Neuropathic pain-alleviating effects of pyrazole-conjugated arylsulfonamides as 5-HT6 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2017.07.031

A novel series of arylsulfonylaminomethyl-3-(1-phenyl-5-isopropyl)pyrazoles was evaluated for serotonin receptor subtype 6 (5-HT₆R) antagonistic effects in vitro. We also investigated their neuropathic pain-alleviating effects in vivo using a rat spinal nerve ligation (SNL) model. Bicyclic aromatic sulfonamino groups, such as naphthalene and quinolin-substituted derivatives, showed good 5-HT₆ inhibitory activity in vitro. Among them, selected compounds, 12 and 13, having 8-quinoylsulfonamino groups, showed potent neuropathic pain-alleviating effects in the rat model.

Full text of DOI:10.1016/j.bmcl.2017.07.031

Accepted Manuscript
Neuropathic pain-alleviating effects of pyrazole-conjugated arylsulfonamides
as 5-HT6 receptor antagonists
Jin Ri Hong, Hyunah Choo, Ghilsoo Nam
PII:
S0960-894X(17)30725-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.07.031
BMCL 25144
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
25 April 2017
28 June 2017
10 July 2017
Please cite this article as: Hong, J.R., Choo, H., Nam, G., Neuropathic pain-alleviating effects of pyrazole-conjugated
arylsulfonamides as 5-HT6 receptor antagonists, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://
dx.doi.org/10.1016/j.bmcl.2017.07.031
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Neuropathic pain-alleviating effects of pyrazole-conjugated arylsulfonamides
as 5-HT6 receptor antagonists
Jin Ri Hong,b Hyunah Choo,a,b Ghilsoo Nama,b*
a Center for Neuro-Medicine, Brain Science Institute, Korea Institutes of Science and
Technology (KIST), Seoul,136-791, Korea.; bSchool of Science, University of Science and
Technology, Daejeon 305-333, Korea.
* Authors to whom correspondence should be addressed: Tel: +82-2-958-5166, Fax: +82-2-958-5189
E-mail: gsnam@kist.re.kr (G. Nam),
1

ABSTRACT
A novel series of arylsulfonylaminomethyl-3-(1-phenyl-5-isopropyl)pyrazoles was evaluated
for serotonin receptor subtype 6 (5-HT6R) antagonistic effects in vitro. We also investigated
their neuropathic pain-alleviating effects in vivo using a rat spinal nerve ligation (SNL)
model. Bicyclic aromatic sulfonamino groups, such as naphthalene and quinolin-substituted
derivatives, showed good 5-HT6 inhibitory activity in vitro. Among them, selected
compounds, 12 and 13, having 8-quinoylsulfonamino groups, showed potent neuropathic
pain-alleviating effects in the rat model.
Key words:
arylsufonylaminomethylpyrazole
neuropathic pain
5-HT6 receptor antagonist
Serotonin receptor subtype 6 (5-HT6R) was identified in humans in 1996.1 It is positively
coupled to Gs protein as a G-protein-coupled receptor (GPCR) and activates cyclic adenosine
monophosphate (cAMP) signaling.2 The 5-HT6R is expressed extensively in the central
nervous system (CNS), including the frontal cortex, stratum, hippocampus, amygdala,
cerebellum, and thalamus,3 and plays an important role in learning and memory functions.
Thus, modulation of 5-HT6R has become a focus in the field of medicinal chemistry as a
potential target for cognitive enhancement in related to Alzheimer’s disease (AD), as well as
obesity.4 Moreover 5-HT6 antagonists showed activity suggesting a potential role in
treatments for pain and irritable bowel syndrome.5 5-HT6 receptor agonists/antagonists at the
peripheral and dorsal ganglia/spinal cord participate in the development and maintenance of
2

secondary mechanical allodynia and hyperalgesia in rats.6 Recently, researchers reported that
a 5-HT6R antagonist attenuated cognitive deficits in a neuropathic pain rat model and
improved the analgesic effects of gabapentinoids (gabapentin and pregabalin) during co-
treatment with gabapentinoids.7 It has been suggested that combined use of neuropathic pain
drugs with 5-HT6R antagonists may constitute a novel strategy for neuropathic pain treatment,
with reduced side effects. Thus, 5-HT6R antagonists may be valuable as neuropathic pain
treatments.
Previously, we published regarding the design and synthesis of a novel series of
3-arylsulfonylamino-5,6-dihydro-6-substituted-1H-pyrazolo[3,4]pyridinones that act as
5-HT6R antagonists, and oral administration of these compounds showed potent 5-HT6
antagonistic effects, including good neuropathic pain-alleviating activity in a rat model.8
Compound A having a naphthylsulfonamide moiety, showed the most potent neuropathic
pain-alleviating effect. A pyrazole and an arylsulfonyl group are found in several 5-HT6R
antagonists, such as AVN-211,9 SAM-531,10 and compound A.8 Although there has been
extensive research aimed toward developing 5-HT6R antagonists for use in therapies for
conditions including AD, obesity, schizophrenia, and neuropathic pain, there is still no Food
and Drug Administration (FDA)-approved drug targeting 5-HT6R. However, idalopirdine 11
has been assessed in a phase III clinical trial of candidate AD treatments.
3

Figure 1. Selected serotonin receptor subtype 6 (5-HT6R) antagonist clinical candidates and
Compound A
In a continued effort to develop neuropathic pain treatments, here we report the synthesis of
pyrazole-conjugated arylsulfonamides and an evaluation of their 5-HT6R antagonistic effects
in vitro. Selected compounds were tested for their neuropathic pain-alleviating effects in an
animal model.
Figure 2. Chemical structure of the target structure
The synthesis of target molecules, conjugated with various aromatic sulfonamides and
(1-phenyl-5-isopropyl)pyrazole, is outlined in Scheme 1.
4

Scheme 1. Reagents and conditions: (a) i) DIBAL-H, DCM, -78°C, ii) hydroxylamine HCl,
TEA, DCM, RT, (b) LAH, DCM, 0°C to RT, (c) ArSO2Cl, TEA, DCM.
Pyrazolyl ester 1, prepared by a previously reported method, was reduced to an aldehyde
using diisobutyl aluminum hydride at -78°C, followed by reaction with hydroxylamine and
transformation into an oxime functionality. Pyrazolymethyl amine 2b was prepared by
reduction of oxime derivatives 2a with lithium aluminum hydride in good yield. Final target
compounds 3-12 were prepared by coupling reaction of 1-phenyl-5-isobutyl-pyrazolmethyl
amine derivatives 2b with arylsulfonyl chlorides, including phenyl and monosubstituted
phenyls such as 3-fluorophenyl, 3-chlorophenyl, 4-tertbutylphenyl, disubstituted phenyl
(2-methyl-3-chlorophenyl, 2,6-dichlorophenyl), 1-naphthyl, 2-naphthyl 8-quinoyl, and
naphthalenylmethyl. Compounds 13 and 14, bearing 4-fluorophenyl at the 1-position of the
pyrazole ring, were synthesized by the same procedure as for compounds 11 and 12, starting
from 1-[(4-fluorophenyl)-5-isopropyl)]pyrazol methyl ester. The synthesized
arylsulfonamides having 1-phenyl-5-isobutyl-pyrazolmethyl (3–12) and 1-[(4-fluorophenyl)-
5-isopropyl)]pyrazol methyl (13, 14) moieties were evaluated for 5-HT6 receptor antagonistic
activity in a functional cyclic adenosine monophosphate (cAMP) assay using HeLa cells
stably expressing the human 5-HT6 receptor.12 The results are summarized in Table 1.
5

Table 1. Percent inhibition and IC50 values for 5-HT6R of the [(1-phenyl-5-iso-
propyl)pyrazol-3-yl]methyl arylsulfonamide derivatives
5-HT6 % Binding
Inhibition (10 µM)
11.3 8.5
5-HT6 Functional cAMP Assay
Compound Ar
IC50 (µM)
Ph
NTa
3
4
3-FPh
NTa
19.5 6.7
3-ClPh
5
40.8 3.1
51,1 2.2
72.3 3.1
59.4 2.4
77.7 3.8
67.8 3.8
28.7 4.3
106.0 14.7
93.4 3.2
10.9 0.30
8.78 0.53
4.01 0.59
5.65 0.45
2.43 0.08
3.3 0.03
NTa
1.42 0.48
1.78 0.61
NTa
4-C(CH3)Ph
(2-Me-3-Cl)Ph
2,6,-Cl2Ph
1-Naphtyl
2-Naphtyl
CH2-1-Naphtyl
8-Quinoyl
8-Quinoyl
6
7
8
9
10
11
12
13
14
A
CH2-1-Naphtyl
36.1 3.7
94.1 3.0
0.31 0.10
a Not tested.
Phenylsulfonamide derivative 3 showed the lowest binding affinity (11.3%) and
8-quinolylsulfonamide derivative 12 the highest (106%). According to these results, we
considered the structure-activity relationship of arylsulfonylaminomethyl-3-(1-phenyl-5-
isopropyl)pyrazole derivatives (3-12). Substituted phenylsulfonamide derivatives displayed
higher inhibitory percentages than phenylsulfonamide derivatives, as shown by the inhibitory
effects of 4 (Ar = 3-FPh), 5 (Ar = 3-ClPh), and 6 (Ar = 4-C(CH3)Ph) of 19.5%. 40.8%, and
51.1%, respectively. Disubstituted phenyl sulfonamide derivatives showed moderate
inhibitory activities, of 59.0–72.3%. Fused aryls, such as naphthyl and quinoyl sulfonamide
derivatives, showed good activities, with IC50 values in the range from 1.42 0.48 to 3.3
0.03. In the case of the naphthyl group, the 1-position substituted derivative was more potent
than the 2-naphtyl derivatives. One methylene unit added to naphthylsulfonamide derivatives
6

11 and 14 decreased their potency, by 29% and 36%, respectively. When a fluorine group
was introduced as a derivative of the pyrazoyl ring, [(1-fluorophenyl-5-isobutyl)pyrazole-3-
yl]methyl-1-naphthylmethylsulfonamide (13, 93.4%) also showed slightly decreased potency
compared with the most active compound, 12 (106%).
The 5-HT6R-antagonizing effect of fused bicyclic sulfonamide derivatives was high.
Those with > 40% binding affinity were selected and evaluated for functional c-AMP activity.
Two compounds, 12 (IC50 = 1.42 0.48) and 13 (IC50 = 1.78 0.61), bearing 8-quinoyl
groups showed high 5-HT6R antagonist activity. This suggests that the nitrogen-containing
bicyclic aromatic ring may play an important role in 5-HT6 inhibitory activity.
The most active compounds, 12 and 13, were evaluated for their inhibitory activity human
of ether-a-go-go-related gene (hERG) channel13 and human CYP450 enzyme14 (Table 2).
The inhibition of hERG is known to induce cytotoxicity. Compound 12 showed lower
inhibition potency (IC50 = 4.76 µM) compare to compound 13 (IC50 = 1.6 µM). The
CYP450 enzyme metabolize used drugs, particularly CYP2D6 and CYP3A4 play more
important roles than CYP2C9 in drug-drug interactions.15 Compound 12 showed higher
remaining percent of all subtypes of tested CYP450 enzymes. According to the results
obtained with hERG and CYP450, compound 12 appeared safer in terms of cytotoxicity and
drug-drug interactions than compound 13.
Table 2. Inhibitory effects of selected compounds on the hERG channel and CYP-450
enzyme
% Control of CYP-450 (10 µM)a
hERG
IC50 (µM)
4.67 0.45
1.60 0.60
Compound
2D6 b
119.15
64.79
2C9 c
58.52
3.87
3A4 d
52.22
34.50
12
13
a Values represent the remaining % activities and the mean SD from triplicate experiments.
b sulfaphenazol, c miconazole, d ketoconazole,
7

The two selected compounds, 12 and 13, were assessed for their in vivo neuropathic pain-
alleviating efficacy. At 14 days after surgical manipulation in the spinal nerve ligation (SNL)
model,16 the test compound was administered orally, and behavioral testing for neuropathic
pain, including temperature hypersensitivity and mechanical allodynia, was performed. The
neuropathic pain-relieving effects of compounds 12 and 13 were examined in a single oral
treatment (100 mg/kg each) in SNL model rats, and compared using gabapentin, which is the
most potent neuropathic pain drug in current clinical use, as a reference. These results are
presented in Figures 3 and 4.
KKJA0564 100mg/kg (n6)
Compound 12, 100 mg/kg (n = 6)
GGaabbaappeennttiinn,110000mmg/gk/gkg(n(=n4=) 4)
B. Mechanical allodynia (%MPE)
A. Mechanical allodynia
100
15
*
80
60
40
20
0
12
9
*
*
*
*
6
3
0
Base
1h
3h
5h
Pre N14D
1h
3h
5h
Experimental time (hour)
Experimental time (day or hour)
D. Heat hypersensitivity (%MPE)
C. Heat hypersensitivy
100
15
*
♣
80
12
*
*
60
♣
9
40
20
0
6
3
0
Base
1h
3h
5h
Pre N14D
1h
3h
5h
Experimental time (hour)
Experimental time (day or hour)
Figure 3. Effects on mechanical allodynia (A and B) and cold allodynia (C and D) after oral
administration of gabapentin (○, 100 mg/kg, n = 4) and compound 12 (●, 100 mg/kg, n = 6)
in neuropathic pain-induced rats. Experimental time is expressed as D (days) after
neuropathic injury (N) and h (hours) after gabapentin or compound 12 administration. *P <
0.05 (gabapentin), *P < 0.05 (compound 12) vs. pre-administration value (paired t-test), ♣P <
8

0.05 gabapentin vs. compound 12 (unpaired t-test).
KKJA0604 100mg/kg (n=4)
Compound 13, 100 mg/kg (n=4)
Gabapentin, 100 mg/kg (n=3)
Gabapentin 100mg/kg (n=3)
B. Mechanical allodynia (%MPE)
A. Mechanical allodynia
100
80
60
40
20
0
15
♣
*
♣
*
*
*
12
9
6
3
0
Base
1h
3h
5h
Pre N14D
1h
3h
5h
Experimental time (hour)
Experimental time (day or hour)
D. Cold allodynia (%MPE)
♣
C. Cold allodynia
120
100
80
60
40
20
100
80
60
40
20
0
*
*
*
0
Base
1h
3h
5h
Pre N14D
1h
3h
5h
Experimental time (hour)
Experimental time (day or hour)
Figure 4. Effects on cold allodynia (A and B) and mechanical allodynia (C and D) after oral
○, 100 mg/kg, ●, 100 mg/kg,
administration of gabapentin ( n = 3) and compound 13 (
n = 4)
in neuropathic pain-induced rats. Experimental time is expressed as D (days) after
neuropathic injury (N) and h (hours) after gabapentin or compound 13 administration. *P <
0.05 (gabapentin), *P < 0.05 (compound 13 vs. pre-administration value (paired t-test), ♣P <
0.05 gabapentin vs. compound 13 (unpaired t-test).
The mechanical allodynia- alleviating effect of compound 12 increased gradually up to 3 h
after administration and decreased slightly at 5 h. However, the increase in mechanical
allodynia remained lower than that seen with gabapentin (Fig. 3A, B). Compound 13
exhibited significant pain relief efficacy after administration up to 3 h. The fast action of
compound 13 yielded 60-fold potency versus gabapentin. However, the increase in
9

mechanical allodynia disappeared by 5 h (Fig. 4A, B). Compounds 12 and 13 both showed a
low ability to alleviate temperature hypersensitivity (Figs. 3 and 4C, D).
In this study, we investigated the neuropathic pain-alleviating effects of a novel
series of arylsulfonylaminomethyl-3-(1-phenyl-5-isopropyl)pyrazole as 5-HT6R antagonists.
The final target compounds (3–13) were synthesized by coupling with 3-(1-phenyl-5-
isopropyl)pyrazol methylamine and various arylsulfonyl chlorides, and their biological
activities against 5-HT6R were evaluated in vitro. Among them, those with a fused bicyclic
aromatic ring, such as naphthalene- and quinolin-containing analogs, had stronger 5-HT6R
inhibitory activities compared with phenyl and mono-/di-substituted phenyl groups. We
identified two compounds (12 and 13) bearing 8-quinolin that exhibited potent 5-HT6R
antagonistic effects, and compound 13, 3-(1-p-fluorophenyl-5-isopropyl)pyrazol-methyl-(8-
quinoyl)sulfonamide, had rapid pain-alleviating efficacy against mechanical allodynia in an
SNL rat model.
Acknowledgments
This work was supported by the Korea Institute of Science and Technology (KIST)
Institutional Program (2E26850), the Original Technology Research Program (NRF-
2016M3C7A1904344) and the Ministry of Science, ICT & Future Planning (NRF-
2016M3C7A1913845), funded by the National Research Foundation of Korea (NRF). We
thank Dr. E.J. Lim for performing the in vivo efficacy tests.
References and notes
1. (a) Rufat, M.; Traiffort, E.; Arrang, J. M.; Tardivel,-Lacombe, J.; Diaz, J.; Leurs, R.;
Schwartz, J. C. Biochem. Biophys. Res. Commun. 1993, 193, 268. (b) Kohen, R.; Metcalf,
10

M. A.; Khan, N.; Druck, T.; Huebner, K.; Lachowicz, J. E.; Meltzer, H. Y.; Sibley, D. R.;
Roth, B. L.; Hamlin, M. W. J. Neurochem. 1996, 66, 46.
2. Monsma, F. J. Jr. ; Shen, Y.; Ward, R. P.; Hamblin, M. W.; Sibly, D. R. Mol. Pharmacol.
1993, 43, 320.
3. (a) Hirst, W. D.; Abrahamsen, B.; Blaney, F. E.; Calver, A. R.; Aloj, L.; Price, G. W.;
Medhurst, A. D. Mol. Pharmacol. 2003, 64, 1295. (b) Perez-Garcia, G.; Meneses, A.
Behav. Brain Res. 2008, 195, 17.
4. (a) Marazziti, D.; Rutigliano, G.; Catena-Dell′Osso, M.; Baroni, S.; Dell′Osso, L. Drugs
of the Future 2014, 39(2), 133-140. (b) Schaffhauser, H.; Rossé G. Curr. Top. Med. Chem.
2010, 10, 207. (c) Wesolowska, A. Pharmacol. Rep. 2010, 62, 564.
5. (a) Bruton, G.; Orlek, B. S.; Stemp, G. WO 2008/113818 A1. 2008. (b) Bruton, G.; Orlek,
B. S.; Stemp, G. US 2010/0041672 A1, 2010.
6. (a) Godinez-Chaparro, B.; Barragán-Iglesias, P.; Castañeda-Corral, G.; Rocha-González,
H. I.; Granados-Soto, V. PAIN, 2011, 12, 687-697. (b) Godinez-Chaparro, B.; LÓpez-
santillan, F. J.; Orduña, P.; Granados-soto, V. Neuroscience, 2012, 222, 379-391.
7. Jayarajan, P.; Nirogi, R.; Shinde, A.; Goura, V.; Babu, V. A,; Yathavakilla, S.;
Bhyrapuneni, G.; Pharmacol. Reports, 2015, 67, 934-942.
8. Devegowda, V. N.; Hong , J-R.; Cho, S.; Lim, E. J.; Choo, H.; Keum, G.; Rhim, H.;. Nam,
G. Bioorg. & Med. Chem. Lett. 2013, 23, 4696-4700.
9. Ivachtchenko, A. V.; Lavrovsky.; Ivanenkov, Y. A. Mol. Pharmaceutics, 2016, 13(3),
945-963.
10. (a) Liu, K. G.; Robichaud, A. J.; Int. Rev. Neurobiol. 2010, 94, 1-34. (b) Codony, X.;
Vela, J. M.; Ramirez, M. J. Curr. Opin. Pharmacol. 2011, 11, 94-100.
11. Galimberti, D.; Scarpini, E. Expert Opin. Investig. Drugs. 2015, 24(7), 981-987.
11

12. Functional cAMP assay was performed by previously reported method. Briefly, the
cAMP dynamic 2 HTRF kit (Cisbio, Avignon, France), which provides a homogeneous
high-throughput assay, was used to analyze cAMP levels. Cells incubated at 37°C in
5% CO2 and 95% air atmosphere were suspended in PBS containing 2 mM IBMX (3-
isobutyl-1-methylxanthine) and stimulated by 100 nM 5-HT for 30 min, with or without
pretreatment with compounds for 10 min. After 30 min, cAMP labeled with the dye d2
and anti-cAMP antibodies labeled with cryptate were added into the cell plates. After
incubated for 1 h at room temperature, the fluorescence intensity of accumulated cAMP
level was measured at 314 nm excitation, and 668 and 620 nm emission using a
Flexstation 3 microplate reader (Molecular Devices, Downingtown, PA).
13. Choi, G-H.; Song, C.; Shin, D.; Park, S.; Biochim. Biophys. Acta. 2011, 1808, 1560.
14. CYP450 remaining test was conducted using the Vivid® CYP450 Screening Kit Protocol
(Invitrogen, Carlsbad, CA).
15. (a) Zhou, S-F.; Liu, J-P.; Lai, X-S. Curr. Med. Chem. 2009, 16, 2661.; (b) Zhou, S-F.;
Curr. Pharm. Des. 2008, 14, 990.
16. Kim, S. H.; Chung, J. M. Pain 1992, 50, 355.
17. Representative spectroscopic data of selected compounds that were evaluated for
biological activity: 12 1H NMR (CDCl3, 400 MHz): δ 8.95−8.94 (m, 1H), 8.43 (d, J = 7.3
Hz, 1H), 8.21 (d, J = 8.3 Hz, 1H), 8.00 (d, J = 8.2 Hz, 1H), 7.60 (t, J = 7.5 Hz, 1H),
7.49−7.46 (m, 1H), 7.09 (d, J = 8.0 Hz, 2H), 6.82 (d, J = 6.3 Hz, 1H), 5.92 (S, 1H), 4,20
(d, J = 6.4 Hz, 2H), 2.30 (d, J = 7.1 Hz, 2H). 1.67−1.59 (m, 1H), 0.75 (d, J = 6.5, 6H) 13C
NMR (CDCl3, 100 MHz): δ 151.1, 148.0, 143.7, 139.5, 136.8, 136.2, 133.2, 130.9, 129,0,
128.7, 127.9, 125.5, 125.4, 122.1, 105.2, 41.6, 34.9, 28.2, 22.3 HRMS (ESI-TOF) m/z
calcd for C23H24N4O2S [M+H]+: 421.1624.. found: 421.1086. 13 1H NMR (CDCl3, 400
MHz): δ 8.99 (dd, J = 4.2, 1.7 Hz, 1H), 8.47 (dd, J = 7.3, 1.3 Hz, 1H), 8.26 (dd, J = 8.3,
12

1.7 Hz, 1H), 8.04 (dd, J = 8.2, 1.3 Hz, 1H), 7.65 (t, J = 7.7 Hz, 1H), 7.52 (dd, J = 8.3, 4.1
Hz, 1H), 7.11 (d, J = 6.6 Hz, 4H), 6.82 (t, J = 6.3 Hz, 1H), 6.0 (s, 1H), 4,20 (d, J = 6.4 Hz,
2H), 2.31 (d, J = 7.1 Hz, 2H). 1.77−1.60 (m, 1H), 0.80 (d, J = 6.6, 6H) 13C NMR (CDCl3,
100 MHz): δ 163.2, 160.3, 151.1, 148.4, 143.9, 143.4, 136.8, 136.4, 135.7, 133.1, 131.0,
128.9, 127.4, 127.3, 125.6, 122.1, 116.0, 115.7, 105.2, 41.6, 34.9, 28.3, 22.3. HRMS
(ESI-TOF) m/z calcd for C23H23FN4O2S [M+H]+: 439.1530.. found: 439.0596.
13

Graphical Abstracts
5-HT6R antagonist
Compound 12 and 13 having quinoline group showed neuropathic pain alleviating effects in
vivo as well as 5-HT6R antagonistic effect in vitro.
14