Discovery of tetrahydropyrido[4,3-d]pyrimidine derivatives for the treatment of neuropathic pain

Article abstract of DOI:10.1016/j.bioorg.2013.11.007

A series of tetrahydropyridopyrimidine derivatives were synthesized and evaluated for neurotoxicity and peripheral analgesic activity followed by assessment of antiallodynic and antihyperalgesic potential in two peripheral neuropathic pain models, the chronic constriction injury (CCI) and partial sciatic nerve ligation (PSNL). Compounds (4b and 4d) exhibiting promising efficacies in four behavioral assays of allodynia and hyperalgesia (spontaneous pain, tactile allodynia, cold allodynia and mechanical hyperalgesia) were quantified for their ED₅₀ values (15.12-65.10 mg/kg). Studies carried out to assess the underlying mechanism revealed that the compounds suppressed the inflammatory component of the neuropathic pain and prevented oxidative and nitrosative stress.

Full text of DOI:10.1016/j.bioorg.2013.11.007

Bioorganic Chemistry 52 (2014) 69–76
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Discovery of tetrahydropyrido[4,3-d]pyrimidine derivatives
for the treatment of neuropathic pain
⇑
Monika Sharma, Vanamala Deekshith, Arvind Semwal, Dharmarajan Sriram, Perumal Yogeeswari
Neuropathic Pain Research Laboratory, Department of Pharmacy, Birla Institute of Technology & Science-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:
Received 25 January 2013
Available online 25 November 2013
A series of tetrahydropyridopyrimidine derivatives were synthesized and evaluated for neurotoxicity and
peripheral analgesic activity followed by assessment of antiallodynic and antihyperalgesic potential in
two peripheral neuropathic pain models, the chronic constriction injury (CCI) and partial sciatic nerve
ligation (PSNL). Compounds (4b and 4d) exhibiting promising efficacies in four behavioral assays of allo-
dynia and hyperalgesia (spontaneous pain, tactile allodynia, cold allodynia and mechanical hyperalgesia)
were quantified for their ED50 values (15.12–65.10 mg/kg). Studies carried out to assess the underlying
mechanism revealed that the compounds suppressed the inflammatory component of the neuropathic
pain and prevented oxidative and nitrosative stress.
Keywords:
Neuropathic pain
Tetrahydropyridopyrimidine
Antiallodynic
Antihyperalgesic
Nitric oxide
Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction
The worldwide prevalence of neuropathic pain has imposed
a burden on healthcare leading to the boom in discovery and
Neuropathic pain is defined as an unpleasant sensory and
emotional experience associated with actual or potential tissue
damage or described in terms of such damage [1]. Neuropathic
pain syndrome includes various diseases with heterogeneous
etiology, e.g. peripheral nerve trauma (trigeminal neuralgia and
phantom limb pain), neurotoxicity (cancer chemotherapy),
infection (postherpetic neuralgia), infarct (stroke), and metabolic
disturbances (diabetic neuropathy) [2]. The complex pathophysiol-
ogy of neuropathic pain involves neuronal hyperexcitability in
damaged areas of the peripheral or central nervous system charac-
terized by allodynia (pain response to non-noxious stimuli) and
hyperalgesia (exaggerated pain sensation) [3,4].
Following nerve injury, a cascade of events leads to complex
inflammatory and immune mechanisms both in the periphery
and the central nervous system [5]. Multiple etiologies, symptoms
and complex underlying mechanisms make the treatment and
management of neuropathic pain challenging [6]. The existing
treatment for neuropathic pain includes anticonvulsants, antide-
pressants, opioids and topical agents which are not specifically
indicated for neuropathic pain, and exhibit inadequate efficacy
and adverse events profile [7–9].
development of many new targets which include various recep-
tors, enzymes, transport systems, and membrane ion channels
for neuropathic pain [10]. Literature review reveals various so-
dium channel blockers, calcium channel blockers, drugs effect-
ing GABAergic system, NMDA, AMPA, and kainate antagonists
under development for the treatment of neuropathic pain
[11–14].
In recent years, numerous heterocycles with tetrahydropyridine
framework have gained considerable attention in the treatment of
neuropathic pain. Various tetrahydropyridopyrazoles [15],
tetrahydropyridopyrimidines [16], tetrahydropyridoindoles [17],
tetrahydropyridoquinazolines [18] have been found to embark
antinociceptive efficacies. Literature reveals several amino and
alkoxy tetrahydro-pyridopyrimidines based PDE10 inhibitors
[19], P2X7 antagonists [20] having neuropathic pain activity.
Several pyridopyrimidine derivatives mediating histaminergic
pathways have also been reported for the alleviation of neuro-
pathic pain.
In our interest to develop newer heterocyclic scaffolds for neu-
ropathic pain, we have recently reported furoic acid hydrazones,
isatin derivatives, piperazinyl derivatives, 1,2,4-triazol-5-ones,
and fused triazolo-thiadiazoles as antinociceptive agents [21–25].
Hence in the course of our efforts towards the identification of
new leads for the treatment of neuropathic pain, and in view of
the above reports on pyridopyrimidines, synthesis of several
substituted tetrahydropyridopyrimidines was accomplished
followed by assessment of neuropathic pain activity and underly-
ing mechanism of action.
Abbreviations: GABA, gamma-aminobytyric acid; NMDA, N-methyl-D-aspartate;
AMPA, 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid; PDE, phos-
phodiesterase; P2X7, purinoreceptor subtype; DCM, dichloromethane; THF, tetra-
hydrofuran; DMF, dimethyl formamide; ELISA, enzyme-linked immunosorbant
assay.
⇑
Corresponding author. Fax: +91 40 66303998.
E-mail address: pyogee@hyderabad.bits-pilani.ac.in (P. Yogeeswari).
0045-2068/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bioorg.2013.11.007

70
M. Sharma et al. / Bioorganic Chemistry 52 (2014) 69–76
2.1.5. Synthesis of 2-amino-6-substituted-5,6,7,8-tetrahydropyridopy-
rimidines(1a–4d)
2. Experimental
2.1. Chemistry
0.2 mmol of substituted acids (1 equiv.) and 2-amino-6-substi-
tuted-5,6,7,8-tetrahydropyridopyrimidin-4-ols (1.0 equiv.) were taken
in DCM (5 mL) followed by addition of triethylamine (1.1 equiv.),
1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride
(EDC) (1.1 equiv.) and 1-hydroxybenzotriazole (HOBT) (1.1 equiv.)
at 0 °C. The reaction mixture was stirred at room temperature over-
night. Following completion, the reaction mixture was washed with
saturated aqueous sodium bicarbonate and brine. The organic layer
was collected, dried over anhydrous sodium sulfate, filtered, and
evaporated in vacuo to give the product in 78–85% yield.
2.1.1. General
The experimental procedures followed were as reported earlier
[29]. Melting points were measured in open capillary tubes on a
Buchi 530 melting point apparatus and are uncorrected. Proton nu-
clear magnetic resonance (1H NMR) spectra were recorded for the
compounds on Brucker Avance (300 MHz) instrument. Chemical
shifts are reported in parts per million (ppm) using tetramethyl si-
lane (TMS) as an internal standard. Mass spectra were measured
with a Shimadzu GC–MS-QP5000 spectrophotometer. Elemental
analyses (C, H and N) were undertaken with a Perkin-Elmer model
240C analyzer and all analyses were consistent with theoretical
values (within 0.4%) unless indicated. The homogeneity of the
compounds was monitored by ascending thin layer chromatogra-
phy (TLC) on silicagel-G (Merck) coated aluminum plates, visual-
ized by iodine vapor and UV light.
2.1.5.1. N-(6-Ethyl-4-hydroxy-5,6,7,8-tetrahydropyrido[4,3-d]pyrimi-
din-2-yl)-2-propyl pentanamide (1a). Yield = 78%; mp = 192–
193 °C; 1H NMR (CDCl3) d: 0.96–1.11 (t, J = 7.2 Hz, 6H), 1.23 (t,
J = 7.6 Hz, 3H), 1.37–1.41 (m, 4H), 1.54–1.59 (q, 4H), 2.65–2.67 (t,
J = 7.6 Hz, 1H), 2.92–3.23 (m, 4H), 3.73 (t, J = 7.2 Hz, 2H), 4.41 (s,
2H), 9.32 (br s, NH, D2O exchangeable). MS (ESI) 320.4 (M+H)+.
Anal. (C17H28N4O2)C,H,N.
2.1.5.2. N-(6-(2,6-Dimethylphenyl)-4-hydroxy-5,6,7,8-tetrahydropyr-
2.1.2. Synthesis of N-substituted piperidin-4-one
ido[4,3-d]pyrimidin-2-yl)-4-nitrobenzamide
(1b). Yield = 67%;
One millimolar of 4-piperidone (1.0 equiv.) was taken in DCM
(15–20 mL). To it, potassium carbonate (2.0 equiv.) was added fol-
lowed by the addition of various alkyl, acid and sulphonyl halides
(1.0 equiv.) at 0 °C. The reaction was stirred at room temperature
overnight, diluted with water (40 mL), and neutralized to pH 6
with 0.1 N acetic acid. The solid was collected by filtration, washed
with water, and dried under vacuum to provide the title
compound.
mp = 201–202 °C; 1H NMR (CDCl3) d: 1.23 (t, J = 7.2 Hz, 3H),
2.92–3.23 (m, 4H), 3.73 (t, J = 7.4 Hz, 2H), 4.41 (s, 2H), 8.11 (d,
J = 7.2 Hz, 2H), 8.44 (d, J = 7.4 Hz, 2H), 9.11 (br s, NH, D2O
exchangeable). MS (ESI) 343.3 (M+H)+. Anal. (C16H17N5O4)C,H,N.
2.1.5.3. N-(6-Ethyl-4-hydroxy-5,6,7,8-tetrahydropyrido[4,3-d]pyrimi-
din-2-yl)-4-phenoxy-benzamide
(1c). Yield = 71%;
mp = 169–
170 °C; 1H NMR (CDCl3) d: 1.24 (t, J = 7.2 Hz, 3H), 2.91–3.21 (m,
4H), 3.71 (t, J = 7.6 Hz, 2H), 4.42 (s, 2H), 7.14–7.17 (m, 3H), 7.32–
7.41 (m, 4H), 8.01 (d, J = 7.6 Hz, 2H), 9.41 (br s, NH, D2O exchange-
able). MS (ESI) 390.4 (M+H)+. Anal. (C22H22N4O3)C,H,N.
2.1.3. Synthesis of methyl N-substituted-4-oxo-piperidine-3-
carboxylate
A 500-mL, three-necked, round-bottomed flask was equipped
with a mechanical stirrer, a reflux condenser, and a pressure-
equalizing dropping funnel bearing a nitrogen inlet. The flask
was flushed with nitrogen and charged with dimethyl carbonate
(8.0 equiv.), 50 mL of anhydrous THF, and sodium hydride
(2.2 equiv.). The suspension was stirred and heated to reflux tem-
perature, when the slow, dropwise addition of N-substituted piper-
idin-4-ones (0.5 mmol) in 20 mL of dry THF was begun. The
addition of N-substituted piperidin-4-ones was continued over a
1 h period. The mixture was stirred and heated at reflux for 6 h,
cooled in an ice bath for 15–20 min, and hydrolyzed by slowly add-
ing 75 mL of 3.0 M aqueous acetic acid. The contents of the flask
were poured into 100 mL of saturated sodium chloride, and the
aqueous mixture was extracted with (4 ꢀ 150) mL portions of
DCM. The organic layers were combined, dried with anhydrous so-
dium sulfate, and concentrated at room temperature with a rotary
evaporator. Distillation of the residual liquid under reduced pres-
sure resulted in N-substituted-4-oxo-piperidine-3-carboxylate
(79–87%) as a yellow liquid.
2.1.5.4. N-(6-Ethyl-4-hydroxy-5,6,7,8-tetrahydropyrido[4,3-d]pyrimi-
din-2-yl)isonicotinamide (1d). Yield = 72%; mp = 193–194 °C; 1H
NMR (CDCl3) d: 1.23 (t, J = 7.4 Hz, 3H), 2.92–3.23 (m, 4H), 3.73 (t,
J = 7.6 Hz, 2H), 4.41 (s, 2H), 7.81 (d, J = 7.2 Hz, 2H), 8.89 (d,
J = 7.4 Hz, 2H), 9.51 (br s, NH, D2O exchangeable). MS (ESI) 390.4
(M+H)+. Anal. (C22H22N4O3)C,H,N.
2.1.5.5. N-(6-(2,6-Dimethylphenyl)-4-hydroxy-5,6,7,8-tetrahydropyr-
ido[4,3-d]pyrimidin-2-yl)-2-propylpentanamide (2a). Yield = 63%;
mp = 231–232 °C; 1H NMR (CDCl3) d: 0.96–1.11 (t, J = 7.8 Hz, 6H),
1.23 (t, J = 7.4 Hz, 3H), 1.37–1.41 (m, 4H), 1.54–1.59 (q, 4H), 2.14
(s, 6H), 2.65–2.67 (t, J = 7.2 Hz, 1H), 2.92–3.23 (m, 4H), 3.73 (t,
J = 7.4 Hz, 2H), 4.41 (s, 2H), 6.76 (t, J = 7.4 Hz, 1H), 7.03 (d,
J = 7.4 Hz, 2H), 9.11 (br s, NH, D2O exchangeable). MS (ESI) 396.5
(M+H)+. Anal. (C23H32N4O2)C,H,N.
2.1.5.6. N-(6-(2,6-Dimethylphenyl)-4-hydroxy-5,6,7,8-tetrahydropyr-
ido[4,3-d]pyrimidin-2-yl)-4-nitrobenzamide (2b). Yield = 65%;
mp = 201–202 °C; 1H NMR (CDCl3) d: 2.13 (s, 6H), 2.91 (t,
J = 7.4 Hz, 2H), 3.71 (t, J = 7.6 Hz, 2H), 4.52 (s, 2H), 6.76 (t,
J = 7.4 Hz, 1H), 7.03 (d, J = 7.2 Hz, 2H), 7.14–7.17 (m, 3H),
7.32–7.41 (m, 4H), 8.01 (d, J = 7.2 Hz, 2H), 9.41 (br s, NH,
D2O exchangeable). MS (ESI) 415.4 (M+H)+. Anal.
(C22H21N5O4)C,H,N.
2.1.4. Synthesis of 2-amino-6-substituted-5,6,7,8-
tetrahydropyridopyrimidin-4-ol
A solution of 0.4 mmol N-substituted-4-oxo-piperidine-3-car-
boxylate (1.0 equiv.) in DMF (5 mL) was treated with guanidine
hydrochloride (2.0 equiv.) followed by the addition of potassium
carbonate (2.0 equiv.), and the mixture was stirred at 110 °C for
16 h. The mixture was cooled to ambient temperature, diluted with
water (40 mL), and neutralized to pH 6 with 0.1 N acetic acid. The
solid was collected by filtration, washed with water, and dried un-
der vacuum to provide the title compound.
2.1.5.7. N-(6-(2,6-Dimethylphenyl)-4-hydroxy-5,6,7,8-tetrahydropyr-
ido[4,3-d]pyrimidin-2-yl)-4-phenoxybenzamide
(2c). Yield = 61%;
mp = 190–191 °C; 1H NMR (CDCl3) d: 2.13 (s, 6H), 2.91 (t,
J = 7.2 Hz, 2H), 3.71 (t, J = 7.4 Hz, 2H), 4.52 (s, 2H), 6.76 (t,
J = 7.6 Hz, 1H), 7.03 (d, J = 7.2 Hz, 2H), 8.11 (d, J = 7.4 Hz, 2H),

M. Sharma et al. / Bioorganic Chemistry 52 (2014) 69–76
71
8.44 (d, J = 7.6 Hz, 2H), 9.11 (br s, NH, D2O exchangeable. MS (ESI)
NH, D2O, exchangeable). MS (ESI) 516.5(M+H)+. Anal.
(C27H24N4O5S)C,H,N.
466.5 (M+H)+. Anal. (C28H26N4O3)C,H,N.
2.1.5.8. N-(6-(2,6-Dimethylphenyl)-4-hydroxy-5,6,7,8-tetrahydropyr-
2.1.5.16. N-(4-Hydroxy-6-tosyl-5,6,7,8-tetrahydropyrido[4,3-d]pyr-
imidin-2-yl)isonicotinamide (4d). Yield = 82%; mp = 205–206 °C;
1H NMR (CDCl3) d: 2.35 (s, 3H), 2.78 (t, J = 7.4 Hz, 2H), 3.69 (t,
J = 7.2 Hz, 2H), 4.42 (s, 2H), 7.41 (d, J = 7.8 Hz, 2H), 7.81–7.85 (m,
4H), 8.89 (d, 4H), 9.54 (br s, NH, D2O exchangeable). MS (ESI)
429.4 (M+H)+. Anal. (C20H19N5O4S)C,H,N.
ido[4,3-d]pyrimidin-2-yl)
isonicotinamide
(2d). Yield = 77%;
mp = 198–199 °C; 1H NMR (CDCl3) d: 2.13 (s, 6H), 2.91 (t,
J = 7.4 Hz, 2H), 3.71 (t, J = 7.2 Hz, 2H), 4.52 (s, 2H), 6.76 (t,
J = 7.8 Hz, 1H), 7.03 (d, J = 7.2 Hz, 2H), 7.81 (d, J = 7.2 Hz, 2H),
8.89 (d, J = 7.2 Hz, 2H), 9.11 (br s, NH, D2O exchangeable). MS
(ESI) 374.4 (M+H)+. Anal. (C21H21N5O2)C,H,N.
2.2. Pharmacology
2.1.5.9. N-(4-Hydroxy-6-(4-methylbenzoyl)-5,6,7,8-tetrahydropyri-
do[4,3-d]pyrimidin-2-yl)-2-propylpentanamide (3a). Yield = 78%;
mp = 189–190 °C; 1H NMR (CDCl3) d: 0.96–1.11 (t, J = 7.8 Hz,
6H), 1.37–1.41 (m, 4H), 1.54–1.59 (q, 4H), 2.34 (s, 3H), 2.53 (q,
1H), 2.98 (t, J = 7.2 Hz, 2H), 3.79 (t, J = 7.4 Hz, 2H), 4.62 (s, 2H),
7.41 (d, J = 7.6 Hz, 2H), 7.93 (d, J = 7.8 Hz, 2H), 9.61 (br s, NH,
The detailed methodology has also been described in earlier
report [29]. Swiss albino mice (either sex) with weights ranging
from 20–25 g were used for the assessment of neurotoxicity, acetic
acid induced writhing and formalin induced flinching model.
Wistar rats of either sex (200–250 g) were used for the inflamma-
tory and neuropathic pain models. All experiments were approved
by the Institutional Animal Ethics Committee of BITS-Pilani, Hyder-
abad campus with a protocol number IAEC/RES/2/1. 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.
D2O
exchangeable).
MS
(ESI)
410.5
(M+H)+.
Anal.
(C23H30N4O3)C,H,N.
2.1.5.10. N-(4-Hydroxy-6-(4-methylbenzoyl)-5,6,7,8-tetrahydropyri-
do[4,3-d]pyrimidin-2-yl)-4-nitrobenzamide
(3b). Yield = 71%;
mp = 209–210 °C; 1H NMR (CDCl3) d: 2.35 (s, 3H), 2.98 (t,
J = 7.4 Hz, 2H), 3.79 (t, J = 7.4 Hz, 2H), 4.62 (s, 2H), 7.41 (d,
J = 7.8 Hz, 2H), 7.91 (d, J = 7.2 Hz, 2H), 8.11 (d, J = 7.4 Hz, 2H),
8.44 (d, J = 7.8 Hz, 4H), 9.54 (br s, NH, D2O exchangeable). MS
(ESI) 433.4 (M+H)+. Anal. (C22H19N5O5)C,H,N.
2.2.1. Motor impairment
Minimal motor impairment was measured in mice by the rota-
rod test. The mice were trained to stay on an accelerating rotarod
that rotates at 10 rpm. The rod diameter was 3.2 cm. Neurotoxicity
was indicated by the inability of the animal to maintain equilib-
rium on the rod for at least 1 min in each of the three trials [26].
2.1.5.11. N-(4-Hydroxy-6-(4-methylbenzoyl)-5,6,7,8-tetrahydropyri-
do[4,3-d]pyrimidin-2-yl)-4-phenoxybenzamide
(3c). Yield = 60%;
mp = 231–232 °C; 1H NMR (CDCl3) d: 2.35 (s, 3H), 2.98 (t,
J = 7.2 Hz, 2H), 3.79 (t, J = 7.6 Hz, 2H), 4.62 (s, 2H), 7.14–7.17 (m,
3H), 7.32–741 (m, 6H), 7.98 (d, J = 7.6 Hz, 2H), 8.01 (d, J = 7.6 Hz,
2H), 9.44 (br s, NH, D2O exchangeable). MS (ESI) 480.5 (M+H)+.
Anal. (C28H24N4O4)C,H,N.
2.2.2. Acetic acid induced writhing
Writhing was induced in a group of mice by an intraperitoneal
injection of 0.1 mL of 2% 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 re-
corded. 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 cal-
culated [27].
2.1.5.12.
N-(4-Hydroxy-6-(4-methylbenzoyl)-5,6,7,8-tetrahydropyrido
[4,3-d]pyrimidin-2-yl) isonicotinamide (3d). Yield = 70%; mp =
163–164 °C; 1H NMR (CDCl3) d: 2.35 (s, 3H), 2.98 (t, J = 7.4 Hz,
2H), 3.79 (t, J = 7.6 Hz, 2H), 4.62 (s, 2H), 7.41 (d, J = 7.4 Hz,
2H), 7.91 (d, J = 7.2 Hz, 2H), 7.81 (d, J = 7.8 Hz, 2H), 8.89 (d,
J = 7.2 Hz, 4H), 9.54 (br s, NH, D2O exchangeable). MS (ESI)
389.4 (M+H)+. Anal. (C21H19N5O3)C,H,N.
2.2.3. Formalin induced flinching
The test involved intra-plantar injection of 25 lL of 1% formalin
into the hind paw of mice, which resulted in flinches in the paw in
the early phase (0–5 min) and the late phase (10–30 min) [28].
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.
2.1.5.13. N-(4-Hydroxy-6-tosyl-5,6,7,8-tetrahydropyrido[4,3-d]pyr-
imidin-2-yl)-2-propyl-pentanamide (4a). Yield = 61%; mp = 180–
181 °C; 1H NMR (CDCl3) d: 0.96–1.11 (t, J = 7.4 Hz, 6H), 1.37–1.41
(m, 4H), 1.54–1.59 (q, 4H), 2.44 (s, 6H), 2.53–2.57 (t, J = 7.6 Hz,
1H),), 2.78 (t, J = 7.4 Hz, 2H), 3.63 (t, J = 7.2 Hz, 2H), 4.42 (s, 2H),
7.40 (d, J = 7.2 Hz, 2H), 7.74 (d, J = 7.2 Hz, 2H), 9.11 (br s, NH, D2O
exchangeable). MS (ESI) 446.5 (M+H)+. Anal. (C22H30N4O4S)C,H,N.
2.2.4. Unilateral mononeuropathy–Chronic constriction nerve injury
(CCI) model
Unilateral mononeuropathy was produced in rats using the CCI
model performed essentially as described by Bennett and Xie [29].
The rats were anesthetized with an intraperitoneal dose of keta-
mine (55 mg/kg) and xylazine (5 mg/kg) with additional doses of
the anesthetic 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 sepa-
rated 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-mat-
tress suture pattern.
2.1.5.14. N-(4-Hydroxy-6-tosyl-5,6,7,8-tetrahydropyrido[4,3-d]pyr-
imidin-2-yl)-4-nitrobenzamide (4b). Yield = 59%; mp = 195–196 °C;
1H NMR (CDCl3) d: 2.35 (s, 3H), 2.78 (t, J = 7.6 Hz, 2H), 3.63 (t,
J = 7.4 Hz, 2H), 4.42 (s, 2H), 7.41 (d, J = 7.6 Hz, 2H), 7.75 (d,
J = 7.2 Hz, 2H), 8.11 (d, J = 7.4 Hz, 2H), 8.44 (d, J = 7.8 Hz, 4H),
9.54 (br s, NH, D2O exchangeable). MS (ESI) 469.47 (M+H)+. Anal.
(C21H19N5O6S)C,H,N.
2.1.5.15. N-(4-Hydroxy-6-tosyl-5,6,7,8-tetrahydropyrido[4,3-d]pyr-
imidin-2-yl)-4-phenoxy benzamide (4c). Yield = 81%; mp = 198–
199 °C; 1H NMR (CDCl3) d: 2.35 (s, 3H), 2.78 (t, J = 7.4 Hz, 2H),
3.69 (t, J = 7.2 Hz, 2H), 4.42 (s, 2H), 7.14–7.17 (m, 3H), 7.32–741
(m, 6H), 7.74 (d, J = 7.6 Hz, 2H), 8.01 (d, J = 7.8 Hz, 2H), 9.44 (br s,

72
M. Sharma et al. / Bioorganic Chemistry 52 (2014) 69–76
2.2.5. Induction of peripheral mononeuropathy–Partial sciatic nerve
ligation model
2.2.7. Carrageenan-induced paw edema
Paw edema was induced in wistar rats by intra-plantar injection
As described by Seltzer et al. [30], in anaesthetized rats, left sci-
atic nerve was exposed at mid-thigh level through small incision,
cleared of adhering muscle tissue, and one-half of the nerve thick-
ness 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.
of 50 lL of 2% carrageenan (k-carrageenan, type IV, Sigma) diluted
in saline. The volume of the paw edema (mL) was determined at 0,
60, 120 and 180 min using a water plethysmometer (Ugo Basile,
Italy) Indomethacin (10 mg/kg, i.p.) was used as positive control
[36]. The percentage protection against inflammation was calcu-
lated as: Vc ꢁ Vd/Vc ꢀ 100, where Vc is the increase in paw volume
in the absence of the test compound (control) and Vd is the increase
of paw volume after administration of the test compound.
2.2.6. Sensory testing using nociceptive assays
Compounds (100 mg/kg, i.p.) were administered at t = 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 as-
sessed in tactile allodynia and mechanical hyperalgesia. Percentage
reversal in spontaneous pain, allodynia or hyperalgesia was calcu-
lated for each animal as defined below [31]
2.2.8. Estimation of total nitrite/nitrate
On 9th day post chronic constriction injury, after 2 h of admin-
istration of test compounds, the total nitrate/nitrite in brain and
sciatic nerve was estimated according to the reported procedure
[37]. The method involved reduction of nitrate to nitrite followed
by calorimetric estimation using Griess reagent. The concentration
of nitrite in the supernatant was calculated using standard curve
and expressed as percentage of control.
ðpost dose value ꢁ pre dose valueÞ
2.2.9. DPPH (1,1-Diphenyl-2-picrylhydrazyl) 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.
%Reversal ¼
ꢀ 100
ðcontralateral paw value ꢁ pre dose valueÞ
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
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
IC50 values. All tests and analyses were run in triplicate and
averaged [38].
lL) were taken in
2.2.6.1. Spontaneous pain. Spontaneous pain was assessed for a to-
tal 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
repositioning was not counted [32].
l
l
Percentage scavenging was calculated according to the
following equation:
2.2.6.2. Tactile allodynia. Paw withdrawal in response to mechani-
cal 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 (0.4–15 g), with
intensities 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 1–2 s
[33]. The weakest force (g) inducing withdrawal of the stimulated
paw at least three times was taken as the paw withdrawal thresh-
old with cut off value at 15 g.
fAbsorbanceðDPPHÞꢁAbsorbanceðDPPHþcompoundÞg
%scavenging ¼
AbsorbanceðDPPHÞ
ꢀ100
3. Results and discussion
3.1. Chemistry
Various alkyl, acid and sulphonyl halides were reacted with 4-
piperidone to form N-substituted piperidin-4-one. Methoxycarb-
onylation of the same in the presence of sodium hydride afforded
the ketoester which underwent base catalyzed reaction with gua-
nidine to form substituted tetrahydropyridopyrimidine [16,18].
Utilizing various alkyl, aryl and hetroaryl acids, the acid amine
coupling was then carried out with with the aid of 1-ethyl-3-
[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) and
1-hydroxy benzotriazole (HOBT) to form the titled compounds
(Scheme 1).
2.2.6.3. Cold allodynia. The operated rat was placed inside an obser-
vation cage that was kept 5 cm from the ground level and was al-
lowed to acclimatize for 10 min or until exploratory behavior
ceased. Few drops (100–200 lL) of freshly dispensed acetone were
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 [34].
3.2. Pharmacology
2.2.6.4. Mechanical hyperalgesia. Mechanical paw withdrawal
thresholds were assessed with a slightly modified version of the
Randall–Selitto method [35] 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 con-
secutive measurements.
Compounds were assessed for neurotoxicity in the rotarod, fol-
lowed by their evaluation in acute models of pain. Among all the
compounds administered at the doses of 30, 100 and 300 mg/kg,
compound 4e exhibited motor impairment at 300 mg/kg dose. In
acetic acid test, compounds 1b, 1c, 3a, 3b, 3d, 4b and 4d sup-
pressed the acetic acid induced writhing response significantly
(p < 0.05) in comparison to the control (Table 1). The standard drug
indomethacin exhibited the highest percentage inhibition
(96.12%). In formalin induced flinching model, significant reversal

M. Sharma et al. / Bioorganic Chemistry 52 (2014) 69–76
73
O
lastly mechanical hyperalgesia. Baseline sensory response values
were measured for each group of animals (n = 4) pre-operatively
and 9 days post-operatively. Animals displaying allodynic and
hyperalgesic responses in both the models, were then adminis-
tered the test compound (single dose of 100 mg/kg) according to
a pre-determined randomization table and testing was re-per-
formed at 30, 60 and 120 min post drug administration. All of
the behavioral responses were timed with a stopwatch.
O
O
O
i
O
ii
N
R
N
H
N
R
O
NH2
R1
In the CCI model, compounds 1c, 1d, 4a, 4b and 4d completely
reversed the spontaneous pain response throughout the time per-
iod of testing (30–120 min) similar to gabapentin. Compounds 1a,
1b, 2a, 2b and 3d exhibited activity up to 60 min. The onset of ac-
tion of compound 2d was at 120 min. Other compounds were inef-
fective in this test (Fig. 1). Two compounds 4b and 4d were active
in attenuating the tactile allodynia throughout the 120 min exper-
iment. The onset of action of compounds 1d, 2b and 3d was at
60 min similar to gabapentin. The duration of action of compounds
1c and 4c was up to 30 min, whereas for 4a duration of action was
up to 60 min. All other compounds were ineffective in this test. In
the cold allodynia produced in CCI rats, significant reversal of paw
withdrawal durations was observed at all time points by the
administration of compounds 1c, 1d, 2a, 4a, 4b and 4d. Gabapentin
was also found to be effective at all the time points. The onset of
action for compounds 2b and 3d was at 60 min. Mechanical hyper-
algesia was significantly attenuated at all the time points by 4b and
4d similar to gabapentin. The onset of action of compounds 1c and
2b was at 60 min. The compounds 1d, 2a and 3a were active in
only first 30 min of the 120 min experiment (Fig. 1).
In the PSNL model, the paw withdrawal durations due to spon-
taneous ongoing pain were significantly reduced by compounds 1c,
2a, 4b and 4d throughout the experiment similar to gabapentin.
The onset of action for compounds 1b, 1d, 2b, 2d and 4a was at
60 min. 1a and 3d exhibited irregular activity pattern, being effec-
tive at only one time point (60 min). The tactile allodynia produced
in PSNL model was effectively reversed by compounds 4b and 4d at
all the time points like gabapentin. Compound 1c was effective up
to 30 min in 120 min experiment. Compound 2d exhibited irregu-
lar activity pattern, being effective at only one time point (60 min).
NH
iv
iii
N
N
N
N
OH
OH
N
R
N
R
R
R1
1
2
3
4
ethyl
a
2-propylpentanyl
4-nitrobenzyl
2,6-dimethylphenyl
4-methylbenzoyll
b
c
4-phenoxyphenyl
isonicotinyl
4-methylbenzsulfonyl
d
Scheme 1. Synthetic route to substituted tetrahydropyridopyrimidines. Reagents
and conditions: (i) CH2Cl2, RX/RCOCl/RSO2Cl, 0 °C-rt; (ii) dimethyl carbonate, NaH,
THF reflux, 6 h; (iii) guanidine, K2CO3, DMF, reflux, 18 h; (iv) R1COOH, Et3N, EDC-
HCl, HOBT, CH2Cl2, 0 °C-rt, 24 h.
of phase-I was exhibited by compounds 2b with 53.0% inhibition.
In Phase-II, all the tested compounds except 1a, 2b, 2d, 3b, 3c,
3d, 4b and 4c showed significant reversal. Compound 4d exhibited
highest reversal of 80.2%, being more efficacious than indometha-
cin (67.14%) (Table 1).
Compounds were further evaluated in two well-established
peripheral neuropathic pain models–the chronic constriction nerve
injury (CCI) and the partial sciatic nerve ligation (PSNL) model,
where pain was assessed by four nociceptive tests namely sponta-
neous pain, tactile allodynia, cold allodynia and mechanical hyper-
algesia. A minimum of 10 min separated the testing procedures to
reduce the influence of prior nociceptive testing. The order of test-
ing was spontaneous pain, tactile allodynia, cold allodynia and
Table 1
Neurotoxicity and acute antinociceptive efficacy of tetrahydropyridopyrimidines.
Compound
Neurotoxicitya
Acetic acid induced writhingb
Formalin induced flinchingb
% Inhibition
0.5 h
4 h
% Inhibition
Phase-I
Phase-II
Control
1a
1b
1c
1d
2a
2b
2c
2d
3a
3b
3c
3d
4a
4b
4c
–
–
–
–
–
–
–
–
–
–
–
–
–
300
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
300
–
–
–
–
–
–
40.5
59.4*
61.3*
46.2
46.3
17.9
11.3
46.2
65.0*
61.3*
46.2
61.8*
21.7
75.4*
46.2
74.5*
96.1*
25.2
30.4
17.4
20.9
25.3
53.0*
4.3
18.3
15.7
14.8
13.9
19.1
43.4
15.7
37.4
28.7
4.7
50.3*
21.0
22.4
43.5
15.6
55.7*
24.5
54.7*
40.8
70.1*
56.5*
64.6*
29.9
70.1*
43.0
80.2*
72.1*
4d
Indomethacinc
–
–
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 h. The line (–) indicates an absence of neurotoxicity at the maximum dose tested.
b
Vehicle treated animals received 30% v/v PEG 400 in water.
Indomethacin was taken as positive control at 5 mg/kg.
c
*
Represents significance (> than 50%) at p < 0.05 compared to vehicle (One way ANOVA followed by Dunnett’s Test, n = 4) at a dose of 100 mg/kg.

74
M. Sharma et al. / Bioorganic Chemistry 52 (2014) 69–76
100
90
80
70
60
50
40
30
20
10
0
A
110
90
B
Vehicle
1a
Vehicle
1c
1b
1c
70
1d
1d
2b
2a
50
3d
2b
4a
2d
4b
3d
30
4c
4a
4d
GBP
4b
4d
10
GBP
0
30
60
120
-10
0
30
60
120
Time (min)
Time (min)
110
90
C
D
100
90
80
70
60
50
40
30
20
10
0
Vehicle
1c
1d
2a
2b
3a
3b
4b
4d
Vehicle
1c
1d
2a
2b
3d
4a
4b
4d
70
50
30
10
GBP
GBP
0
30
60
Time (min)
120
0
30
60
120
-10
Time (min)
Fig. 1. Efficacy of compounds in spontaneous pain (A), tactile allodynia (B), cold allodynia (C) and mechanical hyperalgesia (D) in CCI rats. Each value represents the% reversal
(mean SEM) in spontaneous pain, tactile allodynia, cold allodynia and mechanical hyperalgesia of four rats; ꢂ denotes significant value, in comparison to their respective
vehicle control at p < 0.05 (one-way ANOVA, followed by post-hoc Dunnett’s test).
The onset of action for compounds 1d, 2a, 2b, 2c and 4a was at
60 min. Cold allodynia produced in the PSNL model was com-
pletely reversed by the compounds 1d, 2b, 4b and 4d. Compounds
3b and 3d were effective up to 30 min of the experiment. The onset
of action for compounds 1c, 2a and 4a was at 60 min. Compounds
2b, 4b and 4d significantly reversed mechanical hyperalgesia at all
the time points like gabapentin. Compounds 3a and 3d were effec-
tive in first 30 min of the experiment whereas compounds 1c, 1d
and 2a were effective till 60 min of the 120 min experiment
(Fig. 2).
A closer look of the structure activity relationship studies re-
vealed that isonicotinyl substitution in compounds 1d, 2d, 3d
and 4d resulted in significant attenuation of one or more nocicep-
tive parameters in both CCI and PSNL animals. 4-nitrobenzyl sub-
stitution also proved to be additive to the antinociceptive
efficacies in case of compounds 1b, 2b, 3b and 4b in both CCI
and PSNL animals. Aliphatic substituent (2-propylpentanyl) in
compounds 1a, 2a, 3a and 4a resulted in comparatively moderate
attenuation of nociceptive parameters in neuropathic animals.
However, 4-phenoxyphenyl substitution in compounds 1c, 2c, 3c
and 4c proved to be detrimental for the efficacy. The general trend
in antinociceptive efficacy with respect to R1 substitution was:
isonicotinyl > 4-nitrobenzyl > 2-propylpentanyl > 4-phenoxyphe-
nyl substitution.
Compounds exhibiting more than 90% reversal in one or more
of the nociceptive assays (4b and 4d) were taken further for ED50
studies. In the CCI model, compound 4d reversed spontaneous pain
with an ED50 value of 21.84 mg/kg at 60 min (Table 2). Compound
4b reversed tactile and cold allodynia with an ED50 value of
18.24 mg/kg and 42.73 mg/kg at 120 min and 60 min respectively.
Compound 4b reversed mechanical hyperalgesia with an ED50 va-
lue of 26.29 mg/kg at 60 min. In PSNL model, compound 4b re-
versed spontaneous pain and tactile allodynia with an ED50 value
of 15.12 mg/kg and 41.13 mg/kg both at 60 min respectively. In
cold allodynia and mechanical hyperalgesia, compound 4d came
out to be the most effective compound with an ED50 value of
38.21 mg/kg and 55.53 mg/kg at 60 min and 120 min respectively.
The anti-inflammatory profile of the selected compounds (4b
and 4d) as evidenced in formalin induced flinching test was inves-
tigated in the carrageenan induced paw edema model. 4d showed
a significant reduction in paw edema throughout the period of
180 min (Table 3). Following nerve injury, subsequent generation
of free radicals leads to oxidative and nitrosative stress which
exaggerates pain states. The putative role of nitric oxide (NO) in
the pathophysiology of chronic nerve ligation as evident by signif-
icant increase 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. Compound 4d exhibited signif-
icant reduction (p < 0.05) of nitric oxide in sciatic nerve (Table 3).
None of the compounds significantly attenuated nitrosative stress
in the brain suggesting the inhibition of peripheral nitric oxide. The
free radical scavenging abilities of the compounds (4b and 4d)
were assessed using DPPH method. Compound 4b exhibited IC50
N-alkyaltion of the tetrahydropyridine moiety results in com-
pounds with varied antinociceptive efficacies. The order of bioac-
tivity of various N-alkylated tetrahydropyridopyrimidines (R
substitution) in neuropathic pain model was 4-methylbenzsulfo-
nyl > 4-methylbenzoyl > 2,6-dimethylphenyl > Ethyl.

M. Sharma et al. / Bioorganic Chemistry 52 (2014) 69–76
75
90
A
B
100
90
80
70
60
50
40
30
20
10
0
Vehicle
1a
1b
1c
1d
2a
2b
2d
3d
4a
4b
4d
GBP
80
Vehicle
1d
2a
2b
2c
2d
4a
4b
4d
70
60
50
40
30
20
10
0
GBP
0
30
60
120
0
30
60
120
Time (min)
Time (min)
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
D
C
Vehicle
Vehicle
1c
1c
1d
2a
2b
3b
3d
4a
4b
4d
GBP
1d
2a
2b
3a
3d
4b
4d
GBP
0
30
60
120
0
30
60
Time (min)
120
Time (min)
Fig. 2. Efficacy of compounds in spontaneous pain (A), tactile allodynia (B), cold allodynia (C) and mechanical hyperalgesia (D) in PSNL rats. Each value represents the%
reversal (mean SEM) in spontaneous pain, tactile allodynia, cold allodynia and mechanical hyperalgesia of four rats; ꢂ denotes significant value, in comparison to their
respective vehicle control at p < 0.05 (one-way ANOVA, followed by post-hoc Dunnett’s test.
Table 2
Quantification studies of compounds 4b and 4d.
Treatment
ED50 values (mg/kg) in CCI model
(TPE in min)a
ED50 values (mg/kg) in PSNL model
(TPE in min)a
SP
TA
CA
MH
SP
TA
CA
MH
4b
4d
34.14 (60)
21.84 (60)
18.24 (120)
24.16 (30)
42.73 (60)
54.33 (60)
26.29 (60)
41.26 (120)
15.12 (60)
21.64 (30)
41.13 (60)
43.22 (30)
57.13 (60)
38.21 (60)
65.10 (60)
55.53 (120)
Each value represents median effective dose in spontaneous pain (SP), tactile allodynia (TA), cold allodynia (CA) and mechanical hyperalgesia (MH).
a
TPE represents time of peak effect in min.
Table 3
Percent protection in carrageenan induced paw edema, DPPH scavenging activity and effect of compounds 4b and 4d on nitrosative stress.
Treatment
% Protection in carrageenan induced paw edema
DPPH scavenging activityb
% Inhibition of Nitrosative stress (nitrite)c
60 min
120 min
180 min
IC50(l
M)
Brain
Sciatic nerve
Vehicle
–
–
–
–
–
–
4b
4d
17.4 2.3
67.4 2.3*
50.3 2.6*
11.8 1.2
83.5 4.5*
61.2 3.8*
46.7 1.3
84.2 6.4*
66.9 4.2*
254
213
–
15.2 1.9
18.9 3.1
–
19.0 3.4
50.1 1.3*
–
Indomethacina
*
Represents significance at p < 0.05 compared to vehicle (One way ANOVA followed by Dunnett’s test, n = 4).
a
Indomethacin was tested at the dose of 10 mg/kg i.p.
DPPH radical scavenging activity of the test compounds (values are represented as% scavenging calculated from the average of triplicate experiments).
b
c
Percent inhibition of nitric oxide in brain and sciatic nerve of CCI rats. Compounds were tested at the respective minimal ED50 dose administered i.p.

76
M. Sharma et al. / Bioorganic Chemistry 52 (2014) 69–76
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In the present study, sixteen novel tetrahydropyridopyrimi-
dines were synthesized and assessed for acute antinociceptive,
antiallodynic and antihyperalgesic potential. In acute models of
pain, significant inhibition of acetic acid induced writhings by se-
ven compounds established their role as peripherally acting anal-
gesics. Also, significant suppression of phase-I of the formalin
induced flinching by one compound and phase-II by eight com-
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Compound 4d significantly reduced edema in carrageenan model
at all the time points. Additionally, compound 4d was also found
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Above findings provide evidences to the multifunctional profile
of the tetrahydropyridopyrimidine scaffold and support their
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Acknowledgments
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The authors wish to thank the Council of Scientific and Indus-
trial Research (CSIR), and Department of Science and Technology,
Government of India, New Delhi for funding the project.
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