4,5-Dihydro-1H-pyrazolo[3,4-d]pyrimidine containing phenothiazines as antitubercular agents

Article abstract of DOI:10.1016/j.bmcl.2014.02.012

A series of novel dihydropyrazolo[3,4-d]pyrimidine derivatives bearing a phenothiazine nucleus were synthesized in excellent yields via a modified Biginelli multicomponent reaction. The newly synthesized compounds were characterized by IR, ¹H NMR, ¹³C NMR, Mass spectra and elemental analysis followed by antimycobacterial screening. Among all the screened compounds, compound 4g showed most pronounced activity against Mycobacterium tuberculosis (Mtb) with minimum inhibitory concentration (MIC) of 0.02 μg/mL, making it more potent than first line antitubercular drug isoniazid.

Full text of DOI:10.1016/j.bmcl.2014.02.012

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1493–1495
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
4,5-Dihydro-1H-pyrazolo[3,4-d]pyrimidine containing
phenothiazines as antitubercular agents
b
c
c
Arif B. Siddiqui a, , Amit R. Trivedi , Vipul B. Kataria , Viresh H. Shah
⇑
a Amneal Pharmaceuticals India Pvt Ltd, 882/1-871, Village Rajoda, Tal.: Bavla Dist.: Ahmedabad 382220, Gujarat, India
b Division of Medicinal Chemistry, Department of Chemistry (DST-FIST Sponsored), Mahatma Gandhi Campus, Maharaja Krishnakumarsinhji Bhavnagar University,
Bhavnagar 364002, Gujarat, India
c Department of Chemistry, Saurashtra University, Kalawad Road, Rajkot 360005, Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel dihydropyrazolo[3,4-d]pyrimidine derivatives bearing a phenothiazine nucleus were
synthesized in excellent yields via a modified Biginelli multicomponent reaction. The newly synthesized
compounds were characterized by IR, 1H NMR, 13C NMR, Mass spectra and elemental analysis followed by
antimycobacterial screening. Among all the screened compounds, compound 4g showed most pro-
nounced activity against Mycobacterium tuberculosis (Mtb) with minimum inhibitory concentration
Received 28 August 2013
Revised 1 February 2014
Accepted 4 February 2014
Available online 14 February 2014
(MIC) of 0.02
l
g/mL, making it more potent than first line antitubercular drug isoniazid.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Dihydropyrazolo[3,4-d]pyrimidine
Phenothiazines
Biginelli multicomponent reaction
Cytotoxicity
Antitubercular activity
Tuberculosis (TB) is a highly infectious airborne disease caused
by the pathogenic bacterium Mycobacterium tuberculosis (Mtb).1
According to the latest World Health Organization (WHO) report,
an estimated 8.6 million people developed TB and 1.3 million died
from the disease (including 320,000 deaths among HIV-positive
people) in 2012.2 The majority of cases worldwide in 2012 were
in the South-East Asia (29%), African (27%) and western Pacific
(19%) regions. India and China alone accounted for 26% and 12%
of total cases, respectively.2 The standard antitubercular treatment
regimen, termed DOTS (Directly Observed Therapy, Short-course),3
is based on the co-administration of age-old drugs like isoniazid
(INH), rifampin (RMP), ethambutol (EMB), and pyrazinamide
(PZA) for the first two months, followed by a prolonged treatment
with INH and RMP for additional 4–7 months with no guarantee of
complete sterilization from the infection.4,5 Furthermore, emer-
gence of new virulent forms of TB such as multi drug resistant
(MDR-TB) and extremely drug resistant (XDR-TB), and its synergy
with human immunodeficiency virus (HIV) has fuelled its epidemic
nature.6–9 These reasons make a compelling case for an urgent
need for new and effective antitubercular agents with improved
properties such as enhanced activity against MDR strains, reduced
toxicity, rapid mycobactericidal mechanism of action and the
ability to penetrate host cells and exert antimycobacterial effects
in the intracellular environment.10–12
Phenothiazines are important classes of compounds which have
increasingly attracted attention, owing to their remarkable biolog-
ical and pharmacological properties, such as antibacterial, anti-
fungal, anticancer, antiviral, anti-inflammatory, antimalarial,
antifilarial, trypanocidal, anticonvulsant, analgesic, immunosup-
pressive and multidrug resistance reversal.13 These activities are
the results of the actions exerted by phenothiazines on biological
systems via the interaction of the pharmacophoric substituent (in
some cases of strict length), via the interaction multicyclic ring sys-
tem (p–p interaction, intercalation in DNA) and via the lipophilic
character permitting the penetration through the biological mem-
branes to reach its site of action.13 Further, Phenothiazines have
been shown to exhibit in vitro and in vivo activity against Mtb
and multidrug-resistant Mtb. Some of the phenothiazine derived
antipsychotic agents such as chlorpromazine, trifluoperazine
(TPZ) and thioridazine are found to be effective inhibitors of
Mtb.14–20 Phenothiazines are predicted to target the genetically
validated respiratory chain component type II NADH:quinone oxi-
doreductase (Ndh).21
On the other hand, Pyrazolopyrimidines and related fused het-
erocyclic ring systems which structurally resemble purines are of
great interest as they are reported to encompass an array of
pharmacological activities such as antiviral,22 anticoccidials,23
antimicrobial,24 antitumor,25 CNS agents,26 tuberculostatic,27–29
⇑
Corresponding author. Tel.: +91 9723558040.
E-mail address: drarifsiddiqui2013@gmail.com (A.B. Siddiqui).
http://dx.doi.org/10.1016/j.bmcl.2014.02.012
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

1494
A. B. Siddiqui et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1493–1495
O
H
N
H
N
H
N
CH3
CH3
SCH3
NHNH2
N
N
a
N
b
S
1
S
2
S
3
R
H2N
H
N
c
N
H
N
N
S
4a-k
R= H, 2-OH, 3-OH, 4-OH, 2-Cl, 3-Cl, 4-Cl, 2-NO2, 3-NO2, 4-NO2, 4-OCH3
Scheme 1. Synthetic protocol of title compounds 4a–k. Reagents and conditions: (a) NH2NH2, reflux; (b) ethyl acetoacetate, sodium ethoxide, reflux; (c) guanidine
hydrochloride, aldehyde (R-CHO), P2O5, EtOH, reflux.
antileishmanial,30 antiinflammatory31 and cardiovascular activi-
ties.32 Prompted by above mentioned observations and in continu-
ation of our previous work,33,34 we report herein the synthesis of
some novel structural hybrids by combining phenothiazine and
dihydropyrazolo[3,4-d]pyrimidine pharmacophoric units in single
molecular platform in order to investigate their in vitro antituber-
cular activity.
(TAACF) in BACTEC 12B medium using the microplate alamar blue
assay.35 The results of the antitubercular studies are presented in
Table 1. Compounds exhibiting P90% inhibition in the initial
screening were tested at and below 6.25 lg/mL using 2-fold dilu-
tion to determine the actual MIC. In preliminary screening, five
compounds (4b, 4e, 4g, 4h, 4j) inhibited Mtb with 90–100%. In
the secondary level, one compound (4g) inhibited Mtb with MIC
The reaction sequences employed for synthesis of 3-methyl-1-
(10H-phenothiazin-2-yl)-4-phenyl-6-amino-4,5-dihydro-1H-pyr-
azolo[3,4-d]pyrimidines (4a–k) are outlined in Scheme 1.
Initially, synthesis of intermediates 2 and 3 were accomplished
according to our previously published methods.33,34 Reaction of
3-methyl-1-(10H-phenothiazin-8-yl)-1H-pyrazol-5-(4H)-one (3),
an appropriate aldehyde, guanidine hydrochloride and phospho-
rus pentoxide under reflux conditions afforded the desired com-
pounds 4a–k. The yields of the products were obtained in the
range of 75–90%. Designed series of molecules 4a–k were charac-
terized by IR, 1H NMR, 13C NMR and Mass spectrometry tech-
niques before evaluating for in vitro antitubercular screening. In
the 1H NMR spectra of 4a–k a sharp peak representing the
methine proton of the pyrimidine was observed in the range of
d = 4.90–5.05 ppm confirming the formation of the dihydropyraz-
olo[3,4-d]pyrimidine nucleus.
of <1
l
g/mL and three compounds (4b, 4e, 4j) with MIC of <3
lg/
mL when compared to isoniazid (MIC: 0.03
lg/mL). Among all
the compounds, 4-(4-chlorophenyl)-3-methyl-1-(10H-phenothia-
zin-2-yl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-amine (4g)
was found to be the most active compound in vitro with MIC of
0.02 lg/mL against Mtb. The activity was considerably affected
by substitutions at the phenyl ring of the dihydropyrazolo[3,4-
d]pyrimidine nucleus. It was observed that in a group of com-
pounds having electron-withdrawing halogen and nitro group on
the ortho and para positions of the phenyl ring were influencing
the antitubercular activity significantly (4b, 4e, 4g, 4h, 4j), other
substituents on phenyl ring decreased the antitubercular activity,
alkoxy group remarkably reduced the potency of the test com-
pound (4k).
Having identified a good number of active antimycobacterial
compounds, the next step was to examine the toxicity of the drug
candidates. Compounds exhibiting relatively low MICs were tested
for cytotoxicity (IC50) in VERO cells and a selectivity index (SI),
defined as IC50: MIC was calculated. The compounds 4j and 4b
were somewhat more toxic than 4e, 4g and 4h. Generally
All the newly synthesized compounds 4a–k were initially
screened for their in vitro antimycobacterial activity at 6.25 lg/
mL against Mycobacterium tuberculosis H37Rv strain by the Tuber-
culosis Antimicrobial Acquisition and Coordinating Facility
Table 1
In vitro antitubercular screening data of compounds 4a–k against M. tuberculosis H37Rv
b
Sr. No.
R
% Inhibition
MICa
(lg/mL)
IC50 VERO cells (
lg/mL)
SIc (SI = IC50/MIC)
4a
4b
4c
4d
4e
4f
4g
4h
H
78
92
57
67
95
56
100
94
73
95
42
—
n.d.
2.23
n.d.
n.d.
1.73
n.d.
0.02
1.73
n.d.
n.d.
7.8
n.d.
3.5
n.d.
n.d.
5.8
n.d.
>500
5.8
n.d.
5.2
2-OH
3-OH
4-OH
2-Cl
3-Cl
4-Cl
2-NO2
3-NO2
4-NO2
4-OCH3
—
n.d.
n.d.
>10
n.d.
>10
>10
n.d.
9.1
4i
4j
1.73
n.d.
4k
Isoniazid
n.d.
>1000
n.d.
0.03d
>40,000e
a
b
c
Minimum inhibitory concentration against H37Rv strain of M. tuberculosis (
l
g/mL).
Measurement of cytotoxicity in VERO cells: 50% inhibitory concentrations (
Selectivity index (in vitro): IC50 in VERO cells/MIC against M. tuberculosis.
Data from TAACF.37
lg/mL).
d
e
Data from literature.38 The activity criterion for this assay is an SI >10.

A. B. Siddiqui et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1493–1495
1495
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compound with an MIC 66.25
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g/mL and SI P10 are interesting
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g/mL in novel compound
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class may be considered an excellent lead,36 which makes
3-methyl-4-(4-nitrophenyl)-1-(10H-phenothiazin-2-yl)-4,5-dihy-
dro-1H-pyrazolo[3,4-d]pyrimidin-6-amine (4g) very promising
antimycobacterial compound.
In conclusion, we have conveniently synthesized a series of no-
vel dihydropyrazolo[3,4-d]pyrimidine derivatives bearing a pheno-
thiazine nucleus and evaluated for their in vitro antitubercular
activity with anticipation of generating new structural leads. The
results indicate that the preparative pathway elaborated by us to
the novel dihydropyrazolo[3,4-d]pyrimidine having phenothiazine
nucleus may open a relatively facile route to a new class of antitu-
bercular compounds.
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