Discovery of novel tetrahydro-pyrazolo [4,3-c] pyridines for the treatment of neuropathic pain: Synthesis and neuropharmacology

Article abstract of DOI:10.1016/j.ejmech.2013.05.022

We disclose the discovery of a novel series of tetrahydropyrido-pyrazoles that are potent inhibitors of tumour necrosis factor-alpha (TNF-α), nitric oxide and cannabinoid receptor subtype 1 (CB₁). We report herein the synthesis and neuropharmacological screening results of the titled compounds in two acute pain and two neuropathic pain models in rodents. Particularly the analogue N-(4-bromophenyl)-3-tert-butyl-5-ethyl-4,5,6,7-tetrahydro-1H- pyrazolo[4,3-c]pyridine-1-carboxamide (8a) exhibited pronounced acute antinociceptive efficacy, also being effective in chronic constriction injury (ED₅₀ = 23.8 mg/kg) and partial sciatic nerve injury (ED₅₀ = 29.0 mg/kg) models with CB₁ receptor activity (IC₅₀ = 49.6 nM) and inhibitory effect on TNF-α (86.4% inhibition at 100 mg/kg). These results suggest the importance of the development of this lead as multi-targeted treatment strategy for neuropathic pain.

Full text of DOI:10.1016/j.ejmech.2013.05.022

European Journal of Medicinal Chemistry 66 (2013) 211e220
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of novel tetrahydro-pyrazolo [4,3-c] pyridines for the
treatment of neuropathic pain: Synthesis and neuropharmacology
*
Perumal Yogeeswari , Monika Sharma, Ganesh Samala, Matharasala Gangadhar,
Srirama Karthick, Saketh Mallipeddi, Arvind Semwal, Dharmarajan Sriram
Neuropathic Pain Research Laboratory, Department of Pharmacy, Birla Institute of Technology & Science e Pilani, Hyderabad Campus, R.R. District,
500078 Andhra Pradesh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
We disclose the discovery of a novel series of tetrahydropyrido-pyrazoles that are potent inhibitors of
tumour necrosis factor-alpha (TNF-a), nitric oxide and cannabinoid receptor subtype 1 (CB1). We report
Received 23 January 2013
Received in revised form
11 May 2013
Accepted 18 May 2013
Available online 7 June 2013
herein the synthesis and neuropharmacological screening results of the titled compounds in two acute
pain and two neuropathic pain models in rodents. Particularly the analogue N-(4-bromophenyl)-3-tert-
butyl-5-ethyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-1-carboxamide (8a) exhibited pronounced
acute antinociceptive efficacy, also being effective in chronic constriction injury (ED50 ¼ 23.8 mg/kg) and
partial sciatic nerve injury (ED50 ¼ 29.0 mg/kg) models with CB1 receptor activity (IC50 ¼ 49.6 nM) and
Keywords:
Antihyperalgesic
Antiallodynic
Tetrahydropyrazolo-pyridine
Cannabinoid receptor
Nitric oxide
inhibitory effect on TNF-
development of this lead as multi-targeted treatment strategy for neuropathic pain.
Ó 2013 Elsevier Masson SAS. All rights reserved.
a (86.4% inhibition at 100 mg/kg). These results suggest the importance of the
Antioxidant
1. Introduction
recorded history [6]. Scott et al. elucidated the roles of the two
cannabinoid receptor subtypes, CB1 and CB2 in cannabinoid-
mediated analgesia. CB1 receptors have a widespread distribution
in the central and peripheral nervous systems, and CB2 receptors
are restricted largely to cells of the immune system [7]. Activation
of CB1 receptors suppresses calcium conductance and inhibits in-
ward rectifying potassium conductance, thereby suppressing
neuronal excitability and transmitter release [8]. CB2 receptor
activation stimulates MAPK (mitogen-activated protein kinase)
activity but does not modulate calcium or potassium conductance
and is thought to have anti-inflammatory effect [9]. Several
tetrahydropyrido-pyrazoles have been reported in the past for
various targets directed towards the treatment of neuropathic pain
and other neurological disorders. These include their potential
mechanism of action on cannabinoid receptor [10], cathepsin S
Neuropathic pain is a complex pathophysiological phenomenon
that may arise from various etiologies such as trauma, compression
injury, disease states, viral infection, amputation, or metabolic dis-
turbances such as diabetes [1,2]. While specific symptoms of neuro-
pathic pain may vary widelyamong patients, the hallmark symptoms
include thermal and mechanical hyperalgesia (exaggerated response
to a noxious stimulus), thermal and mechanical allodynia (a noxious
response to normally innocuous stimuli), paroxysmal and sponta-
neous pain [2,3]. The epidemiology of neuropathic pain is not well
described, in part, due to the diversity of associated conditions.
However, preliminary prevalence estimates of common conditions
suggest that neuropathic pain affects 3e8% of the worldwide popu-
lation [4]. Since the size of this population is increasing worldwide, it
is inevitable that neuropathic pain will place a progressively
demanding burden on health care resources [5].
protease [11e15], tumour necrosis factor-a (TNF-a) [16], and orexin
receptors [17].
Evidence for the use of Marijuana (Cannabis sativa) as a treat-
ment option for pain can be traced back to the beginnings of
Lack of effective and well-tolerated therapy to treat neuropathic
pain has resulted in a need for development of the multi-faceted
treatment strategy, shifting the paradigm from the well estab-
lished concept of biological activity at single receptor (magic bul-
lets) to ligands with multiple activities. During the last decade, our
research group has reported various aryl semicarbazones as
important leads in the discovery of anticonvulsant agents [18e21].
* Corresponding author. Tel.: þ91 9705932091; fax: þ91 40 66303998.
E-mail addresses: pyogee@hyderabad.bits-pilani.ac.in, pyogie_2000@rediffmail.com
(P. Yogeeswari).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.05.022

212
P. Yogeeswari et al. / European Journal of Medicinal Chemistry 66 (2013) 211e220
Due to the similar pathophysiology between epilepsy and neuro-
pathic pain, it is not surprising that several antiepileptic drugs are
being used to manage neuropathic pain [22]. In our drug discovery
program we have advanced the aryl semicarbazones as a novel class
of anticonvulsant leads in attenuating neuropathic pain conditions
[23e26]. Also, aryl semicarbazones have shown to act through
GABA mediation [27,28]. In our interest to develop newer hetero-
cyclic scaffolds for neuropathic pain, we have recently reported
furoic acid hydrazones, isatin derivatives, piperazinyl derivatives,
1,2,4-triazol-5-ones, and fused triazolo-thiadiazoles as anti-
nociceptive agents [29e33]. Hence in the course of our efforts to-
wards the identification of new leads for the treatment of
neuropathic pain, we were interested in the integration of aryl
semicarbazide template into the tetrahydropyrido-pyrazoles phar-
macophore. Here we describe the efforts in optimizing a novel series
acting through cannabinoid receptor coupled with inhibition of
(acetyl chloride or pivaloyl chloride) were added to 1 at 0 ꢀC
with subsequent hydrolysis to yield 2 followed by cyclisation in
the next step in presence of equimolar amounts of various aryl
semicarbazides to yield tert-butyl-3-alkyl-1-(substituted phenyl-
carbamoyl)-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carbox-
ylate (3aeh and 4aeh) [10,15]. A deprotecting agent (TFA) was
reacted with a solution of compounds 3aeh and 4aeh in dichloro-
methane providing the corresponding 5aeh and 6aeh. Finally, the
compounds 5aeh and 6aeh were N-alkylated with ethyl iodide to
yield the compounds 7aeh and 8aeh in yields ranging between 62%
and 78%. All the compounds were purified through silica gel column
chromatography. (HPLC purity >95%) and were fully characterized
by 1H NMR, 13C NMR, Electrospray ionization (ESI) and elemental
analysis.
3. Results and discussion
TNF-a and oxidative stress.
Initial biological evaluation at a single dose of 100 mg/kg of the
synthesized tetrahydropyrido-pyrazoles (7aeh and 8aeh) was
carried out using both acute and chronic pain models. To examine
the potential therapeutic value of these newer derivatives in the
treatment of pain, the first part of the study examined the ability of
the derivatives to inhibit writhing and flinching responses in the
acetic acid induced writhing test and formalin-induced flinching
test. The acute neurological toxicity was determined by the rotarod
test (Table 1). All the tested compounds except 7f, 8f and 8h sup-
pressed the acetic acid induced writhing response significantly
(p < 0.01) in comparison to control (Table 1). Compounds 7e, 8a, 8b,
8d and 8e were the most active compounds with percentage
2. Chemistry
The synthesis of 5-ethyl-3-alkyl-N-(mono/disubstitutedphenyl)-
4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-1-carboxamides
(7aeh and 8aeh) was achieved as presented in Scheme 1. The syn-
thesis started with N-Boc-piperidone which was treated with mor-
pholine in the presence of a catalyst p-toluene sulphonic acid (pTSA)
to yield an oily product tert-butyl 4-morpholino-5,6-dihydropyri
dine-1(2H)-carboxylate (1). As described in the literature, the
tetrahydropyrido-pyrazole core (3aeh and 4aeh) was assembled in
two steps from the enamine (1). Alkyl acid chlorides employed
Scheme 1. Synthetic protocol of substituted tetrahydropyrido-pyrazoles.

P. Yogeeswari et al. / European Journal of Medicinal Chemistry 66 (2013) 211e220
213
Table 1
Neurotoxicity and activity of the compounds on acute nociceptive models.
Treatment
R1
R2
Neurotoxicitya
0.5 h
Acetic acid induced writhing
% inhibition
Formalin induced flinching
4 h
% inhibition
Phase I
Phase II
Vehicle
7a
7b
7c
7d
7e
7f
7g
7h
8a
8b
8c
8d
8e
8f
8g
e
e
e
e
e
e
e
H
4-Br
6-CH3
5-CH3
4-CH3
4-F
5-CH3
3-CF3
4-Cl
4-Br
6-CH3
5-CH3
4-CH3
4-F
5-CH3
3-CF3
4-Cl
e
e
59.7c
60.5c
62.2c
56.1c
76.3c
10.5
1.7
12.2
2.6
7.0
7.8
5.2
4.4
1.7
0.0
15.7
0.0
15.7
4.4
7.0
2.6
3.5
4.7
81.6c
78.9c
55.8c
16.3
14.3
15.0
23.1
7.5
2-CH3
2-CH3
2-CH3
2-F
2-F
H
e
e
300
300
e
300
300
e
e
e
e
e
56.1c
55.2c
72.8c
73.7c
63.1c
71.1c
71.9c
17.5
H
H
e
e
e
e
72.1c
68.0c
15.7
59.9c
73.4c
60.5c
55.1c
68.3c
72.1c
2-CH3
2-CH3
2-CH3
2-F
2-F
H
e
e
e
e
e
e
100
e
100
e
e
e
61.4c
15.8
8h
H
e
e
e
Indomethacinb
e
e
e
96.1c
a
Doses of 30, 100, and 300 mg/kg were administered. The figures in the table indicate the minimum dose whereby bioactivity was demonstrated in half or more of the mice
(three in each group). The animals were examined at 0.5 and 4.0 h. The line (e) indicates an absence of neurotoxicity at the maximum dose tested.
b
Indomethacin was taken as positive control at 5 mg/kg.
Represents significance at p < 0.05 compared to vehicle (One way ANOVA followed by Dunnett’s test, n ¼ 4) at a dose of 100 mg/kg.
c
inhibition of 70.0% or above. In the formalin-induced flinching
model, ten compounds (7a, 7b, 7c, 8a, 8b, 8d, 8e, 8f, 8g and 8h)
showed inhibition of flinching response in the second phase while
none of them were effective in the initial phase indicating the effect
of these compounds on the tonic inflammatory pain response.
Compounds 7a, 7b and 8e exhibited greater inhibition (>72.0%)
than the standard drug Indomethacin, while compound 8a was
equipotent with the standard drug. In the acute neurotoxicity assay
all compounds except 7c and 7d did not cause motor-deficient in
the highest dose tested (300 mg/kg). Compound 8e was found to be
neurotoxic at a dose of 100 mg/kg.
In the next phase of screening, two well known peripheral
neuropathic pain models were used that included the chronic
constriction injury (CCI) and partial sciatic nerve ligation (PSNL)
models in rats. In the CCI model, the left sciatic nerve proximal to
the trifurcation point was constricted with four loose ligatures
using 4-0 braided silk thread, while in the PSNL model, left sciatic
nerve was exposed at mid thigh level and one-half of the nerve
thickness was tightly ligated using 7-0 silk suture. Four nociceptive
assays aimed at determining the severity of behavioural neuro-
pathic responses namely allodynia and hyperalgesia were per-
formed. The assays involved measurement of the degree of
spontaneous (ongoing) pain and tests of hind limb withdrawal to
cold and mechanical stimuli (tactile allodynia, cold allodynia and
mechanical hyperalgesia). A minimum of 10 min separated the
testing procedures to reduce the influence of prior nociceptive
testing. The order of testing was as follows: spontaneous pain,
tactile allodynia, cold allodynia and lastly mechanical hyperalgesia.
Baseline sensory response values were measured for each group of
animals (n ¼ 4) pre-operatively and 9 days post-operatively. Ani-
mals displaying allodynic and hyperalgesic responses in both the
models, were then administered the test compound (single dose of
100 mg/kg) according to a pre-determined randomization table and
testing was re-performed at 30, 60 and 120 min post drug
administration. All of the behavioural responses were timed with a
stopwatch. All of the compounds were tested at a single dose of
100 mg/kg.
period of testing (30 mine120 min) similar to gabapentin. Com-
pounds 7f and 8f showed reversal of spontaneous pain only at
120 min (Table 2).
Eight compounds (7a, 7b, 7d, 7e, 8a, 8b, 8d, and 8e) were
effective in attenuating the tactile allodynic response in the CCI
animals. Compounds 7a, 7b, 8a, 8b and 8d were effective
throughout the 120 min time period, while compounds 7d, 7e, 8e
and gabapentin were effective only at two time points. All other
compounds were ineffective. In the cold allodynia produced in CCI
rats, the significant reversal was observed with the administration
of compounds 7a, 7b, 7d, 7e, 8a, 8b, 8d and 8e (Table 3). Compound
8a was found to be effective throughout the 120 min experiment.
The duration of action for compounds 7a, 7d and 8g was found to
be 30 min whereas compounds 7b, 7e, 8b, 8d and 8e were effective
for 60 min in 120 min experiment. Gabapentin showed 58.0%
protection in this test and was found to be effective only at 30 min
time point. All other compounds were found to be ineffective in this
test. Hyperalgesia evoked by a mechanical pin-prick stimulus was
effectively attenuated at all time-points (30 mine120 min) of study
by compounds 7a, 7b, 7d, 7e, 8a, 8b, 8d, and 8e. The duration of
action for all of these compounds was 120 min except for 7b and 8e,
which were found to be effective for 60 min. All other compounds
were ineffective in this test. Overall, it appears that in the CCI model
of neuropathic pain, compounds that showed promising results
include 7a, 7b, 7d, 7e, 8a, 8b, 8d and 8e effective in all the four tests,
8g in two tests and 7g in one test.
In the PSNL model, the paw withdrawal durations due to
spontaneous ongoing pain were significantly reduced by com-
pounds 7b, 8b and 8d throughout the time period (30 mine
120 min) similar to gabapentin. Compounds 7a, 8a and 8e showed
activity upto 60 min duration while compounds 7e, 7f and 8g were
effective for a shorter duration (30 min). All other compounds were
ineffective in this test. Compound 7d, which completely reversed
the spontaneous pain response in the CCI model, was inactive in the
PSNL model (Table 2).
The tactile allodynia produced by the PSNL was effectively
reversed by compound 8d throughout the 120 min duration better
than gabapentin while compounds 7a, 8b and 8e were effective till
60 min. Compounds 7d, 7e, 7g, and 8a showed activity for short
In the CCI model, nine compounds out of sixteen completely
reversed the spontaneous pain response throughout the time

214
P. Yogeeswari et al. / European Journal of Medicinal Chemistry 66 (2013) 211e220
Table 2
Effects of compounds on spontaneous pain and tactile allodynia in CCI and PSNL model.
Study
group
Spontaneous pain
% reversal (CCI)a
Tactile allodynia
% reversal (CCI)b
% reversal (PSNL)a
% reversal (PSNL)b
60 min 120 min
7.7 ꢁ 0.7 12.5 ꢁ 1.2 0.0 ꢁ 0.0
30 min
60 min
4.6 ꢁ 0.6
120 min
30 min
60 min
120 min
1.2 ꢁ 0.1
30 min
60 min
120 min
30 min
Vehicle
7a
7b
7c
7d
7e
7f
7g
7h
8a
8b
8c
8d
8e
8.9 ꢁ 4.6
4.5 ꢁ 0.2
3.2 ꢁ 0.3
2.9 ꢁ 0.3
3.4 ꢁ 0.8
1.3 ꢁ 0.3
4.1 ꢁ 2.1
73.9 ꢁ 0.2c 65.1 ꢁ 0.2c
77.3 ꢁ 7.3c 68.3 ꢁ 6.7c
23.6 ꢁ 4.2 33.3 ꢁ 1.6
72.9 ꢁ 0.8c 67.5 ꢁ 1.3c
69.3 ꢁ 5.5c 63.6 ꢁ 1.1c
15.0 ꢁ 3.3 11.0 ꢁ 5.6
13.3 ꢁ 8.5 15.2 ꢁ 3.0
16.9 ꢁ 4.2 15.0 ꢁ 0.4
90.6 ꢁ 2.1c 85.9 ꢁ 1.3c
95.0 ꢁ 0.7c 90.7 ꢁ 2.1c
21.1 ꢁ 2.4 19.8 ꢁ 2.2
85.4 ꢁ 0.8c 90.9 ꢁ 0.5c
81.6 ꢁ 3.9c 85.9 ꢁ 2.5c
14.4 ꢁ 2.1 20.5 ꢁ 1.8
63.6 ꢁ 1.2c 56.7 ꢁ 3.3c 60.8 ꢁ 4.2c 41.3 ꢁ 1.7 57.7 ꢁ 9.0c 70.5 ꢁ 3.8c 57.7 ꢁ 9.0c 58.3 ꢁ 8.3c 58.3 ꢁ 8.3c 29.2 ꢁ 4.2
53.7 ꢁ 0.4c 70.3 ꢁ 2.2c 66.1 ꢁ 4.5c 58.4 ꢁ 2.2c 55.0 ꢁ 1.5c 58.0 ꢁ 5.0c 65.1 ꢁ 3.4c 18.4 ꢁ 1.6 46.4 ꢁ 7.0 38.3 ꢁ 3.1
27.7 ꢁ 2.1
7.5 ꢁ 2.0 11.1 ꢁ 4.0
4.8 ꢁ 0.4 17.3 ꢁ 1.9
9.9 ꢁ 5.5
2.2 ꢁ 0.2 27.6 ꢁ 2.6 16.0 ꢁ 9.0
3.5 ꢁ 0.3
64.4 ꢁ 5.6c 40.7 ꢁ 5.9 34.4 ꢁ 5.6
5.9 ꢁ 0.3 17.2 ꢁ 4.0 56.6 ꢁ 9.2c 66.1 ꢁ 8.9c 53.3 ꢁ 2.1c 41.9 ꢁ 1.1 30.2 ꢁ 2.3
72.8 ꢁ 5.6c 59.2 ꢁ 9.2c 28.2 ꢁ 1.4 14.7 ꢁ 6.4 39.3 ꢁ 2.3 69.6 ꢁ 1.2c 67.0 ꢁ 2.2c 58.3 ꢁ 8.3c 41.7 ꢁ 8.3 39.2 ꢁ 4.2
51.8 ꢁ 0.2c 57.6 ꢁ 1.3c 26.6 ꢁ 1.5 17.3 ꢁ 7.7
6.6 ꢁ 2.2
9.9 ꢁ 5.5 13.7 ꢁ 9.3 20.2 ꢁ 1.3 15.1 ꢁ 1.5
3.5 ꢁ 0.3
12.5 ꢁ 0.3 20.7 ꢁ 0.7 10.2 ꢁ 3.1 13.8 ꢁ 0.5 4.41 ꢁ 0.1 11.8 ꢁ 7.4 11.8 ꢁ 7.4 69.7 ꢁ 3.3c 11.1 ꢁ 2.0 11.1 ꢁ 2.0
27.4 ꢁ 1.7 13.6 ꢁ 3.1 12.7 ꢁ 3.1 11.0 ꢁ 5.7 13.2 ꢁ 1.6
8.0 ꢁ 1.1 19.7 ꢁ 1.6 18.1 ꢁ 5.0
8.2 ꢁ 1.1 14.2 ꢁ 1.2
83.2 ꢁ 1.4c 90.4 ꢁ 3.7c 55.7 ꢁ 9.4c 43.1 ꢁ 9.8 66.7 ꢁ 3.3c 58.2 ꢁ 5.3c 50.5 ꢁ 5.0c 61.6 ꢁ 5.1c 44.9 ꢁ 1.6 44.9 ꢁ 1.6
88.5 ꢁ 1.5c 93.9 ꢁ 0.6c 78.3 ꢁ 5.0c 57.8 ꢁ 2.2c 90.1 ꢁ 1.5c 54.5 ꢁ 1.5c 69.7 ꢁ 3.3c 89.4 ꢁ 3.5c 61.6 ꢁ 8.1c 46.6 ꢁ 7.0
12.4 ꢁ 4.6
4.9 ꢁ 0.4 14.4 ꢁ 3.8 13.6 ꢁ 3.6
4.5 ꢁ 0.5
9.7 ꢁ 1.9 44.7 ꢁ 5.3 36.4 ꢁ 3.0 16.7 ꢁ 1.6
4.5 ꢁ 0.5
89.5 ꢁ 0.6c 89.9 ꢁ 5.7c 67.3 ꢁ 5.4c 64.2 ꢁ 8.4c 69.7 ꢁ 7.3c 54.5 ꢁ 5.5c 58.0 ꢁ 8.6c 61.6 ꢁ 8.4c 50.4 ꢁ 1.9c 88.4 ꢁ 9.6c
91.6 ꢁ 4.7c 89.3 ꢁ 2.7c 65.0 ꢁ 8.0c 56.9 ꢁ 0.6c 61.2 ꢁ 5.2c 58.3 ꢁ 8.3c 51.7 ꢁ 8.3c 66.7 ꢁ 3.3c 51.7 ꢁ 8.3c 38.3 ꢁ 8.3
8f
8g
8h
GBP
53.9 ꢁ 1.4c 15.5 ꢁ 1.2
9.9 ꢁ 4.4
7.5 ꢁ 3.2 19.7 ꢁ 1.9 12.2 ꢁ 3.1
4.5 ꢁ 0.5 12.2 ꢁ 3.1 12.2 ꢁ 3.1
4.5 ꢁ 0.5
89.5 ꢁ 4.5c 65.6 ꢁ 10.6c 75.2 ꢁ 6.9c 87.3 ꢁ 6.3 42.2 ꢁ 5.4 19.8 ꢁ 4.0
6.7 ꢁ 2.7 13.7 ꢁ 9.3
9.9 ꢁ 5.5 24.7 ꢁ 5.6 13.0 ꢁ 6.1 13.0 ꢁ 6.1
18.3 ꢁ 6.3 22.3 ꢁ 8.5
12.5 ꢁ 2.9 20.9 ꢁ 2.2 13.9 ꢁ 1.4
6.3 ꢁ 1.3 13.8 ꢁ 1.6 13.8 ꢁ 1.6 12.8 ꢁ 1.2 15.1 ꢁ 1.5
3.5 ꢁ 0.4 28.5 ꢁ 2.8
71.2 ꢁ 0.2c 91.2 ꢁ 0.7c
88.8 ꢁ 0.7c 92.9 ꢁ 1.9c 94.2 ꢁ 2.1c 82.4 ꢁ 5.1c 35.4 ꢁ 3.0 59.4 ꢁ 1.7c 50.7 ꢁ 1.4c 43.1 ꢁ 2.5 56.1 ꢁ 2.3c 58.5 ꢁ 6.1c
a
Each value represents the % reversal of spontaneous pain (mean ꢁ SEM) of four rats.
b
c
Each value represents the % reversal of tactile allodynia (mean ꢁ SEM) of four rats.
Denotes significant value, in comparison to their respective vehicle control at p < 0.05 (One-way ANOVA, followed by post-hoc Dunnett’s test).
duration (30 min). Gabapentin reversed the tactile allodynia from
60 to 120 min duration (Table 2).
Overall, it appears that in the PSNL model, compounds that
showed promising results include 7a, 7e, 8a, 8b, 8d, and 8e effective
in all the four tests, 7b, 7d and 8g in three out of four tests and 7f
and 7g in one test. The active compounds that were found to be
effective in both neuropathic pain models include 7a, 7b, 7e, 8a, 8b,
8d and 8e.
Analysis of the structure and activity reveals that in general,
compounds with pivaloyl group (8ae8h) at the pyrazole nucleus
were more active than those with methyl function (7ae7h) and
significantly alleviated one or more nociceptive responses in CCI
and PSNL rats. Mono-substitution of phenyl ring at C-4 position
with chloro or bromo (7a, 7b, 8a and 8d) resulted in compounds
which exhibited pronounced activity against nociceptive parame-
ters in neuropathic animals. Substitution of C-4 of phenyl with
Cold allodynia produced by PSNL model was completely
reversed till 60 min duration by compounds 8a, 8b, 8d, and 8e as
observed with gabapentin. Administration of compounds 7a, 7b,
7d, 7e, and 8g to animals with an allodynic response to cold stimuli,
showed antiallodynic activity only at 30 min period (Table 3). All
other compounds were ineffective in this test. Mechanical hyper-
algesia produced by PSNL was completely reversed significantly at
all time-points by compounds 7e and 8b similar to gabapentin.
Compounds 7a, 7d, 8a and 8d which showed complete reversal
until 120 min in the CCI model, showed activity until 60 min only in
the PSNL model. The duration of action for compounds 7b, 8e and
8g was found to be 60 min (Table 3).
Table 3
Effects of compounds on cold allodynia and mechanical hyperalgesia in CCI and PSNL model.
Study group Cold allodynia
Mechanical hyperalgesia
% reversal (CCI)b
% reversal (CCI)a
% reversal (PSNL)a
% reversal (PSNL)b
30 min
60 min
6.3 ꢁ 1.9
120 min
30 min
60 min
2.5 ꢁ 0.3
120 min
30 min
60 min
120 min
30 min
60 min
2.8 ꢁ 0.3
120 min
Vehicle
7a
7b
7c
7d
7e
7f
7g
7h
8a
8b
8c
8d
8e
0.6 ꢁ 2.7
5.4 ꢁ 1.9
4.1 ꢁ 1.0
1.5 ꢁ 0.5 4.7 ꢁ 2.1
4.9 ꢁ 1.0
5.3 ꢁ 0.5
2.9 ꢁ 0.3
2.9 ꢁ 0.3
67.4 ꢁ 1.7c 38.1 ꢁ 1.0 32.6 ꢁ 7.2 59.7 ꢁ 1.1c 43.7 ꢁ 2.0 41.5 ꢁ 1.0 83.3 ꢁ 1.7c 66.7 ꢁ 0.8c 61.1 ꢁ 5.1c 50.0 ꢁ 1.0c 70.0 ꢁ 1.0d 40.0 ꢁ 1.0
63.4 ꢁ 1.6c 52.6 ꢁ 8.8c 13.4 ꢁ 4.6 61.3 ꢁ 6.2c 41.6 ꢁ 4.9 37.2 ꢁ 5.0 67.0 ꢁ 4.5c 73.2 ꢁ 1.8c 26.1 ꢁ 1.8 57.8 ꢁ 2.2c 69.4 ꢁ 1.9d 44.4 ꢁ 1.8
18.4 ꢁ 4.9 18.7 ꢁ 1.2 14.1 ꢁ 9.4 17.1 ꢁ 5.9 14.8 ꢁ 3.6 12.0 ꢁ 6.4 3.8 ꢁ 1.0 20.7 ꢁ 2.4 16.8 ꢁ 1.4 39.6 ꢁ 2.7 20.8 ꢁ 4.2 14.6 ꢁ 2.1
54.4 ꢁ 5.6c 37.3 ꢁ 3.7 41.8 ꢁ 0.8 60.4 ꢁ 4.2c 14.3 ꢁ 4.1 38.7 ꢁ 2.0 70.8 ꢁ 4.6c 57.5 ꢁ 1.2c 65.0 ꢁ 1.2c 63.3 ꢁ 1.3c 50.5 ꢁ 0.8d 18.7 ꢁ 1.3
72.7 ꢁ 7.4c 53.1 ꢁ 5.4c 40.6 ꢁ 2.2 58.7 ꢁ 5.8c 35.4 ꢁ 3.1 40.0 ꢁ 1.5 62.1 ꢁ 1.7c 58.9 ꢁ 1.9c 63.3 ꢁ 1.3c 62.1 ꢁ 1.7c 58.9 ꢁ 1.9d 63.3 ꢁ 1.3d
15.0 ꢁ 0.3 21.2 ꢁ 8.3
12.1 ꢁ 2.3 13.7 ꢁ 0.7
15.9 ꢁ 2.5 23.0 ꢁ 3.8
2.8 ꢁ 0.3 13.0 ꢁ 7.3 16.0 ꢁ 0.8
7.9 ꢁ 2.3 35.7 ꢁ 1.8 42.9 ꢁ 1.3 14.3 ꢁ 1.2 44.8 ꢁ 1.8 40.2 ꢁ 1.3 18.2 ꢁ 1.8
7.0 ꢁ 3.8 30.2 ꢁ 1.3 15.7 ꢁ 1.2 20.9 ꢁ 1.6 13.4 ꢁ 0.9 20.5 ꢁ 8.0 21.8 ꢁ 6.4 15.6 ꢁ 4.4 11.1 ꢁ 1.1 10.6 ꢁ 0.6
9.5 ꢁ 2.8 13.2 ꢁ 1.9
7.9 ꢁ 2.2 11.0 ꢁ 5.9 14.6 ꢁ 2.1 14.6 ꢁ 2.1
8.3 ꢁ 0.8 17.5 ꢁ 7.5
5.0 ꢁ 0.5 11.3 ꢁ 1.1
73.0 ꢁ 1.0c 65.0 ꢁ 0.5c 56.9 ꢁ 3.5c 55.4 ꢁ 7.4c 50.6 ꢁ 7.1c 48.2 ꢁ 0.1 81.9 ꢁ 6.9c 66.0 ꢁ 2.1c 65.3 ꢁ 9.7c 59.8 ꢁ 2.7c 53.6 ꢁ 3.6d 41.1 ꢁ 6.1
61.4 ꢁ 3.8c 61.2 ꢁ 7.2c 18.8 ꢁ 0.8 58.3 ꢁ 9.9c 56.0 ꢁ 0.9c 10.4 ꢁ 4.8 69.4 ꢁ 1.4c 84.4 ꢁ 4.4c 78.9 ꢁ 1.1c 95.5 ꢁ 1.1c 57.7 ꢁ 2.3d 50.0 ꢁ 1.0d
17.9 ꢁ 1.0 10.1 ꢁ 1.0 40.5 ꢁ 8.2 12.0 ꢁ 3.2 12.7 ꢁ 1.2
9.5 ꢁ 0.7 18.2 ꢁ 9.1 18.2 ꢁ 9.1
9.1 ꢁ 2.0 14.3 ꢁ 7.1
7.1 ꢁ 1.7 14.3 ꢁ 7.1
53.9 ꢁ 1.6c 50.4 ꢁ 1.8c 40.4 ꢁ 4.4 62.1 ꢁ 0.7c 52.7 ꢁ 4.4c 38.2 ꢁ 1.8 93.8 ꢁ 6.3c 80.0 ꢁ 5.8c 67.5 ꢁ 5.5c 62.3 ꢁ 1.2c 56.3 ꢁ 1.2d 46.3 ꢁ 1.6
64.3 ꢁ 1.6c 64.4 ꢁ 1.9c 40.0 ꢁ 7.1 64.3 ꢁ 1.6c 64.4 ꢁ 1.9c 40.0 ꢁ 1.2 70.8 ꢁ 4.2c 72.9 ꢁ 1.4c 11.2 ꢁ 2.4 60.0 ꢁ 6.1c 60.2 ꢁ 1.4d 43.9 ꢁ 1.0
8f
8g
8h
GBP
3.4 ꢁ 0.3
7.1 ꢁ 0.4 12.0 ꢁ 8.2 19.3 ꢁ 0.9 16.4 ꢁ 1.1 19.3 ꢁ 0.9 13.8 ꢁ 6.2 12.7 ꢁ 2.7
8.8 ꢁ 1.2 12.1 ꢁ 2.1 12.1 ꢁ 2.1
8.8 ꢁ 1.2
55.6 ꢁ 1.8c 35.0 ꢁ 2.1
9.3 ꢁ 2.6 52.7 ꢁ 9.3c 19.1 ꢁ 0.0 15.0 ꢁ 0.5 3.6 ꢁ 1.6 12.2 ꢁ 2.2 12.1 ꢁ 0.4 52.8 ꢁ 2.8c 59.5 ꢁ 3.5d 45.8 ꢁ 2.8
17.8 ꢁ 1.0 10.8 ꢁ 4.1 10.5 ꢁ 3.0 18.2 ꢁ 9.5 17.2 ꢁ 8.1 10.6 ꢁ 7.1 20.0 ꢁ 1.3 11.7 ꢁ 5.0
2.2 ꢁ 0.9 11.1 ꢁ 1.0 16.7 ꢁ 5.6 11.1 ꢁ 1.0
58.0 ꢁ 6.4c 42.9 ꢁ 7.4 35.6 ꢁ 1.7 54.1 ꢁ 4.3c 58.2 ꢁ 5.1c 32.1 ꢁ 1.4 92.9 ꢁ 7.1c 78.6 ꢁ 7.1c 75.7 ꢁ 3.1c 86.2 ꢁ 2.1c 91.3 ꢁ 5.2d 55.8 ꢁ 3.8d
a
Each value represents the % reversal of cold allodynia (mean ꢁ SEM) of four rats.
b
c
Each value represents the % reversal of mechanical hyperalgesia (mean ꢁ SEM) of four rats.
Denotes significant value, in comparison to their respective vehicle control at p < 0.05 (One-way ANOVA, followed by post-hoc Dunnett’s test).
denotes significant value, in comparison to their respective vehicle control at p < 0.05 (One-way ANOVA, followed by post-hoc Dunnett’s test).
d

P. Yogeeswari et al. / European Journal of Medicinal Chemistry 66 (2013) 211e220
215
trifluoromethyl (7c and 8c) proved to be detrimental for the ac-
tivity. Compound 7c was also neurotoxic. Hence trifluoromethyl is
not favourable for bioactivity. Based on our previous experience
with dimethyl phenyl analogues [28,31e36] we attempted to
include these substitutions in the current scaffold. The order of
bioactivity in neuropathic pain models, when the phenyl ring was
disubstituted with methyl functions was 2,4-dimethyl (7d and
8d) > 2,5-dimethyl (7e and 8e) > 2,6-dimethyl (7f and 8f). When C-
2 of phenyl ring was substituted with fluoro group in combination
with C-4 fluoro (7g and 8g) or C-5 methyl (7h and 8h), it was found
to be detrimental to bioactivity.
protection suggesting that these compounds act by GABA media-
tion. Radioligand binding assays were performed to assess the
potential of the compounds to modulate cannabinoid (CB) re-
ceptors CB1 in brain and CB2 in spleen of rats. Only compound 8a
showed selective modulation in CB1 assay with an IC50 of 49.6 nM
with no effect on CB2 receptor. The compounds (8a, 8b, and 8d)
were also screened for their inhibitory potential on the cysteine
protease cathepsin S implicated in neuropathic pain using fluo-
rescent peptide substrates and recombinant human cathepsins S
assay kit (SensoLyteÒ 440 Cathepsin S Assay Kit). The compounds
did not show significant inhibition at 50 mM concentration.
Based on all these structure-activity correlations and neuro-
toxicity data, three compounds (8a, 8b and 8d) were chosen for
further quantification and mechanistic studies. In the CCI model,
compound 8b reversed spontaneous pain and tactile allodynia with
an ED50 value of 10.4 mg/kg and 21.7 mg/kg with time to peak effect
(TPE) at 60 min and 30 min respectively, emerging as the most
potent compound in CCI model (Table 4). In the PSNL model,
compound 8d reversed spontaneous pain with an ED50 value of
7.4 mg/kg, at 30 min. The significant suppression of flinching
observed in the second phase of formalin assay (Table 1) by com-
pounds 8a, 8b and 8d suggested the mediation of anti-
inflammatory pathways. The probable role of the selective com-
pounds (8a, 8b and 8d) in inhibition of pro-inflammatory mediator
4. Conclusion
Utilizing pharmacophore hybrid approach, a series of structur-
ally novel tetrahydropyrido-pyrazoles were synthesized and eval-
uated for acute antinociceptive, antiallodynic and antihyperalgesic
potential. The most promising compounds (8a, 8b and 8d) were
found to inhibit TNF-apha, a pro-inflammatory cytokine involved in
the modulation of inflammatory component of neuropathic pain.
The neuroprotection exhibited by these compounds is also associ-
ated with their free radical scavenging abilities and subsequent
attenuation of oxidative and nitrosative stress. Also, the CB1 re-
ceptor affinity exhibited by the compound 8a in nanomolar range
(49.6 nM) and enhancement of peripheral GABAergic neurotrans-
mission by the compounds 8a, 8b and 8d demonstrate the ability of
compounds to modulate several systems. Above findings provide
support to their multifunctional profile and advocate the devel-
opment of tetrahydropyrido-pyrazole scaffold as leads for struc-
turally novel compounds in the treatment of neuropathic pain.
tumour necrosis factor alpha (TNF-
lipopolysaccharide-induced TNF- release in mice and the results
are presented in Table 4. TNF- levels were found to be inhibited by
a) was investigated using
a
a
the tested compound in the range of 59e86% as evidenced with the
pyrrolopyridines in the past [37].
Following nerve injury, subsequent generation of free radicals
occurs and leads to oxidative and nitrosative stress exaggerating
pain states. The putative role of nitric oxide (NO) in the patho-
physiology of chronic nerve ligation as evident by significant in-
crease in nitrite and nitrate levels in both brain and sciatic nerve led
us to estimate the levels of nitrite, metabolite of NO in brain and
sciatic nerve of CCI rats. There was no significant reduction of nitrite
in the brain of CCI rats as compared to vehicle treated animals with
compounds 8a, 8b and 8d. However, a significant reduction was
observed in the sciatic nerve of the CCI animals as compared to
vehicle treated group with the compounds 8b and 8d indicating
inhibition of local NO (Table 5). The compounds also exhibited
free radical scavenging activities in the DPPH (2-diphenyl-1-
picrylhydrazyl) assay thereby providing neuroprotection by the
reduction of oxidative stress (Table 5). Further mechanistic studies
were performed to assess the potential of these compounds on well
established and new targets of neuropathic pain like GABA and
cannabinoid receptors (CB1 and CB2) and Cathepsin S based on the
previous reports on tetrahydropyrido-pyrazoles (Fig. 1) [10,15,
38,39] and aryl semicarbazone scaffolds [40].
5. Experimental section
5.1. Chemistry
Reagents and all solvents were analytically pure and were used
without further purification. All the experiments were monitored
by analytical thin layer chromatography (TLC) performed on silica
gel GF254 pre-coated plates. After elution, plate was visualized
under UV illumination at 254 nm for UV active materials. Melting
points were measured in open capillary tubes on a Buchi 530
melting point apparatus and are uncorrected. Proton nuclear
magnetic resonance (1H NMR) spectra were recorded for the
compounds on Bruker Avance (300 MHz) instruments, respectively.
Chemical shifts are reported in parts per million (ppm) using tet-
ramethyl silane (TMS) as an internal standard. Multiplicities of
NMR signals are designated as s (singlet), d (doublet), t (triplet), q
(quartet), br (broad), m (multiplet, for unresolved lines), etc. 13C
NMR spectra were recorded on 75 MHz spectrometer. Elemental
analyses (C, H and N) were undertaken with a PerkineElmer model
240C analyzer and all analyses were consistent with theoretical
In the subcutaneous picrotoxin-induced seizure (scPIC)
threshold test, all the compounds tested (8a, 8b and 8d) showed
Table 4
Quantification studies and percent inhibition of TNF-a in LPS stimulated mice.
Treatment
ED50 values (mg/kg) in CCI modela
ED50 values (mg/kg) in PSNL modela
(TPE in min)a
% inhibition of
TNF-
productionc
a
(TPE in min)b
SP
TA
CA
MH
SP
TA
CA
MH
8a
8b
8d
27.3 (30)
10.4 (60)
15.5 (60)
23.8 (30)
21.7 (30)
23.4 (60)
41.4 (30)
37.4 (60)
29.8 (60)
37.3 (30)
38.3 (60)
25.7 (60)
29.0 (30)
8.1 (30)
7.4 (30)
37.5 (60)
24.9 (30)
36.1 (120)
68.0 (30)
65.8 (60)
44.4 (60)
29.0 (30)
23.6 (30)
31.8 (30)
86.4 ꢁ 0.7d
77.6 ꢁ 0.4d
58.9 ꢁ 1.7d
a
Each value represents median effective dose (ED-50) in spontaneous pain (SP), mechanical hyperalgesia (MH), cold allodynia (CA) and tactile allodynia (TA).
TPE represents time of peak effect in minutes.
Percent inhibition of soluble TNF-
b
c
a
in LPS stimulated mice as compared to vehicle-treated control.
d
Represents the significance at p < 0.05, compared to vehicle control (One way ANOVA followed by Dunnett’s test, n ¼ 4).

216
P. Yogeeswari et al. / European Journal of Medicinal Chemistry 66 (2013) 211e220
Table 5
Mechanistic studies of compounds 8a, 8b and 8d.
Treatment
% inhibition of nitritea
GABAergic
mechanism
(scPIC)b
CB receptor binding
Cathepsin Sd
% inhibition
DPPH scavenging activitye
c
(IC50
)
Brain
Sciatic nerve
0.5 h
4 h
CB-1
CB-2
IC50
81
8a
8b
8d
16.4 ꢁ 1.5
15.3 ꢁ 0.4
12.5 ꢁ 0.8
47.8 ꢁ 1.3f
68.7 ꢁ 1.5f
65.4 ꢁ 2.1f
100
100
300
100
100
e
49.6 nM
>1
>1
>1
m
m
m
M
M
M
39.86 ꢁ 2.1
34.13 ꢁ 1.2
12.14 ꢁ 1.4
m
M
>1
>1
m
m
M
M
102
134
m
m
M
M
a
Percent inhibition of nitric oxide in brain and sciatic nerve as compared to vehicle treated control.
Doses of 30, 100 and 300 mg/kg (i.p.) were administered. The figures in the table indicate the minimum dose whereby bioactivity was demonstrated in half or more of the
b
mice (n ¼ 3). The animals were examined 0.5 h and 4 h. The line (e) indicates an absence of anticonvulsant activity at the maximum dose tested.
c
Equilibrium-competition binding of compounds vs. [3H] CP55940 using either rat brain (CB1) or rat spleen homogenates (CB2). The data shown are average of triplicates.
Inhibition of recombinant human cathepsin S in a fluorescence assay at 50 mM, employing Z-Val-Val-Arg-AMC as synthetic substrates. Data represent means of two
d
experiments performed in duplicate.
e
DPPH radical scavenging activity of test compounds.
Represents the significance at p < 0.05, compared to vehicle control (One way ANOVA followed by Dunnett’s test, n ¼ 4).
f
values (within ꢁ0.4%) unless indicated. High-resolution mass
spectra were obtained by using Perkin Elmer ESI-QTOF mass
spectrometry. The homogeneity of the compounds was monitored
by ascending thin layer chromatography (TLC) on silicagel-G
(Merck) coated aluminium plates, visualized by iodine vapour
and UV light. Developing solvents were chloroformemethanol
(9:1). Appropriate names for all the new compounds were given
with the help of ChemBioOffice 2010.
N2. The reaction solution was stirred for 3 h followed by addition of
water and stirring was further continued for 1 h. The organic layer
was washed with 0.1 N HCl and brine, dried over Na2SO4, filtered
and concentrated.
5.1.3. General method for the synthesis of tert-butyl 3-alkyl-1-(substi
tuted phenyl carbamoyl)-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-
5(4H)-carboxylate (3aeh,4aeh)
Equimolar quantity of appropriate N-substituted phenyl
hydrazinecarboxamide (aeh) and K2CO3 (1.3 equiv) were added to
a solution of 2 in ethanol. The reaction mixture was refluxed
overnight, and then concentrated. The residue was diluted with
EtOAc, then washed with 1.0 N HCl and brine. The organic
layer was dried over Na2SO4, filtered and concentrated to give a
crude product which was purified on silica gel column to give 3ae
h, 4aeh.
5.1.1. Synthesis of tert-butyl 4-morpholino-5,6-dihydropyridine-
1(2H)-carboxylate (1)
In a round-bottom flask equipped with a DeaneStark trap, a
reflux condenser, and an internal thermocouple, morpholine
(1.05 equiv), and p-toluenesulfonic acid (0.5% equiv) were added to
N-Boc-piperidone (1.0 equiv), in toluene. The reaction solution
was refluxed overnight under N2 atmosphere. The solvent was
evaporated in vacuo to afford 4 tert-butyl 4-morpholino-5,6-
dihydropyridine-1(2H)-carboxylate (1), which was used directly
for the next step. (Colourless oil.)
5.1.4. General method for the deprotection of amino group of 3-alkyl-
N-substitued phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-
1 carboxamide (5aeh, 6aeh)
5.1.2. General method for the synthesis of tert-butyl 3-alkanoyl-4-
oxopiperidine-1-carboxylate (2)
The enamine prepared above was taken in dichloromethane. To
this was added Et3N (1.5 equiv), followed by slow addition of alkyl
acid chlorides (acetyl chloride or pivaloyl chloride for) at 0 ꢀC under
The appropriate compound (3aeh and 4aeh) was dissolved in
10 mL dichloromethane followed by slow addition of 10 mL of
trifluoroacetic acid. The mixture was stirred overnight at room
temperature, then concentrated, partitioned between dichloro-
methane and saturated aqueous NaHCO3 solution. The organic
Fig. 1. Representative pyrazole-based leads for neuropathic pain.

P. Yogeeswari et al. / European Journal of Medicinal Chemistry 66 (2013) 211e220
217
layer was dried (Na2SO4), filtered and concentrated in vacuo
The residue was purified by column chromatography on silica
gel eluting with dichloromethane/MeOH to yield the desired
compound.
113.3, 119.8, 121.1, 122.4, 134.1, 142.6, 144.2, 150.8, 155.4. MS (ESI)
317.17 (M þ H)þ. Anal. (C17H21FN4O)C, H, N.
5.1.5.7. 5-Ethyl-3-methyl-N-(3-(trifluoromethyl)phenyl)-4,5,6,7-
tetrahydro-1H-pyrazolo[4,3-c]pyridine-1-carboxamide (7g). Yield ¼
5.1.5. General method for the synthesis of 5-ethyl-3-alkyl-N-substituted
phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-1-carboxamide
(7aeh, 8aeh)
The compound (5aeh/6aeh) was taken in acetonitrile. To it was
added K2CO3 (1.5 equiv) followed by slow addition of ethyl iodide
(1.5 equiv) in ice cold condition. After stirring reaction at room
temperature for 3e4 h, the reaction mixture was filtered, dried in
vacuo and column purified.
62%; mp ¼ 209 ꢀC; 1H NMR (CDCl3)
1.33 (t, J ¼ 7.6 Hz, 3H), 2.53 (s,
d
3H), 2.92e3.22 (m, 4H), 3.82 (t, J ¼ 7.6 Hz, 2H), 4.91 (s, 2H), 7.38e7.42
(m, 2H), 7.69 (d, J ¼ 7.6 Hz, 1H), 8.12 (s, 1H), 8.74 (s, 1H D2O
exchangeable); 13C NMR
d 12.4,13.5, 24.8, 49.2, 51.4, 53.9, 117.2, 120.3,
124.1, 124.5,125.1, 129.1, 130.1, 136.2, 142.1, 144.7, 151.1. MS (ESI) 353.1
(M þ H)þ. Anal. (C17H21FN4O)C, H, N.
5.1.5.8. N-(4-Chlorophenyl)-5-ethyl-3-methyl-4,5,6,7-tetrahydro-
1H-pyrazolo[4,3-c]pyridine-1-carboxamide (7h). Yield ¼ 68%; mp ¼
5.1.5.1. N-(4-Bromophenyl)-5-ethyl-3-methyl-4,5,6,7-tetrahydro-1H-
187 ꢀC; 1H NMR (CDCl3)
d
1.33 (t, J ¼ 7.6 Hz, 3H), 2.52 (s, 3H), 2.92e
pyrazolo[4,3-c]pyridine-1-carboxamide (7a). Yield ¼ 78%; mp ¼
3.22 (m, 4H), 3.81 (t, J ¼ 7.6 Hz, 2H), 4.91 (s, 2H), 7.44 (dd, J ¼ 7.8Hz,
232 ꢀC; 1H NMR (CDCl3)
d
1.34 (t, J ¼ 7.2 Hz, 3H), 2.52 (s, 3H), 2.91e
2H), 7.61 (dd, J ¼ 7.8 Hz, 2H), 8.67 (s, 1H D2O exchangeable); 13C
3.21 (m, 4H), 3.81 (t, J ¼ 7.6 Hz, 2H), 4.91 (s, 2H), 7.56 (dd, J ¼ 7.8 Hz,
NMR d 12.1, 13.3, 24.9, 49.1, 51.1, 53.2, 117.4, 126.1, 129.6, 133.5,
2H), 7.81 (dd, J ¼ 7.8 Hz, 2H), 8.52 (s, 1H, D2O exchangeable); 13C
134.2, 142.3, 144.7, 150.9. MS (ESI) 320.1 (M
(C16H19ClN4O)C, H, N.
þ
H)þ. Anal.
NMR
d 12.4, 13.3, 25.1, 49.8, 52.4, 54.9, 118.2, 122.1, 123.3, 134.3,
135.1, 142.8, 145.1, 152.4. MS (ESI) 364.0 (M
(C16H19BrN4O)C, H, N.
þ
H)þ. Anal.
5.1.5.9. N-(4-Bromophenyl)-3-tert-butyl-5-ethyl-4,5,6,7-tetrahydro-
1H-pyrazolo[4,3-c]pyridine-1-carboxamide (8a). Yield
¼
62%;
5.1.5.2. N-(2,6-Dimethylphenyl)-5-ethyl-3-methyl-4,5,6,7-tetrahydro-
mp ¼ 210 ꢀC; 1H NMR (CDCl3)
d 1.17 (m, 9H), 1.25e1.21 (m,
1H-pyrazolo[4,3-c]pyridine-1-carboxamide (7b). Yield ¼ 66%; mp ¼
J ¼ 7.2 Hz, 3H), 3.61e3.41 (m, 9H), 7.12 (dd, J ¼ 7.8 Hz, 2H), 7.58 (dd,
241 ꢀC; 1H NMR (CDCl3)
d
1.33 (t, J ¼ 7.2 Hz, 3H), 2.53 (s, 3H), 2.65 (s,
J ¼ 7.8 Hz, 2H), 8.52 (s, 1H, D2O exchangeable); 13C NMR
d 131.6,
6H), 2.91e3.22 (m, 4H), 3.82 (t, J ¼ 7.6 Hz, 2H), 4.92 (s, 2H), 7.08 (t,
131.4, 131.0, 129.4, 129.3, 125.5, 121.8, 120.0, 66.2, 65.9, 45.2,
44.1, 37.9, 27.9, 27.0. MS (ESI) 406.1 (M þ H)þ. Anal. (C16H19BrN4O)
C, H, N.
J ¼ 7.6 Hz, 1H), 7.22 (d, J ¼ 7.8 Hz, 2H), 8.41 (s, 1H, D2O exchange-
able); 13C NMR
d 12.3, 13.4, 17.7, 25.2, 49.1, 52.1, 54.6, 117.8, 126.5,
127.3, 130.1, 134.9, 142.4, 145.2, 151.3. MS (ESI) 313.2 (M þ H)þ. Anal.
(C18H24N4O)C, H, N.
5.1.5.10. 3-tert-Butyl-N-(2,6-dimethylphenyl)-5-ethyl-4,5,6,7-
tetrahydro-1H-pyrazolo[4,3-c]pyridine-1-carboxamide (8b). Yield ¼
5.1.5.3. N-(2,5-Dimethylphenyl)-5-ethyl-3-methyl-4,5,6,7-tetrahydro-
65%; mp ¼ 214 ꢀC; 1H NMR (CDCl3)
d
1.33 (t, J ¼ 7.2 Hz, 3H), 1.87 (s,
1H-pyrazolo[4,3-c]pyridine-1-carboxamide (7c). Yield ¼ 64%; mp ¼
6H), 2.53 (s, 3H), 2.65 (s, 6H), 2.91e3.22 (m, 4H), 3.81 (t, J ¼ 7.6 Hz,
2H), 4.93 (s, 2H), 7.08 (t, J ¼ 7.6 Hz,1H), 7.22 (d, J ¼ 7.8 Hz, 2H), 8.41 (s,
228 ꢀC; 1H NMR (CDCl3)
d
1.34 (t, J ¼ 7.2 Hz, 3H), 2.52 (s, 3H), 2.67 (s,
6H), 2.92e3.22 (m, 4H), 3.81 (t, J ¼ 7.6 Hz, 2H), 4.92 (s, 2H), 7.12 (d,
J ¼ 7.8 Hz, 1H), 7.43 (d, J ¼ 7.6 Hz, 1H), 7.51 (s, 1H), 8.41 (s, 1H, D2O
1H, D2O exchangeable); 13C NMR
d 12.3, 13.4, 17.7, 25.2, 49.1, 52.1,
54.6,117.8,126.5, 127.3,130.1, 134.9, 142.4,145.2, 151.3. MS (ESI) 355.2
exchangeable); 13C NMR
d
12.1, 13.1, 16.7, 21.6, 25.1, 49.1, 51.9, 54.1,
(M þ H)þ. Anal. (C18H24N4O)C, H, N.
112.3, 118.1, 125.1, 128.1, 129.0, 134.2, 135.3, 142.1, 145.5, 151.2. MS
(ESI) 313.2 (M þ H)þ. Anal. (C18H24N4O)C, H, N.
5.1.5.11. 3-tert-Butyl-N-(2,5-dimethylphenyl)-5-ethyl-4,5,6,7-
tetrahydro-1H-pyrazolo[4,3-c]pyridine-1-carboxamide (8c). Yield ¼
5.1.5.4. N-(2,4-Dimethylphenyl)-5-ethyl-3-methyl-4,5,6,7-tetrahydro-
68%; mp ¼ 242 ꢀC; 1H NMR (CDCl3)
d
1.34 (t, J ¼ 7.2 Hz, 3H), 1.89
1H-pyrazolo[4,3-c]pyridine-1-carboxamide (7d). Yield ¼ 71%; mp ¼
(s, 6H), 2.53 (s, 3H), 2.71 (s, 6H), 2.92e3.23 (m, 4H), 3.82 (t,
J ¼ 7.6 Hz, 2H), 4.91 (s, 2H), 7.12 (d, J ¼ 7.8 Hz, 1H), 7.43 (d, J ¼ 7.6 Hz,
213 ꢀC; 1H NMR (CDCl3)
d
1.33 (t, J ¼ 7.2 Hz, 3H), 2.53 (s, 3H), 2.67 (s,
6H), 2.92e3.21 (m, 4H), 3.81 (t, J ¼ 7.6 Hz, 2H), 4.89 (s, 2H), 7.11 (s,
1H), 7.51 (s, 1H), 8.41 (s, 1H, D2O exchangeable); 13C NMR
d 12.1, 13.1,
1H), 7.24 (d, J ¼ 7.4 Hz, 1H), 7.36 (d, J ¼ 7.6 Hz, 1H), 8.52 (s, 1H, D2O
16.7, 21.6, 25.1, 49.1, 51.9, 54.1, 112.3, 118.1, 125.1, 128.1, 129.0, 134.2,
135.3, 142.1, 145.5, 151.2. MS (ESI) 355.2 (M þ H)þ. Anal. (C18H24N4O)
C, H, N.
exchangeable); 13C NMR
d 12.3, 13.5, 17.2, 21.4, 24.6, 49.2, 51.8, 53.8,
114.1, 117.2, 126.2, 131.1, 131.9, 134.4, 142.2, 143.4, 144.4, 150.9. MS
(ESI) 313.2 (M þ H)þ. Anal. (C18H24N4O)C, H, N.
5.1.5.12. 3-tert-Butyl-N-(2,4-dimethylphenyl)-5-ethyl-4,5,6,7-
5.1.5.5. N-(2,4-Difluorophenyl)-5-ethyl-3-methyl-4,5,6,7-tetrahydro-
1H-pyrazolo[4,3-c]pyridine-1-carboxamide (7e). Yield ¼ 68%; mp ¼
tetrahydro-1H-pyrazolo[4,3-c]pyridine-1-carboxamide (8d). Yield ¼
66%; mp ¼ 237 ꢀC; 1H NMR (CDCl3)
d
1.33 (t, J ¼ 7.2 Hz, 3H), 1.88
231 ꢀC; 1H NMR (CDCl3)
d
1.34 (t, J ¼ 7.4 Hz, 3H), 2.52 (s, 3H), 2.91e
(s, 6H), 2.52 (s, 3H), 2.74 (s, 6H), 2.92e3.22 (m, 4H), 3.81 (t,
J ¼ 7.6 Hz, 2H), 4.91 (s, 2H), 7.11 (s,1H), 7.24 (d, J ¼ 7.4 Hz,1H), 7.36 (d,
3.23 (m, 4H), 3.81 (t, J ¼ 7.6 Hz, 2H), 4.91 (s, 2H), 6.83e7.06 (m, 2H),
7.17e7.21 (m, 1H), 8.91 (s, 1H, D2O exchangeable); 13C NMR
J ¼ 7.6 Hz, 1H), 8.52 (s, 1H, D2O exchangeable); 13C NMR
d 12.3, 13.5,
d
12.1,13.3, 24.1, 49.6, 51.2, 53.9,104.2,112.1,114.3,116.9,125.1,142.1,
17.2, 21.4, 24.6, 49.2, 51.8, 53.8, 114.1, 117.2, 126.2, 131.1, 131.9, 134.4,
142.2,143.4,144.4,150.9. MS (ESI) 355.2 (M þ H)þ. Anal. (C18H24N4O)
C, H, N.
144.6, 151.2, 160.4, 164.1. MS (ESI) 321.1 (M
(C16H18F2N4O)C, H, N.
þ
H)þ. Anal.
5.1.5.6. 5-Ethyl-N-(2-fluoro-5-methylphenyl)-3-methyl-4,5,6,7-
5.1.5.13. 3-tert-Butyl-N-(2,4-difluorophenyl)-5-ethyl-4,5,6,7-
tetrahydro-1H-pyrazolo[4,3-c]pyridine-1-carboxamide (7f). Yield
¼
tetrahydro-1H-pyrazolo[4,3-c]pyridine-1-carboxamide (8e). Yield
¼
65%; mp ¼ 174 ꢀC; 1H NMR (CDCl3)
d
1.33 (t, J ¼ 7.6 Hz, 3H), 2.52 (s,
65%; mp ¼ 251 ꢀC; 1H NMR (CDCl3)
d
1.33 (t, J ¼ 7.4 Hz, 3H), 1.89 (s,
3H), 2.91e3.21 (m, 4H), 3.82 (t, J ¼ 7.6 Hz, 2H), 4.92 (s, 2H), 7.01 (d,
J ¼ 7.8, 1H), 7.24e7.32 (m, 1H), 7.61 (s, 1H), 8.64 (s, 1H D2O
6H), 2.53 (s, 3H), 2.91e3.32 (m, 4H), 3.83 (t, J ¼ 7.6 Hz, 2H), 4.92 (s,
2H), 6.83e7.06 (m, 2H), 7.17e7.21 (m, 1H), 8.91 (s, 1H, D2O
exchangeable); 13C NMR
d
12.2, 13.1, 24.1, 25.1, 49.4, 51.6, 54.1, 112.2,
exchangeable); 13C NMR
d 12.1, 13.3, 24.1, 49.6, 51.2, 53.9, 104.2, 112.1,

218
P. Yogeeswari et al. / European Journal of Medicinal Chemistry 66 (2013) 211e220
114.3, 116.9, 125.1, 142.1, 144.6, 151.2, 160.4, 164.1. MS (ESI) 363.2
the early phase (0e5 min) and the late phase (10e30 min). Time
spent in paw licking and biting was monitored in each 5 min and
calculated for both the phases. Test compounds were administered
30 min before the experiment [43].
(M þ H)þ. Anal. (C16H18F2N4O)C, H, N.
5.1.5.14. 3-tert-Butyl-5-ethyl-N-(2-fluoro-5-methylphenyl)-4,5,6,7-
tetrahydro-1H-pyrazolo [4,3-c]pyridine-1-carboxamide (8f). Yield ¼
71%; mp ¼ 235 ꢀC; 1H NMR (CDCl3)
d
1.33 (t, J ¼ 7.6 Hz, 3H), 1.88 (s,
5.2.4. Unilateral mononeuropathy e chronic constriction nerve
injury model
6H), 2.52 (s, 3H), 2.91e3.33 (m, 4H), 3.82 (t, J ¼ 7.6 Hz, 2H), 4.91 (s,
2H), 7.01 (d, J ¼ 7.8, 1H), 7.24e7.32 (m, 1H), 7.61 (s, 1H), 8.64 (s, 1H,
Unilateral mononeuropathy was produced in rats using the CCI
model performed essentially as described by Bennett & Xiao [44].
The rats were anesthetized with an intraperitoneal dose of keta-
mine (55 mg/kg) and xylazine (5 mg/kg) with additional doses of
the anaesthetic given as needed. Under aseptic conditions, a 3-cm
incision was made on the lateral aspect of the left hindlimb at the
mid-thigh level. The left paraspinal muscles were then separated
from the spinous processes and the common left sciatic nerve was
exposed just above the trifurcation point. Four loose ligatures were
made with a 4-0 braided silk suture around the sciatic nerve with
about 1-mm spacing. The wound was then closed by suturing the
muscle using chromic catgut with a continuous suture pattern.
Finally, the skin was closed using silk thread with horizontal-
mattress suture pattern.
D2O exchangeable); 13C NMR
d 12.2, 13.1, 24.1, 25.1, 49.4, 51.6, 54.1,
112.2, 113.3, 119.8, 121.1, 122.4, 134.1, 142.6, 144.2, 150.8, 155.4. MS
(ESI) 359.2 (M þ H)þ. Anal. (C17H21FN4O)C, H, N.
5.1.5.15. 3-tert-Butyl-5-ethyl-N-(3-(trifluoromethyl)phenyl)-4,5,6,7-
tetrahydro-1H-pyrazolo [4,3-c]pyridine-1-carboxamide (8g). Yield ¼
69%; mp ¼ 192 ꢀC; 1H NMR (CDCl3)
d
1.32 (t, J ¼ 7.6 Hz, 3H),
1.87 (s, 6H), 2.52 (s, 3H), 2.91e3.42 (m, 4H), 3.81 (t, J ¼ 7.6 Hz, 2H),
4.90 (s, 2H), 7.38e7.42 (m, 2H), 7.69 (d, J ¼ 7.6 Hz, 1H), 8.12 (s,
1H), 8.74 (s, 1H, D2O exchangeable); 13C NMR
d 12.4, 13.5,
24.8, 49.2, 51.4, 53.9, 117.2, 120.3,124.1, 124.5,125.1, 129.1, 130.1, 136.2,
142.1, 144.7, 151.1. MS (ESI) 395.2 (M þ H)þ. Anal. (C17H21FN4O)C, H, N.
5.1.5.16. 3-tert-Butyl-N-(4-chlorophenyl)-5-ethyl-4,5,6,7-tetrahydro-
1H-pyrazolo[4,3-c]pyridine-1-carboxamide (8h). Yield
¼
68%;
5.2.5. Induction of peripheral mononeuropathy-Partial sciatic nerve
ligation model
mp ¼ 190 ꢀC; 1H NMR (CDCl3)
1.33 (t, J ¼ 7.6 Hz, 3H), 1.89 (s, 6H),
d
2.52 (s, 3H), 2.86e3.39 (m, 4H), 3.83 (t, J ¼ 7.6 Hz, 2H), 4.91 (s, 2H),
7.44 (dd, J ¼ 7.8 Hz, 2H), 7.61 (dd, J ¼ 7.8 Hz, 2H), 8.67 (s, 1H D2O
As described by Seltzer [45], in anaesthetized rats, left sciatic
nerve was exposed at mid thigh level through small incision,
cleared of adhering muscle tissue, and one-half of the nerve
thickness was tightly ligated using 7.0 silk suture. The wound was
closed and dusted with neomycin powder. The animals were then
transferred to their home-cages and left for recovery.
exchangeable); 13C NMR
d 12.1, 13.3, 24.9, 49.1, 51.1, 53.2, 117.4,
126.1, 129.6, 133.5, 134.2, 142.3, 144.7, 150.9. MS (ESI) 362.1
(M þ H)þ. Anal. (C16H19ClN4O)C, H, N.
5.2. Pharmacological methods
5.2.6. Sensory testing using nociceptive assays
Swiss albino mice (either sex) with weights ranging from 20 to
25 g were used for the assessment of neurotoxicity, acetic acid
induced writhing model, formalin induced flinching model and LPS
Compounds (100 mg/kg, i.p.) were administered at t ¼ 0, in 30%
v/v PEG 400. The control group of rats received only the vehicle.
Gabapentin (100 mg/kg, i.p.) was used as positive control. Paw
withdrawal duration (PWD) was observed in spontaneous pain and
cold allodynia and paw withdrawal threshold (PWT) was assessed
in tactile allodynia and mechanical hyperalgesia. Percentage
reversal in spontaneous pain, allodynia or hyperalgesia was calcu-
lated for each animal as defined below [46]
induced TNF-a release. Wistar rats of either sex (200e250 g) were
used for the inflammatory and neuropathic pain models. All ex-
periments were approved by the Institutional Animal Ethics Com-
mittee. Animals were housed six (mice) and four (rats) per cage at
constant temperature under a 12 h light/dark cycle (lights on at
7:00 AM), with food and water ad libitum. All of the animals were
acclimatized to the housing conditions for a period of one week
before the start of experiments.
ðpost dose value ꢂ pre dose valueÞ
% Reversal ¼
ꢃ 100
The various pharmacological tests performed are as follows,
ðcontralateral paw value ꢂ pre dose valueÞ
5.2.1. Motor impairment
Minimal motor impairment was measured in mice by the
rotarod test. The mice were trained to stay on an accelerating
rotarod that rotates at 10 revolutions per minute. The rod diameter
was 3.2 cm. Neurotoxicity was indicated by the inability of the
animal to maintain equilibrium on the rod for at least 1 min in each
of the three trials [41].
5.2.6.1. Spontaneous pain. Spontaneous pain was assessed for a
total time period of 5 min as described previously by Choi et al. The
operated rat was placed inside an observation cage that was kept
5 cm from the ground level. An initial acclimatization period of
10 min was given to each of the rats. The test consisted of noting the
cumulative duration for which the rat holds its ipsilateral paw off
the floor. The paw lifts associated with locomotion or body repo-
sitioning was not counted [47].
5.2.2. Acetic acid induced writhing
Writhing was induced in a group of mice by an intraperitoneal
injection of 0.1 mL of 2.0% v/v acetic acid. Test group mice received
acetic acid half an hour after the administration of test compounds.
The number of writhings occurring for a period of 30 min was recor-
ded. For scoring purposes, a writhe was indicated by stretching of the
abdomen with simultaneous stretching of at least one hind limb. The
percentage inhibition of the writhing response was calculated [42].
5.2.6.2. Tactile allodynia. Paw withdrawal in response to mechan-
ical stimuli was measured using von Frey filaments (UGO Basile,
Italy). Each rat was placed on a metallic mesh floor covered with a
plastic box. A set of von Frey monofilaments (0.4e15 g), with in-
tensities of mechanical stimulation increasing in graded manner
with successively greater diameter filaments, were applied to the
plantar surface of the hind paw five times at intervals of 1e2 s [48].
The weakest force (g) inducing withdrawal of the stimulated paw at
least three times was taken as the paw withdrawal threshold with
cut off value at 15 g.
5.2.3. Formalin induced flinching
The test involved intra plantar injection of 25
mL of 1.0% formalin
into the hind paw of mice, which resulted in flinches in the paw in

P. Yogeeswari et al. / European Journal of Medicinal Chemistry 66 (2013) 211e220
219
5.2.6.3. Cold allodynia. The operated rat was placed inside an
observation cage that was kept 5 cm from the ground level and was
allowed to acclimatize for 10 min or until exploratory behaviour
IC50 values. All tests and analyses were run in triplicate and aver-
aged [54].
Percentage scavenging was calculated according to the
following equation.
ceased. Few drops (100e200 mL) of freshly dispensed acetone were
fAbsorbanceðDPPHÞ ꢂ AbsorbanceðDPPH þ compoundÞg
AbsorbanceðDPPHÞ
% Scavenging ¼
ꢃ 100
squirted as a fine mist onto the midplantar region of the affected
paw. A cold allodynic response was assessed by noting down the
duration of paw-withdrawal response. For each measurement, the
paw was sampled three times and a mean calculated. At least 3 min
elapsed between each test [49].
5.2.11. Radioligand binding assay for cannabinoid (CB1 and CB2)
receptors
Packard TopCount NXT v2.50 instrument was used for the as-
says. CP55940 [3H] was used as hot ligand. Rat whole-brain
membrane was used as a protein at the concentration of 100
mg/
well (1 mg/mL) for radioligand binding of CB1 and rat spleen
5.2.6.4. Mechanical hyperalgesia. Mechanical paw withdrawal
thresholds were assessed with a slightly modified version of the
RandalleSelitto method [50] using analgesymeter (UGO Basile,
Italy). The instrument exerts a force that increases at a constant
rate. This force was applied to the hind paw of the rat, which was
placed on a small plinth under a cone-shaped pusher with a
rounded tip (1.5 mm in diameter) until the animal withdrew its
paw. A cut off of 250 g was used to avoid injury. Mechanical paw
withdrawal thresholds were calculated as the average of two
consecutive measurements.
membrane was used as protein in the concentration of 100
(1 mg/mL) for radioligand binding assay of CB2.
mg/well
For the preparation of rat brain and spleen membranes, rats
were killed by decapitation and then brain and spleen were
removed and rapidly homogenized in ice-cold tris buffer (50 mM
tris, 100 mM NaCl, 10 mM MgCl2, pH 7.4) with a glass/teflon ho-
mogenizer. The homogenates were centrifuged at 20,000ꢃ g at
41 ꢀC for 10 min and the pellet re-suspended in tris buffer. Ho-
mogenization and centrifugation were repeated twice and the final
pellet was re-suspended in storage buffer (50 mM tris, 1 mM EDTA,
pH 7.4) to make a protein concentration of 1 mg/mL [55].
5.2.7. Determination of the median effective dose (ED50
)
Radioligand assays were done as reported by Leggett et al. [56]
Briefly 96 well plates were prepared in triplicate to a final volume of
1 mL containing 0.5 nM [3H] CP55940 (PerkineElmer Life Sciences
Inc., U.S.A.) and 250 mg of protein diluted in tris buffer (50 mM Tris.
HCl, 2 mM EDTA, 5 mM MgCl2 also containing 0.2 mg/mL bovine
serum albumin (BSA), pH 7.4). Stock drugs were diluted in the same
buffer containing 5 mg/mL BSA. Plates were incubated for 90 min at
19.1 ꢀC. Specific binding was calculated by subtracting nonspecific
To determine ED50 as an estimate of the compounds’ potency, a
doseeresponse curve in various sensory tests was plotted. ED50 was
the dose that yielded 50% of the response [51]. This value was
linearly interpolated between the dose just above and just below
the ED50 value.
5.2.8. LPS induced TNF-a release in mice
Test compounds (100 mg/kg) were administered by i.p. route
in a volume of 10 mL/kg body weight. Thirty minutes later, ani-
mals were challenged with LPS (1.0 mg/kg in sterile PBS,
administered ip). After 90 min, blood samples were collected;
plasma was prepared [52] and analysed for levels of TNF by
ELISA assay (R&D Systems) according to the manufacturer’s
directions.
binding (NSB), determined in the presence of 1
mM of WIN for CB1
and 1 M of SR144528 (Amersham, U.K.) for CB2.
m
5.2.12. Subcutaneous picrotoxin-induced seizure threshold test (scPIC)
The test compounds were evaluated for their ability to antago-
nize scPIC-induced convulsions in mice after i.p. administration.
After 30 min of drug administration the in-house validated
convulsive dose of picrotoxin (3.15 mg/kg) was injected subcuta-
neously in a volume of 0.01 mL/g body weight into each of the mice.
The mice were placed in isolated cages and observed for the next
45 min for the presence or absence of threshold convulsion.
Absence of a threshold convulsion was taken as the end point,
which indicates that the test substance has the ability to elevate the
picrotoxin seizure threshold [57].
5.2.9. Estimation of total nitrite/nitrate
On 9th day post chronic constriction injury, after 2 h of
administration of test compounds, the total nitrate/nitrite in brain
and sciatic nerve was estimated according to the reported proce-
dure [53]. The method involved reduction of nitrate to nitrite fol-
lowed by calorimetric estimation using Griess reagent. The
concentration of nitrite in the supernatant was calculated using
standard curve and expressed as percentage of control.
5.2.13. Cathepsin S assay
A microplate-based screening procedure was applied to identify
potential Cathepsin S inhibitors using fluorescent peptide sub-
strates and recombinant human cathepsins S [58]. The activity of
human recombinant Cathepsin S was measured using Benzylox-
ycarbonyl-valine-valinearginine-7-amido-4-methylcoumarin (Z-
VVR-AMC) as the substrate. All the test compounds were dissolved
5.2.10. DPPH assay
A solution of DPPH was prepared by dissolving 5 mg of DPPH in
2 mL of methanol, and the solution was kept in the dark at 4 ꢀC.
Varying concentrations of test compounds (200
96-well microplate. Then, 5 L of methanolic DPPH solution (final
concentration 300 M) was added to each well. After 20 min of
mL) were taken in
m
m
in DMSO to a final concentration of 50 mM and incubated with re-
incubation, absorbance of the solution was read using an ELISA
Reader (EL340 Biokinetic reader, Bio-Tek Instrumentation, USA) at
a wavelength of 517 nm. A methanolic solution of DPPH served as
a control. A dose response curve was plotted to determine the
combinant activated enzyme at 37 ꢀC for 10 min in the assay buffer
containing 0.1 M sodium acetate (pH 7.0), 1 mM EDTA, 0.01% Triton
X-100, 1 mM DDT before initiating the reaction with the addition of
substrate Z-Val-Val-Arg-AMC (Km ¼ 1.7 M). After 30 min, the

220
P. Yogeeswari et al. / European Journal of Medicinal Chemistry 66 (2013) 211e220
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reaction was stopped by addition of E-64 and the fluorescence
generated by release of methylcoumarin (nMec) from the substrate
was read in Perkin Elmer LS50B luminescence spectrophotometer
(Perkin Elmer, Shelton, CT, USA) with excitation and emission
wavelengths of 354 nm and 442 nm, respectively.
5.3. Statistical analysis
All data are expressed as means ꢁ standard error of mean (SEM).
The data were analysed using Student’s t test only when two means
were compared (acute pain assay). In the case of neuropathic pain
studies, statistical significance was determined for drug effects by
one-way ANOVA, and Bonferroni’s post hoc test was used for in-
dividual comparisons with vehicle values. Significance was
assigned to a p value of less than 0.05. The statistical software
package PRISM (Graphpad Software Inc., San Diego, CA) was used
for the analysis.
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Acknowledgements
The authors wish to thank the Department of Science & Tech-
nology (DST), Government of India, New Delhi for funding the
project.
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