Novel dihydropyrimidines as a potential new class of antitubercular agents

Article abstract of DOI:10.1016/j.bmcl.2010.08.046

A small library of 30 dihydropyrimidines was synthesized and evaluated for their in vitro antitubercular activity against Mycobacterium tuberculosis H ₃₇Rv. Two compounds, ethyl 4-[3-(4-fluorophenyl)-1-phenyl-1H-pyrazol- 4-yl]-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5 carboxylate 4a and ethyl 4-[3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl]-6-methyl-2-oxo-1,2,3, 4-tetrahydropyrimidine-5-carboxylate 4d were found to be the most active compounds in vitro with MIC of 0.02 μg/mL against MTB and were more potent than isoniazid.

Full text of DOI:10.1016/j.bmcl.2010.08.046

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6100–6102
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel dihydropyrimidines as a potential new class of antitubercular agents
⇑
Amit R. Trivedi, Vimal R. Bhuva, Bipin H. Dholariya, Dipti K. Dodiya, Vipul B. Kataria, Viresh H. Shah
Department of Chemistry, Saurashtra University, Kalawad Road, Rajkot 360 005, Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A small library of 30 dihydropyrimidines was synthesized and evaluated for their in vitro antitubercular
activity against Mycobacterium tuberculosis H37Rv. Two compounds, ethyl 4-[3-(4-fluorophenyl)-1-phe-
nyl-1H-pyrazol-4-yl]-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5 carboxylate 4a and ethyl 4-[3-(4-
nitrophenyl)-1-phenyl-1H-pyrazol-4-yl]-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate 4d
Received 11 May 2010
Revised 9 August 2010
Accepted 10 August 2010
Available online 14 August 2010
were found to be the most active compounds in vitro with MIC of 0.02
potent than isoniazid.
lg/mL against MTB and were more
Keywords:
Dihydropyrimidines
Ó 2010 Elsevier Ltd. All rights reserved.
Antitubercular activity
Antimycobacterial activity
Mycobacterium tuberculosis H37Rv
DHFR (dihydrofolate reductase)
Tuberculosis (TB) is a worldwide pandemic caused by different
species of mycobacteria. The latest statistics reveals that around
2 million people throughout the world die annually from tubercu-
losis and there are around 8 million new cases each year, out of
which developing countries show major share.1 Among HIV-in-
fected people with weakened immune system, TB is a leading killer
epidemic. Every year about 2 million people living with HIV/AIDS
die from TB.2 Furthermore, in recent times the appearance of
multidrug-resistant TB (MDR-TB), a form of TB that does not
respond to the standard treatments, is more common. It is a shock-
ing revelation that MDR-TB is present in almost all countries as per
the recent survey, made by the World Health Organization (WHO)
and its partners. A recent estimation by WHO has revealed that
within next 20 years approximately 30 million people will be
infected with the bacillus.3 Keeping in view of the above statistics,
WHO declared TB as a global health emergency and aimed at saving
14 million lives between 2006 and 2015.4
Despite the efforts of academic institutions and the pharmaceu-
tical companies engaged in the design, synthesis, and development
of new antitubercular regimens, the current TB therapeutic arsenal
is poor. A number of compounds belonging to different prototypes
have shown moderate to excellent antitubercular activities.5 As on
today at least 10 molecules including fluoroquinolones (gatifloxa-
cin and moxifloxacin),6–9 diarylquinoline (TMC207),10 nitroimidaz-
oles (OPC67683 and PA824),11,12 pyrrole (LL3858),13 1,2-diamine
(SQ109)14 are in different stages of clinical trial.15 All the above
facts reveal that there is an urgent need for development of new
drugs with divergent and unique structure and with a mechanism
of action possibly different from that of existing drugs.
In this context, dihydropyrimidines are potential inhibitors of
dihydrofolate reductase (DHFR),16 which is a promising drug target
for treatment of mycobacterial infections. DHFR is a key enzyme in
the folate cycle17,18 which supplies one-carbon units, derived from
the action of serine hydroxymethyltransferase19,20 on
L-serine, for
the biosynthesis of deoxythymidine monophosphate (dTMP). Inhi-
bition of the folate cycle leads to interruption of the supply of thy-
midine and thus to inhibition of DNA biosynthesis and inhibition of
proliferation of cells. Inhibition of proliferation is a useful goal in
the therapy of bacterial and protozoal infections.21 Although DHFR
does not represent a new target, there is still enthusiasm for the
development of DHFR inhibitors, particularly with regard to myco-
bacteria.22–26 A unique feature of DHFR is the selectivity that is
possible in the design of inhibitor. This makes it an ideal ‘old’ target
for rational and effective drug design for antimycobacterial agents.
Dihydropyrimidines are not represented in the current clinical
antitubercular regimens, suggesting that this class of compounds
may target new biochemical mechanisms, potentially allowing
treatment of MDR-TB and there are very few investigatory reports
on dihydropyrimidines as antitubercular agents.27,28 Recognizing
these facts and in continuation of work on pyrimidine deriva-
tives,29–31 we set upon a programme of making antitubercular
agents, using the central dihydropyrimidine as the template and
adding versatile substituents on the various positions of dihydro-
pyrimidine ring. The synthetic route for the preparation of dihy-
dropyrimidines derivatives (4a–e to 9a–e) is outlined in Scheme
1. Various 3-(aryl)-1-phenyl-1H-pyrazole-4-carbaldehydes (1a–e)
bearing a range of electron withdrawing and electron releasing
substituents, viz., 4-F; 4-Cl; 4-Br; 4-NO2; 4-CH3 were prepared
⇑
Corresponding author. Tel.: +91 0281 2589443.
E-mail address: shah_v_h@yahoo.com (V.H. Shah).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.046

A. R. Trivedi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6100–6102
6101
N N
N N
R2
CHO
O
1a-e
O
R2
R1
NH
R1
NH2
X
a
+
H3C
N
H
X
H3C
O
H2N
2
4a-e to 8a-e
3
4a-e X = O, R1 = OC2H5, R2 = 4-F, 4-Cl, 4-Br, 4-NO2, 4-CH3
5a-e X = S, R1 = OC2H5, R2 = 4-F, 4-Cl, 4-Br, 4-NO2, 4-CH3
6a-e X = NH, R1 = OC2H5, R2 = 4-F, 4-Cl, 4-Br, 4-NO2, 4-CH3
7a-e X = S, R1 = OCH3, R2 = 4-F, 4-Cl, 4-Br, 4-NO2, 4-CH3
8a-e X = O, R1 = OCH3, R2 = 4-F, 4-Cl, 4-Br, 4-NO2, 4-CH3
N N
N N
O
O
R2
b
R2
R1
NH
R1
NH
CH3
H3C
N
H
S
H3C
N
S
5a-e
9a-e
9a-e X = SCH3, R1 = OC2H5, R2 = 4-F, 4-Cl, 4-Br, 4-NO2, 4-CH3
Scheme 1. Synthetic protocol of title compounds 4a–e to 9a–e. Reagents and conditions: (a) concd HCl, EtOH, reflux; (b) DMS, EtOH, 40% NaOH, stirring.
according to the previously reported procedure.32 All the dihydro-
pyrimidines derivatives (4a–e to 9a–e) were synthesized by the
multi-component Biginelli reaction involving substituted alde-
hydes, ethyl/methyl acetoacetate and urea derivatives. Dihydro-
pyrimidines 5a–e on S-methylation with dimethylsulfate in the
presence of 40% NaOH afforded S-methylated adducts 9a–e. The
compounds 4a–e to 6a–e, and 9a–e have carbethoxy group in
the 5-position, while compounds 7a–e to 8a–e have carbmethoxy
group in the 5-position. The yields of the products were obtained in
the range of 60–74%. Designed series of molecules were character-
ized by 1H NMR, 13C NMR, and Mass spectrometry techniques
before evaluating for antimycobacterial activity.
In the preliminary screening, nine compounds (4a, 4d, 5d, 5e,
6b, 7d, 8c, 8e, and 9e) inhibited MTB with 90–100%. In the second-
ary level, two compounds (4a and 4d) inhibited MTB with MIC of
<1
<2
l
l
g/mL and three compounds (5e, 8e, and 9e) with MIC of
g/mL. Among all the compounds, compounds 4a and 4d having
4-F and 4-NO2 substituents at the 4-phenyl ring of pyrazolyl sub-
stitution were the most potent compounds (MIC of 0.02 g/mL
and SI >500) of the series. These compounds are better than the
existing drug INH (MIC: 0.03 g/mL) in the in vitro studies. There-
l
l
fore, these compounds provide an excellent lead for further devel-
opment as novel antitubercular molecule. Substituents with
different electronic properties at fourth position of C-3 phenyl ring
of pyrazolyl substitution exhibited high inhibitory activity against
MTB, indicating that the electronic properties of the substituents
have only minor influence on the antimycobacterial activity. With
the sizable number of compounds no definite conclusion could be
drawn from this study, yet the detailed SAR, in vivo evaluation and
mode of action of the series would be undertaken in due course.
Having identified good number of active antimycobacterial
dihydropyrimidines, the next step was to examine the toxicity of
the drug candidates. Compounds exhibiting reasonably low MICs
1H NMR spectra of dihydropyrimidines derivatives (4a–e to
9a–e) showed singlets at d 4.96–5.75 ppm corresponding to
methine group and d 8.69–9.01 ppm corresponding to NH group
of pyrimidine ring confirming the formation of dihydropyrimidine
nucleus. Further, formation of S-methylated adducts 9a–e was con-
firmed by a singlet at d 3.51–3.61 ppm corresponding to S-methyl
group. On the other hand, 13C NMR spectra displayed chemical
shift at d 38.7–46.8 ppm for carbon of methine proton. Also,
S-methyl carbon was observed at d 12.3–14.3 ppm for compounds
9a–e. The elemental analysis results were within 0.4% of the the-
oretical values.
All compounds were initially screened for their in vitro antimy-
cobacterial activity at 6.25 lg/mL against MTB H37Rv strain by the
Tuberculosis Antimicrobial Acquisition and Coordinating Facility
(TAACF) in BACTEC 12B medium using the Microplate Alamar Blue
Assay.33 Compounds exhibiting P90% inhibition in the initial
(from 0.02 to 6.25 lg/mL) were tested for cytotoxicity (IC50) in
VERO cells, and a selectivity index (SI), defined as IC50: MIC, was
calculated. The IC50 and SI values are shown in Table 1. The com-
pounds 5e, 7d and 9e were somewhat more toxic than the 4a,
4d, 5d, 6b, 8c, and 8e. Generally, compounds with an MIC 6
6.25
lg/mL and an SI P 10 are interesting compounds, and an
MIC 6 1
l
g/mL in a novel compound class is considered an
screen were retested at and below 6.25
dilution to determine the actual MIC.
lg/mL using two-fold
excellent lead,34 which makes the ethyl 4-[3-(4-fluorophenyl)-
1-phenyl-1H-pyrazol-4-yl]-6-methyl-2-oxo-1,2,3,4-tetrahydro

6102
A. R. Trivedi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6100–6102
Table 1
In vitro antitubercular screening data of dihydropyrimidines 4a–e to 9a–e
Sr. No.
R1
R2
X
% Inhibition
MIC
l
g/mL
IC50 VERO cells
SI (SI = IC50/MIC)
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
OC2H5
OC2H5
OC2H5
OC2H5
OC2H5
OC2H5
OC2H5
OC2H5
OC2H5
OC2H5
OC2H5
OC2H5
OC2H5
OC2H5
OC2H5
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OC2H5
OC2H5
OC2H5
OC2H5
OC2H5
—
4-F
4-Cl
4-Br
4-NO2
4-CH3
4-F
O
O
O
O
O
S
S
S
100
49
33
100
68
30
75
87
92
96
18
94
51
58
78
73
68
43
91
46
62
75
90
39
98
8
0.02
n.d.
n.d.
0.02
n.d.
n.d.
n.d.
n.d.
3.13
1.56
n.d.
3.13
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
3.13
n.d.
n.d.
n.d.
6.25
n.d.
1.56
n.d.
n.d.
n.d.
n.d.
1.56
0.03
>10
n.d.
n.d.
>10
n.d.
n.d.
n.d.
n.d.
>10
8.9
n.d.
>10
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
9.6
n.d.
n.d.
n.d.
>10
n.d.
>10
n.d.
n.d.
n.d.
n.d.
7.4
>500
n.d.
n.d.
>500
n.d.
n.d.
n.d.
n.d.
>3.2
5.7
n.d.
>3.2
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
3.0
n.d.
n.d.
n.d.
>1.6
n.d.
>6.4
n.d.
n.d.
n.d.
n.d.
4.7
4-Cl
4-Br
4-NO2
4-CH3
4-F
S
S
NH
NH
NH
NH
NH
S
S
S
S
S
4-Cl
4-Br
4-NO2
4-CH3
4-F
4-Cl
4-Br
4-NO2
4-CH3
4-F
O
O
O
O
4-Cl
4-Br
4-NO2
4-CH3
4-F
O
SCH3
SCH3
SCH3
SCH3
SCH3
—
4-Cl
13
13
37
97
—
4-Br
4-NO2
4-CH3
—
Isoniazid
—
—
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Acknowledgments
This work was funded by CSIR through grant no. 01(02348)/09/
EMR-II. The authors wish to thank CSIR for its financial support.
The authors are indebted to Tuberculosis Antimicrobial Acquisition
and Coordinating Facility (TAACF/USA) for biological tests. A.R.T. is
thankful to CSIR, New Delhi for senior research fellowship.
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