Facile transformation of Biginelli pyrimidin-2(1H)-ones to pyrimidines. In vitro evaluation as inhibitors of Mycobacterium tuberculosis and modulators of cytostatic activity

Article abstract of DOI:10.1016/j.ejmech.2011.03.010

A series of pyrimidine derivatives bearing amine substituents at C-2 position were obtained from Biginelli 3,4-dihydropyrimidin-2(1H)-ones and the effect of structural variation on anti-TB activity against Mycobacterium tuberculosis H₃₇Rv strai

Full text of DOI:10.1016/j.ejmech.2011.03.010

European Journal of Medicinal Chemistry 46 (2011) 2290e2294
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Facile transformation of Biginelli pyrimidin-2(1H)-ones to pyrimidines.
In vitro evaluation as inhibitors of Mycobacterium tuberculosis and modulators
of cytostatic activity
,
a
b
b
c
d
Kamaljit Singh a , Kawaljit Singh , Baojie Wan , Scott Franzblau , Kelly Chibale , Jan Balzarini
*
a Organic Synthesis Laboratory, Department of Applied Chemical Sciences and Technology, Guru Nanak Dev University, Amritsar 143005, India
b Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612-723, USA
c Department of Chemistry and Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
d Rega Institute for Medical Research, Katholieke Universiteit Leuven, 10 Minderbroedersstraat, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pyrimidine derivatives bearing amine substituents at C-2 position were obtained from Bigi-
nelli 3,4-dihydropyrimidin-2(1H)-ones and the effect of structural variation on anti-TB activity against
Mycobacterium tuberculosis H37Rv strain and antiviral activity in a series of cell cultures was evaluated.
While the compounds were found to possess structure dependent cytostatic activity, these were not
found to be efficient inhibitors of M. tuberculosis nor did they inhibit a broad variety of DNA or RNA
viruses in cell culture.
Received 18 January 2011
Received in revised form
2 March 2011
Accepted 4 March 2011
Available online 15 March 2011
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Biginelli compounds
Pyrimidines
Tuberculosis
MABA
Inhibitors
Cytostatic activity
1. Introduction
replication inhibitors and depict a variety of other biological effects.
Although a large number of DHPM derivatives have been prepared
The nitrogen heterocycles in general and pyrimidines in
particular are found in several biologically active natural products
and depict considerable therapeutic potential [1]. In view of wide
spectrum biological activities such as anti allergic [2], antitumor
[3], antipyretic [4], anti-inflammatory [4] and antiparasitic [5]
activities, exhibited by synthetic pyrimidine based scaffolds,
a number of analogues have garnered considerable attention.
During a screening effort for antiviral agents, we found that mul-
tifunctionalized tetrahydropyrimidines derivatives bearing bulky
C-2 alkyl substituents depict cytostatic activity and inhibit prolif-
eration of murine leukemia, murine mammary carcinoma, human
T-lymphocyte and human cervix carcinoma cells [6]. 3,4-Dihy-
dropyrimidin-2-(1H)-ones (DHPMs) and their appropriately func-
tionalized derivatives have interesting pharmacological profiles [7].
These are potent antihypertensive agents, mitotic kinesin inhibi-
tors, a1a-adrenergic receptor antagonists, or hepatitis B virus
in a single-pot Biginelli multi component reaction [8] (MCR) and its
variants [9], very useful and convincing structural variability of
these interesting heterocycles have been achieved through chem-
ical functionalization of all the six positions around the DHPM core
[10]. Recently, we reported [6] on the facile conversion of DHPMs
into tetrahydropyrimidines. As part of an ongoing, multi-faceted
program aimed toward development of small molecules as thera-
peutic agents, herein we report a straightforward conversion of
Biginelli DHPMs into C-2 amine substituted pyrimidine derivatives
and their screening as inhibitors of Mycobacterium tuberculosis and
their inhibitory effect against the proliferation of some cell cultures.
2. Chemistry
Oxidation of DHPM derivatives 1 (Scheme 1) to 1,2-dihy-
dropyrimidin-2-one derivatives 2 was readily achieved using pyr-
idinium chlorochromate (PCC) as reported by us earlier [11].
Subsequent treatment of appropriate 2 with phosphorous oxy-
chloride at 105 ꢀC furnished corresponding 2-chloro derivatives
3aec (Scheme 1), in 85%, 91% and 93% yield, respectively. Further
* Corresponding author. Tel.: þ91 183 2258853; fax: þ91 183 2258819 20.
E-mail address: kamaljit19in@yahoo.co.in (K. Singh).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.010

K. Singh et al. / European Journal of Medicinal Chemistry 46 (2011) 2290e2294
2291
O
R1
O
R1
R1
O
R1
O
R2
R2
R2
R2
N
PCC
R3XH
O
N
O
O
NH
POCl3
105 0C
O
N
N
H
O
N
XR3
N
H
O
N
Cl
1
2
3
4
a: R1 = H, R2 = Et
b: R1 = C6H5, R2 = Et
c: R1 = C6H5, R2 = Me
Scheme 1. Synthesis of 2-substituted pyrimidine-5-carboxylate 4 derivatives.
reaction of 3aec with N- and O-nucleophiles, furnished corre-
sponding 2-substituted pyrimidine derivatives 4, in very good
yields (Table 1). This fairly simple and efficacious protocol can be
visualized as a general protocol for obtaining a range of C-2
substituted pyrimidine analogues, for obtaining C-2 diversified
pyrimidine derivatives.
Replacing 3-hydroxypropyl amine substituent from C-2 position of
4f with n-butylamino substituent (4h) saw both an increase in %
inhibition as well as marginal decrease in MIC. However, none of
these derivatives were found to be markedly active compared to the
reference compounds.
While screening pyrimidine derivatives for their activity against
a broad variety of DNA and RNA viruses (including HIV) in the
appropriate cell culture models, we observed that while none of the
C-2 alkyl/aryl/amine substituted dihydropyrimidinone derivatives
showed appreciable activity against any of the investigated viruses
at subtoxic concentrations, C-2 amine substituted pyrimidine
derivatives 4e and 4d proved [18] markedly cytostatic against
3. Result and discussion
Tuberculosis (TB) is a deadly contagious disease that spreads
through the air. Tuberculosis frequently attacks lungs (as pulmo-
nary TB) but can also affect the central nervous system, the circu-
latory system, genitourinary system, bones, joints and the
lymphatic system. In 2008, there were estimated 8.9e9.9 million
incident cases of TB, 9.6e13.3 million prevalent cases and 1.1e1.7
million deaths among HIV infected individuals. If not treated, of the
more than 2 billion total individuals infected with TB, the ones
infected with active pulmonary TB are potentially contagious and
pose serious threat [12]. TB is a disease of poverty affecting mostly
young adults and more than half of all deaths occur in Asia. The
main reasons for the rising number of people infected with TB
bacterium can be attributed to wide spread intake of immuno-
suppressive drugs, HIV/AIDS, substance abuse, neglect or inefficient
implementation of TB control programmes, poor management of
TB infected patients, emergence of drug-resistant strains etc. A
cumulative total of 36 million TB patients were successfully treated
and over 6 million deaths were averted in Directly Observed
Treatment, Short-course (DOTS) during 1995e2005 and Stop TB
strategy programmes since 2006. Almost 30,000 cases of multi-
drug-resistant TB (MDR-TB) and extensively drug-resistant TB
(XDR-TB) were notified in 2008 [12].
MDCK cell cultures (IC50: 0.9
mg/mL and 1.2
mg/mL, respectively).
Compound 4e was more cytotoxic (MCC: 4e10
mg/mL) to confluent
cell cultures (i.e., human embryonic lung cells, feline Crandell
kidney cells) than 4d (MCC: ꢁ100 mg/mL) in these cell cultures.
Therefore, representative members of the newly synthesized
compounds 4 were evaluated for their inhibitory effect against the
proliferation of murine leukemia (L1210), murine mammary
carcinoma (FM3A), human T-lymphocyte (CEM), and human cervix
carcinoma (HeLa) cells (Table 3). Whereas, pyrimidine derivatives
bearing amino (4aec, 4feh), morpholino (4n) and ethoxy (4o)
substituents at the C-2 position (Table 1) depicted a marginal
cytostatic activity of the test compounds (IC50: 86e500 mM), more
bulky aryl group containing derivatives such as 4iel, bearing
groups such as tryptamine (4i), 2-hydroxyaniline (4j), 4-hydrox-
yaniline (4k) and 2-aminoaniline (4l) rendered a significantly
higher antiproliferative activity (IC50: 13e58 mM) (Table 3). The C-2
piperidine substituted pyrimidine derivative 4m also showed
higher cytostatic activity. 4-Hydroxyanilne substituted pyrimidine
derivatice 4k showed highest cytostatic activity (IC50: 13
mM)
against CEM cells.
As the literature precedence attests [13] there is only one report
on the evaluation of aniline pyrimidines [14] as antituberculosis
agents but no report of the testing of 2-aminosubstituted pyrimi-
dine 5-carboxyalte derivatives such as 4. While isoniazid (Table 2)
acts through inhibition of cell-wall synthesis and inhibits InhA -
a NADH specific enoylase-reductase involved in the biosynthesis of
fatty acids in mycobacteria [15], moxifloxacin is a broad spectrum
antibacterial agent that acts through inhibition of the top-
oisomerase II (DNA gyrase) [16] and topoisomerase IV, required for
bacterial DNA replication, transcription, repair, and recombination.
PA-824 kills nonreplicating M. tuberculosis by intracellular NO
release [17]. These examples led us to test selected derivatives 4
(Table 2) for their antituberculosis activity against M. tuberculosis.
From the MICs in Table 2, it can be deduced that the presence of
an N-benzyl group at C-2 position (4d) or better a 3-aminoaniline
substituent (4l) seems to be useful for significant antitubercular
activity. The presence of an ethyl ester, rather than methyl ester
substituent at C-5 position of the pyrimidine core (4d vs 4e) led to
improvement in MIC values. Replacing the 3-aminoaniline
substituent in 4l with 2-hydroxyaniline, piperidine or morpholine
substituent at C-2 position to form 4j, 4m and 4n, respectively, only
raised the MIC, without a significant effect on % inhibition.
Table 1
Synthesis of C-2 elaborated pyrimidine 4 derivatives.
S. No.
Compound
R1
R2
X
R3
Yield of 4 (%)
1.
2.
3.
4.
5.
6.
7.
8.
9.
4aa
4ba
4cb
4db
4eb
4fb
H
C6H5
H
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C2H5
C2H5
C2H5
C2H5
CH3
C2H5
C2H5
C2H5
C2H5
NH
NH
NH
NH
NH
NH
NH
NH
NH
H
H
75
80
82
92
85
75
87
97
65
C6H5CH2
C6H5CH2
C6H5CH2
(CH2)3OH
CH(CH3)2
n-C4H9
4gb
4hb
4ib
CH2CH2
(1H-indol-3-yl)
2-OHC6H4
4-OHC6H4
3-NH2C6H4
e(CH2)2eCH2e
(CH2)2e
e(CH2)2eOe
(CH2)2e
C2H5
10.
11.
12.
13.
4jb
C6H5
C6H5
C6H5
C6H5
C2H5
C2H5
C2H5
C2H5
NH
NH
NH
N
85
94
75
68
4kb
4lb
4mb
14.
4nb
4ob
C6H5
C6H5
C2H5
C2H5
N
O
70
87
15.
a
NH3 gas, THF, rt.
EtOH, 80ꢀ C.
b

2292
K. Singh et al. / European Journal of Medicinal Chemistry 46 (2011) 2290e2294
Fourier-transform spectrophotometer in the range 400e4000 cmꢂ1
using chloroform as medium. Melting points were determined in
open capillaries and are uncorrected. For monitoring the progress
of a reaction and for comparison purpose, thin layer chromato-
graphy (TLC) was performed on pre-coated aluminium sheets
Merck (60F254, 0.2 mm) using an appropriate solvent system. The
chromatograms were visualized under UV light. For column chro-
matography silica gel (60e120 mesh) was employed and eluents
were ethyl acetate/hexane mixtures.
Table 2
The structure and anti-TB activity (MIC) against Mycobacterium tuberculosis for
selected pyrimidine-5-carboxylate 4 derivatives.
Compound
% Inhibition at 128
mM
MIC(
63.8
>128
>128
117.7
121.8
31.2
>128
125.3
0.12
m
M)
4d
4e
4f
4h
4j
4l
4m
4n
93
3
57
99
92
99
86
96
e
5.2. General procedure for the synthesis of compound 3
Isoniazid
Moxifloxacin
PA-824
e
0.47
0.48
A solution of compound 2 (10 mmol) in phosphorous oxy-
chloride (10 mL) was heated under reflux for 30 min. The resulting
reaction mixture was distilled under reduced pressure to remove
excess phosphorous oxychloride. Last traces of phosphorous oxy-
chloride were removed by azeotrophic distillation with dry
benzene and crude product was purified by column chromato-
graphy to afford pure product 3.
e
4. Conclusion
In summary, a simple and efficient method for the trans-
formation of Biginelli dihydropyrimidin-2(1H)-ones to pyrimidine
derivatives bearing mainly amine substituents at C-2 position has
been devised. The methodology holds potential for the introduc-
tion of a number of tailor-made substituents at the C-2 position of
DHPMs in a synthetically useful manner. While none of the
compounds tested for inhibition of M. tuberculosis displayed useful
activity, the nature of the C-2 substituent was found to modulate
the cytostatic activity in cell culture.
5.2.1. 2-Chloro-4-methylpyrimidine-5-carboxylic acid ethyl ester
(3a)
Viscous liquid. Rf: 0.6 (25% EtOAc/hexane). Yield: 85%. IR (KBr):
nmax 3350, 1750, 1650, 650 cmꢂ1. 1H NMR (300 MHz, CDCl3, 25 ꢀC):
d
1.44 (t, J ¼ 7.2 Hz, 3H, estereCH3), 2.84 (s, 3H, C6eCH3), 4.42 (q,
J ¼ 7.2 Hz, 2H, estereCH2), 9.02 (s, 1H, C4eH). 13C NMR (75 MHz,
CDCl3, 25 ꢀC):
d 14.0, 24.6, 63.6, 78.5, 162.0, 162.8, 164.7, 172.5. Anal.
Calcd. for C8H9N2O2Cl: C, 47.88; H, 4.49; N, 13.96; Found: C, 47.40;
H, 4.10; N, 14.10. MS: m/z 200.5 (Mþ).
5. Experimental section
5.2.2. 2-Chloro-4-methyl-6-phenylpyrimidine-5-carboxylic acid
ethyl ester (3b)
5.1. General
Viscous liquid. Rf: 0.8 (25% EtOAc/hexane). Yield: 91%. IR (KBr):
All liquid reagents were dried/purified following recommended
drying agents and/or distilled over 4 Å molecular sieves. THF was
dried (Naebenzophenone ketyl) under nitrogen. 1H NMR
(300 MHz) and 13C (75 MHz) NMR spectra were recorded in CDCl3
on a multinuclear Jeol FT-AL-300 spectrometer with chemical shifts
nmax 3245, 1740, 1655, 715 cmꢂ1. 1H NMR (300 MHz, CDCl3, 25 ꢀC):
d
1.07 (t, J ¼ 7.2 Hz, 3H, estereCH3), 2.61 (s, 3H, C6eCH3), 4.20 (q,
J ¼ 7.2 Hz, 2H, estereCH2), 7.42e7.67 (m, 5H, ArH). 13C NMR
(75 MHz, CDCl3, 25 ꢀC):
d 13.5, 22.4, 62.1, 124.3, 128.3, 128.5, 130.6,
136.2, 160.4, 166.1, 166.9, 168.5. Anal. Calcd. for C14H13N2O2Cl: C,
60.76; H, 4.70; N, 10.13; Found: C, 60.36; H, 4.35; N, 10.02. MS: m/z
276.8 (Mþ).
being reported in parts per million (
d
) relative to internal tetra-
methylsilane (TMS,
d
0.0, 1H NMR) or (CDCl3,
d
77.0, 13C NMR). Mass
spectra were recorded from Indian Institute of Integrative Medicine
(CSIR), Jammu, under electron impact at 70 eV on a Bruker Dal-
tonics Esquire 3000 spectrometer. Elemental analysis was per-
formed on FLASH EA 112 (Thermoelectron Corporation) analyzer at
Department of Chemistry, Guru Nanak Dev University, Amritsar
and the results are quoted in %. IR recorded on FTIR Shimadzu 8400
5.2.3. 2-Chloro-4-methyl-6-phenylpyrimidine-5-carboxylic acid
methyl ester (3c)
Viscous liquid. Rf: 0.7 (25% EtOAc/hexane). Yield: 93%. IR (KBr):
nmax 3245, 1710, 1610, 665 cmꢂ1. 1H NMR (300 MHz, CDCl3, 25 ꢀC):
d
2.61 (s, 3H, C6eCH3), 3.58 (s, 3H, estereCH3), 7.41e7.61 (m, 5H,
ArH). 13C NMR (75 MHz, CDCl3, 25 ꢀC):
d
22.4, 52.7, 124.0, 128.2,
Table 3
128.6, 130.8, 136.0, 160.5, 166.0, 167.5, 168.5. Anal. Calcd. for
C13H11N2O2Cl: C, 59.43; H, 4.19; N, 10.67; Found: C, 59.23; H, 4.30;
N, 10.20.MS: m/z 262.5 (Mþ).
Inhibitory effect against the proliferation of murine leukemia (L1210), murine
mammary carcinoma (FM3A), human T-lymphocyte (CEM) and human cervix
carcinoma (HeLa) cells.
a
Compound
IC50
(m
M)
5.3. Synthesis of compounds 4a and b
L1210
FM3A
CEM
HeLa
4a
4b
4c
4f
4g
4h
4i
4j
4k
4l
245 ꢃ 34
402 ꢃ 139
378 ꢃ 83
238 ꢃ 9
132 ꢃ 28
164 ꢃ 13
28 ꢃ 6
432 ꢃ 96
336 ꢃ 177
306 ꢃ 6
213 ꢃ 0
174 ꢃ 2
192 ꢃ 12
34 ꢃ 4
207 ꢃ 45
382 ꢃ 166
480 ꢃ 29
150 ꢃ 33
118 ꢃ 44
162 ꢃ 40
31 ꢃ 3
183 ꢃ 23
216 ꢃ 29
147 ꢃ 13
213 ꢃ 14
86 ꢃ 45
138 ꢃ 2
23 ꢃ 9
Compound 3a and b (1.9 mmol) was treated with THF saturated
with ammonia gas (evolved by warming 30% aqueous ammonia
solution) at room temperature for 45 min. The resulting reaction
mixture was distilled under reduced pressure to remove excess THF
and crude product was purified through crystallization to afford
compound 4a and 4b.
58 ꢃ 4
52 ꢃ 4
43 ꢃ 3
50 ꢃ 11
24 ꢃ 5
24 ꢃ 9
24 ꢃ 2
13 ꢃ 4
5.3.1. 2-Amino-4-methylpyrimidine-5-carboxylic acid ethyl ester
(4a)
46 ꢃ 3
43 ꢃ 1
46 ꢃ 8
50 ꢃ 2
4m
4n
4o
43 ꢃ 2
55 ꢃ 1
72 ꢃ 41
ꢁ500
47 ꢃ 3
>500
>500
ꢁ500
White solid. Rf: 0 6 (60% EtOAc/hexane). Yield: 75%. M.p.
250e253 ꢀC (Methanol). IR (KBr): nmax 3300, 1730, 1700, 1450,
251 ꢃ 45
239 ꢃ 54
120 ꢃ 66
135 ꢃ 71
50% inhibitory concentration.
1260 cmꢂ1. 1H NMR (300 MHz, DMSO, 25 ꢀC):
d
1.26 (t, J ¼ 6.9 Hz,
a

K. Singh et al. / European Journal of Medicinal Chemistry 46 (2011) 2290e2294
2293
3H, estereCH3), 2.49 (s, 3H, C6eCH3), 4.19 (q, J ¼ 6.9 Hz, 2H,
estereCH2), 7.34 (br, 2H, NH), 8.61 (s, 1H, C4eH). 13C NMR (75 MHz,
DMSO, 25 ꢀC):
14.1, 24.1, 59.9, 111.4, 160.8, 163.7, 164.8, 169.6. Anal.
Calcd. for C8H11N3O2: C, 53.04; H, 6.04; N, 23.20; Found: C, 53.32; H,
5.86; N, 22.90. MS: m/z 182 (Mþ1).
5.4.4. 2-(3-Hydroxypropylamino)-4-methyl-6-phenylpyrimidine-
5-carboxylic acid ethyl ester (4f)
Viscous liquid. Rf: 0.3 (30% EtOAc/hexane). Yield: 75%. IR (KBr):
nmax 3431, 2925, 1717, 1597, 1261, 726 cmꢂ1 1H NMR (300 MHz,
CDCl3, 25 ꢀC):
d
.
d
0.94 (t, J ¼ 7.2 Hz, 3H, estereCH3), 1.71e1.78 (m, 2H,
CH2NH), 2.50 (s, 3H, C6eCH3), 3.59e3.68 (m, 4H, 2ꢄCH2), 4.04 (q,
5.3.2. 2-Amino-4-methyl-6-phenylpyrimidine-5-carboxylic acid
ethyl ester (4b)
J ¼ 7.2 Hz, 2H, estereCH2), 5.59 (br, 1H, NH), 7.39e7.53 (m, 5H, ArH).
13C NMR (75 MHz, CDCl3, 25 ꢀC):
d 13.5, 22.8, 33.1, 37.3, 58.3, 61.1,
White solid; Rf: 0.7 (80% EtOAc/hexane). Yield: 80%. M.p.
127.7,128.4,129.6,138.7,161.7. Anal. Calcd. for C17H21N3O3: C, 64.76;
H, 6.67; N, 13.33; Found: C, 64.20; H, 6.20; N, 12.90. MS: m/z 338
(Mþ23).
290e292 ꢀC (Methanol). IR (KBr): nmax 3409, 3177, 1700, 1649, 1550,
1276 cmꢂ1. 1H NMR (300 MHz, CDCl3, 25 ꢀC):
d
0.94 (t, J ¼ 7.2 Hz, 3H,
estereCH3), 2.48 (s, 3H, C6eCH3), 4.05 (q, J ¼ 7.2 Hz, 2H, estereCH2),
5.70 (br, 2H, NH), 7.39e7.53 (m, 5H, ArH). 13C NMR (75 MHz, CDCl3,
5.4.5. 2-Isopropylamino-4-methyl-6-phenylpyrimidine-5-
carboxylic acid ethyl ester (4g)
25 ꢀC):
d 13.4, 22.6, 61.1, 116.4, 127.7, 128.3, 129.5, 138.6, 161.8, 166.5,
167.5, 168.4. Anal. Calcd. for C14H15N3O2: C, 65.37; H, 5.84; N, 16.34;
Found: C, 65.20; H, 5.50; N, 15.90. MS: m/z 258 (Mþ1).
Viscous liquid. Rf: 0.9 (20% EtOAc/hexane). Yield: 87%. IR (KBr):
nmax 3250, 1705, 1590, 1256, 1382 cmꢂ1. 1H NMR (300 MHz, CDCl3,
25 ꢀC):
d
0.94 (t, J ¼ 7.2 Hz, 3H, estereCH3), 1.25 (d, J ¼ 6.6 Hz, 6H,
5.4. General procedure for the synthesis of compound 4ceo
(CH3)2CH), 2.47 (s, 3H, C6eCH3), 4.04 (q, J ¼ 7.2 Hz, 2H, estereCH2),
4.23e4.32 (m, 1H, CH(CH3)2), 5.40 (br, 1H, NH), 7.38e7.53 (m, 5H,
To a solution of compound 3 (1.9 mmol) in absolute ethanol
(20 mL) was added respective amine (2.85 mmol) and heated at
80 ꢀC until reaction get completed (2e3 h, TLC). The resulting
reaction mixture was distilled under reduced pressure to remove
excess ethanol and crude product was purified by column chro-
matography to afford pure product 4cen in good yield. Compound
4o was prepared by heating compound 3b (1.9 mmol) in absolute
ethanol (20 mL) for 2 h at 80 ꢀC. The resulting reaction mixture was
distilled under reduced pressure to remove excess ethanol and
crude product was purified by column chromatography to afford
pure product 4o.
ArH). 13C NMR (75 MHz, CDCl3, 25 ꢀC):
d 13.4, 22.7, 22.9, 42.7, 60.9,
114.8, 127.8, 128.1, 129.3, 139.2, 160.4, 166.1, 167.2, 168.8. Anal. Calcd.
for C17H21N3O2: C, 68.23; H, 7.02; N, 14.05; Found: C, 68.10; H, 6.85;
N, 13.70.MS: m/z 300 (Mþ1).
5.4.6. 2-Butylamino-4-methyl-6-phenylpyrimidine-5-carboxylic
acid ethyl ester (4h)
Viscous liquid. Rf: 0.8 (20% EtOAc/hexane). Yield: 97%. IR (KBr):
nmax 3310, 1730, 1560, 1382, 720 cmꢂ1 1H NMR (300 MHz, CDCl3,
.
25 ꢀC):
d 0.92e0.97 (m, 6H, CH3 and estereCH3), 1.37e1.47 (m, 2H,
CH2), 1.54e1.64 (m, 2H, CH2), 2.48 (s, 3H, C6eCH3), 3.46e3.52 (m,
2H, CH2NH), 4.04 (q, J ¼ 7.2 Hz, 2H, estereCH2), 5.31 (br, 1H, NH),
5.4.1. 2-Benzylamino-4-methylpyrimidine-5-carboxylic acid ethyl
ester (4c)
7.38e7.53 (m, 5H, ArH). 13C NMR (75 MHz, CDCl3, 25 ꢀC):
d 13.5,13.7,
19.9, 23.0, 31.7, 41.0, 60.9, 114.9, 127.8, 128.1, 129.3, 139.2, 161.2,
166.1, 168.9. Anal. Calcd. for C18H23N3O2: C, 69.00; H, 7.35; N, 13.42;
Found: C, 68.82; H, 6.93; N, 13.11. MS: m/z 336 (Mþ23).
White solid. Rf: 0.8 (20% EtOAc/hexane). Yield: 82%. M.p.
93e95 ꢀC (DCM/Hexane). IR (KBr): nmax 3200, 3115, 1720, 1545,
1225 cmꢂ1. 1H NMR (300 MHz, CDCl3, 25 ꢀC):
d
1.36 (t, J ¼ 7.2 Hz, 3H,
estereCH3), 2.66 (s, 3H, C6eCH3), 4.31 (q, J ¼ 7.2 Hz, 2H,
estereCH2), 4.70 (d, J ¼ 6.0 Hz, 2H, CH2NH), 5.94 (br, 1H, NH),
7.24e7.36 (m, 5H, ArH), 8.85 (s, 1H, C4eH). 13C NMR (75 MHz,
5.4.7. 2-[2-(1H-Indol-3-yl)-ethylamino]-4-methyl-6-
phenylpyrimidine-5-carboxylic acid ethyl ester (4i)
Brownish solid. Rf: 0.5 (30% EtOAc/hexane). Yield: 65%. M.p.
CDCl3, 25 ꢀC):
d 14.3, 24.7, 29.6, 45.3, 60.4, 127.4, 127.5, 128.6, 138.4,
115e117 ꢀC (DCM/Hexane). IR (KBr): nmax 3364, 3259, 1693, 1556,
161.1, 162.2, 165.4. Anal. Calcd. for C15H17N3O2: C, 66.42; H, 6.27; N,
15.50; Found: C, 66.90; H, 6.01; N, 15.18. MS: m/z 272 (Mþ1).
1261, 697 cmꢂ1.1H NMR (300 MHz, CDCl3, 25 ꢀC):
d
0.94 (t, J ¼ 7.2 Hz,
3H, estereCH3), 2.48 (s, 3H, C6eCH3), 3.08 (t, J ¼ 6.9 Hz, 2H, CH2),
3.84 (q, J ¼ 6.9 Hz, 2H, CH2NH), 4.04 (q, J ¼ 7.2 Hz, 2H, estereCH2),
5.45 (br,1H, NH), 7.03e7.68 (m,10H, ArH), 8.04 (br,1H, NH).13C NMR
5.4.2. 2-Benzylamino-4-methyl-6-phenylpyrimidine-5-carboxylic
acid ethyl ester (4d)
(75 MHz, CDCl3, 25 ꢀC):
d 13.5, 22.9, 25.4, 41.5, 61.0,111.1,113.0,115.1,
White solid. Rf: 0.7 (20% EtOAc/hexane). Yield: 92%. M.p.
118.9, 119.3, 122.0, 127.3, 127.9, 128.2, 129.3, 136.3, 139.1, 162.1, 166.1,
168.9, 172.5. Anal. Calcd. for C24H24N4O2: C, 72.00; H, 6.00; N, 14.00;
Found: C, 71.84; H, 5.84; N, 13.83. MS: m/z 423 (Mþ23).
70e72 ꢀC (DCM/Hexane). IR (KBr): nmax 3258, 3126, 1715, 1558,
1255 cmꢂ1. 1H NMR (300 MHz, CDCl3, 25 ꢀC):
d
0.95 (t, J ¼ 7.2 Hz,
3H, estereCH3), 2.49 (s, 3H, C6eCH3), 4.05 (q, J ¼ 7.2 Hz, 2H,
estereCH2), 4.71 (d, J ¼ 6.0 Hz, 2H, CH2NH), 5.63 (br, 1H, NH),
5.4.8. 2-(2-Hydroxyphenylamino)-4-methyl-6-phenylpyrimidine-
5-carboxylic acid ethyl ester (4j)
7.25e7.55 (m, 10H, ArH). 13C NMR (75 MHz CDCl3, 25 ꢀC):
d 13.5,
22.9, 45.3, 61.0, 115.5, 127.2, 127.5, 127.9, 128.2, 128.5, 129.4, 138.9,
139.0, 161.0, 168.8. Anal. Calcd. for C21H21N3O2: C, 72.62; H, 6.05; N,
12.10; Found: C, 72.30; H, 5.83; N, 11.92. MS: m/z 348 (Mþ1).
Yellowish solid. Rf: 0.5 (30% EtOAc/hexane). Yield: 85%. M.p.
145e147 ꢀC (DCM/Hexane). IR (KBr): nmax 3361, 1715, 1562, 1258,
737 cmꢂ1. 1H NMR (300 MHz, CDCl3, 25 ꢀC):
d
0.98 (t, J ¼ 7.2 Hz, 3H,
estereCH3), 2.57 (s, 3H, C6eCH3), 4.09 (q, J ¼ 7.2 Hz, 2H,
5.4.3. 2-Benzylamino-4-methyl-6-phenylpyrimidine-5-carboxylic
acid methyl ester (4e)
estereCH2), 6.84e7.58 (m, 9H, ArH), 7.27 (br, 1H, NH), 10.23 (br, 1H,
OH). 13C NMR (75 MHz, CDCl3, 25 ꢀC):
d 13.5, 22.7, 61.5, 120.1, 120.5,
Light brownish solid. Rf: 0.7 (30% EtOAc/hexane). Yield: 85%. M.p.
122.2, 126.0, 127.2, 127.8, 128.5, 130.2, 135.0, 137.6, 148.6, 158.6,
167.6, 186.8. Anal. Calcd. for C20H19N3O3: C, 68.77; H, 5.44; N, 12.03;
Found: C, 68.32; H, 5.14; N, 11.82. MS: m/z 372 (Mþ23).
103e105 ꢀC (DCM/Hexane). IR (KBr): nmax 3261, 3127, 1721, 1558,
1249 cmꢂ1. 1H NMR (300 MHz, CDCl3, 25 ꢀC):
d
2.47 (s, 3H, C6eCH3),
3.58 (s, 3H, estereCH3), 4.71 (d, J ¼ 6.0 Hz, 2H, CH2NH), 5.68 (br, 1H,
NH), 7.23e7.55 (m,10H, ArH).13C NMR (75 MHz, CDCl3, 25 ꢀC):
22.9,
d
5.4.9. 2-(4-Hydroxyphenylamino)-4-methyl-6-phenylpyrimidine-
5-carboxylic acid ethyl ester (4k)
45.3, 51.9, 115.2, 127.2, 127.5, 127.9, 128.2, 128.5, 129.5, 138.8, 138.9,
161.1, 169.4. Anal. Calcd. for C20H19N3O2: C, 72.07; H, 5.70; N, 12.61;
Found: C, 72.23; H, 5.90; N, 12.30. MS: m/z 334 (Mþ1).
Viscous liquid. Rf: 0.3 (30% EtOAc/hexane). Yield: 94%. IR (KBr):
nmax 3365, 1710, 1560, 1250, 730 cmꢂ1. 1H NMR (300 MHz, CDCl3,

2294
K. Singh et al. / European Journal of Medicinal Chemistry 46 (2011) 2290e2294
25 ꢀC):
d
0.98 (t, J ¼ 7.2 Hz, 3H, estereCH3), 1.65 (s, 1H, OH), 2.54 (s,
with test samples in 7H12 medium [19] using the microplate Ala-
mar Blue assay [20] (MABA). The MIC was defined as the lowest
concentration effecting a reduction of ꢁ90% in fluorescence relative
to untreated controls.
3H, C6eCH3), 4.09 (q, J ¼ 7.2 Hz, 2H, estereCH2), 6.78e7.60 (m, 9H,
ArH), 7.20 (br, 1H, NH). 13C NMR (75 MHz, CDCl3, 25 ꢀC):
d
13.5, 22.8,
61.3, 115.5, 116.6, 121.7, 121.8, 127.9, 128.3, 129.9, 131.7, 138.6, 151.8,
158.8, 158.9, 166.0, 167.3, 168.6. Anal. Calcd. for C20H19N3O3: C,
68.77; H, 5.44; N, 12.03; Found: C, 68.90; H, 5.30; N, 11.90. MS: m/z
372 (Mþ23).
5.6. Cytostatic activity assays
Murine leukemia L1210, murine mammary carcinoma FM3A,
human T-lymphocyte CEM and human cervix carcinoma HeLa cells
were suspended at 300,000e500,000 cells/mL in RPMI-1640
culture medium supplemented with 10% fetal bovine serum and
5.4.10. 2-(3-Aminophenylamino)-4-methyl-6-phenylpyrimidine-5-
carboxylic acid ethyl ester (4l)
Viscous liquid. Rf: 0.3 (20% EtOAc/hexane). Yield: 75%. IR (KBr):
nmax 3550, 1705, 1550, 1250, 735 cmꢂ1. 1H NMR (300 MHz, CDCl3,
2 mM
L-glutamine, and 100 ml of the cell suspensions were added to
25 ꢀC):
d
0.98 (t, J ¼ 7.2 Hz, 3H, estereCH3), 2.56 (s, 3H, C6eCH3),
100 l of an appropriate dilution of the test compounds in 96-well-
m
microtiter plates. After incubation at 37 ꢀC for two (L1210 and
FM3A) or three (CEM and HeLa) days, the cell number was deter-
mined using a Particle Counter ZI (Coulter, Analis, Ghent, Belgium).
The number of the suspension cells could be counted directly; the
number of the monolayer HeLa cells was counted after detachment
of the cells upon trypsinization. The IC50 was defined as the
compound concentration required for inhibiting cell proliferation
by 50%.
4.09 (q, J ¼ 7.2 Hz, 2H, estereCH2), 6.38e6.41 (m, 1H, ArH),
6.97e7.62 (m, 8H, ArH), 7.32 (br, 1H, NH). 13C NMR (75 MHz, CDCl3,
25 ꢀC):
d 13.5, 22.9, 61.2, 105.9, 109.7, 109.8, 117.0, 128.0, 128.3, 129.6,
138.6, 140.1, 146.8, 158.6, 165.8, 167.1, 168.4. Anal. Calcd. for
C20H20N4O2: C, 68.97; H, 5.75; N, 16.09; Found: C, 68.93; H, 5.40; N,
15.93. MS: m/z 349 (Mþ1).
5.4.11. 2-(Piperidin-1-yl)-4-methyl-6-phenylpyrimidine-5-
carboxylic acid ethyl ester (4m)
Yellowish solid. Rf: 0.5 (10% EtOAc/hexane). Yield: 68%. M.p.
Acknowledgments
80e82 ꢀC (DCM/Hexane). IR (KBr): nmax 3412, 2932, 2852, 1718,
1558, 700 cmꢂ1 1H NMR (300 MHz, CDCl3, 25 ꢀC):
. d 0.94 (t,
The authors are grateful for the financial support from UGC
(F. No. 37-188/2009(SR)), CSIR (01(2364)/10/EMR-II) and K.U. Leuven
(GOA no. 10/14). KS thanks CSIR for a senior research Fellowship.
J ¼ 7.2 Hz, 3H, estereCH3), 1.56e1.70 (m, 3ꢄ2H, CH2epipe), 2.48 (s,
3H, C6-CH3), 3.88 (t, J ¼ 4.8 Hz 2ꢄ2H, NCH2epipe), 4.03 (q,
J ¼ 7.2 Hz, 2H, estereCH2), 7.36e7.58 (m, 5H, ArH). 13C NMR
(75 MHz, CDCl3, 25 ꢀC):
d 13.4, 23.1, 24.7, 25.7, 44.5, 60.7,113.1,128.0,
References and notes
129.1, 139.7, 160.1, 165.6, 166.8, 169.1. Anal. Calcd. for C19H23N3O2: C,
70.15; H, 7.08; N, 12.92; Found: C, 69.91; H, 6.95; N, 12.63. MS: m/z
326 (Mþ1).
[1] D.T. Hurst, Introduction to the Chemistry and Biochemistry of Pyrimidines,
Purines and Pteridines. John Wiley & Sons, New York, 1980.
[2] M. Ban, H. Taquchi, T. Katsushima, S. Akoki, A. Wantanbe, Bioorg. Med. Chem.
6 (1998) 1057e1067.
[3] G.E. Wright, J.J. Gombino, J. Med. Chem. 27 (1984) 181e185.
[4] L.F. Jalander, J.E. Longquist, Heterocycles 48 (1998) 743e747.
[5] S.K. Srivastva, A. Agarwal, P.K. Murthy, P.M.S. Chauhan, S.K. Agarwal,
A.P. Bhaduri, S.N. Singh, N. Fatima, R.K. Chatterjee, J. Med. Chem. 42 (1999)
1667e1672.
[6] K. Singh, D. Arora, J. Balzarini, Tetrahedron 66 (2010) 8175e8180.
[7] (a) C.O. Kappe, Eur. J. Med. Chem. 35 (2000) 1043e1052;
(b) K. Singh, D. Arora, K. Singh, S. Singh, Mini Rev. Med. Chem. 9 (2009)
95e106.
[8] P. Biginelli, Gazz. Chim. Ital. 23 (1893) 360e416.
[9] C.O. Kappe, Tetrahedron 49 (1993) 6937e6963.
[10] (a) K. Singh, D. Arora, E. Poremsky, J. Lowery, R.S. Moreland, Eur. J. Med. Chem.
44 (2009) 1997e2001;
5.4.12. 2-Morpholino-4-methyl-6-phenylpyrimidine-5-carboxylic
acid ethyl ester (4n)
Yellowish solid. Rf: 0.3 (10% EtOAc/hexane). Yield: 70%. M.p.
120e122 ꢀC (DCM/Hexane). IR (KBr): nmax 2972, 1718, 1517,
770 cmꢂ1. 1H NMR (300 MHz, CDCl3, 25 ꢀC):
d
0.95 (t, J ¼ 7.2 Hz, 3H,
estereCH3), 2.49 (s, 3H, C6eCH3), 3.75 (t, J ¼ 4.5 Hz 2ꢄ2H,
NCH2emorp), 3.92 (t, J ¼ 4.5 Hz, 2ꢄ2H, OCH2emorp), 4.05 (q,
J ¼ 7.2 Hz, 2H, estereCH2), 7.38e7.58 (m, 5H, ArH). 13C NMR
(300 MHz, CDCl3, 25 ꢀC):
d 13.4, 23.1, 44.0, 60.9, 66.8, 114.4, 128.0,
129.3, 139.2, 160.1, 165.6, 166.9, 168.9. Anal. Calcd. for C18H21N3O3:
C, 66.06; H, 6.42; N, 12.84; Found: C, 65.72; H, 6.21; N, 12.43.
MS: m/z 328 (Mþ1).
(b) K. Singh, D. Arora, D. Falkowski, Q. Liu, R.S. Moreland, Eur. J. Org. Chem.
(2009) 3258;
(c) K. Singh, S. Singh, Tetrahedron 65 (2009) 4106e4112 and references cited
therein.
5.4.13. 2-Ethoxy-4-methyl-6-phenylpyrimidine-5-carboxylic acid
ethyl ester (4o)
[11] K. Singh, K. Singh, Aust. J. Chem. 61 (2008) 910e913.
[12] (a) Global Tuberculosis Report: A Short Update to the 2009 Report. WHO,
Geneva, Switzerland, 2009;
Viscous liquid. Rf: 0.3 (10% EtOAc/hexane). Yield: 87%. IR (KBr):
nmax 3300, 1730, 1700, 1150, 1410 cmꢂ1. 1H NMR (300 MHz, CDCl3,
(b) D. Maher, M.C. Raviglionem, in: D. Schlossberg (Ed.), Tuberculosis and
Nontuberculosis, Mycobacterial Infections, fourth ed. Saunders, Philadelphia,
1999, p. 104;
25 ꢀC):
d
1.03 (t, J ¼ 6.9 Hz, 3H, estereCH3), 1.45 (t, J ¼ 6.9 Hz, 3H,
CH3), 2.57 (s, 3H, C6eCH3), 4.15 (q, J ¼ 7.2 Hz, 2H, estereCH2), 4.50
(c) B.R. Bloom, C.J.L. Murray, Science 257 (1992) 1055e1064.
[13] Y.L. Janin, Bioorg. Med. Chem. 15 (2007) 2479e2513.
[14] J. Morgan, R. Haritakul, P.A. Keller, Bioorg. Med. Chem. Lett. 13 (2003)
1755e1757.
[15] K. Johnsson, D.S. King, P.G. Schultz, J. Am. Chem. Soc. 117 (1995) 5009e5010.
[16] K. Drlica, X. Zhao, Microbiol. Mol. Biol. Rev. 61 (1997) 377e392.
[17] R. Singh, U. Manjunatha, H.I.M. Boshoff, Y.H. Ha, P. Niyomrattanakit,
R. Ledwidge, C.S. Dowd, I.Y. Lee, P. Kim, L. Zhang, S. Kang, T.H. Keller, J. Jiricek,
C.E. Barry, Science 322 (2008) 1392e1395.
(q, J ¼ 7.2 Hz, 2H, OCH2), 7.40e7.64 (m, 5H, ArH). 13C NMR (75 MHz,
CDCl3, 25 ꢀC):
d 13.4, 14.3, 22.6, 61.4, 63.5, 119.6, 128.1, 129.9, 137.7,
163.9, 166.3, 168.2, 168.5. Anal. Calcd. for C16H18N2O3: C, 67.13; H,
6.29; N, 9.79; Found: C, 66.82; H, 6.10; N, 9.45. MS: m/z 309 (Mþ23).
5.5. Antituberculosis activity assays
[18] K. Singh, K. Singh, J. Balzarini, Unpublished results.
[19] K. Falzari, Z. Zhu, D. Pan, H. Liu, P. Hongmanee, S.G. Franzblau, Antimicrob.
Agents Chemother. 49 (2005) 1447e1454.
[20] L. Collins, S.G. Franzblau, Antimicrob. Agents Chemother. 41 (1997)
1004e1009.
Minimum inhibitory concentrations (MIC) against replicating
cultures of M. tuberculosis ATCC 27294 (American Type Culture
Collection, Rockville, MD) were determined after 7 days incubation