were evaluated for their in vitro antitubercular activity against Mycobacterium tuberculosis H37Rv, multidrug resistance tuberculosis and extensively drug resistance tuberculosis by agar diffusion method and tested for the cytotoxic action on peripheral blood mononuclear cells by MTT assay. Among all the tested compounds in the series, compounds 7 and 11 emerged as promising antitubercular agents at 16μg/mL against multidrug resistance tuberculosis and over 64μg/mL against extensively drug resistance tuberculosis. The conformational features and supramolecular assembly of the promising compounds 7 and 11 were determined by single crystal X-ray study.

Full text of DOI:10.1111/cbdd.12065

Chem Biol Drug Des 2013; 81: 219–227
Research Article
Synthesis and Antitubercular Activity of 2-(substituted
phenyl⁄benzyl-amino)-6-(4-chlorophenyl)-5-
(methoxycarbonyl)-4-methyl-3,6-dihydropyrimidin-1-ium
Chlorides
Venugopala K. Narayanaswamy1, Susanta K.
Tuberculosis (TB) is an infectious disease caused by
Mycobacterium tuberculosis. It affects lungs and also
other parts of the body and has lead to death of 1.5 mil-
lion HIV-positive people from tuberculosis in 2010a. HIV-
Nayak2, Melendhran Pillay3, Renuka Prasanna4,
Yacoob M. Coovadia3 and Bharti Odhav1,*
1Department of Biotechnology and Food Technology,
Durban University of Technology, Durban 4001, South
infected individuals are up to 37 times more susceptible to
develop TBb. The emergence of multidrug-resistant tuber-
Africa
culosis (MDR-TB), extensively drug-resistant tuberculosis
(XDR-TB) (1), totally drug-resistant tuberculosis (2) and co-
infections with acquired immuno deficiency virus (AIDS) (3)
has been responsible for this serious situation. Further-
more, current drugs used for the treatment of TB are cyto-
toxic and showing decreasing efficacy due to the
development of resistant organisms (4). Therefore, new
and improved anti-TB drugs with new targets and mecha-
nism of action are urgently needed to gain sustained suc-
cessful treatment of MDR-TB, XDR-TB and totally drug-
resistant tuberculosis-infected patients (5).
2Center for Nano Science and Technology@Polimi, Istituto
Italiano di Tecnologia, Via Pascoli 70 ⁄ 3-20133 Milan, Italy
3Department of Microbiology, National Health Laboratory
Services, KZN Academic Complex, Inkosi Albert Luthuli
Central Hospital, Durban 4001, South Africa
4Department of Polymer Science & Technology, Sri
Krishnadevaraya University, Anantapur 515055, India
*Corresponding author: Bharti Odhav, odhavb@dut.ac.za
A series of 2-(substituted phenyl ⁄ benzyl-amino)-6-(4-
chlorophenyl)-5-(methoxycarbonyl)-4-methyl-3,6-dihy-
dropyrimidin-1-ium chlorides 7–13 and 15 was synthe-
sized in their hydrochloride salt form. The title
compounds were characterized by FT-IR, NMR (1H
and 13C) and elemental analysis. They were evaluated
for their in vitro antitubercular activity against Myco-
bacterium tuberculosis H37Rv, multidrug resistance
tuberculosis and extensively drug resistance tubercu-
losis by agar diffusion method and tested for the cyto-
toxic action on peripheral blood mononuclear cells by
MTT assay. Among all the tested compounds in the
series, compounds 7 and 11 emerged as promising
antitubercular agents at 16 lg ⁄ mL against multidrug
resistance tuberculosis and over 64 lg ⁄ mL against
extensively drug resistance tuberculosis. The confor-
mational features and supramolecular assembly of the
promising compounds 7 and 11 were determined by
single crystal X-ray study.
In 1893, the synthesis of multifunctionalized 3,4-dihydro-
pyrimidine-2(1H)-ones was reported by Pietro Biginelli (6).
Among pharmacologically important heterocyclic com-
pounds, dihydropyrimidine analogues have been reported
to have several pharmacological activities (7) such as, an-
tiviral (8), anticancer (9), antihypertensive (10), antitubercular
(11), antimicrobial (12), anti-inflammatory (13) and calcium
channel blocking action (14). Dihydropyrimidines are poten-
tial inhibitors of dihydrofolate reductase, which interrupts
the supply of thymidine in the folate cycle. This inhibits
DNA biosynthesis and influences cell proliferation (15)
which may be a mechanism to inhibit MDR or XDR-TB.
Keeping all these observations in mind and as a part of
our research work on the development of heterocyclic
compounds for pharmacological activity (16–18), we syn-
thesized and characterized 3,6-dihydropyrimidine ana-
logues 7–13 and 15 in their hydrochloride salt form
(Scheme 1) following Lipinski’s rule of five (19). These ana-
logues were then investigated for their in vitro activity
against M. tuberculosis reference strain H37RV, MDR-TB
strain and a well-characterized XDR-TB strain. To under-
stand the safety of the molecules, the title compounds
were tested for their cytotoxic potential against peripheral
blood mononuclear cells (PBMC) by evaluating their viabil-
Key words: anti-TB activity, cytotoxicity, dihydropyrimidine,
single crystal X-ray crystallography
Abbreviations: Et-OH, ethanol; MDR-TB, multidrug resistance
tuberculosis; MIC, minimum inhibitory concentration; XDR-TB,
extensively drug resistance tuberculosis.
Received 2 August 2012, revised 20 September 2012 and
accepted for publication 24 September 2012
ª 2012 John Wiley & Sons A/S. doi: 10.1111/cbdd.12065
219

Narayanaswamy et al.
Cl
Cl
O
O
O
NH2
HCl
Ethanol,
reflux, 8 h
POCl3
reflux, 16 h
O
NH
+
+
O
H2N
O
N
H
O
H
O
1
3
4
2
Cl
Cl
NH2
R4
O
O
O
R1
R2
Cl-
H
Isopropanol
reflux, 16 h
R3
R2
+
N
O
N+
R4
N
H
Cl
N
H
N
H
R3
R1
7 - 13
5
6
7 = R1 = H, R2 = Br, R3, R4 = H
8 = R1 = OH, R2 = H, R3 = NO2, R4 = H
1
= R = H, R2 = Br, R3 = F, R4 = H
9
10 = R1 = H, R2 = H, R3 = H, R4 = CN
11 = R1 = H, R2 = SCF33, R3 = H, R4 = H
1
2
4
12
= R = H, R = H, R = Br, R = H
13 = R1 = H, R2 = Cl, R3 = H, R4 = OH
Cl
Cl
H2N
O
O
O
OCH3
Cl-
Isopropanol
reflux, 16 h
O
N+
H
OCH3
N
+
N
H
N
H
N
H
Cl
OCH3
OCH3
5
14
15
Scheme 1: Synthesis of 3,6-dihydropyrimidine salts (7–13 and 15).
ity after exposure to the compounds using the MTT assay
(20). In addition, we report herein the single crystal X-ray
studies of two of the active molecules 7 and 11 from the
series to understand their conformational features and
supramolecular assembly.
solvent system and visualization with UV-light. Melting
points were determined on a Bu¨ chi Melting Point B-545
apparatus. The IR spectra were recorded on a Nicolet 6700
FT-IR spectrometry. 1H and 13C NMR spectra were
recorded on Bruker AVANCE III 400 MHz instruments in
DMSO as a solvent. Chemical shifts (d) were indicated in
parts per million downfield from tetramethylsilane and the
coupling constants (J) are recorded in Hertz. Splitting pat-
tern is abbreviated as follows; s, singlet; d, doublet; m, mul-
tiplet. Mass spectra were recorded using LC-MS-Agilent
1100 series with MSD (Ion trap) using 0.1% aqueous trifluo-
roacetic acid in acetonitrile system on C18-BDS column.
Elemental analysis was performed on Thermo Finningan
FLASH EA 1112 CHN analyzer, Italy.
Methods and Materials
All the chemicals were obtained from Sigma Aldrich, South
Africa and Merck, South Africa chemical company. Reac-
tions were monitored by thin layer chromatography (TLC).
Thin layer chromatography was performed on Merck 60 F-
254 silica gel plates with ethyl acetate and n-hexane (6:4) as
220
Chem Biol Drug Des 2013; 81: 219–227

Synthesis and Antitubercular Activity of 3,6-Dihydropyrimidin-1-ium Chlorides
Synthesis of methyl 4-(4-chlorophenyl)-6-methyl-2-
676. 1H-NMR (400 MHz, DMSO-d6) d 2.41 (s, 3H), 3.62
(s, 3H), 5.43 (s, 1H), 7.19–7.51 (m, 8H), 10.20 (s, 1H),
11.14 (s, 2H). 13C-NMR (400 MHz, DMSO-d6) d 17.53,
51.68, 103.31, 122.17, 123.19, 126.87, 128.46, 128.93,
129.70, 131.67, 132.98, 136.24, 140.00, 144.90, 148.71,
164.61. Anal. calcd for C19H18BrCl2N3O2 (471.2): C,
48.43; H, 3.85; N, 8.92%; found C, 48.39; H, 3.84; N,
8.93%.
oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (4)
A solution of methyl acetoacetate (0.12 mol), 4-chloro-
benzaldehyde (0.1 mol) and urea (0.1 mol) was refluxed in
the presence of concentrated hydrochloric acid (0.05 mol)
for 8 h in 5 mL of ethanol (Et-OH; Scheme 1). Completion
of reaction was monitored on TLC. The reaction mixture
was then cooled to room temperature, and the pure pre-
cipitate was collected by filtration. The solid obtained was
washed with cold Et-OH, dried and recrystallized using Et-
OH solvent. Appearance: yellow solid; yield 66%. mp 205–
206 ꢀC. IR (KBr) m ⁄ cm 3240, 3110, 1724, 1704, 1649,
1490, 1461, 781; 1H-NMR (400 MHz, DMSO-d6) d 2.30
(s, 3H), 3.92 (s, 3H), 5.43 (s, 1H), 7.13 (d, 2H,
J = 9.05 Hz), 7.51 (s, 1H), 7.86 (d, 2H, J = 9.05 Hz), 9.01
(s, 1H). 13C-NMR (400 MHz, DMSO-d6) d 18.66, 52.05,
54.35, 109.58, 113.20, 128.23, 136.26, 148.25, 153.40,
159.18, 167.76. MS: m ⁄ z 281 [M + H]+.
6-(4-Chlorophenyl)-2-(2-hydroxy-4-
nitrophenylamino)-5-(methoxycarbonyl)-4-methyl-
3,6-dihydropyrimidin-1-ium chloride (8)
Appearance: yellow solid; yield 67%. mp 172–173 ꢀC. IR
(KBr) m ⁄ cm 3356, 3193, 3067, 1707, 1674, 1588, 1492,
769. 1H-NMR (400 MHz, DMSO-d6) d 2.40 (s, 3H), 3.61
(s, 3H), 5.39 (s, 1H), 7.17 (d, J = 9.00 Hz, 1H), 7.34 (d,
J = 8.48 Hz, 2H), 7.45 (d, J = 8.44 Hz, 2H), 8.10–8.15 (m,
2H), 9.75 (s, 1H), 10.21 (s, 1H), 11.01 (s, 1H), 11.93 (s,
1H). 13C-NMR (400 MHz, DMSO-d6)
d 19.87, 50.77,
Synthesis of methyl 2-chloro-4-(4-chlorophenyl)-6-
methyl-1,4-dihydropyrimidine-5-carboxylate (5)
A solution of compound 4 (1 mol) in POCl3 (5 mol) was
refluxed for 16 h (Scheme 1). The reaction was monitored
by TLC. Unreacted POCl3 was evaporated completely and
the remaining residue was taken in ethyl acetate and
washed with 10% sodium bicarbonate solution followed
by water and finally brine solution. The ethyl acetate layer
was dried over sodium sulphate and evaporated to obtain
a solid which was recrystallized using Et-OH solvent.
Appearance: brown solid; yield 72%. mp 218–219 ꢀC. IR
(KBr) m ⁄ cm 3244, 3108, 1708, 1641, 1491, 1466, 786.
1H-NMR (400 MHz, DMSO-d6) d 2.32 (s, 3H), 3.91 (s, 3H),
5.44 (s, 1H), 7.13–7.86 (m, 4H), 9.02 (s, 1H). 13C-NMR
(400 MHz, DMSO-d6) d 18.72, 53.67, 54.35, 105.41,
113.20, 128.54, 135.29, 141.20, 145,67, 158.39, 167.76.
MS m ⁄ z 299 [M + H]+.
52.68, 99.77, 116.06, 116.92, 120.21, 128.20, 128.42,
129.57, 131.99, 138.23, 143.60, 148.79, 151.05, 157.11,
163.69, 165.94. Anal. calcd for C19H18Cl2N4O5 (453.3): C,
50.35; H, 4.00; N, 12.36%; found: C, 50.36; H, 4.04; N,
12.35%.
2-(3-Bromo-4-fluorophenylamino)-6-(4-
chlorophenyl)-5-(methoxycarbonyl)-4-methyl-3,6-
dihydropyrimidin-1-ium chloride (9)
Appearance: white solid; yield 71%. mp 236–236 ꢀC. IR
(KBr) m ⁄ cm 3191, 3032, 1718, 1676, 1583, 1487, 769,
699. 1H-NMR (400 MHz, DMSO-d6) d 2.41 (s, 3H), 3.59
(s, 3H), 5.35 (s, 1H), 7.32–7.77 (m, 7H), 9.63 (s, 1H),
10.68 (s, 1H), 11.25 (s,1H). 13C-NMR (400 MHz, DMSO-
d6)
d 17.57, 51.56, 51.97, 102.85, 116.22, 116.44,
120.50, 120.76, 122.59, 122.69, 128.70, 128.79, 129.38,
131.35, 131.44, 132.88, 140.16, 144.58, 149.52, 160.10,
162.58, 164.60. Anal. calcd for C19H17BrCl2FN3O2
(489.2): C, 46.65; H, 3.50; N, 8.59%; found C, 46.65; H,
3.51; N, 8.58%.
General procedure for the synthesis of 7–13 and 15
A solution of compound 5 (1 mmol) and aromatic substi-
tuted amine (1 mmol) in isopropanol (10 mL) was refluxed
for 16 h (Scheme 1). The reaction completion was moni-
tored by TLC. The reaction medium was cooled to room
temperature, and the product obtained was filtered,
washed with cold isopropanol and dried to get the pure
product. The product obtained was purified by column
chromatography using ethyl acetate and n-hexane (6:4) as
eluent (60–120 silica gel). Compounds 7–13 and 15 were
achieved as hydrochloride salts.
6-(4-Chlorophenyl)-2-(4-cyanophenylamino)-5-
(methoxycarbonyl)-4-methyl-3,6-dihydropyrimidin-
1-ium chloride (10)
Appearance: yellow solid; yield 63%. mp 116–117 ꢀC. IR
(KBr) m ⁄ cm 3194, 3063, 2226, 1673, 1562, 1489, 770.
1H-NMR (400 MHz, DMSO-d6) d 2.38 (s, 3H), 3.60 (s, 3H),
5.41 (s, 1H), 7.35 (t, J = 8.62 Hz, 4H), 7.44 (d,
J = 8.48 Hz, 2H), 7.80 (d, J = 8.60 Hz, 2H), 9.88 (s, 1H),
10.11 (s, 1H), 10.58 (s, 1H). 13C-NMR (400 MHz, DMSO-
2-(3-Bromophenylamino)-6-(4-chlorophenyl)-5-
(methoxycarbonyl)-4-methyl-3,6-dihydropyrimidin-
1-ium chloride (7)
Appearance: white solid; yield 65%. mp 217–218 ꢀC. IR
(KBr) m ⁄ cm 3199, 3068, 1707, 1671, 1582, 1475, 787,
d6) d 18.22, 51.37, 52.13, 102.30, 106.66, 118.81,
122.48, 128.10, 128.37, 128.80, 132.60, 133.70, 141.23,
147.73, 165.05. Anal. calcd for C20H18Cl2N4O2 (417.3): C,
57.57; H, 4.35; N, 13.43%; found C, 57.58; H, 4.35; N,
13.44%.
Chem Biol Drug Des 2013; 81: 219–227
221

Narayanaswamy et al.
6-(4-Chlorophenyl)-5-(methoxycarbonyl)-4-methyl-
2-(3-(trifluoromethylthio) phenylamino)-3,6-
dihydropyrimidin-1-ium chloride (11)
d 17.69, 51.29, 51.46, 55.23, 55.42, 98.47, 104.28,
115.86, 128.42, 128.70, 129.60, 132.71, 140.53, 149.67,
158.01, 160.64, 164.78. Anal. calcd for C22H25Cl2N3O4
(466.3): C, 56.66; H, 5.40; N, 9.01%; found C, 56.68; H,
5.36; N, 9.07%.
Appearance: pale yellow solid; yield 69%. mp 220–
221 ꢀC. IR (KBr) m ⁄ cm 3191, 3043, 1714, 1671, 1585,
1472, 1088, 766. 1H-NMR (400 MHz, DMSO-d6) d 2.42
(s, 3H), 3.62 (s, 3H), 5.44 (s, 1H), 7.36 (d, J = 8.48 Hz,
2H), 7.42–7.63 (m, 6H), 10.29 (s, 1H), 11.09 (s, 2H). 13C-
NMR (400 MHz, DMSO-d6) d 17.56, 25.44, 51.61, 51.69,
103.32, 124.31, 124.33, 126.67, 127.85, 128.43, 128.91,
130.80, 131.28, 132.97, 133.78, 136.35, 140.03, 148.62,
164.65. Anal. calcd for C20H18Cl2F3N3O2S (492.3): C,
48.79; H, 3.69; N, 8.53%; found C, 48.80; H, 3.68; N,
8.54%.
Single crystal X-ray crystallographic study
A particular size of single crystals 7 and 11 was deter-
mined by X-ray diffraction study using Bruker KAPPA
APEX II DUO diffractometer using graphite-monochroma-
˚
tor, Mo-Ka radiation (k = 0.71073 A; Table 1). Single crys-
tals of compounds 7 and 11 were grown from acetone by
slow evaporation method at room temperature for X-ray
study. Data collection was carried out at 100 (2) K temper-
ature using liquid Nitrogen (N2) cryo-system attached with
Oxford Cryostat. Cell refinement and data reduction were
2-(4-Bromophenylamino)-6-(4-chlorophenyl)-5-
(methoxycarbonyl)-4-methyl-3,6-dihydropyrimidin-
1-ium chloride (12)
c
performed using the program SAINT . The crystal structure
solution was worked out by full matrix least-squares
method using SHELXL97 and absorption correction per-
formed using SADABS (21). All the non-hydrogen atoms and
some of hydrogen atoms were located in difference Fou-
rier maps and were refined anisotropically and the remain-
ing hydrogen atoms were fixed geometrically and refined
isotropically. Graphical presentations were drawn using Or-
tep-3 and Mercury (22,23). The crystal structure solution
Appearance: pale yellow solid; yield 66%. mp 227–
228 ꢀC. IR (KBr) m ⁄ cm 3184, 3042, 1707, 1671, 1571,
1488, 771, 688. 1H-NMR (400 MHz, DMSO-d6) d 2.41 (s,
3H), 3.61 (s, 3H), 5.40 (s, 1H), 7.16 (d, J = 8.64 Hz, 2H),
7.34 (d, J = 8.48 Hz, 2H), 7.47 (d, J = 8.44 Hz, 2H), 7.64
(d, J = 8.64 Hz, 2H), 9.93 (s, 1H), 10.76 (s, 1H), 11.13 (s,
1H). 13C-NMR (400 MHz, DMSO-d6)
d 19.49, 50.84,
52.39, 99.86, 114.98, 123.88, 128.25, 128.54, 131.88,
131.99, 140.90, 143.41, 148.00, 150.94, 165.80. Anal.
calcd for C19H18BrCl2N3O2 (471.2): C, 48.43; H, 3.85; N,
8.92%; found C, 48.45; H, 3.86; N, 8.94%.
Table 1: Crystal data and measurement details for compounds 7
and 11
Crystal data
Compound 7
19H18BrCl2N3O2
884242
471.2
Needle
0.18 · 0.07 · 0.05
100 (2)
Compound 11
Formula
C
C20H18Cl2F3N3O2S
884243
492.33
Needle
0.17 · 0.08 · 0.04
100 (2)
2-(3-Chloro-5-hydroxyphenylamino)-6-(4-
chlorophenyl)-5-(methoxycarbonyl)-4-methyl-3,6-
dihydropyrimidin-1-ium chloride (13)
CCDC Number
Formula weight
Crystal morphology
Crystal size(mm)
Temperature ⁄ K
Radiation
Appearance: white solid; yield 67%. mp 198–199 ꢀC. IR
(KBr) m ⁄ cm 3646, 3198, 3062, 1719, 1677, 1581, 1481,
787. 1H-NMR (400 MHz, DMSO-d6) d 2.40 (s, 3H), 3.58
(s, 3H), 5.32 (s, 1H), 6.77–7.32 (m, 5H), 7.43 (d,
J = 8.48 Hz, 2H), 9.41 (s, 1H), 10.30 (s, 2H), 10.80 (s,
Mo-Ka
Mo-Ka
˚
Wavelength (A)
0.71073
Monoclinic
P 21 ⁄ c
7.7294(9)
23.290(3)
11.0671(1)
90
97.608(3)
90
1974.7(1)
4
0.71073
Monoclinic
C 2 ⁄ c
28.174(3)
7.7682(7)
21.941(2)
90
112.101(2)
90
4449.2(7)
8
Crystal system
Space group
1H). 13C-NMR (400 MHz, DMSO-d6)
d 17.68, 51.46,
˚
a (A)
52.03, 102.35, 115.15, 116.31, 118.94, 128.63, 128.67,
130.67, 132.72, 140.60, 145.33, 148.79, 157.38, 164.78.
Anal. calcd for C19H18Cl3N3O3 (442.7): C, 51.55; H, 4.10;
N, 9.49%; found C, 51.57; H, 4.08; N, 9.50%.
˚
b (A)
˚
c (A)
a (ꢀ)
b (ꢀ)
c (ꢀ)
3
˚
Volume (A )
Z
6-(4-Chlorophenyl)-2-(2,4-dimethoxybenzylamino)-
5-(methoxycarbonyl)-4-methyl-3,6-
dihydropyrimidin-1-ium chloride (15)
Density (gm ⁄ cm3)
l (1 ⁄ mm)
1.58
2.372
951.8
1.47
0.433
2015.7
F (000)
Appearance: pale yellow solid; yield 71%. mp 221–
222 ꢀC. IR (KBr) m ⁄ cm 3222, 3029, 1714, 1680, 1585,
1468, 762. 1H-NMR (400 MHz, DMSO-d6) d 2.37 (s, 3H),
3.60 (s, 3H), 3.74 (s, 6H), 4.30 (d, J = 14.72 Hz, 1H), 4.45
(d, J = 14.32 Hz, 1H), 5.43 (s, 1H), 6.41 (d, J = 7.68 Hz,
1H), 6.54 (s, 1H), 7.17 (d, J = 8.36 Hz, 1H), 7.30 (d,
J = 8.48 Hz, 2H), 7.41 (d, J = 8.40 Hz, 2H), 8.50 (s, 1H),
10.26 (s, 1H), 11.11 (s, 1H). 13C-NMR (400 MHz, DMSO-d6)
h (min, max)
Total number of refln
No. Unique refln
No. of parameters
R_ obs, wR2_obs
1.8, 27.2
14372
4366
258
0.042, 0.085
0.910,)0.758
2.0, 26.4
14087
4559
294
0.044, 0.086
0.263, )0.295
Dqmin(eA)3),
˚
)3
˚
Dqmax(eA
)
GooF
1.012
0.989
222
Chem Biol Drug Des 2013; 81: 219–227

Synthesis and Antitubercular Activity of 3,6-Dihydropyrimidin-1-ium Chlorides
was worked out by full matrix least-squares method using
24 h in a 5% CO2 humidified incubator. After 24 h, 10 lL
of MTT salt (5 mg ⁄ mL) was added to each well and incu-
bated for a further 4 h in a 37 ꢀC humidified incubator.
The purple formazan precipitate formed was dissolved in
50 lL of 2% (v ⁄ v) dimethyl sulphoxide ⁄ water and incu-
bated for another 30 min at room temperature. The absor-
bance was read at 570 nm with a reference wavelength of
650 nm in an ELISA microplate reader. The results were
evaluated against untreated cells. All the experiments were
conducted in triplicate.
SHELXL97 and absorption correction performed using SAD-
d
ABS . All the non-hydrogen atoms and some hydrogen
atoms were located in difference Fourier maps and were
refined anisotropically, and other hydrogen atoms were
fixed geometrically and refined isotropically.
Antitubercular activity
All the mycobacterial culture preparations were performed
in a Level II Biosafety Laboratory using personal protective
equipment. H37Rv (ATCC No. 25177), a MDR-TB strain
and XDR-TB strains were clinical isolates (Department of
Microbiology, Inkosi Albert Luthuli Central Hospital,
National Health Laboratory Services, Durban) tested for
antimycobacterial activity in Middlebrook 7H10 agar
enriched with OADC (0.005%, v ⁄ v, oleic acid; 0.5%, 171
w ⁄ v, BSA; 0.2%, w ⁄ v, glucose; 0.02%, v ⁄ v, catalase and
0.085%, w ⁄ v, NaCl) and incubated at 37 ꢀC for 3 weeks.
Rifampcin, isoniazid, ofloxacin and kannamycin were used
as controls. Title compounds 7–13 and 15 were dissolved
in distilled water to yield concentration ranging from 64 to
4 lg ⁄ mL and these were sterilized using a 0.22 l polycar-
bonate filter. Tests were conducted in triplicate.
Results and Discussion
Chemistry
The 3,6-dihydropyrimidine analogues 7–13 and 15 were
synthesized as outlined in Scheme 1 starting from methyl
4-(4-chlorophenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimi-
dine-5-carboxylate 4 which has been synthesized by Bigi-
Determination of minimum inhibitory
concentration
Minimum inhibitory concentration (MIC) was performed
according to the agar dilution method as previously
described (24) with slight modifications. Mycobacterium
tuberculosis reference strains H37Rv, MDR and XDR were
cultured in Middlebrook 7H10 medium (25). Cultures with
confluent growth were vortexed thoroughly in a sterile tube
containing 4.5 mL phosphate buffer, 0.05% tween 80 with
glass beads (5 mm diameter) to remove clumps. After set-
tling for 45 min, the clear bacterial supernatant was stan-
dardized to a McFarland Number 1 with sterile water. This
Figure 1: ORTEP diagram with atom labelling of 7 with displace-
ment ellipsoids drawn at the 50% probability level except hydro-
gen atoms as arbitary circle. Quaternary chloride salt of N1
(pyrimidine nitrogen) is shown as proton transfer.
gave
a
bacterial concentration of approximately
1 · 107 cfu ⁄ mL. The bacterial suspension was diluted 10-
fold with sterile water and 100 lL of the appropriate dilu-
tion was spotted onto Middlebrook 7H10 agar plates con-
taining 4–64 lg ⁄ mL of the drug compounds. The plates
were read after a 3-week incubation period at 37 ꢀC and
the MIC was defined as the minimum drug concentration
to inhibit growth of the organism when compared to the
drug-free controls.
Cytotoxicity evaluation
The cytotoxic effect of the compounds was tested using
the standard protocol (20). Stock solution of 1 mg ⁄ mL of
title compounds 7–13 and 15 was dissolved in water and
further twofold dilutions were made to obtain concentra-
tions of 15.625–1000 lg ⁄ mL. Ficoll separated PBMC
(1 · 106 cells ⁄ mL) were seeded in 96-well plates contain-
ing 180 lL of media and 20 lL of each sample was
added to each well and this was incubated at 37 ꢀC for
Figure 2: ORTEP diagram with atom lebelling of 11 with dis-
placement ellipsoids drawn at the 50% probability level except
hydrogen atoms as arbitary circle. Quaternary chloride salt of N1
(pyrimidine nitrogen) is shown as proton transfer.
Chem Biol Drug Des 2013; 81: 219–227
223

Narayanaswamy et al.
nelli reaction by employing Lewis acid according to the
procedure described in our previous study (26). The purifi-
cation of the product was accomplished using Et-OH by
recrystallization method and the yield of the product was
66%. The formation of methyl 4-(4-chlorophenyl)-6-methyl-
by IR, NMR (1H and 13C) and elemental analysis. In infra-
red spectra of 7–13 and 15, the secondary amino group
and ester carbonyl group are observed in the range 3184–
A
2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
4
was
confirmed by its 1H-NMR spectrum, in which the appear-
ance of methyl singlet peak at d = 2.31 ppm indicated the
presence of methyl group on heterocyclic ring due to less
deshielding effect and methoxy singlet peak in ester group
at d = 3.92 ppm due to more deshielding effect. In 13C-
NMR, d = 159.18 ppm was accounted for the less de-
shielded carbonyl carbon on heterocyclic ring and
d = 167.76 ppm was accounted for more deshielded car-
bonyl carbon in ester group. On LC-MS molecular mass of
the compound was in good agreement with the molecular
weight of compound 4. Synthesis of methyl 2-chloro-4-(4-
chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxyl-
ate 5 was achieved by treating compound 4 with phos-
phorous oxychloride for 16 h. The yield of compound 5
was 72% after recrystallization using Et-OH. Formation of
methyl 2-chloro-4-(4-chlorophenyl)-6-methyl-1,4-dihydro-
pyrimidine-5-carboxylate 5 was confirmed by proton NMR
spectrum, where the disappearance of a proton from the
nitrogen atom that is adjacent to para chlorophenyl ring.
This can be attributed in 13C-NMR spectrum in which
missing of less deshielded carbonyl carbon is observed
that is due to the transformation of heterocyclic carbonyl
carbon to heterocyclic carbon chlorine single bond. Its for-
mation is further confirmed by LC-MS spectral data in
which molecular ion peak is observed at m ⁄ z 299 due to
M + H peak. Title compounds 7–13 and 15 were achieved
as hydrochloride salt by refluxing equimolar proportion of
compound 5 and mono ⁄ disubstituted aromatic amines in
isopropanol medium. The yield of the compounds was
found to be in the range of 63–71% after purification by
column chromatography. The structures of novel 3,6-dihy-
dropyrimidine analogues 7–13 and 15 were characterized
B
Figure 3: N-HÁ Á ÁCl hydrogen bonds form infinite molecular chain,
and C-HÁ Á ÁO and C-HÁ Á ÁCl weak hydrogen bonds lead dimer for-
mation for 7 (A) and 11 (B). Interactions are shown as black dot-
ted lines.
Table 2: Intermolecular interactions of compounds 7 and 11
˚
˚
˚
˚
D-XÁ Á ÁA(A)
D-X (A)
XÁ Á ÁA (A)
DÁ Á ÁA (A)
—D-XÁ Á ÁA (ꢀ)
Symmetry code
Compound 7
Compound 11
N1-H1Á Á ÁCl2
N2-H2Á Á ÁCl2
N3-H3Á Á ÁCl2
C11-H11Á Á ÁCl2
C15-H15Á Á ÁO2
Cg1Á Á ÁCg2
0.77 (3)
0.86 (4)
0.76 (4)
0.93
2.39 (3)
2.24 (4)
2.57 (4)
2.81
3.100 (3)
3.070 (3)
3.268 (3)
3.678 (3)
3.290 (4)
–
3.148 (2)
3.101 (2)
3.198 (2)
3.628 (3)
3.298 (4)
3.649 (3)
3.391 (3)
3.898 (3)
–
154 (3)
164 (3)
152 (3)
156
151
–
149 (2)
162 (3)
155 (3)
163
132
163
1)x,)y,1)z
2)x,)y,1)z
2)x,)y,1)z
x,y,1 + z
2)x,)y,1)z
2)x,)y,2)z
1 ⁄ 2)x,3 ⁄ 2)y,)z
1 ⁄ 2)x,1 ⁄ 2)y,)z
1 ⁄ 2)x,1 ⁄ 2)y,)z
)1 ⁄ 2 + x,1 ⁄ 2 + y,z
)x,1)y,)z
0.93
2.45
–
3.675 (2)
2.44 (2)
2.28 (3)
2.45 (2)
2.68
2.59
2.73
2.54
2.97
N1-H1Á Á ÁCl2
N2-H2Á Á ÁCl2
N3-H3Á Á ÁCl2
C1-H1CÁ Á ÁCl2
C15-H15Á Á ÁO2
C18-H18Á Á ÁCl2
C8-H8Á Á ÁF3
0.80 (2)
0.86 (3)
0.81 (2)
0.98
0.95
0.95
x,y,z
0.95
0.95
148
167
–
x,1)y,1 ⁄ 2 + z
x,1)y,1 ⁄ 2 + z
1 ⁄ 2)x,3 ⁄ 2)y,)z
C9-H9Á Á ÁCg1
Cg1Á Á ÁCg1
–
3.996 (2)
Cg1 = Centroid of six-member ring C14–C19; Cg2 = Centroid of six-member ring C7–C12.
224
Chem Biol Drug Des 2013; 81: 219–227

Synthesis and Antitubercular Activity of 3,6-Dihydropyrimidin-1-ium Chlorides
3222 and 1705–1719 per cm, respectively. The nitrile
and which evidence the hydrochloride salt formation with
protonation of N1 (pyrimidine nitrogen) atom and Cl2 (chlo-
ride ion) in the crystal structure.
stretching is observed at 2226 per cm for compound 10.
The 1H-NMR spectrum of 7–13 and 15 indicated the
chemical shift of the methyl group on heterocyclic ring and
ester methoxy group in the range of d = 2.33–2.42 ppm
and d = 3.54–3.62 ppm, respectively. Heterocyclic me-
thine proton is observed in the range of d = 5.26–
5.44 ppm. Salt form of the title compounds were con-
firmed by proton NMR in which proton on quaternary
nitrogen that was involved in the formation of chloride salt
was observed in the range of d = 10.18–10.98 ppm and
also supported by single crystal X-ray studies. In 13C-
NMR, ester carbonyl carbon is observed in the range of
d = 164.60–165.94 ppm. Elemental analysis results were
within 0.04% of the calculated values of the proposed
title compounds 7–13 and 15. The IR, NMR (1H & 13C)
and elemental analysis data are discussed in detail under
experimental section.
The molecular packing of 7 is mainly based on all the
three N-H groups that participate in hydrogen bond with
the chloride ion (Table 2), additional weak interactions of
C-HÁ Á ÁCl, C-HÁ Á ÁO and pÁ Á Áp enhance the stability of
three-dimensional molecular assembly (Figure 3A, Table 2).
Similarly, in case of 11, the N-HÁ Á ÁCl hydrogen bonds form
infinite molecular chain along with weak C-HÁ Á ÁO hydrogen
bonds that form dimer (Figure 3B, Table 2).
Further, additional weak intermolecular interactions of C-
HÁ Á ÁF (27), C-HÁ Á Áp (28,29) along with pÁ Á Áp (30) (Table 2)
short contacts in 11 structure do not alter much in basic
three-dimensional structure in comparison with 7 structure.
However, the molecular assembly is preferred as zig-zag
arrangement with all the intermolecular interactions that
are shown along a-axis and b-axis of the unit cell for the 7
and 11, respectively (Figure 4A,B).
Single crystal X-ray studies
Crystallographic details are listed in Table 1. Figures 1 and
2 provide the thermal ellipsoid plot with atom labelling of 7
and 11 drawn at the 50% probability level, respectively,
Pharmacology
3,6-Dihydropyrimidine analogues 7–13 and 15 were
screened for their in vitro antitubercular and cytotoxicity
activity following standard protocol. The purity of the com-
pounds was ascertained by HPLC and it was found to be
more than 99% and their structures have been elucidated
by spectral interpretation. To investigate the role of the
substitution types and patterns on the aryl ⁄ benzyl ring
which is attached to dihydropyrimidine ring through sec-
ondary amine group (-NH-) for in vitro antitubercular and
cytotoxicity activity, various mono and disubstituted com-
pounds were prepared. Compounds 7–13 and 15 were
A
Table 3: In vitro antitubercular activity of 3,6-dihydropyrimidine
analogues 7–13 and 15
B
Minimum inhibitory concentration
(lg ⁄ mL)
H37Rv(ATCC
Compounds
25177)
MDR-TB
XDR-TB
7
8
9
10
11
12
13
15
4
8
8
8
4
16
32
16
2
1
0.5
2
16
32
32
32
16
64
32
32
8
>64
>64
>64
>64
>64
>64
>64
>64
8
Standards Isoniazid
Rifampicin
Ofloxacin
Kannamycin
32
16
32
64
16
32
Figure 4: The hydrogen bonds and weak intermolecular interac-
tions (as black dotted lines) lead to zig-zag molecular packing in
the unit cell for 7 structure along a-axis (A) and for the 11 struc-
ture along b-axis (B).
MDR-TB, multidrug resistance tuberculosis; XDR-TB, extensively
drug resistance tuberculosis.
Chem Biol Drug Des 2013; 81: 219–227
225

Narayanaswamy et al.
evaluated for their in vitro antitubercular activity against
H37Rv, MDR-TB and XDR-TB strains. The results of the
antitubercular activity are tabulated in Table 3. Com-
pounds 7 and 11 exhibited activity at 4 lg ⁄ mL against
H37Rv and were emerged as promising molecules against
MDR-TB at 16 lg ⁄ mL. Compound 7 having electron with-
drawing bromine atom at third position of the aryl group
that is connected to dihydropyrimidine nucleus through
secondary amine bridge exhibited significant antitubercular
activity at 16 lg ⁄ mL against MDR-TB. Compound 11 also
revealed similar activity at 16 lg ⁄ mL having trifluoromethyl-
thio group at third position of the aryl ring which is con-
nected to dihydropyrimidine through secondary amine
bridge. However, compound 12 having bromine atom at
fourth position of the aryl ring exhibited moderate activity
at 64 lg ⁄ mL against MDR-TB. Compound 10 having
cyano group at fourth position exhibited activity similar to
that of compound 12. Analogues such as 8, 9, 13 and 15
that are having disubstituted phenyl amino group on dihy-
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Appendix S1. Spectral details such as IR and NMR (1H
and 13C) of compounds 7–13 and 15, single-crystal X-ray
data of compounds 7 and 11 (CCDC number 884242 and
884243, respectively).
Chem Biol Drug Des 2013; 81: 219–227
227