Synthesis and anti-tubercular evaluation of some novel pyrazolo[3,4-d] pyrimidine derivatives

Article abstract of DOI:10.1007/s00044-011-9712-3

A series of phenothiazine clubbed pyrazolo[ 3,4-d]pyrimidines have been synthesized by using the Biginelli multi-component cyclocondensation reaction and their ability to inhibit growth of Mycobacterium tuberculosis in vitro have been determined. The results show that compounds 4b, 4d, and 4f exhibited excellent anti-tubercular activity with percentage inhibition of 93, 91, and 96, respectively, at a minimum inhibitory concentration (MIC) of < 6.25 lg/ml, whereas compounds 4a, 4c, 4e, 4g, and 4h exhibited moderate to good anti-tubercular activity with percentage inhibition of 75, 68, 74, 54, and 63, respectively at a MIC of >6.25 lg/ml. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9712-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:1887–1891
DOI 10.1007/s00044-011-9712-3
ORIGINAL RESEARCH
Synthesis and anti-tubercular evaluation of some novel
pyrazolo[3,4-d]pyrimidine derivatives
•
Amit R. Trivedi Bipin H. Dholariya Chintan P. Vakhariya
•
•
•
Dipti K. Dodiya Haresh K. Ram Vipul B. Kataria
•
•
•
Arif B. Siddiqui Viresh H. Shah
Received: 22 July 2010 / Accepted: 15 June 2011 / Published online: 28 June 2011
Ó Springer Science+Business Media, LLC 2011
Abstract A series of phenothiazine clubbed pyrazol-
o[3,4-d]pyrimidines have been synthesized by using the
Biginelli multi-component cyclocondensation reaction and
their ability to inhibit growth of Mycobacterium tubercu-
losis in vitro have been determined. The results show that
compounds 4b, 4d, and 4f exhibited excellent anti-tuber-
cular activity with percentage inhibition of 93, 91, and 96,
respectively, at a minimum inhibitory concentration (MIC)
of\6.25 lg/ml, whereas compounds 4a, 4c, 4e, 4g, and 4h
exhibited moderate to good anti-tubercular activity with
percentage inhibition of 75, 68, 74, 54, and 63, respectively
at a MIC of [6.25 lg/ml.
Keywords Pyrazolopyrimidines ꢀ Phenothiazines ꢀ
Anti-tubercular activity ꢀ BACTEC assay
Introduction
Tuberculosis is an infectious pulmonary disease caused by
the pathogen Mycobacterium tuberculosis that is respon-
sible for infecting 8–10 million people globally and three
million deaths every year according to a World Health
Organization report. Owing to the rapid spread of tuber-
culosis strains resistant to all first-line anti-tubercular drugs
such as isoniazid, rifamcin, and ethionamide, and due to
the toxicity of second-line drugs, such as ethionamide,
aminosalicylic acid, cycloserine, amikacin, kanamycin, and
capreomycin, the discovery of new anti-tubercular agents
with potential activity, less toxicity, broader spectrum, and
safer therapeutic profiles, is an urgent need (Scior et al.,
2002; Tripathi et al., 2005).
A. R. Trivedi ꢀ B. H. Dholariya ꢀ C. P. Vakhariya ꢀ
D. K. Dodiya ꢀ H. K. Ram ꢀ V. B. Kataria ꢀ
A. B. Siddiqui ꢀ V. H. Shah (&)
Department of Chemistry, Saurashtra University,
Rajkot 360005, India
e-mail: shah_v_h@yahoo.com
In this context, Pyrazole and pyrimidine derivatives
attracted organic chemists very much due to their biolog-
ical and chemotherapeutic importance. Pyrazolopyrimi-
dines and related fused heterocycles are of interest as
potential bioactive molecules. They are known to exhibit
pharmacological activities such as CNS depressant (Julino
and Stevens, 1998; Ibrahim Abdou et al., 2004), neuro-
leptic (Filler, 1974), and tuberculostatic (Ghorab et al.,
2004). Pyrazolo[3,4-d]pyrimidines were identified as a
general class of adenosine receptors (Davies et al., 1984).
In the literature, we have found that the replacement of
1H of pyrazole of pyrazolo[3,4-d]pyrimidine ring system
by some other bioactive moiety drastically alters its phar-
macological properties. Keeping this in mind, we have
contemplated on the synthesis of pyrazolo[3,4-d]pyrimi-
dine derivatives bearing a phenothiazine moiety.
A. R. Trivedi
e-mail: damitdada@gmail.com
B. H. Dholariya
e-mail: bipin_dholariya@yahoo.com
C. P. Vakhariya
e-mail: chintan.vakharia@gmail.com
D. K. Dodiya
e-mail: diptid85@gmail.com
H. K. Ram
e-mail: ram_haresh2007@yahoo.co.in
V. B. Kataria
e-mail: vipkatkat@gmail.com
A. B. Siddiqui
e-mail: jmdesai@yahoo.com
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Med Chem Res (2012) 21:1887–1891
Moreover, phenothiazines form an important class of
oil bath. The reaction mixture was poured into ice-cold
water and product was recrystallized from ethanol. Yield
82%, mp 122°C, Rf-value 0.60; IR (KBr, cm-1): 3335
(NH), 653 (C–S–C). 1H NMR (300 MHz, CDCl3) d:
7.45–7.76 (m, 7H, Ar–H), 9.15 (s, 1H, NH), 7.86–7.95 (m,
3H, NHNH2). 13C NMR (CDCl3) d: 105, 105.6, 109, 113,
118.2, 124.1, 126.2, 129.4, 143, 144.3, 151.5; MS m/z (%)
229. Anal. Calcd. for C12H11N3S: C, 62.86; H, 4.84; N,
18.33. Found: C, 62.72; H, 4.72; N, 18.26%.
heterocyclic compounds possessing wide spectrum diverse
biological activities like sedative, tranquilizers, anti-
epileptic, anti-tubercular, bactericides, and parasiticides
(Mietzsch, 1954; Okafor, 1977; Albery et al., 1979).
Interestingly, phenothiazines are able to cleave DNA upon
photochemical induction (Nishiwaki et al., 1990). Fairly
early, it was recognized that due to the low oxidation
potential of this class of tricyclic nitrogen–sulfur hetero-
cycles, their propensity to form stable radical-cations play
a key role in their physiological activities (Moutet and
Reverdy, 1983). More recently, owing to their reversible
oxidation (Mcintyre and Gerischer, 1984), phenothia-
zine derivatives have become attractive supramolecular
(Duesing etal., 1990; Brunet al., 1991) and material scientific
motifs (Knorr and Daub, 1995; Spreitzer and Daub, 1996).
Keeping in mind the biomedical applications, as a
continuation of our previous study (Trivedi et al., 2008a, b)
and with a view to further assess the pharmacological
profile of this class of compounds, we envisioned our
approach toward the synthesis of a novel series of pyraz-
olo[3,4-d]pyrimidine derivatives by incorporating the
phenothiazine and pyrazolo[3,4-d]pyrimidine in a single
molecular framework with a potential spectrum of bio-
responses. The anti-tubercular activities of the newly syn-
thesized compounds against M. tuberculosis H37Rv strain
were studied using radiometric BACTEC and broth dilu-
tion assay methods.
Synthesis of 3-methyl-1-(10H-phenothiazin-8-yl)-1H-
pyrazol-5(4H)-one (3)
A mixture of 1-(10H-phenothiazin-8-yl)hydrazine (2.29 g,
0.01 mol) and ethyl acetoacetate (1.3 ml, 0.01 mol) in
sodium ethoxide (20 ml) was heated under refluxed con-
dition for 12 h. The reaction mixture was poured into
ice-cold water, and the product was recrystallized from
ethanol. Yield 68%, mp 113°C, Rf-value 0.59; IR (KBr,
cm-1): 3330 (NH), 650 (C–S–C). 1H NMR (300 MHz,
CDCl3) d: 2.47 (s, 3H, CH3), 4.26 (s, 2H, CH2), 7.66–7.90
(m, 7H, Ar–H), 9.03 (s, 1H, NH). 13C NMR (CDCl3) d:
15.8, 41.1, 112.5, 112.9, 113.2, 115, 117.1, 121.7, 128.2,
129.3, 137.8, 142.3, 163.4, 175.6; MS m/z (%) 295. Anal.
Calcd. for C16H13N3OS: C, 65.06; H, 4.44; N, 14.23.
Found: C, 64.92; H, 4.29; N, 14.31%.
General procedure for the synthesis 4,5-Dihydro-4-(aryl)-
3-methyl-1-(10H-phenothiazin-8-yl)-1H-pyrazolo[3,4-
d]pyrimidine-6-thiols (4a–h)
Experimental
An equimolar mixture of 3-methyl-1-(10H-phenothiazin-8-
yl)-1H-pyrazol-5(4H)-one (3), an appropriate aldehyde
(0.01 mol), and thiourea (0.01 mol) was heated under
reflux in ethanol (30 ml) in the presence of phosphorus
pentoxide (200 mg) as a catalyst for 5 h. Then, the reaction
mixture was kept at room temperature for 2 h. The yellow
crystalline products so obtained was isolated and recrys-
tallized from ethanol.
General
Melting points were determined in open capillary tubes and
are uncorrected. Thin layer chromatography (TLC) was
performed on Silica gel G (Merck) in the solvent system–
ethyl acetate:hexane (3:7, v/v); iodine chamber was used for
1
visualization of TLC spots. H NMR was determined in
CDCl3 solution on a Bruker DPX 300 MHz spectrometer.
13C NMR (75 and 125 MHz) spectra were registered on a
Bruker AC 200, DPX 300, and ARX 500, at 25°C, in
CDCl3. IR spectra were recorded on Shimadzu 8400 spec-
trometer in KBr. Elemental analysis of the newly synthe-
sized compounds was carried out on Carlo Erba 1108
analyzer and are found within the range of theoretical value.
4,5-Dihydro-4-(phenyl)-3-methyl-1-(10H-phenothiazin-8-
yl)-1H-pyrazolo[3,4-d]pyrimidine-6-thiol (4a)
Yield 72%, mp 1218C; Rf-value 0.66; IR (KBr, cm-1):
1
3333, 1614, 1640, 650. H NMR (300 MHz, CDCl3) d:
2.40 (s, 3H, CH3), 4.93 (s, 1H, CH), 7.37–7.53 (m, 12H,
Ar–H), 8.38 (s, 1H, NH), 9.02 (s, 1H, NH). 13C NMR
(CDCl3) d: 25.8, 40.7, 50.2, 60.5, 101.8, 102.4, 105.2,
115.1, 116.3, 122.5, 126, 127.3, 127.9, 128.2, 128.8, 135.3,
142.5, 144.1, 155.8, 164.7; MS m/z (%): 441. Anal. Calcd.
for C24H19N5S2: C, 65.28; H, 4.34; N, 15.86. Found: C,
65.15; H, 4.16; N, 15.74%.
Chemistry
Synthesis of 1-(10H-phenothiazin-8-yl)hydrazine (2)
A mixture of 2-(methylthio)-10H-phenothiazine (0.01 mol)
and hydrazine hydrate (10 ml) was refluxed for 8 h on an
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1889
4,5-Dihydro-4-(2-hydroxyphenyl)-3-methyl-1-(10H-
phenothiazin-8-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-thiol
(4b)
for C24H18N5S2Cl: C, 60.56; H, 3.81; N, 14.71. Found: C,
60.47; H, 3.87; N, 14.64%.
4,5-Dihydro-4-(2-nitrophenyl)-3-methyl-1-(10H-
phenothiazin-8-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-thiol
(4f)
Yield 78%, mp 157°C; Rf-value 0.62; IR (KBr, cm-1):
3330, 1610, 1642, 655. H NMR (300 MHz, CDCl3) d:
1
2.42 (s, 3H, CH3), 4.95 (s, 1H, CH), 7.48–7.72 (m, 11H,
Ar–H), 8.40 (s, 1H, NH), 9.11 (s, 1H, NH), 10.08 (s, 1H,
OH). 13C NMR (CDCl3) d: 26.2, 34.1, 52.3, 60, 103, 103.6,
105.3, 114.2, 115.5, 116.3, 121.7, 122.6, 127.2, 127.5,
127.9, 128.6, 142.3, 143.8, 155.1, 155.8, 163.4; MS m/z
(%):457. Anal. Calcd. for C24H19N5OS2: C, 63.00; H, 4.19;
N, 15.31. Found: C, 62.91; H, 4.26; N, 15.21%.
Yield 83%, mp 2328C; Rf-value 0.63; IR (KBr, cm-1):
3338, 1608, 1648, 658. H NMR (300 MHz, CDCl3) d:
1
2.46 (s, 3H, CH3), 4.94 (s, 1H, CH), 7.28–7.55 (m, 11H,
Ar–H), 8.28 (s, 1H, NH), 9.21(s, 1H, NH). 13C NMR
(CDCl3) d: 26.5, 36.1, 51.1, 59.2, 103.1, 103.5, 105, 114.7,
116.7, 122.3, 124.8, 126.3, 127, 127.8, 128.1, 128.9, 134.3,
135.5, 142.7, 143.2, 148.5, 155.5, 163.3; MS m/z (%): 486.
Anal. Calcd. for C24H18N6O2S2: C, 59.24; H, 3.73; N,
17.27. Found: C, 59.15; H, 3.81; N, 17.18%.
4,5-Dihydro-4-(4-hydroxyphenyl)-3-methyl-1-(10H-
phenothiazin-8-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-thiol
(4c)
4,5-Dihydro-4-(4-nitrophenyl)-3-methyl-1-(10H-
phenothiazin-8-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-thiol
(4g)
Yield 81%, mp 171°C; Rf-value 0.55; IR (KBr, cm-1):
3328, 1612, 1642, 645. H NMR (300 MHz, CDCl3) d:
1
2.44 (s, 3H, CH3), 4.98 (s, 1H, CH), 7.13–8.10 (m,
11H,Ar–H), 8.35 (s, 1H, NH), 9.09 (s, 1H, NH), 10.15 (s,
1H, OH). 13C NMR (CDCl3) d: 26.8, 41.5, 52.3, 59.3,
103.2, 103.8, 105.2, 114.4, 116, 122.3, 127.2, 127.5, 128,
128.4, 142.3, 143.1, 155.8, 156, 163.2; MS m/z (%): 457.
Anal. Calcd. for C24H19N5OS2: C, 63.00; H, 4.19; N,
15.31. Found: C, 62.93; H, 4.25; N, 15.21%.
Yield 74%, mp 1968C; Rf-value 0.67; IR (KBr, cm–1):
3335, 1610, 1650, 654. H NMR (300 MHz, CDCl3) d:
1
2.33 (s, 3H, CH3), 4.97 (S, 1H, CH), 7.76–7.93 (m, 11H,
Ar–H), 8.32 (s, 1H, NH), 9.17 (s, 1H, NH). 13C NMR
(CDCl3) d: 26.3, 40.2, 52.5, 59.3, 103.1, 103.8, 105.2,
114.3, 116.3, 121.8, 122.5, 127.3, 127.8, 128.2, 129.9,
134.2, 140.5, 142.3, 143.8, 147.6, 155.4, 163.8; MS m/z
(%): 486. Anal. Calcd. for C24H18N6O2S2: C, 59.24; H,
3.73; N, 17.27. Found: C, 59.18; H, 3.60; N, 17.14%.
4,5-Dihydro-4-(2-chlorophenyl)-3-methyl-1-(10H-
phenothiazin-8-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-thiol
(4d)
4,5-Dihydro-4-(4-methoxyphenyl)-3-methyl-1-(10H-
phenothiazin-8-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-thiol
(4h)
Yield 76%, mp 1648C; Rf-value 0.60; IR (KBr, cm-1):
3325, 1615, 1643, 648. H NMR (300 MHz, CDCl3) d:
1
2.35 (s, 3H, CH3), 4.88 (S, 1H, CH), 7.68–7.80 (m, 11H,
Ar–H), 8.38 (s, 1H, NH), 9.13 (s, 1H, NH). 13C NMR
(CDCl3) d: 26.1, 36.2, 52, 60.3, 103.5, 103.8, 105, 114.3,
116.7, 122.1, 126.7, 127.5, 127.9, 128.2, 129, 129.5, 133.6,
139.2, 142.8, 143.5, 155.1, 162.4; MS m/z (%): 476. Anal.
Calcd. for C24H18N5S2Cl: C, 60.56; H, 3.81; N, 14.71.
Found: C, 60.49; H, 3.70; N, 14.62%.
Yield 77%, mp 2028C; Rf-value 0.58; IR (KBr, cm-1):
3332, 1613, 1654, 652. H NMR (300 MHz, CDCl3) d:
1
2.37 (s, 3H, CH3), 3.70 (s, 3H, OCH3), 4.91 (s, 1H, CH),
7.31–7.98 (m, 11H, Ar–H), 8.30 (s, 1H, NH), 9.07 (s, 1H,
NH). 13C NMR (CDCl3) d: 26.5, 40.2, 52.4, 60.3, 103.2,
103.9, 104.9, 114.5, 116.5, 122.8, 126.1, 127.8, 128.6,
129.1, 133.3, 136, 142.3, 149.3, 155.9, 164.1; MS m/z (%):
471. Anal. Calcd. for C25H21N5OS2: C, 63.67; H, 4.49; N,
14.85. Found: C, 63.56; H, 4.52; N, 14.79%.
4,5-Dihydro-4-(4-chlorophenyl)-3-methyl-1-(10H-
phenothiazin-8-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-thiol
(4e)
Yield 79%, mp 1448C; Rf-value 0.57; IR (KBr, cm-1):
3327, 1611, 1648, 650. H NMR (300 MHz, CDCl3) d:
Results and discussion
1
2.38 (s, 3H, CH3), 4.90 (s, 1H, CH), 7.32–8.23 (m, 11H,
Ar–H), 8.43 (s, 1H, NH), 9.05 (s, 1H, NH). 13C NMR
(CDCl3) d: 26, 41.2, 52.3, 59.6, 103.3, 104, 105.6, 114.7,
116.2, 122.3, 127.2, 127.9, 128, 128.5, 128.9, 132.1, 134.5,
142.3, 143.6, 155.7, 162.5; MS m/z (%): 476. Anal. Calcd.
Chemistry
Classical Biginelli reaction was reported in 1893. Some of
the drawbacks of Classical Biginelli reaction are that it
requires high acidic condition, very long reaction time, and
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Med Chem Res (2012) 21:1887–1891
Biological activity
poor yield of product. In last decades, research work on the
synthesis of Biginelli compounds has been generally
increased due to their high pharmacological activity.
In order to promote conditions that would favor higher
yields of products, we have recently performed Biginelli
condensation using different catalysts such as PPA, AlCl3,
BF3, etc. In this way, we have found that using phosphorus
pentoxide as a catalyst in Biginelli’s one-pot protocol, a
significant increase in the yields of DHPMs was observed,
especially for systems that give only moderate yields using
traditional Biginelli conditions.
In vitro evaluation of the anti-tubercular activity was car-
ried out at the Tuberculosis Acquisition Antimicrobial
Coordinating Facility (TAACF) screening program, Ala-
bama, USA. Minimum inhibitory concentration (MIC) was
determined against M. tuberculosis H37Rv by using the
radiometric BACTEC (Collins and Franzblau, 1997;
Franzblau et al., 1998) and broth dilution (Yajko et al.,
1995; Suling et al., 2000) assay methods. The result of anti-
tubercular activity is presented in Table 1.
The activity is considerably affected by substitutions at
the phenyl ring of the pyrazolo[3,4-d]pyrimidine nucleus.
It has been observed that compounds 4b, 4d, and 4f having
methoxy, chloro, and nitro group at second position,
showed excellent anti-tubercular activity with percentage
inhibition of 93, 91, and 96, respectively, at a MIC of
\6.25 lg/ml, whereas the presence of same substituents at
any other position of phenyl ring remarkably reduced the
anti-tubercular activity.
Different pyrimidine derivatives containing a phenothi-
azine nucleus were synthesized under reflux temperature.
Reaction of 3-methyl-1-(10H-phenothiazin-8-yl)-1H-pyra-
zol-5(4H)-one (3), an appropriate aldehyde and thiourea, in
the presence of catalytic amount of phosphorus pentoxide
under reflux condition afforded 4,5-dihydro-4-(aryl)-3-
methyl-1-(10H-phenothiazin-8-yl)-1H-pyrazolo[3,4-d]pyrim-
idine-6-thiols (4a–h). The yields of the products were
found to be excellent (80–90%). The structures of the
synthesized compounds were assigned on the basis of IR,
1H NMR spectra, 13C NMR, and mass spectra, and purity
1
Conclusions
was proven by elemental analysis. In H NMR spectra of
(4a–h), a sharp peak representing methine proton of
pyrimidine was observed in the range of 4.88–4.97 dppm
confirming the formation of pyrazolo[3,4-d]pyrimidine
nucleus (Scheme 1).
In the present article, we report the synthesis, spectral stud-
ies, and anti-tuberculosis activity of a novel series pyrazolo
[3,4-d]pyrimidine containing phenothiazine nucleus. The
Scheme 1 Reaction scheme
H
N
H
SCH3
N
NHNH2
DMF
Ethyl acetoacetate
R
NH2NH2
S
S
H
HS
2
1
N
N
O
H
N
H
N
CH3
CH3
N
N
N
N
Thiourea
R-CHO
S
S
3
4a-h
R= H, 2-OH, 2-Cl, 4-Cl, 2-NO2, 4-NO2, 4-OCH3
Table 1 In vitro antitubercular
screening data of 4a–h
Compound
R
Molecular formula
C24H19N5S2
MIC (lg/ml)
Inhibition (%)
4a
4b
4c
4d
4e
4f
Phenyl
[6.25
\6.25
[6.25
\6.25
[6.25
\6.25
[6.25
[6.25
75
93
68
91
74
96
54
63
2-Hydroxyphenyl
4-Hydroxyphenyl
2-Chlorophenyl
4-Chlorophenyl
2-Nitrophenyl
C24H19N5OS2
C24H19N5OS2
C24H18N5S2Cl
C24H18N5S2Cl
C24H18N6O2S2
C24H18N6O2S2
C25H21N5OS2
4g
4h
4-Nitrophenyl
4-Methoxyphenyl
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Med Chem Res (2012) 21:1887–1891
1891
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preliminary in vitro anti-tuberculosis screening of these
novel series of 4,5-dihydro-4-(aryl)-3-methyl-1-(10H-phe-
nothiazin-8-yl)-1H-pyrazolo[3,4-d]pyrimidines 6-thiols has
evidenced that substitutions at second position on the phenyl
ring of the pyrazolo[3,4-d]pyrimidine nucleus have emerged
as potential compounds endowed with excellent anti-tuber-
culosis activity. On the contrary, substituents at any other
position of phenyl ring showed remarkable decrease in the
anti-tuberculosis activity. The possible decrease of anti-
tuberculosis activity of this basic pyrazolo[3,4-d]pyrimidine
structure through modulation of ring substituents warrants
further investigations. In summary, we have identified a
novel series of pyrazolo[3,4-d]pyrimidine containing phe-
nothiazine nucleus, which may develop into the potential
class of anti-tubercular agents.
Acknowledgments This study was funded by the CSIR through
Grant No. 01(02348)/09/EMR-II. The authors gratefuly acknowledge
the CSIR for its financial support. The authors are indebted to the
Tuberculosis Antimicrobial Acquisition and Coordinating Facility
(TAACF/USA) for biological tests. Amit Trivedi is thankful to CSIR,
New Delhi for fellowship.
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