Synthesis and in–vitro anti–HIV–1 evaluation of novel pyrazolo[4,3–c]pyridin–4–one derivatives

Article abstract of DOI:10.1016/j.ejmech.2019.111714

In our continuing efforts to find novel anti–HIV compounds, we have synthesized sixteen novel pyrazolo[4,3–c]pyridin–4–one derivatives. All the synthesized compounds were screened for anti–HIV activity against HIV–1_VB59 (R5, subtype C). Compounds 12a–12c and 12e were also tested against HIV–1_UG070 (X4, subtype D) in TZM–bl cell line. Compound 12c was found to be the most active against HIV–1_VB59 and HIV–1_UG070 with IC₅₀ value 3.67 μM and 2.79 μM, and therapeutic indices 185 and 243, respectively. The lead compound 12c inhibited the HIV-192/BR/018 (R5, subtype B) and drug resistant isolates, NIH-119 (X4/R5, subtype B) and NARI-DR (R5, subtype C) effectively. The activity of the lead compound was further confirmed by PBMC assays. The molecular docking data showed that the most active compound 12c binds in the non-nucleoside binding pocket of HIV-1 reverse transcriptase, which was confirmed by the ToA assay. Thus the study indicated that 12c may be considered as a NNRTI and further explored as a lead for anti-HIV drug development.

Full text of DOI:10.1016/j.ejmech.2019.111714

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Synthesis and in–vitro anti–HIV–1 evaluation of novel pyrazolo[4,3–c]pyridin–4–one
derivatives
Sanjay Kumar, Shiv Gupta, Leila Fotooh Abadi, Shraddha Gaikwad, Dipen Desai,
Kamlesh Kumar Bhutani, Smita Kulkarni, Inder Pal Singh
PII:
S0223-5234(19)30866-9
DOI:
https://doi.org/10.1016/j.ejmech.2019.111714
EJMECH 111714
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 December 2018
Revised Date: 23 August 2019
Accepted Date: 16 September 2019
Please cite this article as: S. Kumar, S. Gupta, L.F. Abadi, S. Gaikwad, D. Desai, K.K. Bhutani, S.
Kulkarni, I.P. Singh, Synthesis and in–vitro anti–HIV–1 evaluation of novel pyrazolo[4,3–c]pyridin–
4–one derivatives, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
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Synthesis and In–vitro Anti–HIV–1 Evaluation of Novel Pyrazolo[4,3
c]pyridin one Derivatives
–
–4–
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Sanjay Kumar1, Shiv Gupta1, Leila Fotooh Abadi2, Shraddha Gaikwad2, Dipen Desai2, Kamlesh
Kumar Bhutani1#, Smita Kulkarni2*, Inder Pal Singh1*
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1Department of Natural Products, National Institute of Pharmaceutical Education and
Research (NIPER), Sector–67, S. A. S. Nagar–160 062, Punjab, India
2Department of Virology, ICMR–National AIDS Research Institute (NARI), Bhosari, Pune–
411 026, Maharashtra, India
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*Address correspondence to this author at the Department of Natural Products, National Institute
of Pharmaceutical Education & Research, S.A.S. Nagar, Punjab–160 062, India; Tel: +91–172–
229–2144; Department of Virology, ICMR–National AIDS Research Institute (NARI), Bhosari,
Pune–411 026, Maharashtra, India; Tel: +91–20–27331207/27331200; E–mails:
ipsingh@niper.ac.in; ipsingh67@yahoo.com; skulkarni@icmr.org.in; skulkarni.nari@gov.in
# 25th December 1951 – 6th January 2018
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ABSTRACT
In our continuing efforts to find novel anti–HIV compounds, we have synthesized sixteen novel
pyrazolo[4,3–c]pyridin–4–one derivatives. All the synthesized compounds were screened for
anti–HIV activity against HIV–1VB59 (R5, subtype C). Compounds 12a–12c and 12e were also
tested against HIV–1UG070 (X4, subtype D) in TZM–bl cell line. Compound 12c was found to be
the most active against HIV–1VB59 and HIV–1UG070 with IC50 value 3.67 µM and 2.79 µM, and
therapeutic indices 185 and 243, respectively. The lead compound 12c inhibited the HIV-
192/BR/018 (R5, subtype B) and drug resistant isolates, NIH-119 (X4/R5, subtype B) and
NARI-DR (R5, subtype C) effectively. The activity of the lead compound was further confirmed
by PBMC assays. The molecular docking data showed that the most active compound 12c binds
in the non-nucleoside binding pocket of HIV-1 reverse transcriptase, which was confirmed by
the ToA assay. Thus the study indicated that 12c may be considered as a NNRTI and further
explored as a lead for anti-HIV drug development.
Key words: Pyrazolo[4,3–c]pyridin–4–one, Anti–HIV–1, QED, ADMET, TZM–bl.
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INTRODUCTION
Acquired immunodeficiency syndrome (AIDS) is a result of advanced stage infection by human
immunodeficiency virus (HIV). It is one of the world's most significant public health challenges,
particularly in low– and middle–income countries. In 2017, approximately 36.9 million people
(35.1 million adults) were living with HIV and 1.8 million people became newly infected,
globally. Nearly 1 million people died from AIDS related illness in 2017 [1]. An estimated 21.7
million people were receiving HIV treatment in 2017. However, globally, only 59% of the 36.9
million people living with HIV were receiving antiretroviral therapy (ART). In 2017, 8 out of 10
pregnant women living with HIV received antiretrovirals (ARVs) to prevent and eliminate
mother–to–child transmission and to keep mothers alive [2].
There are various antiretroviral drugs available in the market such as entry or fusion inhibitors,
nucleoside or non–nucleoside reverse transcriptase inhibitors (NRTI/NNRTI), integrase
inhibitors (IN), protease inhibitors (PI) and maturation inhibitors [3]. The resistance of virus to
the available antiretroviral drugs is the biggest challenge for ART and the discovery of new anti–
HIV agents to overcome this resistance is continually required.
Pyrazole is an important class of heterocyclic nucleus. Some of the pyrazole and fused–pyrazole
containing drugs were approved for treatment of various diseases, including celecoxib (1) and
lonazolac (2), COX–2 selective nonsteroidal anti–inflammatory drugs (NSAID) [4,5]; fipronil
(3), an insecticide; dipyrone (4), a potent analgesic and antipyretic agent [6]; sildenafil (5) [7]
used to treat erectile dysfunction (Figure 1).
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Figure 1. FDA approved pyrazole and fused–pyrazole containing drugs.
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Fused–pyrazoles are reported for various biological activities including antiviral [8],
antimicrobial [9,10], antiprotozoal [11], anticancer [12,13] and anti–inflammatory etc [14–16].
Some fused–pyrazole compounds were also reported for anti–HIV activity. The 1H–
pyrazolo[3,4–b]pyridine–3–yl derivatives were reported for anti–HIV–1 activity, which were
shown to act through reverse transcriptase inhibition [17,18] (Figure 2). Savant et al. reported
novel
3–amino–4,5–dihydro–6–methyl–4–oxo–N–aryl–1H–pyrazolo[4,3–c]pyridine–7–
carboxamide derivatives for anti–HIV activity against HIV–1 (IIIB) and HIV–2 (ROD) [19]
(Figure 2). Here, we report sixteen new N–substituted 1H–pyrazolo[4,3–c]pyridine–4–one
derivatives (Figure 3) and their evaluation for anti–HIV–1 activity. However, the N-substituted
compounds are not yet known for these activities. The aim of this work was to evaluate the effect
of introducing various N-substitutions on the fused pyrazolo-pyridine skeleton.
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CIC95 (cell culture inhibitory concentration): The concentration at which the spread of the virus is inhibited by
>95%; WT: wild type; FBS: fetal bovine serum; NHS: normal human serum.
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Figure 2. Synthetic fused–pyrazole derivatives for anti–HIV activity.
R
N
N
O
N
H
12a-12p
R = H, halogen, trifluoromethyl, alkyl
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Figure 3. Structure of designed N–substituted 1H–pyrazolo[4,3–c]pyridine–4–one scaffold.
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RESULTS AND DISCUSSIONS
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2.1 In silico QED and ADMET properties
Drug–likeness assesses the oral bioavailability of a chemical compound in the early stages of
drug discovery. Lipinski’s rule of 5 along with other similar rules gives qualitative impression of
drug–likeness. But these rules fail during the prioritization of chemicals based on their drug–
likeness. Bickerton et al. addressed this problem and proposed Quantitative Estimation of Drug–
likeness (QED) for the lead prioritization. They also suggested that compound with QED greater
than 0.5 can be considered as a good lead for further drug development. In this work, sixteen
new compounds were designed for synthesis by modifying the N-substituents and fused pyrazole
ring and rationalized for their drug-like properties through in silico approach. QED of all
designed compounds (12a– 12p) was calculated according to the reported method (Table 1) [20].
In order to make comparison, QED value was calculated for two known drugs i.e. nevirapine
(QED = 0.860) and zidovudine (QED= 0.422) [20–22]. QED value of all synthesized compounds
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was greater than 0.5, which indicated that the compounds possess drug-like properties.
Table 1. QED of pyrazolo[4,3–c]pyridin–4–one derivatives(12a–12p)
Compound No.
QED
0.654
0.634
0.670
0.670
0.700
0.687
Compound No
QED
0.687
0.674
0.693
0.693
0.694
0.694
Compound No
12m
12n
12o
12p
QED
0.655
0.672
0.672
0.717
0.860
0.420
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
Nevirapine
Zidovudine
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All the compounds were further evaluated for in silico ADMET properties using admetSAR tool
(Table 2) [23]. In order to validate the in silico protocol, we also evaluated in silico ADMET
properties of two known drugs (nevirapine and zidovudine). The results indicated that, all the
tested compounds along with nevirapine and zidovudine may cross blood brain barrier (BBB)
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and absorb in human intestine (HIA) (Table 2). Results also showed all tested compounds along
with nevirapine are permeable for Caco–2 cells, whereas, zidovudine showed negative result for
Caco–2 cell permeability. The above predicted result for known drugs is also evident from the
fact that zidovudine capsule and solution has oral bioavailability of less than 64% whereas oral
bioavailability of nevirapine tablet was approximately 91% [24]. CYP450 isozymes are involved
in the metabolism of drugs which in turn helps to excrete the drug out of the body and reduce the
effect of the drug. These isozymes also play a major role in drug–drug interaction. All the tested
compounds along with nevirapine were predicted as substrate for CYP450 3A4, whereas
zidovudine is nonsubstrate for CYP450 3A4. The above results are in line with the literature
reports that drug–drug interaction shown by nevirapine is due to the induction of CYP450 3A4
enzyme whereas zidovudine does not cause inhibition of CYP450 enzymes. Inhibition of
CYP450 isozyme results in drug–drug interaction. Most of the designed compounds were found
to be non–inhibitor of these enzymes. Compounds 12e, 12f and 12g were predicted to inhibit
enzyme CYP450 1A2 and CYP450 2C19. All the tested compounds along with nevirapine and
zidovudine were predicted to be non–inhibitors for enzymes CYP450 2C9, CYP450 2D6 and
CYP450 3A4. The in silico results also showed these compounds are noncarcinogenic and
nonmutagenic.
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Table 2. In silico ADMET properties of designed pyrazolo[4,3–c]pyridin–4–one derivatives (12a–12p)
12a 12b 12c 12d 12e 12f 12g 12h 12i 12j 12k 12l 12m 12n 12o 12p Nevirapine Zidovudine
BBB
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
HIA
Caco–2
permiability
P–GPS
p–GPI
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
–
NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS
NI NI NI NI NI NI NI NI NI NI NI NI
S
I
I
I
I
I
NI
Renal
cation transport
organic
NI NI NI NI NI NI NI NI NI NI NI NI NI
NI NI NI NI
NI
CYP450 2C9
substrate
NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS
NS
CYP450 2D6
NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS
S
NS
NS
substrate
CYP450 3A4
S
S
S
S
S
I
S
I
S
I
S
I
S
I
S
I
S
I
S
I
S
I
S
I
S
I
S
I
S
I
substrate
CYP450 1A2
inhibitor
CYP450 2C9
inhibitor
CYP450 2D6
inhibitor
CYP450 2C19
inhibitor
CYP450 3A4
inhibitor
NI NI NI NI
NI
NI
NI
NI
NI
NI NI NI NI NI NI NI NI NI NI NI NI NI
NI NI NI NI NI NI NI NI NI NI NI NI NI
NI NI NI NI
NI NI NI NI
NI NI NI NI
NI NI NI NI
NI NI NI
I
I
I
I
NI NI NI NI NI NI
NI NI NI NI NI NI NI NI NI NI NI NI NI
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Toxicity
Carcinogen
NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT
T
T
NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC
NC
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Blood–Brain Barrier (BBB): don’t cross BBB (–); cross BBB (+), Human Intestinal Absorption (HIA): not absorbed (–); absorbed (+),Caco–2 Permeability: not
permeable (–); permeable (+), Cytochrom 450 (CYP450): S = substrate for enzyme; NS = not a substrate for enzyme, I = enzyme inhibitor; NI = not enzyme
inhibitor, NT: nontoxic; T: toxic, NC: noncarcinogenic.
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2.2 Chemistry
All the designed compounds, a total of sixteen pyrazolo[4,3–c]pyridin–4–one derivatives were
synthesized using Scheme 1. Compound 6–Methyl–2–oxo–1,2–dihydropyridine–3–carbonitrile
(14) was synthesized using 3–ketobutyraldehyde dimethyl acetal (13) and cyanoacetamide as
starting materials [25]. The nitrile group of compound 14 was then converted into formyl group
using DIBAL–H to give compound 15 [26]. The structures of compounds 14 and 15 were
confirmed by comparing melting point, 1H NMR and 13C NMR with the literature values [25,26].
The pyrazolo[4,3–c]pyridin–4–one derivatives were synthesized from compound 15 in two steps.
In the first step, compound 15 and substituted phenylhydrazine hydrochlorides were stirred in
ethanol at room temperature to yield intermediate hydrazones, which were used in second step
without purification. Only one intermediate hydrazone, 6–methyl–3–((2–(p-tolyl) hydrazono)
methyl) pyridin–2(1H)–one (16a) [22] obtained from the reaction of 15 with p–tolylhydazine
hydrochloride was purified by repeated washing with ethanol for characterization. Spectral data
of compound 16a was matched with reported data [22]. In the second step, the intermediate
hydrazones were converted in to pyrazolo[4,3–c]pyridin–4–one derivatives (12a–12p) in boiling
nitrobenzene (Scheme 1) [27]. The structures of synthesized compounds (Figure 4) were
confirmed by spectral data i.e. IR, mass, 1H NMR, 13C NMR etc. (Supplementary information).
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Scheme 1. (a) Cyanoacetamide, piperidine acetate, 80 °C, 24 h, 80%; b) HMDS, toluene, 110 °C, 3 h; c) DIBAL–H,
toluene, 0 °C, 8 h, (combined yield of steps b and c: 75%); d) R–NH–NH2.HCl, ethanol, rt, 2 h; e) Nitrobenzene,
210 °C, 15 min (combined yield of steps d and e: 41–64%).
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Figure 4. Structures of pyrazolo[4,3–c]pyridin–4–one derivatives (12a–12p).
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2.3 Anti–HIV activity
Prior to screening the synthesized pyrazolo[4,3–c]pyridin–4–one derivatives (12a–12p) for anti–
HIV activity, the compounds were tested for cytotoxicity using MTT cell viability assay and
CC50 values were determined. The compound 12c showed CC50 value 679 µM which is closer to
CC50 value of Zidovudine i.e. 872 µM. Further all the compounds were screened for anti–HIV
activity against HIV–1VB59 (R5, subtype C). The compounds 12a–12d and 12e were further
tested against both HIV–1VB59 (R5, subtype C) and HIV–1UG070 (X4, subtype D) in TZM–bl cell
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line. The compounds 12d and 12f–12p were not tested against HIV–1UG070 (X4, subtype D)
because of IC50 of these compounds against HIV–1VB59 (R5, subtype C) was less than CC50
value. The IC50 values and therapeutic indices (TI) were calculated for each of them and
compared with the drug control (Table 3). The compound 12c was the most active compound
against HIV–1VB59 and HIV–1UG070 among all the tested pyrazolo[4,3–c]pyridin–4–one
derivatives showing IC50 values of 3.67 µM and 2.79 µM, respectively, against these two strains.
The compound 12c also showed good therapeutic indices 185 and 243 against HIV–1VB59 and
HIV–1UG070, respectively. Subsequently, the active lead compound 12c was also tested against
HIV-192/BR/018 (R5, subtype B) and exhibited IC50 value7.42±2.3
µM. Furthermore, the compound
was also effective against two nevirapine drug resistant isolates HIV-1N119 (X4/R5, subtype B)
and HIV-1NARI-DR (R5, subtype C) with IC50 values 3.24±0.9
(Table 4).
µM and 2.53±0.2 µM, respectively
Substitutions of N–1 phenyl ring with electron withdrawing groups like –F, –Cl, –CF3 and –
OCH3 lead to loss of the anti–HIV activity, whereas, substitution with methyl group leads to
increased anti–HIV activity. Further, compound 12c with 3,5–dimethyl substitution possessed
greater anti–HIV activity as compared to the 4–methyl and 3,4–dimethyl substituted compounds.
Similarly, substitutions of N–1 phenyl ring with electron withdrawing groups like –F, –Cl, –CF3
and –OCH3 resulted in increased toxicity and substitution with methyl ring led to reduced
toxicity.
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Table 3. Anti–HIV–1 activity of pyrazolo[4,3–c]pyridin–4–one derivatives (12a–12p)
Sr. Compound
No No.
Cytotoxicity
CC50 (µM)
Anti–HIV–1 testing data
IC50 HIV1VB59 TI
IC50
TI
(R5) (
µ
M)
HIV1VB59
(R5)
6.6
5.86
185
0.74
2.24
1.00
NA
NA
NA
NA
0.58
0.52
NA
NA
NA
HIV1UG070
(X4) (µM)
228.79
444.44
2.79
NT
>196.08
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
HIV1UG070
(X4)
1.67
NA
243
NT
NA
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
1
2
3
4
5
6
7
8
383.18
708.00
679.00
93.40
552.94
34.06
100.35
260.25
93.50
93.50
79.80
72.57
158.72
133.45
129.54
45.91
872
58.08
120.80
3.67
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
12o
12p
Zidovudine
126.44
246.78
127.78
>170.65
>193.05
>170.65
>170.65
138.28
9
10
11
12
13
14
15
16
17
139.47
>205.76
>191.57
>191.57
>177.94
0.03
NA
29067
0.027
32296
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183
184
185
CC50: 50% cytotoxic concentration; IC50: 50% inhibitory concentration; Therapeutic index (TI): CC50/IC50; NA: Not
applicable; NT: Not tested
Table 4. Anti–HIV–1 activity of lead compound 12c against various primary isolates of HIV-1
HIV-1 primary isolates
HIV-1 VB59
CC50 IC50
M) M) TI
679 3.67
HIV-1 UG070
IC50
HIV-1 92/BR/018
IC50
HIV-1 NARI-DR
IC50
HIV-1N119
IC50
Co
mp.
(
µ
(
µ
(
µ
M) TI
(
µ
M)
TI
(
µ
M)
TI
(
µ
M)
TI
2.79
7.42
2.53
3.24
±223 ±2.3
185
±1.8
243
±2.3
91
±0.2
268
±0.9
210
12c
Zido
vudi
ne
Nev
irapi
ne
872± 0.02±
13.8 0.01
0.01±
43600 0.0
0.016
87200 0.005
±
0.02
±
0.02
±
0.01
0.01
54500
1990
43600
2.6
43600
597
0.71
0.53
±0.3
0.30
±0.1
232.3
±13.7
140.0
±52.6 4.3
±63
±0.1
840
1126
186
CC50: 50% cytotoxic concentration; IC50: 50% inhibitory concentration; Therapeutic index (TI): CC50/IC50
187
The data represents Mean ± SD of three independent assays.
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The lead compound 12c exhibited a CC50 of 592 µM and IC50 value of 8.65 µM in the
confirmatory assays carried out in PBMCs against HIV–1VB51 (Table 5). Compounds 12a and
12c were also tested for the inhibition of reverse transcriptase (RT) enzyme. Compound 12c
exhibited an IC50 value 30.80±9.65
µM, while compound 12a had an IC50 value of 82.90±5.58
µ
M . Zidovudine and Nevirapine were used as controls in the assay and inhibited the HIV–1 RT
with an IC50 of 0.02 ± 0.01 µM and 13.14±1.88 µM, respectively.
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Table 5. Anti–HIV–1 activity of 12c in PBMCs
Anti–HIV–1 testing data
Sr. Compound
No No.
Average Cytotoxicity
CC50 ( M)
(
µ
IC50 HIV1VB51 (R5)
(
µ
M)
TI HIV1VB51 (R5)
1
2
3
592±124
1071±25
1003±16
8.65±2.09
0.05±0.01
0.19±0.03
68
21420
5278
12c
Zidovudine
Nevirapine
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CC50: 50% cytotoxic concentration; IC50: 50% inhibitory concentration; Therapeutic index (TI) = CC50/IC50.
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2.3.1: Time-of addition experiment
To identify the target of the drug action, the time-of-addition assay (ToA) was carried out using
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the lead compound 12c along with known anti-retrovirals (ARVs). It was observed that the
inhibition of the lead compound 12c declined after 10 h post infection similar to NVP (Figure 5).
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Figure 5. Time-of-addition analysis.
The target of lead compound 12c was compared to known antiretroviral drugs. Final compound
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concentrations were 5-fold higher than their IC50 values. D.S. (0.25 g/mL), AZT (0.05
µ
µM),
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212
213
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NVP (0.94 M), RAL (0.16 M), RTV (59.39 M) and 12c (1.42 µM). Test compounds were
µ
µ
µ
added at different time points (0, 1, 2, 3, 4, 6, 8, 10, 18, and 24 h) at or after infection. The
percent infection (RLU) was determined. Data represent mean±SD calculated from three
independent experiments. The results showed that 12c followed an inhibition pattern similar to
AZT upto 3 h which continued up to 10 h like NVP. It was noted that the activity of compound
12c depended on the concentration tested. It blocked the viral reverse transcription process at
low concentrations, whereas, at higher concentrations, it showed inhibition for extended
duration. Thus the ToA assay enabled to discriminate between NRTI versus NNRTI.
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2.4
Molecular docking
Blind docking calculations were carried out to find the potential binding sites of 12c in the
reverse transcriptase enzyme. The results showed that 12c binds within 48 pockets of the reverse
transcriptase (Figure 6). The highest energy cluster (-10.5 kcal/mol) of 12c occupies the
allosteric binding site of non-nucleoside reverse transcriptase inhibitors. Further, docking studies
of all the synthesized compounds (12a–12p) were performed within the non-nucleoside binding
pocket (NNBP) to study the binding modes. The binding mode analysis showed that the phenyl
ring of 12a, 12b, 12d–12l and 12n–12p (Figure 7a) and pyridinone ring of compounds 12c and
12m (Figure 7b) occupies the aromatic-rich region of NNBP. Interestingly, 12f and 12c showed
maximum binding affinities of -10.9 and -10.5 kcal/mol, respectively (Table 6). The binding
affinity of 12c is translated into the anti-HIV activity contrary to 12f. The analysis of the protein-
219
220
221
222
223
224
225
226
227
16

228
229
230
231
232
233
234
235
236
237
238
239
240
241
ligand interaction showed that the pyridinone moiety of 12c occupies a hydrophobic aromatic-
rich pocket formed mainly by the side chains of Tyr181, Tyr188, Phe227, and Trp229. The
hydrophobic contacts were found between the pyridinone ring of 12c and residue Y181 and
Y188 (Figure 8b). These hydrophobic interactions were absent in 12f (Figure 8a). Furthermore,
the phenyl ring of 12f occupies the hydrophobic pocket formed by the side chains of Tyr181,
Leu100. The hydrogen bonding in 12f increases the binding affinity but in effect, it reduces the
hydrophobic interactions with residue Y181 and Y188 (Figure 8a). Compound 12m also interacts
with the residue Y181 and Y188 but its binding affinity (-9.5 kcal/mol) is low. No other
compound showed interaction with both of the residues i.e Y181 and Y188. The NNBP does not
exist in the absence of an inhibitor; rather, binding of these inhibitors causes the side chains of
Y181 and Y188 to flip from a ‘down’ to an ‘up’ orientation which generates this NNBP [28].
Therefore, the interaction of inhibitors with these residues is considered important for the
activity. These results support the hypotheses about the importance of hydrophobic contacts for
NNBP.
17

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243
Figure 6. Clustered blind docking results.
244
245
246
247
Figure 7. Binding mode of compounds 12a–12p a) Compound 12a, 12b, 12d–12l and 12n–12p b) Compound 12c
and 12f .
18

248
249
250
251
252
Figure 8. Binding modes of 12f (8a) and 12c (8b) showing various interactions with reverse transcriptase enzyme
(Atoms of protein involved in hydrophobic interactions are shown as grey ball and atoms of protein involved in
hydrogen bond are shown as black ball. Residue involved in pi-stacking is shown as grey stick).
253
Table 6. Binding affinities and interactions of compounds 12a–12p
254
Binding Affinity
(kcal/mol)
Hydrogen
Bond
Compound
12a
Hydrophobic interaction
pi-Stacking
Halogen Bond
P95, L100, K103, V106,
Y181, L234, Y318
-9.8
-9.6
H235, Y318 W229
H235, Y318 W229
–
–
L100, K103, V106, Y181,
Y318
12b
P95, L100, V106, V179,
-10.5
Y181, Y188, F227, W229, –
L234
–
–
12c
P95, L100, K103, V106,
Y181, L234, Y318
-10.3
-9.7
H235, Y318 W229
H235, Y318 W229
H235, Y318 W229
–
–
–
12d
12e
12f
L100, K103, V106, Y181,
Y318
L100, K103, V106, Y181,
L234, Y318
-10.9
-10.4
-9.7
V106, V179, Y181, F227
Y318
W229
–
–
12g
12h
L100, K103, V106, L234, H235, Y318 W229
19

Y318
L100, K103, V106, L234,
Y318
-10.1
-9.6
H235, Y318 W229
–
12i
12j
12k
12l
L100, K103, V106, Y181,
L234, Y318
P236, Y318
–
–
V179
L100, V106, V179, Y181,
F227, L234
-9.3
W229
–
–
L100, K103, V106, Y181,
L234, Y318
-10.1
H235, Y318 W229
L100, V106, Y181, Y188,
F227, L234
-9.5
-10.3
-10
K101
W229
–
–
–
12m
12n
12o
L100, K103, V106, Y318
H235, Y318 W229
H235, Y318 W229
L100, K103, V106, Y181,
L234, Y318
L100, K103, V106, Y181,
L234, Y318
-10
H235, Y318 W229
–
12p
255
256
257
258
259
260
261
262
263
264
265
266
3
CONCLUSIONS
Sixteen new pyrazolo[4,3–c]pyridin–4–one derivatives were synthesized. All synthesized
compounds were predicted to be drug-like as indicated by QED value greater than 0.5. Also,
synthesized compounds were predicted to have favorable ADMET parameters. Compound 12c
was found to be the most active against HIV–1VB59 and HIV–1UG070 with IC50 value 3.67 µM and
2.79 µM, and therapeutic indices of 185 and 243, respectively. The compound could also inhibit
nevirapine drug resistant viruses and has been shown to inhibit reverse transcriptase assay. The
molecular docking study showed that the most active compound 12c binds in the non-nucleoside
binding pocket of HIV–1 reverse transcriptase, which was confirmed by ToA assay. The study
indicated that 12c may be considered as a NNRTI can be further explored as a lead for anti–
HIV–1 drug development.
20

267
4
EXPERIMENTAL
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270
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274
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276
277
278
279
280
281
282
283
284
4.1 General
All chemicals were purchased from Sigma–Aldrich, Hyderabad, India, or Alfa Aeser,
Hyderabad, India, and used without further purification for the synthesis of pyrazolo[4,3–
c]pyridin–4–one derivatives. Solvents used for the synthesis were of LR (laboratory reagent)
grade and used without further purification. Precoated silica gel aluminum sheets (TLC Silica gel
60 F254, 0.25 mm thickenss, Merck, Germany) were used for TLC and visualized under UV light
and derivatized using Dragendorff reagent. Compounds were purified using Silica gel (#230–
400, Merck, Germany) column chromatography. Purity of synthesized compounds was checked
using high–performance liquid chromatography (HPLC, Shimadzu Corporation, Kyoto, Japan).
All the newly synthesized pyrazolo[4,3–c]pyridin–4–one derivatives were characterized by using
1
1
by H NMR, 13C NMR, mass spectrometer and High–Resolution Mass Spectra (HRMS). H
13
NMR and C NMR spectra were recorded on 400 and 100 MHZ (Bruker FT–NMR Avance II,
USA) spectrometer, respectively, using tetramethylsilane as an internal standard. The chemical
shifts are reported in
δ units. Melting points were recorded on capillary melting point apparatus
(Buchi). Infra–red (IR) spectra were recorded by using Perkin Elmer–Spectrum II instrument.
Mass spectra were recorded on Thermo LTQ–XL mass spectrometer (Thermo, USA). HRMS
were recorded on maxisTM ESI–Q–TOF (Bruker, Germany).
285
286
287
288
289
4.2 Synthesis of 6–methyl–2–oxo–1,2–dihydropyridine–3–carbonitrile (14)
The compound 14 was synthesized using a method reported by Walton et al. [25]. 3–
Ketobutyraldehyde dimethyl acetal (13) (38 mmol, 5.0 g, 5 mL) and cyanoacetamide (41.8
mmol, 3.52 g) were mixed in 250 mL round bottom flask (RBF). The piperidinium acetate
solution (25% v/v, 15 mL) was added with stirring to dissolve the mixture. The resulted reaction
21

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291
292
293
mixture was transferred at 80 °C and stirred continuously for 24 h. The reaction mixture was
cooled to room temperature and filtered to collect the precipitate, which was washed with cold
water three times and dried under vacuum to give white compound 14 (3.7 g, 73 % yield)
(Scheme 1).
294
295
296
297
298
299
300
301
302
303
304
305
306
307
4.3 Synthesis of 6–methyl–2–oxo–1,2–dihydropyridine–3–carbaldehyde (15)
Compound 15 was synthesized using the method described by Showalter et al. [26]. In brief,
compound 14 (14.9 mmol, 2 g) and hexamethyldisilazane (14.9 mmol, 2.4 g, 3.1 mL) were
mixed in 100 mL round bottom flask (RBF) under nitrogen. To above mixture, dry toluene (10
mL) was added and mixture was stirred at 120 °C for 2 h to give clear solution. The reaction
mixture was cooled to room temperature followed by removal of the solvent under vacuum to
obtain yellowish oil. Dry toluene (30 mL) was added to the oily residue under nitrogen with
continuous stirring at 0 °C. Di-isobutylaluminium hydride (DIBAL–H) (25% w/w in toluene) (14
mL) was added dropwise for 1 h to above solution. The reaction mixture was stirred at 0 °C for 8
h. After completion, the reaction solution was transferred in to 250 mL RBF and acidified with
3N HCl (50 mL) to give yellow precipitate. The reaction mixture was extracted with the
dichloromethane (DCM) three times. Combined DCM extract was treated with activated
charcoal and concentrated under vacuum after filtration to give yellowish compound 14 (1.7 g,
82 % yield) (Scheme 1).
308
309
310
311
312
4.4 Synthesis of pyrazolo[4,3–c]pyridin–4–one derivatives (12a–12q)
The pyrazolo[4,3–c]pyridin–4–one derivatives were synthesized by using compound 15 as
starting material in two steps. In first step, the compound 15 (1 mmol, 137 mg) was dissolved in
ethanol (20 mL) and substituted phenylhydrazine hydrochloride (1.1 equivalent) was added. The
reaction mixture was stirred at room temperature for 2 h. The completion of reaction was
22

313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
monitored by TLC. The resulted reaction mixture was allowed to cool to room temperature. The
solvent was evaporated under vacuum; ethanol (5 mL) was added and sonicated for 30 seconds
to produce suspension. The resulted suspensions were kept in refrigerator for 1 h to settle down
the suspended particles. The suspended particles was filtered out and used for second step
without further purification. In second step, the resulted solid was poured in boiling nitrobenzene
at 210 °C and stirred at same temperature for 15 minutes [27]. The completion of reaction was
monitored by TLC. After the completion, the reaction mixture was allowed to cool to room
temperature followed by silica gel column chromatography to isolate pure pyrazolo[4,3–
c]pyridin–4–one derivatives (12a–12p)
6–Methyl–1–( –tolyl)–1 –pyrazolo[4,3–c]pyridin–4(5
51%; mp = 263–265 °C; FTIR (CHCl3)
.
p
H
H
)–one
(
12a): Light brown solid; Yield
1
υ
cm–13418,2923, 2851, 1673, 1463, 1377; H NMR
(CDCl3, 400 MHz):
δ
11.46 (s, 1H), 8.31 (s, 1H), 7.49 (d,
J
= 7.0 Hz, 2H), 7.33 (d,
J
= 6.9 Hz,
2H), 6.36 (s, 1H), 2.44 (s, 6H); 13C NMR (CDCl3, 100 MHz):
δ
161.0, 144.7, 142.5, 137.9,
137.5, 136.7, 130.1 (2C), 123.3 (2C), 112.5, 92.2, 21.1, 19.6; HRMS (ESI) m/z cacld for
C14H13N3O [M+Na]+: 262.0956, found: 262.0944.
6–Methyl–1–phenyl–1
H
–pyrazolo[4,3–c]pyridin–4(5
H)–one
(12b): Tint white solid; Yield
1
58%; mp = 236–240 °C; FTIR (CHCl3)
υ
cm–1 3443,2922, 2851, 1651, 1458, 1377, 758; H
NMR (CDCl3, 400 MHz):
δ 11.30 (s, 1H), 8.33 (s, 1H), 7.62 (dd, J = 8.6, 1.1 Hz, 2H), 7.57–7.53
(m, 2H), 7.43 (t,
J
= 7.4 Hz, 1H), 6.41 (s, 1H), 2.44 (s, 3H); 13C NMR (CDCl3, 100MHz):
δ
161.0, 148.4, 144.7, 142.7, 139.1, 137.7, 129.6 (2C), 127.9, 123.4 (2C), 112.7, 92.3, 19.7;
HRMS (ESI) m/z cacld for C13H11N3O [M+Na]+: 248.0800, found: 248.0792.
1–(3,5–Dimethylphenyl)–6–methyl–1
H–pyrazolo[4,3–c]pyridin–4(5
H)–one
(12c): White
solid; Yield 64%; mp = 286–289 °C; FTIR (CHCl3)
υ
cm–1 3432,2917, 1633, 1459, 1259, 749;
23

336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
1H NMR (CDCl3, 400 MHz):
1H), 2.45 (s, 3H), 2.41 (s, 6H); 13C NMR (CDCl3, 100 MHz):
δ 11.46 (s, 1H), 8.31 (s, 1H), 7.22 (s, 2H), 7.06 (s, 1H), 6.38 (s,
δ
161.1, 144.7, 142.5, 139.5,
139.0, 137.4, 129.5 (2C), 121.2 (2C), 112.5, 92.4, 21.3 (2C), 19.7; HRMS (ESI) m/z cacld for
C15H15N3O [M+Na]+: 276.1113, found: 276.1104.
1–(3,4–Dimethylphenyl)–6–methyl–1
solid; Yield 50%; mp = 248–250 °C; FTIR (CHCl3)
NMR (CDCl3, 400 MHz): 11.36 (s, 1H), 8.30 (s, 1H), 7.39 (d,
H–pyrazolo[4,3–c]pyridin–4(5
H)–one
(12d): Brown
1
υ
cm–1 3433,2923, 1638, 1459, 1016; H
δ
J = 1.9 Hz, 1H), 7.31 (dd, J =
8.1, 2.0 Hz, 1H), 7.29 (s, 1H), 6.36 (s, 1H), 2.43 (s, 3H), 2.36 (s, 3H), 2.34 (s, 3H); 13C NMR
(CDCl3, 100 MHz):161.0, 144.7, 142.4, 138.2, 137.4, 136.9, 136.6, 130.4, 124.7, 120.7, 112.5,
92.3, 19.9, 19.7, 19.5; HRMS (ESI) m/z cacld for C15H15N3O [M+Na]+: 276.1113, found:
276.1105.
1–(3–Methoxyphenyl)–6–methyl–1H–pyrazolo[4,3–c]pyridin–4(5
H)–one
(12e): Brown solid;
1
Yield 52%; mp = 184–189 °C; FTIR (CHCl3)
υ
cm–1 3433,2923, 1638, 1466, 1017; H NMR
(CDCl3, 400 MHz):
δ 10.99 (s, 1H), 8.31 (s, 1H), 7.44 (m, 1H), 7.20–7.18 (m, 2H), 6.97 (m, 1H),
6.43 (s, 1H), 3.89 (s, 3H), 2.43 (s, 3H); 13C NMR (CDCl3, 100 MHz):
δ
160.6, 144.7, 142.6,
140.2, 137.7, 130.2, 115.4, 113.7, 109.2, 106.3, 101.5, 92.4, 55.6, 19.7; HRMS (ESI) m/z cacld
for C14H13N3O2 [M+Na]+: 278.0905, found: 278.0895.
6–Methyl–1–(3–(trifluoromethyl)phenyl)–1
H–pyrazolo[4,3–c]pyridin–4(5
H)–one
(12f):
Light brown solid; Yield 50%; mp = 233–235 °C; FTIR (CHCl3)
υ
cm–1 3390,2921, 2850, 1671,
1465, 1275, 1120, 750; 1H NMR (CDCl3, 400 MHz):
δ
11.52 (s, 1H), 8.36 (s, 1H), 7.93 (s, 1H),
13
7.84 (m, 1H), 6.69 (m, 2H), 6.40 (s, 1H), 2.48 (s, 3H); C NMR (CDCl3, 100 MHz):
δ 160.9,
144.7, 143.5, 139.7, 138.4, 132.1, 130.3, 126.2, 124.8, 124.4, 120.2, 114.1, 91.8, 19.8; MS (ESI)
m/z 294 [M + 1], 316 [M+Na]+ for C14H10F3N3O.
24

359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
6–Methyl–1–(4–(trifluoromethyl)phenyl)–1
H
–pyrazolo[4,3–c]pyridin–4(5
H
)–one
(12g):
Brown solid; Yield 46%; mp = 262–264 °C; FTIR (CHCl3)
1330, 1108, 1062; 1H NMR (CDCl3, 400 MHz):
6.44 (s, 1H), 2.45 (s, 3H); 13C NMR (CDCl3, 100 MHz):
υ
cm–1 3435, 2920, 2851, 1669,
δ
10.97 (s, 1H), 8.35 (s, 1H), 7.83–7.78 (m, 4H),
δ
160.6, 144.8, 143.3, 142.0, 138.5,
129.5, 126.9 (2C), 123.1 (2C), 122.4, 113.2, 91.9, 19.8; MS (ESI) m/z 294 [M+H], 316 [M+Na]+
for C14H10F3N3O.
1–(4–Chlorophenyl)–6–methyl–1
H–pyrazolo[4,3–c]pyridin–4(5
H)–one (12h): Light brown
solid; Yield 52%; mp = 247–249 °C; FTIR (CHCl3)
1377, 1262, 751; 1H NMR (CDCl3, 400 MHz):
υ
cm–1 3433, 2955, 2922, 2852, 1663, 1461,
δ
10.88 (s, 1H), 8.31 (s, 1H), 7.58–7.50 (m, 4H),
6.36 (s, 1H), 2.42 (s, 3H); 13C NMR (CDCl3, 100 MHz): 156.3, 144.7, 142.8, 138.0, 137.7,
134.7, 133.6, 129.8 (2C), 124.5 (2C), 114.9, 92.0, 19.7; HRMS (ESI) m/z cacld for C13H10ClN3O
[M+Na]+: 282.0410, found: 282.0404.
1–(3,4–Dichlorophenyl)–6–methyl–1H–pyrazolo[4,3–c]pyridin–4(5
H)–one
(12i):
Light
brown solid; Yield 50%; mp = 273–275 °C; FTIR (CHCl3)
υ
cm–1 3435, 2919, 2850, 1634, 1487,
1
750; H NMR (DMSO–d6, 400 MHz):
δ
11.35 (s, 1H), 8.27 (s, 1H), 7.93 (d,
J = 2.2 Hz, 1H),
7.83 (d,
(DMSO–d6, 100 MHz):
J = 8.7 Hz, 1H), 7.69 (dd, J
= 8.7, 2.3 Hz, 1H), 6.55 (s, 1H), 2.26 (s, 3H); 13C NMR
δ
159.1, 144.9, 139.0, 138.6, 132.6, 132.0, 130.4, 125.0, 123.3, 113.2,
91.2, 79.5, 19.4; MS (ESI) m/z 294 [M+H]+, 296 [M+2+H]+, 298 [M+4+H]+, 316 [M+Na]+, 318
[M+2+Na]+, 320 [M+4+Na]+ for C13H9Cl2N3O.
1–(3,5–Dichlorophenyl)–6–methyl–1H–pyrazolo[4,3–c]pyridin–4(5
H)–one
(12j):
Light
brown solid; Yield 55%; mp = 242–244 °C; FTIR (CHCl3)
υ
cm–13435, 2920, 2850, 1671, 1460,
1
766; H NMR (DMSO–d6, 400 MHz):
δ
11.38 (s, 1H), 8.28 (s, 1H), 7.74 (s, 2H), 7.72 (s, 1H),
159.0, 145.1, 145.0, 141.2, 138.8,
6.56 (s, 1H), 2.28 (s, 3H); 13C NMR (DMSO–d6, 100 MHz):
δ
25

382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
135.4 (2C), 127.6, 121.9 (2C), 113.3, 91.2, 19.4; MS (ESI) m/z 294 [M+H]+, 296 [M+2+H]+, 298
[M+4+H]+, 316 [M+Na]+, 318 [M+2+Na]+, 320 [M+4+Na]+ for C13H9Cl2N3O.
1–(4–Bromophenyl)–6–methyl–1
H–pyrazolo[4,3–c]pyridin–4(5
H)–one
(12k): Brown solid;
1
Yield 48%; mp = 270–271 °C; FTIR (CHCl3)
υ
cm–13401,2917, 1669, 1494, 1349, 566; H
NMR (CDCl3, 400 MHz):
δ 10.63 (s, 1H), 8.31 (s, 1H), 7.67 (dd, J = 6.8, 1.9 Hz, 2H), 7.51 (dd,
J
= 6.9, 1.8 Hz, 2H), 6.35 (s, 1H), 2.41 (s, 3H); 13C NMR (CDCl3, 100 MHz):
δ 160.4, 152.0,
144.6, 142.7, 138.2, 138.1, 132.7 (2C), 124.8 (2C), 121.4, 92.0, 19.8; MS (ESI) m/z 326
[M+Na]+, 328 [M+2+Na]+ for C13H10BrN3O.
1–(3–Bromophenyl)–6–methyl–1H–pyrazolo[4,3–c]pyridin–4(5
H)–one
(12l): Brown solid;
1
Yield 42%; mp = 278–281 °C; FTIR (CHCl3)
υ
cm–1 3419,2921, 1633, 750; H NMR (CDCl3,
400 MHz):
δ
10.28 (s, 1H), 8.31 (s, 1H), 7.81 (br s, 1H), 7.58–8.55 (m, 2H), 7.41 (t,
J
= 8.0 Hz,
1H), 6.38 (s, 1H), 2.42 (s, 3H); 13C NMR (CDCl3, 100 MHz):
δ
160.3, 146.4, 144.7, 142.8,
140.2, 138.2, 130.9, 130.8, 126.4, 123.1, 121.8, 91.9, 19.8; MS (ESI) m/z 326 [M+Na]+, 328
[M+2+Na]+ for C13H10BrN3O.
1–(3–Fluorophenyl)–6–methyl–1
H–pyrazolo[4,3–c]pyridin–4(5
H)–one
(12m): White; Yield
1
51%; mp = 230–232 °C; FTIR (CHCl3)
υ
cm–1 3429,2921, 1660, 1465, 789; H NMR (CDCl3,
400 MHz):
δ
10.75 (s, 1H), 8.32 (s, 1H), 7.54–7.48 (m, 1H), 7.44 (d, J = 8.2 Hz, 1H), 7.39 (dd, J
13
= 9.5, 2.0 Hz, 1H), 7.16–7.11 (m, 1H), 6.42 (s, 1H), 2.44 (s, 3H); C NMR (CDCl3, 100 MHz):
δ
160.4, 144.7, 142.9, 140.6, 140.5, 138.2, 130.9, 130.8, 118.7, 118.7, 114.8, 114.6, 113.0, 111.0,
110.8, 92.0, 19.8; MS (ESI) m/z 265.98 [M+Na]+ for C13H10FN3O.
1–(3,4–Difluorophenyl)–6–methyl–1
Yield 41%; mp = 288–289 °C; FTIR (CHCl3)
(DMSO–d6, 400 MHz):
H
–pyrazolo[4,3–c]pyridin–4(5
H
)–one
(
12n): White;
1
υ
cm–1 3401,2918, 1673, 1469, 770; H NMR
δ
11.33 (s, 1H), 8.24 (s, 1H), 7.81–7.76 (m, 1H), 7.68–7.61 (m, 1H),
26

13
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
7.54–7.52 (m, 1H), 6.52 (s, 1H), 2.25 (s, 3H); C NMR (DMSO–d6, 100 MHz): δ 159.2, 144.8,
144.7, 138.2, 120.4, 119.0, 118.8, 113.4, 113.2, 112.9, 102.1, 91.2, 19.4; MS (ESI) m/z 262
[M+H]+ for C13H9F2N3O.
1–(3,5–Difluorophenyl)–6–methyl–1
H
–pyrazolo[4,3–c]pyridin–4(5
H)–one
(
12o):
Light
1
brown; Yield 41%; mp = 281–284 °C; FTIR (CHCl3)
υ
cm–1 3435,2921, 1625, 747; H NMR
(DMSO–d6, 400 MHz):
δ
11.39 (s, 1H), 8.28 (s, 1H), 7.48 (d,
J
= 6.2 Hz, 2H), 7.39 (tt,
J = 13.9,
13
2.3 Hz, 1H), 6.66 (s, 1H), 2.28 (s, 3H); C NMR (DMSO–d6, 100 MHz):
δ
164.4, 164.3, 162.0,
161.8, 159.0, 145.0 (2C), 144.9, 141.4, 138.7 (2C), 113.4, 107.0, 106.9, 106.7, 103.3, 91.3, 19.4;
MS (ESI) m/z 284.06 [M+Na]+ for C13H9F2N3O.
1–(4–(tert–Butyl)phenyl)–6–methyl–1
H
–pyrazolo[4,3–c]pyridin–4(5
H)–one
(12p): Light
1
brown; Yield 53%; mp = 246–247 °C; FTIR (CHCl3)
υ
cm–1 3435,2923, 1660, 1520, 779; H
NMR (CDCl3, 400 MHz):
δ 11.48 (s, 1H), 8.31 (s, 1H), 7.56–7.51 (m, 4H), 6.41 (s, 1H), 2.43 (s,
3H), 1.38 (s, 9H); 13C NMR (CDCl3, 100 MHz):
δ
161.1, 151.1, 144.7, 142.5, 137.5, 136.6,
126.4 (2C), 123.0 (2C), 112.5, 92.4, 34.8, 31.3 (3C), 19.6 HRMS (ESI) m/z cacld for C17H19N3O
[M+Na]+: 304.1426, found: 304.1424.
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422
423
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425
426
427
4.5 In silico prediction of QED and ADMET properties
The quantitative estimation of drug likeness (QED) of synthesized fused–pyrazole derivatives
were calculated. The molecular descriptors such as molecular weight (Mr), lipophilicity
(ALOGP), number of hydrogen bond donors (HBDs), number of hydrogen bond acceptors
(HBAs), polar surface area (PSA), number of aromatic ring (nAROM) and number of ratable
bond (ROTBs) were used for QED calculation which were calculated using Dragon 5.4 software
[29]. List of 105 toxicophores (notably nitro, conjugated nitrile, azido and thiocyanate) given by
Bickerton et al. was used to identify the number of ALERTs [20]. Absorption, distribution,
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metabolism, excretion (ADME) properties and carcinogenicity and mutagenicity were predicted
using online admetSAR tool [23].
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431
432
433
434
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436
437
438
439
440
441
442
443
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445
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449
4.6 Biological studies
The synthesized fused–pyrazole derivatives were evaluated for their biological activity at
ICMR–National AIDS Research Institute, Pune. The compounds were screened for cytotoxicity
and anti–HIV activity using TZM–bl assay [30,31]. The anti-HIV potential of the most active
lead(s) was further assessed on drug resistant viruses. The activity of the leads identified in
screening assays was confirmed by assessing their anti–HIV–1 potential in Peripheral Blood
Mononuclear Cells (PBMCs).
Cells
The TZM–bl cell line (genetically engineered HeLa cell line), obtained from the National
Institutes of Health (NIH), USA under the AIDS Research Reference Reagent Program (NIH
ARRRP) were used to assess the cytotoxicity and anti–HIV–1 activity. These cells are capable of
expressing CD4, CXCR4, and CCR5 receptors that are necessary for HIV infection. Moreover,
they are engineered with Tat–responsive reporter genes like firefly luciferase (Luc) and
Escherichia coli β–galactosidase enzymes downstream of HIV–1 LTR. The cells were
maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco–Invitrogen, USA)
supplemented with 10% fetal bovine serum (FBS; Moregate Biotech, Australia), penicillin 100
U/mL and streptomycin 100 mg/mL (Gibco–Invitrogen, USA) and 25 mM HEPES buffer
solution (Gibco–Invitrogen, USA). The cell culture was incubated at 37 °C in a humidified 5%
CO2 atmosphere and used when 80% confluency was achieved [32].
Viruses
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