Identification and development of pyrazolo[4,3-c]pyridine carboxamides as Mycobacterium tuberculosis pantothenate synthetase inhibitors

Article abstract of DOI:10.1039/C6NJ02671K

The purpose of this study was to prepare various 1-((1-(substituted)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamides using click chemistry. The synthesized compounds were characterized using various analytical techniques like NMR spectroscopy, mass spectrometry, and elemental analysis and screened for in vitro antitubercular activity against Mycobacterium tuberculosis (MTB) H37Rv strain and two ‘wild’ strains Spec. 210 and Spec. 192 using a classical test-tube method of successive dilution in Youmans' modification of the Proskauer and Beck liquid medium and were evaluated for a MTB pantothenate synthetase (PS) inhibition study as well. The final analogues exhibited minimum inhibitory concentration ranging from 24.72 to >200 μM. Among the compounds, 1-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7d) was found to be the most active compound with IC₅₀ 1.01 ± 0.32 μM against MTB PS; it inhibited MTB with MIC 24.72 μM and it was non-cytotoxic at 50 μM in the RAW 267 cell line. Further, 7d was docked into the crystal structure of MTB PS to investigate its binding interaction pattern.

Full text of DOI:10.1039/C6NJ02671K

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ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
rsc.li/njc

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Identification and development of 1-((1-(substituted)-1H-1,2,3-
triazol-4-pyrazolo[4,3-c]pyridine-5(4H)-carboxamides as
Mycobacterium tuberculosis Pantothenate synthetase inhibitors
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
b
a
DOI: 10.1039/x0xx00000x
Amaroju Suresh, Kalaga Mahalakshmi Naidu, Singireddi Srinivasarao, Napiórkowska Agnieszka,
b
c
c
Augustynowicz-Kopeć Ewa, Sankaranarayanan Murugesan, Subhash Chander, Rangan Krishnan
and Kondapalli Venkata Gowri Chandra Sekhar *
www.rsc.org/
a
The purpose of this study was to prepare various 1-((1-(substituted)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-
dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamides using click chemistry. The synthesized compounds were
characterized using various analytical techniques like NMR, Mass, Elemental analysis and screened for in vitro
antitubercular activity against Mycobacterium tuberculosis (MTB) H37Rv strain and two ‘wild’ strains Spec. 210 and Spec.
1
92 using a classical test-tube method of successive dilution in Youmans’ modification of the Proskauer and Beck liquid
medium and were evaluated for MTB pantothenate synthetase (PS) inhibition study as well. The final analogues exhibited
minimum inhibitory concentration ranging from 24.72 - >200 μM. Among the compounds, 1-((1-(4-methoxyphenyl)-1H-
1
,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7d), was found to be
the most active compound with IC50 1.01±0.32 µM against MTB PS; it inhibited MTB with MIC 24.72 µM and it was non-
cytotoxic at 50 μM in RAW 267 cell line. Further, 7d was docked to crystal structure of MTB PS to know its binding
interactionpattern.
Introduction
Tuberculosis (TB) is contagious and airborne. It has major impact on
the society due to the emergence of multi-drug resistant strains of
Mycobacterium tuberculosis (MTB). In addition, multidrug-resistant
Mycobacterium tuberculosis (MDR-TB) and especially, extensively
drug resistant TB (XDR-TB) are now increasing in incidence in many
In order to overcome the issue of resistance there is a challenging
need to develop new drugs using novel targets of MTB.
The biosynthetic pathway of pantothenate involves four steps
7
1
catalyzed by panB, panC, panD, and panE genes. The last step of
areas around the world. Around 9.6 million people fell ill with TB in
pantothenate biosynthesis viz., the ATP-dependent condensation of
D-pantoate and β-alanine to form pantothenate is catalyzed by
PanC. Pantothenate is essential for several processes such as cell
signaling, fatty acid metabolism, and synthesis of non-ribosomal
2
014, including 1.2 million cases among people living with HIV.
Globally in 2014, an estimated 0.48 million developed MDR-TB and
1
there were an estimated 0.19 million deaths from MDR-TB.
Therefore, the development of new therapeutic drugs targeting
MDR-TB and XDR-TB is important due to lack of effective drug
8
peptides and polyketides. The biosynthesis of pantothenate is
2
essential for the growth of MTB. The pantothenate biosynthesis
pathway is a latent drug target and hence is important for
determined growth and virulence of MTB and PS is very good target
treatments.
The treatment of TB infections is generally carried out through the
use of a combination of drugs including isoniazid (INH), rifampicin
9
, 10
for developing new therapeutics to treat TB.
3
-6
(
RMP), pyrazinamide, ethambutol (ETB) and streptomycin.
Till date many MTB PS inhibitors are reported (Figure 1), which
include
5-tert-butyl-N-pyrazol-4-yl-4,5,6,7-
8
tetrahydrobenzo[d]isoxazole-3-carboxamide derivatives (a), 3-
phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine
derivatives
1
1
(
b),
2,6-disubstituted 4,5,6,7-tetrahdrothieno[2,3-c]pyridine-3-
1
2
13
carboxamide derivatives (c),
naphthoyl)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (e),
and imidazo[2,1-b]thiazole and benzo[d]imidazo[2,1-b]thiazole
nafronyl oxalate (d),
N’-(1-
1
4
1
5
derivatives (f).
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In the present work, we designed and developed novel MTB PS
inhibitors by molecular hybridization approach using the two known
antimycobacterial agents.
Design and chemistry
The molecular hybridization approach involves rational design of
new ligands or the recognition of pharmacophoric sub-units in the
molecular structure of two or more known bioactive derivatives
which, through the adequate combination of these subunits, lead to
the design of new hybrid architectures that maintain preselected
characteristics of the original templates. In this work, we designed
novel MTB inhibitors by hybridizing reported MTB PS inhibitor (1-
benzoyl-N-(4-nitrophenyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-
Figure 1 Structures of literature reported MTB PS inhibitors.
12
c]pyridine-5(4H)-carboxamide) (AA)
and 3-(4-((1-(4-bromo-3-
(
trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-
Pyrazole derivatives are known to contain broad spectrum of
34
yl)benzo[d]isoxazole (BB),
anticipating a new lead in the
1
6
17
pharmacological properties such as antifungal, antidiabetic,
development of novel MTB PS inhibitors with potential MTB MIC
Figure 3). BB is our most active compound from our previous work
1
8
19, 20
antitumor, antibacterial.
Pyrazoles play an essential role in
(
biologically active compounds and therefore signify an interesting
and when screened for MTB PS it exhibited panC IC50 58.4 µM.
2
1
template for medicinal chemistry. Mamolo et al., reported 5-aryl-
-isonicotinoyl-3-(pyridin-2-yl)-4,5-dihydro-1H-pyrazole derivatives
1
which exhibited an interesting in vitro antimycobacterial activity
against MTB, with minimum inhibitory concentration (MIC) values
22
ranging from 8 to 16 µg/mL. Ravindra et al., reported 3-(4-
chlorophenyl)-4-substituted pyrazole derivatives with MIC values
2
3
ranging from 0.35 to 3.15 µg/mL against MTB H37Rv. Chovatia et
al., reported 1-acetyl-3,5-diphenyl-4,5-dihydro-(1H)-pyrazole
derivatives which were screened against MTB H37Rv. Series of N-
phenyl-3-(4-fluorophenyl)-4-substituted pyrazole derivatives
exhibited significant antimycobacterial activity with IC50 values
2
4
25
ranging from 0.47 to 118.0 µM against MTB H37Rv.
Figure 3 Design strategy of the title compounds.
2
6
Triazoles have several medicinal applications. It is quite obvious
that, the favorable properties of 1,2,3-triazole ring like moderate
dipole character, hydrogen bonding capability, rigidity and stability
under in vivo conditions are responsible for their enhanced
Results and Discussion
The designed molecules were synthesized in six steps (Scheme 1).
Initially we prepared 1,3-dicarbonyl intermediate (2) using 1-Boc-4-
piperidone (1), morpholine, p-toluenesulfonic acid (catalytic) and
benzoyl chloride (stark-enamine reaction conditions). Treatment of
2
7, 28
biological activities.
For instance, a variety of 1H-1,2,3-triazole
compounds have been known to exhibit antitubercular activity (g-j)
2
9-33
(
Figure 2).
3
5
2
with hydrazine hydrate yielded pyrazole ring (3). Compound 3
on reacting with propargyl bromide in the presence of CS CO
2
3
formed N-alkyl product (4). Compound 4 was then deprotected
using trifluoroacetic acid to yield compound (5). With weak base
TEA, more nucleophilic amine of aliphatic ring reacted with
phenylisocyanate yielding urea derivative (6). The free acetylene
group was converted to various 1H-1,2,3-triazoles using different
3
3
aromatic azides via click chemistry method. The purity of
compounds synthesized was checked by LC-MS and elemental
analyses. Structures of the compounds were confirmed by spectral
1
13
data. In H NMR and C NMR, the signals of the respective protons
and carbons were verified on the basis of their chemical shifts,
multiplicities, and coupling constants. The results of elemental
analysis were within ± 0.05 of the theoretical values.
Figure
2 Heterocyclic hybrids of [1,2,3] triazole with
antitubercular activity.
2
| J. Name., 2012, 00, 1-3
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7
d
e
4-Methoxyphenyl
4-Fluorophenyl
4-Chlorophenyl
4-Bromophenyl
4-Iodophenyl
24.72
>200
24.72
>200
24.72
>200
7
7
f
196.08
90.18
196.08
90.18
196.08
90.18
7
7
g
h
166.26
192.10
166.26
192.10
166.26
192.10
7
7
i
4-Nitrophenyl
4
-
j
91.98
196.08
>200
91.98
196.08
>200
91.98
196.08
>200
Trifluoromethylphenyl
7
k
2-Chlorophenyl
Scheme 1 Synthetic protocol of titled compounds.
7
l
2-Fluorophenyl
2-Bromophenyl
2-Iodophenyl
Reagents and conditions: (i) (a) Morpholine (1.0 eq), PTSA
(0.01 eq), toluene, 100 °C, 16 h; (b) Benzoyl Chloride (1.1 eq),
7m
180.36
166.26
192.10
98.04
180.36
166.26
192.10
98.04
180.36
166.26
192.10
98.04
TEA (2.0 eq), DCM, 0 °C - rt, 4 h. (ii) N H (1.0 eq), EtOH, 0 °C -
2
4
rt, 4 h (iii) Propargyl bromide (80% in toluene) (1.2 eq), Cs CO
2
3
7n
7o
(1.5 eq), DMF, rt, 16 h. (iv) CF COOH, DCM, rt, 16 h. (v) Phenyl
3
isocyanate (1.3 eq), TEA (3.0 eq), DMF, rt, 4 h. (vi) Substituted
aromatic azides, CuSO .5H O (10 mol %), Sodium ascorbate (10
2-Nitrophenyl
4
2
7
7
p
q
3-Chlorophenyl
3-Methoxyphenyl
2,4-dichlorophenyl
3,4-dichlorophenyl
3,5-dichlorophenyl
mol %), H O:t-BuOH (1:2), rt, 4 h.
2
98.89
98.89
98.89
In-vitro MTB screening
7
r
183.67
183.67
91.83
183.67
183.67
91.83
183.67
183.67
91.83
All the synthesized compounds were tested for their capacity to
inhibit the growth of MTB. In assay three different M.
tuberculosis strains were used. One of them was reference strain M.
tuberculosis H37Rv ATTC 25618 and the others were ‘wild’ strains
7
s
7
t
3
4, 36
isolated from tuberculosis patients.
MTB strain spec. 210 was
3
-Chloro-4-
resistant to p-aminosalicylic acid (PAS), INH, ETB and RMP and
another (Spec. 192) fully sensitive to the administrated
7u
7v
189.40
195.49
189.40
189.40
Fluorophenyl
3
7
tuberculostatics. In this study three different strains were used for
screening as we wanted to know the kind of activity synthesized
compounds showed against the reference strain as well as against
the strains isolated from TB patients. In this study the influence of
the compound on the growth of mycobacteria at a certain
concentration: 3.1, 6.2, 12.5, 25, 50 and 100 μg/mL were evaluated
INH was used as reference drug. The in vitro antimycobacterial
results of title compounds are arranged in Table 1 as MIC (µM) and
the activity ranged from 24.72 - >200 µM.
3,4-difluorophenyl
195.49
186.70
195.49
186.70
7w
3,4-dimethoxyphenyl 186.70
4
-bromo-3
7
x
160.65
160.65
160.65
trifluoromethylphenyl
7
y
3,4-dimethylphenyl
198.57
192.47
≤22.60
198.57
192.47
≤22.60
198.57
192.47
≤91.14
7
z
benzo[d][1,3]dioxole
INH
-
Table 1 Result of Antimycobacterial screening of title compounds
Among the twenty six compounds screened, eight compounds (7b,
7
c, 7d, 7g, 7j, 7p, 7q and 7t) showed activity against MTB with MIC
MIC
MIC (µM) MIC (µM)
˂100 µM. Three compounds (7b, 7c, and 7d) inhibited MTB with
(
µM)
against
MTB
against
MTB
MIC <50 µM. Compound 7d, (1-((1-(4-methoxyphenyl)-1H-1,2,3-
triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H-pyrazolo[4,3-
c]pyridine-5(4H)carboxamide) was found to be the most active
compound with in vitro MIC 24.72 µM. In addition, compounds 7b,
7c and 7d with MIC <50 µM were further subjected to toxicity
studies on normal cell line to analyze the selectivity profile.
Entry
R
against
MTB
Spec. 192 Spec. 210
H37Rv
7
a
b
Phenyl
105.14
25.53
49.64
105.14
25.53
49.64
105.14
51.06
49.64
7
4-Methylphenyl
4-Ethylphenyl
Amongst synthesized derivatives, electron donating group
containing substituent play major impact in exhibiting anti-TB
activity. Structural activity relationship studies are explained based
7
c
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th
on activity of compound 7a. Structural changes at 4 position alter
the activity. Compound 7a was inhibiting 99% growth of MTB H37Rv
strain at 105.14 µM. In this series, introduction of electron donating
7p
-7.69
-4.25
-6.07
-7.83
-5.99
-5.37
-7.37
-4.96
-4.44
-6.60
-4.84
-70.79
-65.23
-67.82
-69.26
-63.35
-63.08
-64.55
-64.16
-57.95
-56.87
-73.86
1.14±0.19
7.78±0.56
1.29±0.14
0.91±0.32
4.76±0.02
6.43±0.12
1.56±0.11
8.21±0.03
6.54±0.21
6.91±0.07
8.39±0.14
7q
7
r
th
groups at 4 position on phenyl ring increased the activity.
7
s
Introduction of electron donating ethyl group (7c) increased the
activity by two fold with MIC 49.64 µM. Presence of electron
donating methyl group (7b) increased the activity by four folds with
MIC 25.53 µM. With introduction of methoxy group (7d) activity
increased by four folds (MIC 24.72 µM), with same methoxy group
at meta position on phenyl ring (7q, MIC 98.89 µM) activity
remained unaltered compared to compound 7a. Presence of
electron withdrawing groups viz., F, Cl, Br and I resulted in decrease
in activity compared to compounds 7b, 7c, and 7d. Even, presence
of electron withdrawing groups at ortho or meta position decreased
7
t
7u
7v
7
w
7
x
y
7
7
z
the activity. Introduction of electron donating methoxy group at Docking study
meta and para position (7w, MIC 186.70 µM) yielded decrease in All the final compounds were docked into the crystal structure of
activity by two folds compared to compound 7a. In conclusion, MTB PS protein (PDB ID: 3IUB) to know the exact binding pattern
presence of electron donating methoxy group at para position on with the receptor. Validation of docking protocol revealed that, the
phenyl ring enhanced the activity. Amongst the synthesized value of RMSD obtained between experimental binding mode of co-
derivatives, 7d emerged to be the most active compound.
crystallized ligand (as in X-ray) and its re-docked pose (Figure 4) was
found to be 0.76, which suggested that, docking procedure could be
relied on for further docking studies.
Pantothenate synthetase enzyme inhibition studies
PS enzyme inhibition studies are carried on synthesized compounds
+
7, 15
+
by estimating the amount of NAD produced.
The NAD
produced can be examined spectrophotometrically at 340 nm. In
the initial screening at 50 μM, all compounds exhibited more than
5
0% inhibition against MTB PS and their IC ’s were further
50
determined. Most of the compounds showed good IC50 ranging
from 0.91±0.32 to 8.97±0.05 µM (Table 2). Seven compounds (7b,
7
d, 7h, 7p, 7r, 7s and 7v) inhibited MTB PS with IC50 ˂2.00 µM.
Compounds 7d and 7s emerged as the most active compounds with
IC50 1.01±0.32 and 0.91±0.32 µM respectively.
Table 2 Docking scores and MTB PS assay
Figure 4 Superimposed view of co-crystallized ligand (green)
Compound
In-silico
In-vitro
with its X-ray pose (red) in 3IUB
Entry
XP GScore Glide energy MTB PanC IC50 µM
Co-LIGAND
-8.32
-7.19
-5.69
-6.44
-8.19
-6.61
-3.25
-5.24
-7.61
-3.80
-4.70
-4.62
-4.46
-6.31
-5.24
-6.33
-78.66
-71.33
-58.61
-64.89
-66.25
-67.18
-69.57
-65.40
-73.33
-62.50
-58.79
-65.89
-61.60
-71.76
-60.42
-72.18
--
Further, in the docking studies, molecules exhibited good
binding energy in the range of -3.25 to -8.19 kcal/mol and
exhibited good fitness with the MTB PS protein. Several
compounds displayed hydrogen bonding interaction with HIE47,
HIE44, Met40, Ser196, Tyr82 and Ser197, amino acid residues.
One of the most active ligand, 1-((1-(4-methoxyphenyl)-1H-
7
a
b
2.13±0.12
1.54±0.22
2.56±0.04
1.01±0.32
3.41±0.08
6.36±0.12
2.46±0.07
1.04±0.55
8.97±0.05
5.04±0.54
2.78±0.07
5.93±0.64
3.54±0.55
3.92±0.19
4.94±0.03
7
7
c
7
d
e
7
1
,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H-
7f
pyrazolo[4,3-c]pyridine-5(4H)carboxamide (7d) with IC50
7
7
g
1.01±0.32 µM showed docking score of -8.19 kcal/mol. The
active site in the hydrophobic pocket is within the vicinity of
Leu146, Val187, Val142, Met195, Ala42, Leu50, Pro38, Ile168
and Met40 some polar amino acid residues HIE47, Ser196,
Ser197 and Thr82 respectively. The ligand also exhibited
hydrogen bonding interaction with Met40, Ser197, Val187
h
7i
7j
7
k
[
15]
7l
residues. The binding pattern of 7d with MTB PS is shown in
Figure 5
.
7m
7
n
o
7
4
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crystallographic data are shown in Table 4. The molecular
structure of the compound with methanol solvent of
crystallization is given as an ORTEP diagram in Figure 6. The part
of the molecule containing p-bromophenyl ring is directly
attached to triazole ring nitrogen. These two rings are not
coplanar; the phenyl ring plane is deviated from the triazole
plane by around 30°. The torsional angle between these two
rings with selected bonds C5-C4-N1-N2
=
= 30.90 and C3-C4-N1-C7
31.71 degrees. The deviation is comparatively less in the other
part of the molecule. Pyrazole ring plane with respect to the
attached phenyl ring plane deviated by around 7.5°, the
corresponding dihedral angle for the selected four bonds are N5-
C12-C13-C18
=
7.54, C11-C12-C13-C14
=
7.34 degrees.
Crystallographic data for the compound 7g is deposited to the
Cambridge Crystallographic Data Center and corresponding
deposition number is CCDC 1500428.
Figure 5 Docked pose of compound 7d inside the 3IUB,
showing two-dimensional interactive diagram
In vitro cytotoxicity studies
The active compounds 7b, 7c and 7d were evaluated for
Promega Cell Titer 96 non-radioactive cell proliferation assay to
analyze the selectivity profile against mouse macrophage
3
8
(
RAW264.7) cell lines. The IC50 values and selectivity index (SI)
values are tabulated in Table 3. The most active compound (7d
)
showed SI value 13.76. The results imply that the compounds
are suitable for further investigation in TB.
Table 3 Cytotoxicity results of the active compounds
*
a
MIC (µM) in
IC50
SI values
Entry
MTB H37Rv
approximation
IC50/MIC
Figure 6 ORTEP diagram showing the X-ray crystal structure of
the compound 7g with a methanol solvent of crystallization.
7
b
25.53
49.64
24.72
335.19
370.12
340.15
13.12
7.45
7
c
Table 4 Crystal data and structure refinement for 7g
7
d
13.76
a
Empirical Formula
Formula Weight
28 24 7 3
C H BrN O. CH OH
Selectivity index;
*
units in µM
586.49
Crystal Color, Habit
Crystal Dimensions
Light yellow
0.1 mm x 0.1 mm x 0.1 mm
Triclinic
Single Crystal X-ray Crystallographic Structure of Compound 7g:
T
he suitable crystals of the compound 7g for X-ray Crystal System
crystallographic study were grown from methanol solution. The Lattice Type
single crystal X-ray diffraction measurement of the molecule Lattice Parameters
Primitive
a = 7.8845(3) A°
b = 11.9643(4) A°
c = 14.9423(4) A°
α = 102.268(2) A°
β = 101.916(2) A°
γ = 95.003(3) A°
(
C28H24N OBr.CH OH) was on Rigaku XtaLAB P200 diffract meter
7 3
using graphite monochromated Mo-Kα radiation (λ = 0.71073 Å)
on 0.1mm x 0.1mm x 0.1mm pale yellow crystal. Data were
collected and processed using CrysAlisPro (Rigaku Oxford
Diffraction). The data were collected at a temperature of -180 ±
1
collected, 6164 were unique (Rint
reflections were merged. The diffraction data were refined and
structure was solved using Crystal Structure 4.2.2 software
program. The structure was solved by direct methods and
expanded using Fourier techniques. The non-hydrogen atoms
°C to a maximum 2
θ
value of 58.2°. Of the 19673 reflections
0.0791) and equivalent
3
δ = 1334.95(8) A°
=
Space Group
Z value
P-1 (#2)
2
3
Dcalc
1.459 g/cm
F
604.00
000
-
1
were refined anisotropically. Hydrogen atoms were refined using µ(MoKα)
the riding model. The compound crystallized into a triclinic Radiation
crystal system with P-1 space group. In a single unit cell four
15.855 cm
Mo-Kα(λ = 0.71073 A°)
Graphite
Radiation monochromator
Voltage, Current
partially occupying molecules along with two methanol solvent
of crystallization are observed with
This journal is © The Royal Society of Chemistry 20xx
50kV, 40mA
Z=2. The basic
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ARTICLE
Journal Name
Temperature
-180.0 °C
58.2°
atmosphere for 16 h. The solvent was evaporated and the crude
reaction mixture was dissolved in DCM (40 mL) and then
Maximum 2θ
Number of measured reflections
Number of Unique reflections
Number of parameters
19673°
2
triethylamine (5.35 mL, 37.65 mmol) was added at 0 °C, under N ,
6164 (Rint = 0.0791)
380
benzoyl chloride (2.9 mL, 25.1 mmol) was added over 10 min, the
ice bath was then removed and the reaction solution was stirred at
room temperature for 4 h. Once completion of the reaction, as
2
Goodness-of-fit on F
1.00
-
3
Δρmax,mix(e /A° )
1.89, -0.78
16.22
indicated by TLC, the reaction was quenched with NaHCO
and extracted with DCM. The organic layers were collected, washed
with saturated brine solution, dried over anhydrous Na SO and
3
solution
Reflection/Parameter Ratio
Residuals: R1 (I>2.00σ(I))
Residuals: R (All reflections)
Residuals: wR2 (All reflections)
Crystal refinement
0.0479
2
4
0.0538
concentrated in vacuo. The resultant crude product was purified by
column chromatography [ethyl acetate / hexane (10 - 15%)] to get
the 1,3-dicarbonyl compound 2 (7.0 g, 92%) as a colorless liquid.
0.1931
CrystalStructure 4.2.2
+
ESI-MS found 304 (M+H) .
Conclusions
tert-butyl 3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-
carboxylate (3)
In this work, we designed novel 1-((1-(substituted)-1H-1,2,3-triazol-
A
stirred solution of tert-butyl 3-benzoyl-4-oxopiperidine-1-
4
-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-
(4H)-carboxamide derivatives by molecular hybridization approach
carboxylate (2) (7.6 g, 25.05 mmol) in ethanol was cooled to 0 °C
and hydrazine hydrate (0.8 mL, 25.05 mmol) was added and stirred
for 4h. Once completion of the reaction, as indicated by TLC the
reaction mixture was concentrated under reduced pressure and the
crude residue was purified by column chromatography to get
compound 3 (6.9 g, 92%) as an off-white solid. ESI-MS found 300
5
using reported MTB PS inhibitor and substituted 1H-1,2,3-triazole
antitubercular compounds. Twenty six compounds were
synthesized and well characterized. One of the compound 7d
showed better MTB PS inhibition and MTB MIC than one of the lead
compound AA. Thus, this 1-((1-(substituted)-1H-1,2,3-triazole
scaffold could be further optimized to develop MTB PS specific
agents. In conclusion, it has been shown that, the potency and low
cytotoxicity of the title compounds make them suitable leads for
synthesizing new compounds with better anti-tubercular activity.
+
1
(
M+H) . H NMR (400 MHz, CDCl
3
) δ 9.48 (s, 1H), 7.58-7.19 (m, 5H),
4
9
1
.82 (s, 2H), 3.91 (t, J = 7.6 Hz, 2H), 2.83 (t, J = 7.8 Hz, 2H), 1.34 (s,
1
3
H); C NMR (100 MHz, CDCl
32.75, 129.31, 127.39, 117.45, 81.67, 43.91, 36.89, 31.49, 27.79;
: (%) C, 68.20; H, 7.07; N, 14.04, Found: C,
3
) 163.54, 146.99, 143.56, 136.64,
Anal. calcd for C17
21 3 2
H N O
6
8.24; H, 7.09; N, 14.13.
Experimental
Materials and methods
tert-butyl-3-phenyl-1-(prop-2-yn-1-yl)-6,7-dihydro-1H-
pyrazolo[4,3-c]pyridine-5(4H)-carboxylate (4)
Chemicals and solvents were procured from commercial source.
The solvents and reagents were of LR grade and if necessary
purified before use. Thin-layer chromatography (TLC) was carried
out on aluminium-supported silica gel plates (Merck 60 F254) with
visualization of components by UV light (254 nm). Column
chromatography was carried out on silica gel (Merck 100-200
A
solution of tert-butyl 3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-
c]pyridine-5(4H)-carboxylate (3) (5.0 g, 16.70 mmol) in DMF was
cooled to 0 °C and Cs CO (8.16 g, 25.05 mmol) and propargyl
2
3
bromide (80% in toluene) (1.64 mL, 21.17 mmol) were added and
allowed to reach room temperature and stirred for 16 h. Once
completion of the reaction, as indicated by TLC, the reaction was
quenched with cold water and extracted with diethyl ether. The
organic layers were collected, washed with saturated brine
1
13
mesh). H NMR and C NMR spectra were recorded at 400 MHz
and 101 MHz respectively using a Bruker AV 400 spectrometer
(
Bruker CO., Switzerland) in CDCl
tetramethylsilane as the internal standard and chemical shift values
δ) were given in ppm. Melting points were determined on an
3 6
and DMSO-d solution with
2 4
solution, dried over anhydrous Na SO and concentrated in vacuo.
(
The resultant crude product was purified by column
chromatography [ethyl acetate / hexane (20 - 30%)] to get the
compound 4 (4.6 g, 83%) as a semisolid. ESI-MS found 338.15
electro thermal melting point apparatus (Stuart-SMP30) in open
capillary tubes and are uncorrected. Elemental analyses were
performed by Elementar Analysensysteme GmbH vario MICRO cube
CHN Analyzer. Mass spectra (ESI-MS) were recorded on Schimadzu
MS/ESI mass spectrometer. Purity of all tested compounds were
determined by LC-MS/MS on Schimadzu and was greater than 95%.
+
1
(
M+H) . H NMR (400 MHz, CDCl
3
) δ 7.60-7.23 (m, 5H), 4.80 (s, 2H),
4
2
1
4
.56 (s, 2H), 3.92 (t, J = 7.4 Hz, 2H), 2.81 (t, J = 7.6 Hz, 2H), 2.72 (s,
13
H), 1.31 (s, 9H); C NMR (100 MHz, CDCl
36.76, 132.87, 129.38, 127.40, 117.51, 81.7, 77.96, 69.05, 43.91,
2.07, 36.77, 31.59, 27.90; Anal. calcd for C20 : (%) C, 69.79;
3
) 163.57, 147.64, 143.65,
23 3 2
H N O
Chemistry
H, 6.96; N, 13.20, Found: C, 69.80; H, 7.01; N, 13.23.
tert-butyl 3-benzoyl-4-oxopiperidine-1-carboxylate (2)
In a two neck 100 mL round-bottom flask equipped with a Dean-
stark trap, a reflux condenser and an internal thermocouple,
compound 1 (5.0 g, 23.15 mmol), toluene (50 mL), morpholine (2.1
mL, 23.15 mmol), and p-toluenesulfonic acid (catalytic) were added
3
-phenyl-1-(prop-2-yn-1-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-
c]pyridine (5)
A solution of tert-butyl 3-phenyl-1-(prop-2-yn-1-yl)-6,7-dihydro-1H-
pyrazolo[4,3-c]pyridine-5(4H)-carboxylate (4) (4.0 g, 11.85 mmol) in
sequentially. The reaction mixture was refluxed under
N
2
2 2 3
CH Cl was cooled to 0 °C and CF COOH (4.5 mL, 59.27 mmol) was
6
| J. Name., 2012, 00, 1-3
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DOI: 10.1039/C6NJ02671K
ARTICLE
added drop wise and stirred at room temperature for 16 h. The 2H), 7.48 – 7.42 (m, 4H), 7.40 – 7.30 (m, 3H), 7.26 – 7.20 (m, 2H),
reaction mixture was concentrated under reduced pressure and the 6.94 (t, J = 6.8 Hz, 1H), 5.47 (s, 2H), 4.71 (s, 2H), 3.83 (t, J = 5.6 Hz,
13
crude residue was washed with hexane and diethyl ether to get 2H), 2.97 (t, J = 5.3 Hz, 2H), 2.36 (s, 3H). C NMR (101 MHz, DMSO-
compound 5 (2.6 g, 92%) as an off-white solid. ESI-MS showed ) δ 155.68, 145.54, 144.42, 140.91, 139.16, 138.77, 134.74,
133.95, 130.71, 129.21, 128.82, 127.78, 126.48, 122.38, 122.24,
20.49, 120.47, 111.99, 44.46, 42.18, 41.66, 22.29, 21.06. EI-MS
d
6
+
238.10 (M+H) .
1
+
N,3-diphenyl-1-(prop-2-yn-1-yl)-6,7-dihydro-1H-pyrazolo[4,3-
c]pyridine-5(4H)-carboxamide (6)
m/z 490.23 (M+H) ; Anal. calcd for C28
25 7
H N O: (%) C, 71.15; H, 5.56;
N, 20.03; Found: C, 71.16; H, 5.58; N, 20.06.
Phenylisocyanate (1.3 mL, 10.95 mmol), was added to the stirred
solution of Compound 5 (2.0 g, 8.42 mmol) and Et
mmol) in DMF at 0 °C under N atm, and stirred at room 6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7c)
temperature for 4 h. Once completion of the reaction, as indicated Off white solid (89%); m.p. 128-130 °C; IR (KBr) ʋmax / cm 3478,
3
N (3.5 mL, 25.26 1-((1-(4-ethylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-
2
-1
1
6
by TLC, the reaction was quenched with cold water then solid was 3031, 2913, 1635, 1422, 1341, 1056. H NMR (400 MHz, DMSO-d ) δ
filtrated and washed with water and hexane to get the key 8.82 (s, 1H), 8.75 (s, 1H), 7.78 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 7.3 Hz,
intermediate 6 (2.7 g, 90%) as an off-white solid. ESI-MS showed 2H), 7.48 – 7.42 (m, 4H), 7.40 – 7.30 (m, 3H), 7.26 – 7.20 (m, 2H),
+
1
3
57.15 (M+H) . H NMR (400 MHz, DMSO-d
J = 8.6 Hz, 2H), 7.78 (d, J = 8.8 Hz, 2H), 7.67 (d, J = 7.6 Hz, 2H), 7.72 – 2H), 2.97 (t, J = 5.4 Hz, 2H), 2.76 (m, 2H), 1.36 (t, 3H). C NMR (101
.64 (m, 2H), 7.28 – 7.18 (m, 2H), 5.47 (s, 2H), 4.71 (s, 2H), 3.86 (t, J MHz, DMSO-d ) δ 155.17, 145.09, 144.67, 144.21, 143.62, 140.38,
7.6 Hz, 2H), 2.90 (t, J = 7.5 Hz, 2H), 2.69 (s, 2H); C NMR (100 138.66, 133.47, 129.93, 128.66, 128.22, 127.25, 126.04, 121.89,
MHz, CDCl ) 164.58, 155.57, 143.57, 139.51, 136.69, 132.85, 121.84, 121.77, 119.95, 114.78, 48.12, 44.19, 41.67, 28.23, 21.85,
6
) δ 8.74 (s, 1H), 7.88 (d, 6.94 (t, J = 6.8 Hz, 1H), 5.47 (s, 2H), 4.71 (s, 2H), 3.83 (t, J = 5.6 Hz,
1
3
7
6
1
3
=
3
+
1
29.38, 128.92, 128.12, 127.40, 121.16, 117.52, 81.73, 77.94, 69.0, 14.45. EI-MS m/z 504.25 (M+H) ; Anal. calcd for C30
29 7
H N O: (%) C,
4
4.01, 42.12, 23.94; Anal. calcd for C22 O: (%) C, 74.14; H, 5.66; 71.55; H, 5.80; N, 19.47; Found: C, 71.56; H, 5.82; N, 19.48.
20 4
H N
N, 15.72, Found: C, 74.16; H, 5.67; N, 15.73.
1
-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-
N,3-diphenyl-1-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-6,7-
dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7a-z)
diphenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-
carboxamide (7d)
-
1
A
solution of N,3-diphenyl-1-(prop-2-yn-1-yl)-6,7-dihydro-1H- Brown solid (90%); m.p. 115-117 °C; IR (KBr) ʋmax / cm 3442, 3027,
1
pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (6) (0.20 g, 1.0 equiv.) is 2832, 1645, 1424, 1365, 1034, 1020. H NMR (400 MHz, DMSO-d
reacted with substituted phenyl azides (1.2 equiv.) in the presence 8.76 (s, 1H), 8.73 (s, 1H), 7.81 – 7.78 (m, 2H), 7.68 – 7.64 (m, 2H),
of sodium ascorbate (0.01 equiv.), CuSO .5H O (0.02 equiv.) and t- 7.47 – 7.41 (m, 4H), 7.35 – 7.31 (m, 1H), 7.25 – 7.21 (m, 2H), 7.11 (d,
BuOH: H O (2:1), at rt for 4 h. Once completion of the reaction, as J = 9.1 Hz, 2H), 6.95 (d, J = 7.4 Hz, 1H), 5.46 (s, 2H), 4.70 (s, 2H), 3.83
indicated by TLC, the reaction was quenched with cold water and (s, 3H), 3.82 (t, J = 7.1 Hz, 2H), 2.97 (t, J = 5.4 Hz, 2H). C NMR (101
extracted with DCM. The DCM layers were collected, washed with MHz, DMSO-d ) δ 156.47, 153.18, 145.02, 143.72, 140.36, 138.63,
saturated brine solution, dried over anhydrous Na SO and 133.49, 129.91, 128.64, 128.24, 127.27, 126.01, 121.88, 121.86,
concentrated in vacuo. The resultant crude product was purified by 121.77, 119.98, 114.78, 111.48, 55.51, 48.12, 44.93, 41.67, 22.13.
6
) δ
4
2
2
13
6
2
4
+
27 7 2
column chromatography [MeOH / DCM (1 -3%)] to yield the title EI-MS m/z 506.20 (M+H) ; Anal. calcd for C29H N O : (%) C, 68.89;
compounds 7a-z.
H, 5.38; N, 19.39; Found: C, 68.91; H, 5.39; N, 19.41.
N,3-diphenyl-1-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-6,7-
1-((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-
dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7a)
6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7e)
-1
-1
Off white solid (83%); m.p. 221-223 °C; IR (KBr) ʋmax / cm 3490, Off white solid (82%); m.p. 158-160 °C; IR (KBr) ʋmax / cm 3490,
1
1
3
8
7
1
4
021, 2843, 1650, 1410, 1340, 1060. H NMR (400 MHz, DMSO-d
6
) δ 3021, 2843, 1655, 1410, 1340, 1120, 1060. H NMR (400 MHz,
) δ 8.85 (s, 1H), 8.74 (s, 1H), 8.00 – 7.90 (m, 2H), 7.71 –
.88 (s, 1H), 8.75 (s, 1H), 7.94 – 7.86 (m, 2H), 7.69 – 7.66 (m, 2H), DMSO-d
6
.58 (dd, J = 8.6, 7.1 Hz, 2H), 7.50 – 7.42 (m, 5H), 7.38 – 7.28 (m, 7.63 (m, 2H), 7.50 – 7.29 (m, 7H), 7.28 – 7.17 (m, 2H), 6.94 (tt, J =
H), 7.28 – 7.15 (m, 2H), 6.94 (t, J = 7.2, 1.2 Hz, 1H), 5.49 (s, 2H), 7.3, 1.2 Hz, 1H), 5.48 (s, 2H), 4.71 (s, 2H), 3.82 (t, J = 5.7 Hz, 2H),
13
13
.72 (s, 2H), 3.83 (t, J = 5.8 Hz, 2.98 (t, J = 5.8 Hz, 2H). C NMR (101 2.97 (t, J = 5.4 Hz, 2H). C NMR (101 MHz, DMSO-d
6
) δ 161.01,
MHz, DMSO-d
6
) δ 155.67, 145.55, 144.46, 140.85, 139.15, 136.98, 155.21, 145.02, 143.72, 140.36, 138.63, 133.49, 129.91, 128.64,
1
33.97, 130.32, 129.20, 129.14, 128.74, 127.78, 126.51, 122.38, 128.24, 127.27, 126.01, 121.88, 121.86, 121.77, 119.98, 114.78,
+
122.39, 120.61, 120.48, 112.00, 44.40, 42.17, 41.16, 40.59, 22.33. 111.48, 50.19, 48.93, 41.87, 24.23. EI-MS m/z 494.20 (M+H) ; Anal.
+
EI-MS m/z 476.20 (M+H) ; Anal. calcd for C28
H
25
N
7
O: (%) C, 70.72; calcd for C28
H24FN
7
O: (%) C, 68.14; H, 4.90; N, 19.87; Found: C,
H, 5.30; N, 20.62; Found: C, 70.74; H, 5.31; N, 20.63.
68.16; H, 4.91; N, 19.89.
N,3-diphenyl-1-((1-(p-tolyl)-1H-1,2,3-triazol-4-yl)methyl)-1,4,6,7-
1-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-
tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxamide (7b)
1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxamide (7f)
-1
-1
Light yellow solid (87%); m.p. 204-206 °C; (KBr) ʋmax / cm 3455, White solid (89%); m.p. 145-147 °C; (KBr) ʋmax / cm 3428, 3027,
1
1
3
025, 2867, 1645, 1420, 1348, 1062. H NMR (400 MHz, DMSO-d
6 6
) δ 2834, 1647, 1412, 1343, 1043, 615. H NMR (400 MHz, DMSO-d ) δ
8.81 (s, 1H), 8.75 (s, 1H), 7.77 (d, J = 8.5 Hz, 2H), 7.67 (d, J = 7.2 Hz, 8.82 (s, 1H), 8.73 (s, 1H), 7.80 (d, J = 9.2 Hz, 2H), 7.66 (d, J = 8.5 Hz,
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Journal Name
2
H), 7.43 (t, J = 7.6 Hz, 4H), 7.37 (t, J = 7.5 Hz, 1H), 7.27 – 7.21 (m, δ 9.03 (s, 1H), 8.75 (s, 1H), 8.17 (d, J = 8.4 Hz, 2H), 7.97 (d, J = 8.5 Hz,
H), 7.18 (d, J = 9.5 Hz, 2H), 6.91 (t, J = 7.8 Hz, 1H), 5.46 (s, 2H), 4.74 2H), 7.69 – 7.64 (m, 2H), 7.47 – 7.42 (m, 4H), 7.37 – 7.29 (m, 1H),
2
1
3
(s, 2H), 3.83 (t, J = 5.7 Hz, 2H), 2.96 (t, J = 5.2 Hz, 2H). C NMR (101 7.27 – 7.20 (m, 2H), 6.96 – 6.92 (m, 1H), 5.51 (s, 2H), 4.72 (s, 2H),
1
3
MHz, DMSO-d
6
) δ 155.28, 145.12, 143.92, 141.36, 138.63, 134.12, 3.83 (t, J = 5.7 Hz, 2H), 2.97 (t, J = 6.7 Hz, 2H). C NMR (101 MHz,
) δ 155.66, 145.62, 144.86, 140.84, 139.75, 139.18, 133.94,
21.77, 119.88, 114.78, 111.48, 52.19, 48.83, 41.67, 24.83. EI-MS 129.14, 128.73, 127.80, 127.60, 127.60, 126.51, 122.91, 122.71,
1
33.39, 129.91, 128.64, 128.24, 127.27, 126.11, 121.88, 121.89, DMSO-d
6
1
+
7
m/z 510.20 (M+H) ; Anal. calcd for C28H24ClN O: (%) C, 65.94; H, 122.38, 121.06, 120.47, 112.02, 44.33, 42.16, 41.14, 22.30. EI-MS
+
4.74; N, 19.23; Found: C, 65.16; H, 4.75; N, 19.89.
m/z 544.19 (M+H) ; Anal. Calcd for C29
24 3 7 3
H F N O : (%) C, 64.08; H,
4
.45; N, 18.04; Found: C, 64.10; H, 4.46; N, 18.05.
1
-((1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-
,7-dihydro-1H-pyrazolo[4,3-c] pyridine-5(4H)-carboxamide (7g) 1-((1-(2-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-
6
-1
Pale yellow solid (87%); m.p. 150-152 °C; IR (KBr) ʋmax / cm 3492, 6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide
(7k)
1
-1
3022, 2913, 1657, 1422, 1332, 1055, 680. H NMR (400 MHz, DMSO- Off white solid (88%); m.p. 137-139 °C; IR (KBr) ʋmax / cm 3523,
1
d
6 6
) δ 8.90 (s, 1H), 8.75 (s, 1H), 7.88 (d, J = 8.6 Hz, 2H), 7.78 (d, J = 8.8 3032, 2923, 1675, 1445, , 1052, 602. H NMR (400 MHz, DMSO-d )
Hz, 2H), 7.67 (d, J = 7.6 Hz, 2H), 7.45 (dt, J = 7.8, 3.6 Hz, 4H), 7.33 (t, δ 8.87 (s, 1H), 8.75 (s, 1H), 7.81 (d, J = 9.2 Hz, 2H), 7.69 (d, J = 8.5 Hz,
J = 7.4 Hz, 1H), 7.23 (t, J = 7.8 Hz, 2H), 6.94 (t, J = 7.4 Hz, 1H), 5.48 (s, 2H), 7.40 (t, J = 7.6 Hz, 5H), 7.37 (t, J = 7.5 Hz, 1H), 7.27 – 7.21 (m,
1
3
2
H), 4.71 (s, 2H), 3.82 (t, J = 5.6 Hz, 2H), 2.96 (t, J = 6.0 Hz, 2H).
C
1H), 7.19 (d, J = 9.5 Hz, 2H), 6.99 (t, J = 7.8 Hz, 1H), 5.42 (s, 2H), 4.64
1
3
NMR (101 MHz, DMSO-d
6
) δ 155.66, 145.58, 144.65, 140.84, (s, 2H), 3.84 (t, J = 5.7 Hz, 2H), 2.96 (t, J = 5.2 Hz, 2H). C NMR
) δ 155.28, 145.22, 142.92, 141.36, 138.53,
22.51, 122.43, 122.38, 121.92, 120.42, 112.05, 44.33, 42.24, 41.24, 136.23, 134.12, 133.39, 129.91, 128.64, 128.24, 127.27, 126.11,
1
1
2
39.15, 136.17, 133.95, 133.19, 129.14, 128.74, 127.79, 126.45, (101MHz, DMSO-d
6
+
+2
2.22. EI-MS m/z 554.12(M+H) ; 556.10 (M+H) ; Anal. Calcd for 121.88, 121.89, 121.77, 120.21, 119.88, 114.78, 111.48, 52.19
+
28 7
C H24BrN O: (%) C, 60.66; H, 4.36; N, 17.68; Found: C, 60.68; H, 48.83, 41.67, 24.83. EI-MS m/z 510.15 (M+H) ; EI-MS m/z 510.15
+
4.37; N, 17.69.
(M+H) ; Anal. calcd for C28
7
H24ClN O: (%) C, 65.94; H, 4.74; N, 19.23;
Found: C, 65.16; H, 4.75; N, 19.89.
1
-((1-(4-iodophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-
,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide
6
(7h) 1-((1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-
-
1
Light brown solid (80%); m.p.147-149 °C; ; IR (KBr) ʋmax / cm 3485, 6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide
(7l)
1
-1
30311, 2905, 1658, 1402, 1043, 560. H NMR (400 MHz, DMSO-d
6
)
White solid (91%); m.p. 156-158 °C; IR (KBr) ʋmax / cm 3576, 3031,
1
δ 8.85 (s, 1H), 8.76 (s, 1H), 7.99 (d, J = 9.2 Hz, 2H), 7.76 (d, J = 8.5 Hz, 2925, 1665, 1421, 1330, 1050. H NMR (400 MHz, DMSO-d
6
) δ 8.76
H), 7.46 (t, J = 7.8 Hz, 4H), 7.35 (t, J = 7.5 Hz, 1H), 7.27 – 7.21 (m, (s, 1H), 8.68 (s, 1H), 7.83 (t, J = 7.8 Hz, 1H), 7.68 (d, J = 7.6 Hz, 2H),
H), 7.12 (d, J = 9.3 Hz, 2H), 6.92 (t, J = 7.8 Hz, 1H), 5.47 (s, 2H), 4.69 7.64 – 7.51 (m, 2H), 7.51 – 7.38 (m, 5H), 7.33 (t, J = 7.4 Hz, 1H), 7.24
2
2
1
3
(s, 2H), 3.83 (t, J = 5.8 Hz, 2H), 2.97 (t, J = 5.4 Hz, 2H). C NMR (101 (t, J = 7.7 Hz, 2H), 6.95 (t, J = 7.4 Hz, 1H), 5.51 (s, 2H), 4.72 (s, 2H),
1
3
MHz, DMSO-d
6
) δ 155.48, 145.02, 144.23, 143.72, 140.36, 138.63, 3.83 (t, J = 6.1 Hz, 2H), 2.98 (t, J = 6.3 Hz, 2H). C NMR (101 MHz,
) δ 155.67, 153.06, 145.56, 143.95, 140.85, 139.19, 133.96,
19.98, 116.78, 115.48, 95.15, 45.23, 42.13, 41.67, 23.93. EI-MS 131.82, 129.16, 128.75, 127.80, 126.51, 125.99, 125.74, 125.13,
1
29.91, 128.64, 128.24, 127.27, 126.01, 121.88, 121.86, 121.77, DMSO-d
6
1
+
m/z 602.12 (M+H) ; Anal. Calcd for C28
7
H24IN O: (%) C, 55.92; H, 4.03; 122.38, 120.48, 117.66, 117.47, 112.00, 44.20, 42.16, 41.16, 22.34.
+
N, 16.30; Found: C, 55.94; H, 4.31; N, 16.32.
EI-MS m/z 494.20 (M+H) ; Anal. calcd for C28
7
H24FN O: (%) C, 68.14;
H, 4.90; N, 19.87; Found: C, 68.16; H, 4.91; N, 19.89.
1
-((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-
,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide
6
(7i) 1-((1-(2-bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-
-1
Light yellow solid (81%); m.p. 144-146 °C; IR (KBr) ʋmax / cm 3505, 6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7m)
1
-1
3031, 2912, 1675, 1532, 1372, 1027. H NMR (400 MHz, DMSO-d
6
) δ White solid (90%); m.p. 119-121 °C; IR (KBr) ʋmax / cm 3490, 3021,
1
9.08 (s, 1H), 8.74 (s, 1H), 8.48 – 8.40 (m, 2H), 8.28 – 8.19 (m, 2H), 2843, 1650, 1410, 1340, 570. H NMR (400 MHz, DMSO-d
6
) δ 8.75
7.71 – 7.63 (m, 2H), 7.50 – 7.39 (m, 4H), 7.40 – 7.27 (m, 1H), 7.28 – (s, 1H), 8.71 (s, 1H), 7.81(d, J = 9.1 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H),
7
.18 (m, 2H), 6.94 (tt, J = 7.3, 1.2 Hz, 1H), 5.51 (s, 2H), 4.71 (s, 2H), 7.44 (t, J = 7.9 Hz, 5H), 7.33 (t, J = 7.4 Hz, 1H), 7.26 – 7.20 (m, 1H),
1
3
3.83 (t, J = 5.7 Hz, 2H), 2.97 (t, J = 5.4 Hz, 2H). C NMR (101 MHz, 7.11 (d, J = 9.1 Hz, 2H), 6.94 (t, J = 7.8 Hz, 1H), 5.46 (s, 2H), 4.73 (s,
1
3
DMSO-d
6
) δ 155.65, 147.17, 145.65, 145.10, 141.20, 140.83, 139.19, 2H), 3.82 (t, J = 5.6 Hz, 2H), 2.97 (t, J = 5.4 Hz, 2H). C NMR
) δ 155.21, 145.02, 144.23, 143.72, 140.36,
21.14, 120.47, 112.03, 44.29, 42.15, 41.13, 22.28. EI-MS m/z 139.21, 138.63, 134.24, 133.49, 133.67, 129.91, 128.64, 128.24,
1
1
5
2
33.92, 129.13, 128.74, 127.81, 126.50, 125.96, 122.89, 122.38, (101MHz, DMSO-d
6
+
21.19 (M+H) ; Anal. Calcd for C28
24 8 3
H N O : (%) C, 64.61; H, 4.65; N, 127.27, 126.01, 121.88, 121.86, 121.77, 120.08, 118.12, 48.12,
+
+2
1.53; Found: C, 64.63; H, 4.66; N, 21.55.
44.73, 41.67, 22.83 EI-MS m/z 554.12(M+H) , 556.10 (M+H) ; Anal.
Calcd for C28 O: (%) C, 60.66; H, 4.36; N, 17.68; Found: C,
H24BrN
7
N,3-diphenyl-1-((1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-
yl)methyl)-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-
carboxamide (7j)
60.68; H, 4.37; N, 17.69.
1-((1-(2-iodophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-
-1
White solid (76%); m.p. 246-248 °C; IR (KBr) ʋmax / cm 3505, 3029, 6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide
(7n)
1
-1
2
903, 1650, 1402, 1357, 1279, 1045. H NMR (400 MHz, DMSO-d
6
)
White solid (87%); m.p. 188-120 °C; (KBr) ʋmax / cm 3512, 3025,
8
| J. Name., 2012, 00, 1-3
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Journal Name
NP lee wa s eJ od uo r nn oa tl aod fj uC s ht em ma ri sg it nr sy
View Article Online
DOI: 10.1039/C6NJ02671K
ARTICLE
1
2
843, 1650, 1408, 1344, 500. H NMR (400 MHz, DMSO-d
6
) δ 8.79 1-((1-(2,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-
(s, 1H), 8.72 (s, 1H), 7.99 (d, J = 9.2 Hz, 2H), 7.76 (d, J = 8.5 Hz, 2H), diphenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-
7
.46 (t, J = 7.8 Hz, 5H), 7.35 (t, J = 7.5 Hz, 2H), 7.19 (d, J = 9.3 Hz, carboxamide (7r)
H), 6.98(t, J = 7.8 Hz, 1H), 5.47 (s, 2H), 4.70 (s, 2H) , 3.82 (t, J = 5.6 White solid (82%); m.p. 139-141 °C; IR (KBr) ʋmax / cm 3505, 3029,
-1
2
1
3
1
6 6
Hz, 2H), 2.97 (t, J = 5.4 Hz, 2H). C NMR (101 MHz, DMSO-d ) δ 2840, 1654, 1415, 1342, 1070, 740. H NMR (400 MHz, DMSO-d ) δ
1
1
1
55.46, 145.02, 144.23, 143.72, 140.36, 138.63, 130.23, 129.91, 8.77 (s, 1H), 8.73 (s, 1H), 7.80 (d, J = 9.2 Hz, 2H), 7.66 (d, J = 8.5 Hz,
28.64, 128.24, 127.61, 127.27, 126.01, 121.88, 121.86, 121.77, 2H), 7.69 (s, 1H), 7.62 (d, J = 9.5 Hz, 2H), 7.50 (t, J = 7.6 Hz, 2H), 7.41
19.98, 116.78, 115.48, 95.15, 48.23, 44.83, 41.67, 22.93. EI-MS (t, J = 7.5 Hz, 3H), 6.91 (t, J = 7.8 Hz, 1H), 5.45 (s, 2H), 4.69 (s, 2H),
+
13
m/z 602.12 (M+H) ; Anal. Calcd for C28
H
24IN
7
O: (%) C, 55.92; H, 4.03; 3.84 (t, J = 5.7 Hz, 2H), 2.96 (t, J = 5.2 Hz, 2H). C NMR (101 MHz,
DMSO-d ) δ 155.31, 145.53, 144.16, 142.72, 141.48, 139.56, 136.29,
35.74, 133.40, 129.99, 129.24, 128.44, 127.25, 126.21, 123.58,
123.49, 121.57, 120.21, 119.88, 114.78, 46.21 44.63, 41.67, 22.23.
N, 16.30; Found: C, 55.94; H, 4.31; N, 16.32.
6
1
1-((1-(2-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-
+
6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7o)
2 7
EI-MS m/z 544.13 (M+H) ; Anal. calcd for C28H23Cl N O: (%) C, 61.77;
-1
Off white solid (86%); m.p. 118-120 °C; IR (KBr) ʋmax / cm 3505, H, 4.26; N, 18.01; Found: C, 61.79; H, 4.27; N, 18.03.
1
3027, 2846, 1655, 1525, 1410, 1360, 1025. H NMR (400 MHz,
6
DMSO-d ) δ 9.07 (s, 1H), 8.75 (s, 1H), 8.49 – 8.41 (m, 2H), 8.28 – 1-((1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-
8.20 (m, 2H), 7.73 – 7.62 (m, 2H), 7.50 – 7.39 (m, 4H), 7.40 – 7.27 diphenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-
(
m, 1H), 7.28 – 7.18 (m, 2H), 6.94 (tt, J = 7.3, 1.2 Hz, 1H), 5.47 (s, carboxamide (7s)
1
3
-1
2
H), 4.69 (s, 2H), 3.82 (t, J = 5.6 Hz, 2H), 2.97 (t, J = 5.4 Hz, 2H).
C
Off white solid (83%); m.p. 228-230 °C; IR (KBr) ʋmax / cm 3503,
1
NMR (101 MHz, DMSO-d
6
) δ 155.18, 147.87, 147.15, 145.12, 3029, 2842, 1654, 1410, 1345, 1066, 745. H NMR (400 MHz, DMSO-
143.72, 141.36, 138.63, 135.23, 134.56, 133.49, 129.91, 128.64,
6
d ) δ 8.78 (s, 1H), 8.74 (s, 1H), 7.81 (d, J = 9.2 Hz, 2H), 7.77 (s, 1H),
1
28.24, 127.27, 126.01, 121.88, 121.86, 121.77, 120.98, 112.03, 7.66 (d, J = 8.6 Hz, 2H), 7.62 (d, J = 9.4 Hz, 2H), 7.54 (t, J = 7.5 Hz,
+
44.29, 42.15, 41.13, 22.28. EI-MS m/z 521.19 (M+H) ; Anal. Calcd 2H), 7.44 (t, J = 7.4 Hz, 3H), 7.01 (t, J = 7.9 Hz, 1H), 5.43 (s, 2H), 4.63
1
3
for C28
H
24
N
8
O
3
: (%) C, 64.61; H, 4.65; N, 21.53; Found: C, 64.63; H, (s, 2H), 3.82 (t, J = 5.7 Hz, 2H), 2.98 (t, J = 5.4 Hz, 2H). C NMR (101
MHz, DMSO-d ) δ 155.18, 146.41, 143.80, 141.43, 138.26, 136.49,
34.31, 133.41, 129.90, 128.52, 128.41, 127.43, 125.32, 121.78,
121.59, 121.47, 120.20, 119.82, 115.78, 111.78, 46.65, 46.24, 40.97,
4.66; N, 21.55.
6
1
1
-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-
,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide
+
6
2 7
(7p) 22.73. EI-MS m/z 544.20 (M+H) ; Anal. calcd for C28H23Cl N O: (%) C,
-1
White solid (86%); m.p. 147-149 °C; IR (KBr) ʋmax / cm 3497, 3029, 61.77; H, 4.26; N, 18.01; Found: C, 61.79; H, 4.27; N, 18.03.
1
2847, 1650, 1444, 1306, 1032, 753. H NMR (400 MHz, DMSO-d
6
) δ
8.80 (s, 1H), 8.75 (s, 1H), 7.89 (s, 1H), 7.81 (d, J = 9.2 Hz, 2H), 7.69 1-((1-(3,5-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-
(d, J = 8.5 Hz, 2H), 7.40 (t, J = 7.6 Hz, 4H), 7.37 (t, J = 7.5 Hz, 1H), diphenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-
7
.27 – 7.21 (m, 1H), 7.19 (d, J = 9.5 Hz, 2H), 6.93 (t, J = 7.8 Hz, 1H), carboxamide (7t)
-1
5.42 (s, 2H), 4.64 (s, 2H), 3.84 (t, J = 5.7 Hz, 2H), 2.96 (t, J = 5.2 Hz, White solid (85%); m.p. 139-141 °C; IR (KBr) ʋmax / cm 3505, 3027,
13
1
6 6
2H). C NMR (101 MHz, DMSO-d ) δ 155.38, 145.42, 142.82, 2846, 1650, 1411, 1345, 1030, 752. H NMR (400 MHz, DMSO-d ) δ
141.39, 138.55, 136.23, 134.32, 133.30, 129.96, 128.74, 128.29, 8.98 (s, 1H), 8.74 (s, 1H), 8.08 (d, J = 1.8 Hz, 2H), 7.78 – 7.64 (m, 3H),
1
27.20, 126.51, 121.98, 121.80, 121.75, 121.34, 120.31, 119.78, 7.45 (t, J = 7.4 Hz, 4H), 7.38 – 7.16 (m, 3H), 6.94 (t, J = 7.3 Hz, 1H),
+
118.78, 45.29 42.80, 41.66, 22.73. EI-MS m/z 510.15 (M+H) ; Anal. 5.49 (s, 2H), 4.71 (s, 2H), 3.83 (t, J = 5.8 Hz, 2H), 2.95 (t, J = 5.9 Hz,
1
3
calcd for C28
H24ClN
7
O: (%) C, 65.94; H, 4.74; N, 19.23; Found: C, 2H C NMR (101 MHz, DMSO-d
139.15, 138.57, 135.63, 133.93, 129.13, 128.73, 127.80, 126.50,
22.83, 122.38, 120.47, 119.25, 112.02, 44.35, 42.16, 41.12, 22.26.
6
) δ 155.64, 145.60, 144.79, 140.84,
65.95; H, 4.76; N, 19.24.
1
+
1-((1-(3-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-
EI-MS m/z 544.13 (M+H) ; Anal. calcd for C28
2 7
H23Cl N O: (%) C, 61.77;
diphenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-
H, 4.26; N, 18.01; Found: C, 61.79; H, 4.27; N, 18.03.
carboxamide (7q)
-
1
Brown solid (79%); m.p. 135-136 °C; IR (KBr) ʋmax / cm 3502, 3023, 1-((1-(3-chloro-4-fluorophenyl)-1H-1,2,3-triazol-4-yl) methyl)-N,3-
1
2
875, 1657, 1454, 1350, 1090. H NMR (400 MHz, DMSO-d
s, 1H), 8.75 (s, 1H), 7.80 (d, J = 9.1 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H), carboxamide (7u)
.47 – 7.41 (m, 4H), 7.34 (t, J = 7.4 Hz, 1H), 7.26 – 7.20 (m, 2H), 7.11 White solid (77%); m.p. 240-242 °C; IR (KBr) ʋmax / cm 3505, 3022,
6
) δ 8.78 diphenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-
(
-1
7
1
(d, J = 9.1 Hz, 2H), 6.94 (t, J = 7.8 Hz, 1H), 5.48 (s, 2H), 4.71 (s, 2H), 2840, 1654, 1410, 1345, 1178, 1020, 780. H NMR (400 MHz, DMSO-
1
3
3
.83 (t, J = 5.7 Hz, 2H), 2.96 (t, J = 5.5 Hz, 2H). C NMR (100 MHz,
) δ 156.25, 154.08, 145.22, 143.87, 141.36, 138.63, 133.49, 7.92 (m, 1H), 7.71 – 7.62 (m, 3H), 7.49 – 7.39 (m, 4H), 7.38 – 7.28
29.91, 128.64, 128.24, 127.27, 126.01, 125.32, 121.88, 121.76, (m, 1H), 7.28 – 7.18 (m, 2H), 6.98 – 6.89 (m, 1H), 5.48 (s, 2H), 4.70
6
d ) δ 8.90 (s, 1H), 8.73 (s, 1H), 8.23 (dd, J = 6.4, 2.7 Hz, 1H), 8.01 –
DMSO-d
6
1
1
2
6
13
21.77, 120.14, 119.98, 114.78, 111.42, 53.51, 46.12, 44.73, 41.87, (s, 2H), 3.82 (t, J = 5.8 Hz, 2H), 2.95 (t, J = 5.8 Hz, 2H). C NMR (101
+
2.21. EI-MS m/z 506.20 (M+H) ; Anal. calcd for C29
H
27
N
7
O
2
: (%) C, MHz, DMSO-d
6
) δ 155.28, 145.09, 144.14, 140.34, 138.63, 133.46,
8.89; H, 5.38; N, 19.39; Found: C, 68.91; H, 5.39; N, 19.41.
128.62, 128.22, 127.27, 126.00, 122.44, 122.30, 121.87, 120.95,
1
20.77, 120.58, 119.99, 118.13, 117.90, 111.51, 43.88, 41.67, 40.64,
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
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New Journal of Chemistry
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DOI: 10.1039/C6NJ02671K
ARTICLE
Journal Name
+
2
1.79. EI-MS m/z 528.17 (M+H) ; Anal. calcd for C28
7
H23ClFN O: (%) 7.20 (m, 2H), 6.93 (t, J = 6.9 Hz, 1H), 5.47 (s, 2H), 4.72 (s, 2H), 3.82
1
3
C, 63.70; H, 4.39; N, 18.57; Found: C, 63.72; H, 4.40; N, 18.59.
(t, J = 5.7 Hz, 2H), 2.97 (t, J = 5.5 Hz, 2H), 2.36 (s, 6H). C NMR (101
MHz, DMSO-d ) δ 159.25, 155.18, 145.02, 143.72, 140.36, 138.63,
6
1-((1-(3,4-difluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-
136.89, 136.12 133.49, 129.91, 128.64, 128.24, 127.27, 126.01,
diphenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-
121.88, 121.86, 121.77, 119.98, 114.78, 111.48, 45.51, 44.02, 41.57,
+
carboxamide (7v)
22.23, 20.92. EI-MS m/z 504.25 (M+H) ; Anal. calcd for C30
H
29
N
7
O:
-1
Off white solid (80%); m.p. 226-228 °C; IR (KBr) ʋmax / cm 3501, (%) C, 71.55; H, 5.80; N, 19.47; Found: C, 71.56; H, 5.82; N, 19.49.
1
3
021, 2846, 1659, 1412, 1335, 1130, 1060. H NMR (400 MHz,
6
DMSO-d ) δ 8.91 (s, 1H), 8.73 (s, 1H), 8.24 (dd, J = 6.5, 2.6 Hz, 1H), 1-((1-(benzo[d][1,3]dioxol-5-yl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-
8.01 – 7.91 (m, 1H), 7.70 – 7.62 (m, 3H), 7.46 – 7.40 (m, 4H), 7.35 (t, diphenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-
J = 7.4 Hz, 1H), 7.20 (t, J = 7.9 Hz, 2H), 7.04 (t, J = 7.3 Hz, 1H), 5.48 (s, carboxamide (7z)
1
3
-1
2H), 4.70 (s, 2H), ), 3.82 (t, J = 5.6 Hz, 2H), 2.95 (t, J = 5.4 Hz, 2H).
C
Brown solid (78%); m.p. 168-170 °C; IR (KBr) ʋmax / cm 3504, 3025,
1
NMR (101 MHz, DMSO-d
6
) δ 155.21, 149.84, 149.21, 145.02, 2840, 1655, 1416, 1345, 1080. H NMR (400 MHz, DMSO-d
6
) δ 8.77
1
1
4
C
43.72, 141.78, 140.36, 138.63, 133.49, 130.12, 129.91, 128.64, (s, 1H), 8.72 (s, 1H), 7.81 (d, J = 9.1 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H),
28.24, 127.27, 126.01, 124.12, 121.88, 121.86, 121.77, 119.98, 7.44 (t, J = 7.9 Hz, 4H), 7.33 (t, J = 7.4 Hz, 1H), 7.26 (m, 1H), 7.11 (d,
+
5.22, 42.13, 40.81, 23.53. EI-MS m/z 528.17 (M+H) ; Anal. calcd for J = 9.1 Hz, 2H), 6.94 (s, 1H), 5.94 (s, 2H), 5.46 (s, 2H), 4.70 (s, 2H),
13
28 23 2 7
H F N O: (%) C, 65.74; H, 4.53; N, 19.17; Found: C, 65.76; H, 3.82 (d, J = 7.1 Hz, 2H), 2.97 (t, J = 5.4 Hz, 2H). C NMR (101 MHz,
4
.54; N, 19.19.
DMSO-d
29.89, 129.54, 128.44, 127.45, 126.81, 123.98, 123.69, 121.77,
120.81, 119.88, 114.78, 109.98, 101.21, 52.21, 48.63, 41.67, 24.73.
6
) δ 156.31, 148.74, 147.53, 139.76, 136.99, 135.44, 133.49,
1
1-((1-(3,4-dimethoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-
+
diphenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-
EI-MS m/z 520.20 (M+H) ; Anal. calcd for C29
25 7 3
H N O : (%) C, 67.05;
carboxamide (7w)
H, 4.85; N, 18.87; Found: C, 67.06; H, 4.87; N, 18.89.
-1
Brown solid (88%); m.p. 207-209 °C; IR (KBr) ʋmax / cm 3500, 3020,
1
2845, 1654, 1410, 1345, 1080. H NMR (400 MHz, DMSO-d
6
) δ 8.78 Biological activity
(s, 1H), 8.72 (s, 1H), 7.82 (d, J = 9.1 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H), MTB PS screening
7.44 (t, J = 7.9 Hz, 4H), 7.33 (t, J = 7.4 Hz, 1H), 7.26 (m, 1H), 7.11 (d, The MTB panC gene (Rv3602c) encoding the pantothenate
J = 9.1 Hz, 2H), 6.94 (s, 1H), 5.46 (s, 2H), 4.72 (s, 2H), 3.84 (s, 6H), synthetase was cloned and changed into BL21 (DE3) cells and the
1
3
7, 15
3
.82 (d, J = 7.2 Hz, 2H), 2.97 (t, J = 5.4 Hz, 2H). C NMR (101 MHz, protien was expressed.
For the assay, to each well of a 96-well
) δ 156.31, 153.21, 148.74, 139.76, 136.99, 135.44, 129.89, plate, 60 μL of PS reaction mixture containing 0.4 mM NADH, 10
, 5 mM β-alanine, 5 mM pantoic acid, 1 mM potassium
19.88, 118.78, 114.25, 109.13, 101.21, 55.86, 46.90, 44.63, 40.32, phosphoenolpyruvate,10 mM ATP, and 20 μL of enzyme mixture
DMSO-d
6
1
1
2
6
29.54, 128.44, 127.45, 126.81, 123.98, 123.69, 121.77, 120.81, mM MgCl
2
+
2.09. EI-MS m/z 536.20 (M+H) ; Anal. calcd for C30
H
29
N
7
O
3
: (%) C, consisting of 18 units/mL each of chicken muscle myokinase, rabbit
muscle lactate dehydrogenase and rabbit muscle pyruvate kinase,
diluted in 100 mM HEPES buffer were added. The reaction mixture
-((1-(4-bromo-3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4- and enzyme mixture were added to the plate to a final volume of
7.27; H, 5.47; N, 18.31; Found: C, 67.29; H, 5.48; N, 18.33.
1
yl)methyl)-N,3-diphenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-
(4H)-carboxamide (7x)
100 μL with 100 mM HEPES buffer (pH 7.8). Concentration of
enzyme was determined based on the range finding experiments by
5
-1
White solid (79%); m.p. 221-223 °C; IR (KBr) ʋmax / cm 3500, 3022, varying the concentration of enzymes. Compound solutions were
1
2
854, 1660, 1405, 1343, 1119, 1070. H NMR (400 MHz, DMSO-d
δ 8.91 (s, 1H), 8.73 (s, 1H), 8.24 (dd, J = 6.5, 2.6 Hz, 1H), 8.01 – 7.82 the reaction began with the addition of 10 μL of 4.32 pM of MTB PS,
m, 1H), 7.69 – 7.53 (m, 3H), 7.46 – 7.41 (m, 4H), 7.36 (t, J = 7.4 Hz, diluted in buffer. The test plate was immediately moved to a
H), 7.21 (t, J = 7.9 Hz, 2H), 7.04 (t, J = 7.3 Hz, 1H), 5.48 (s, 2H), 4.70 microplate reader and the reduction of NADH was quantified at 340
6
)
then added to the plates (from 50 μM to lower concentration) and
(
1
1
3
(
s, 2H), ), 3.82 (t, J = 5.6 Hz, 2H), 2.95 (t, J = 5.4 Hz, 2H). C NMR nm. The reaction elements except MTB PS were mixed in the well
101 MHz, DMSO-d ) δ 155.18, 145.02, 143.72, 140.36, 139.10, and the background reaction was calculated; MTB PS was then
38.63, 136.89, 136.12, 133.49, 130.23, 129.91, 128.64, 128.24, added and the reaction kinetics was monitored. Reactions were
(
6
1
1
4
27.27, 126.01, 121.88, 121.86, 121.77, 120.98, 120.01, 114.78, carried out at 37 °C in a heat controlled Perkin Elmer Victor X3
+
8.01, 46.32, 44.83, 22.19. EI-MS m/z 622.20, 624.15 (M+1, M+2) ; Spectrophotometer. % inhibitions were calculated using following
Anal. calcd for C30
3 7
H23BrF N O: (%) C, 55.96; H, 3.72; N, 15.75; formula: 100 x [(1 - compound rate - background rate) / (full
7
, 15
Found: C, 55.98; H, 3.74; N, 15.76.
reaction rate - background rate)].
1-((1-(3,4-dimethylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-
In vitro MTB screening
diphenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-
The antimycobacterial activities of the compounds 7a-z were
evaluated against MTB H37Rv strain and two “wild” strains
carboxamide (7y)
-1
Off white solid (84%); m.p. 221-223 °C; IR (KBr) ʋmax / cm 3505, extracted from tuberculosis patients: one strain is Spec. 210
1
3
022, 2844, 1658, 1412, 1345, 1076. H NMR (400 MHz, DMSO-d
6
)
resistant to PAS, INH, ETB and RMP and the other strain is Spec. 192
δ 8.82 (s, 1H), 8.79 (s, 1H), 7.78 (d, J = 8.6 Hz, 2H), 7.66 (d, J = 7.3 Hz, fully sensitive to the administrated anti-TB agents. In vitro anti-TB
2H), 7.47 – 7.42 (m, 3H), 7.44 (s, 1H), 7.41 – 7.31 (m, 2H), 7.27 – activity is performed by a classical test-tube method of successive
1
0 | J. Name., 2012, 00, 1-3
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Page 11 of 13
Journal Name
NP lee wa s eJ od uo r nn oa tl aod fj uC s ht em ma ri sg it nr sy
View Article Online
DOI: 10.1039/C6NJ02671K
ARTICLE
dilution in Youmans’ modification of the Proskauer and Beck liquid (SR/FST/CSI-240/2012) Government of India, New Delhi is great
3
6, 37
medium containing 10% of bovine serum.
Bacterial respites was fully acknowledged. A.S. thanks DST for providing junior research
prepared from 14 days old cultures of gradually growing strains. fellowship and S.S. thank CSIR for providing junior research
Solutions of compounds in DMSO were tested. Stock solutions fellowship.
contained 10 mg of compounds in 1 mL. Dilutions (in geometric
37
progression) were prepared in Youmans’ medium. The medium is
without compounds and containing INH as reference drug was used
for comparison. Incubation was performed at 37 °C. The MIC values
were determined as MIC inhibiting the growth of tested TB strains
in relation to the probe with no tested compound. The influence of
the compound on the growth of bacteria at concentrations of 3.12,
Notes and references
1
World Health Organization (WHO) - Global Tuberculosis Control
report 2015,
http://www.who.int/tb/publications/global_report/gtbr15_mai
n_text.pdf.
6
.25, 12.5, 25, 50 and 100 μg/mL was evaluated.
2
3
P. M. S. Chauhan, N. Sunduru, M. Sharma, Future. Med. Chem.,
2
010, 2, 1469.
D. L. Combs, R. J. O’Brien, L. J. Geiter, Ann. Intern. Med., 1990,
12, 397.
In vitro cytotoxicity screening
Compounds 7b, 7c and 7d were further tested for toxicity in a RAW
1
2
64.7 cell line at the concentration of 50 μM. After 72 h of
4
5
6
7
8
M. A. Stratton, M. T. Reed, Clin. Pharm., 1986, 5, 977.
D. A. Mitchison, Tubercle, 1985, 66, 219.
exposure, viability was evaluated on the basis of cellular translation
of MTT into a formazan product by the Promega Cell Titer 96
M. Yan, S. Ma, ChemMedChem, 2012, 7, 2063.
38, 39
nonradioactive cell proliferation assay.
R. Zheng, J. S. Blanchard, Biochemistry, 2001, 40, 12904.
S. Velaparthi, M. Brunsteiner, R. Uddin, B. Wan, S. G. Franzblau,
P. A. Petukhov, J. Med. Chem. 2008, 51, 1999.
Docking Study
Docking studies of the title compounds (7a-z) was performed using
Glide 5.9 (Extra Precision) running on maestro version 9.4, in order
to investigate their in-silico biding affinity as well as their binding
9
1
S. Wang, D. Eisenberg, Prot. Sci. 2003, 12, 1097.
0 Y. Yang, P. Gao, Y. Liu, X. Ji, M. Gan, Y. Guan, X. Hao, Z. Li, C.
Xiao, Bioorg. Med. Chem. Lett., 2011, 21, 3943.
4
0
pattern with enzyme Pantothenate synthetase. Enzyme used for
the docking study was retrieved from RCSB Protein Data Bank (PDB
ID: 3IUB) in complex with co-crystallised ligand (indole-2-
carboxamide derivative). Selected protein consist of two chains
asymmetric units (A and B), both consist of co-crystallized ligand, in
the current study unit A was separated and used further for docking
studies. Protein preparation wizard of Schrödinger suite was used
for preparation of selected protein. Protein was pre-processed
separately by deleting the substrate co-factor as well as the
crystallographically observed water molecules (water without H
bonds), followed by optimization of hydrogen bonds. After
assigning charge and protonation position, finally energy was
minimized with root mean square deviation (RMSD) value of 0.30 Å
using optimized potentials for liquid simulations-2005 (OPLS-2005)
1
1
1
1
2
3
G. Samala, P. B. Devi, R. Nallangi, J. P. Sridevi, P. Yogeeswari, D.
Sriram, Eur. J. Med. Chem., 2013, 69, 356.
G. Samala, P. B. Devi, R. Nallangi, J. P. Sridevi, P. Yogeeswari, D.
Sriram, Bioorg. Med. Chem., 2014, 22, 1938.
E. L. White, K. Southworth, L. Ross, S. Cooley, R. B. Gill, M. I.
Sosa, A. Manouvakhova, L. Rasmussen, C. Goulding, D.
Eisenberg, T. M. Fletcher, J. Biomol. Screen., 2007, 12, 100.
G. Samala, R. Nallangi, P. B. Devi, S. Saxena, R. Yadav, J. P.
Sridevi, P. Yogeeswari, D. Sriram, Bioorg. Med. Chem., 2014, 22,
1
4
5
4
223.
1
1
1
G. Samala, P. B. Devi, S. Saxena, N. Meda, P. Yogeeswari, D.
Sriram, Bioorg. Med. Chem., 2016, 24, 1298.
6 P. P. Deohate, J. P. Deohate, B. N. Berad, Asian. J. Chem., 2004,
6, 255.
1
4
1
force field. Finally energy minimized protein and co-crystallized
ligand was used to build energy grids using the default value of
protein atom scaling (1.0 Å) within a cubic box of 14 Å dimensions,
7 K. L. Kees, J. J. Fitzgerald, K. E. Steiner, J. F. Mattes, B. Mihan, T.
Tosi, D. Moondoro, M. L. McCaleb, J. Med. Chem., 1996, 39,
3
920.
4
2
centered on the centroid of the X-ray ligand pose. The structures
of 7a-z were drawn using ChemSketch and converted to 3D
structure with the help of 3D optimization tool. Using LigPrep 2.6
module, the drawn ligands were geometry optimized; partial atomic
1
8 (a) P. Diana, A. Carbone, P. Barraja, A. Martorana, O. Gia, L.
DallaVia, G. Cirrincione, Bioorg. Med. Chem. Lett., 2007, 17,
6
134; (b) H. J. Park, K. Lee, S. J. Park, B. Ahn, J. C. Lee, H. Y. Cho,
K. I. Lee, Bioorg. Med. Chem. Lett., 2005, 15, 3307.
43
charges were computed using OPLS-2005 force field. Finally,
prepared ligands were docked with prepared protein using Glide 5.9
module, in extra precision mode (XP). The leading docked pose
1
2
9 R. Aggarwal, S. P. Singh, V. Kumar, P. Tyagi, Bioorg. Med. Chem.,
2
006, 14, 1785.
0 (a) N. Harikrishan, A. M. Isloor, K. Ananda, A. Obaid, H. K. Fun,
New J. Chem., 2016, 40, 73; (b) N. K. Piyush, P. S. Shailesh and K.
R. Dipak, New J. Chem., 2014, 38, 2902; (c) R. Manikannan, R.
Venkatesan, S. Muthusubramanian, P. Yogeeswari, D. Sriram,
Bioorg. Med. Chem. Lett., 2010, 20, 6920.
(with lowest Glide score value) found from Glide was analyzed.
RMSD value was calculated between the experimental binding
mode of co-crystallized ligand as in X-ray and re-docked pose to
ensure accuracy and reliability of the docking procedure.
2
2
1 D. Raffa, B. Maggio, M. V. Raimondi, S. Cascioferro, F. Plescia, G.
Cancemi, G. Daidone, Eur. J. Med. Chem., 2015, 97, 732.
2 M. G. Mamolo, D. Zampieri, V. Falagiani, L. Vio, E. Banfi, Il
Farmaco, 2001, 56, 593.
Acknowledgements
The financial assistance provided by Department of Science and
Technology (DST), (F.No.SB/S1/OC-60/2013) and DST FIST grant
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 11
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DOI: 10.1039/C6NJ02671K
ARTICLE
Journal Name
2
2
2
2
2
3 R. B. Pathak, P. T. Chovatia, H. H. Parekh, Bioorg. Med. Chem.
Lett., 2012, 22, 5129.
4 P. T. Chovatia, J. D. Akabari, P. K. Kachhadia, P. D. Zalavadia, H.
S. Joshi, J. Serb. Chem. Soc., 2007, 71, 713.
5 R. C. Khunt, V. M. Khedkar, R. S. Chawda, N. A. Chauhan, A. R.
Parikh, E. C. Coutinho, Bioorg. Med. Chem. Lett., 2012, 22, 666.
6 A. Suresh, N. Suresh, S. Misra, M. M. Krishna Kumar, K. V. G.
Chandra Sekhar, ChemistrySelect, 2016, 1, 1705.
7 (a) M. Bhat, G.K. Nagarjuna, R. Kayarmar, S.K. Peethamber, R.
Mohammed Shafeeulla, RSC Adv., 2016, 6, 59375; (b) H. C. Kolb,
K. B. Sharpless, Drug. Discov. Today., 2003, 8, 1128.
8 D. Kumar, G. B. Khare, S. Kidwai, A. K. Tyagi, R. Singh, D. S.
Rawat, Eur. J. Med. Chem., 2014, 81, 301.
2
2
9 S. R. Patpi, L. Pulipati, P. Yogeeswari, D. Sriram, N. Jain, B.
Sridhar, R. Murthy, T. Anjana Devi, S. V. Kalivendi, S. Kantevari,
J. Med. Chem., 2012, 55, 3911.
30
N. Boechat, V. F. Ferreira, S. B. Ferreira, M. L. G. Ferreira, F. C.
da Silva, M. M. Bastos, M. S. Costa, M. S. Lourenço, A. C.
Pinto, A. U. Krettli, A. C. Aguiar, B. M. Teixeira, N. V. da Silva, P.
R. C. Martins, F. F. M. Bezerra, A. S. Camilo, G. P. da Silva, C. C.
P. Costa, J. Med. Chem., 2011, 54, 5988.
3
1 S. Kim, S. Cho, T. Oh, P. Kim, Bioorg. Med. Chem. Lett., 2012, 22,
844.
6
32
P. Shanmugavelan, S. Nagarajan, M. Sathishkumar, A.
Ponnuswamy, P.Yogeeswari, D. Sriram, Bioorg. Med. Chem.
Lett., 2011, 21, 7273.
3
3
3
3 H. N. Nagesh, K. Mahalakshmi Naidu, D. Harika Rao, J. P. Sridevi,
D. Sriram, P. Yogeeswari, K. V. G. Chandra Sekhar, Bioorg. Med.
Chem. Lett., 2013, 23, 6805.
4 K. Mahalakshmi Naidu, S. Srinivasarao, N. Agnieszka, A. Ewa, M.
M. Krishna Kumar, K. V. G. Chandra Sekhar, Bioorg. Med. Chem.
Lett., 2016, 26, 2245.
5 X. M. Ye, A. W. Konradi, J. Smith, D. L. Aubele, A. W. Garofalo, J.
Marugg, M. L. Neitzel, C. M. Semko, H. L. Sham, M. Sun, A. P.
Truong, J. Wu, H. Zhang, E. Goldbach, J. M. Sauer, E. F. Brigham,
M. Bova, G. S. Basi, Bioorg. Med. Chem. Lett., 2010, 20, 2195.
6 G. P. Youmans, Am. Rev. Tuberc., 1947, 56, 376.
3
3
3
7 G.P. Youmans, A.S. Youmans, J. Bactriol., 1949, 58, 247.
8 P. B. Devi, G. Samala, J. P. Sridevi, S, Saxena, M, Alvala, E. G,
Salina, D, Sriram, P. Yogeeswari, Chemmedchem, 2014, 9, 2538.
9 D. Gerlier, N. Thomasset, Immunol. Methods, 1986, 94, 57.
0 Glide, Schrödinger, LLC, New York, 2013 version 5.9.
1 W. L. Jorgensen, D. S. Maxwell, R. J. Tirado, J. Am. Chem. Soc.,
3
4
4
1996, 118, 11225.
42 S. Chander, A. Penta, S. Murugesan, Journal of Pharmacy
Research, 2014, 8, 552.
43 Lig-Prep, Schrödinger, LLC, New York, 2013 version 2.6.
1
2 | J. Name., 2012, 00, 1-3
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New Journal of Chemistry
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DOI: 10.1039/C6NJ02671K
Graphical Text
Novel series of pyrazolo[4,3-c]pyridine carboxamides was synthesized and characterized using various spectroscopic
techniques. The synthesized compounds were evaluated for their antitubercular activity, pantothenate synthetase enzyme
inhibition study and were docked to 3IUB crystal structure of MTB PS.