Synthesis and the 5-HT6 receptor antagonistic effect of 3-arylsulfonylamino-5,6-dihydro-6-substituted pyrazolo[3,4]pyridinones for neuropathic pain treatment

Article abstract of DOI:10.1016/j.bmcl.2013.05.100

A novel series of 3-arylsulfonylamino-5,6-dihydro-6-substituted-1H- pyrazolo[3,4-c]pyridine-7-ones was designed and synthesized as 5-HT₆ ligands. Among the derivatives synthesized, the lead compound, 12b, having piperidine functionality at the 6-position and (1-naphthyl)sulfonamino at the 3-position of the core structure showed the most potent 5-HT₆ inhibitory activity in vitro, good stability without CYP liability, and good neuropathic pain alleviation activity in a rat animal model.

Full text of DOI:10.1016/j.bmcl.2013.05.100

Bioorganic & Medicinal Chemistry Letters xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and the 5-HT6 receptor antagonistic effect
of 3-arylsulfonylamino-5,6-dihydro-6-substituted
pyrazolo[3,4]pyridinones for neuropathic pain treatment
Vani Nelamane Devegowda a, Jin-Ri Hong b, Sungjin Cho c, Eun Jeong Lim a, Hyunah Choo a,
Gyochang Keum a, Hyewon Rhim c, , Ghilsoo Nam a,
⇑
⇑
a Center for Neuro-Medicine, Brain Science Institute, Korea Institutes of Science and Technology (KIST), Seoul 136-791, Republic of Korea
b School of Science, University of Science and Technology, Daejeon 305-333, Republic of Korea
c Center for Neuroscience, Brain Science Institute, Korea Institutes of Science and Technology (KIST), Seoul 136-791, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 3-arylsulfonylamino-5,6-dihydro-6-substituted-1H-pyrazolo[3,4-c]pyridine-7-ones was
designed and synthesized as 5-HT6 ligands. Among the derivatives synthesized, the lead compound, 12b,
having piperidine functionality at the 6-position and (1-naphthyl)sulfonamino at the 3-position of the
core structure showed the most potent 5-HT6 inhibitory activity in vitro, good stability without CYP lia-
bility, and good neuropathic pain alleviation activity in a rat animal model.
Received 24 April 2013
Revised 28 May 2013
Accepted 30 May 2013
Available online xxxx
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Arylsufonylamino-5,6-dihydro-
pyrazolo[3,4-c]pyridine-7-one
Synthesis
5-HT6 receptor antagonist
Neuropathic pain
The 5-HT6 receptor is a novel target for cognition enhancement,
Alzheimer disease (AD), obesity, anxiety, depression, and neuro-
pathic pain.1 This receptor was discovered in 1993 by several
groups as a serotonin receptor that is positively coupled to Gs pro-
tein and activates c-AMP.2 The 5-HT6 receptor is distributed mainly
in the central nervous system (CNS) such as the stratum, nucleus
accumbens, olfactory tubercle, and cortex, with moderate expres-
sion in the hippocampus, amygdala, cerebellum, and thalamus.3
Several rodent behavioral model studies have demonstrated that
blockade of the 5-HT6 receptor function improves cognition.4
Therefore, many pharmaceutical companies and research groups
have developed selective 5-HT6 antagonists for the treatment of
cognitive impairments such as AD and schizophrenia. Recently,
some patents have described that 5-HT6 antagonists showed po-
tent activity as treatments for pain and irritable bowel syndrome.5
5-HT6 receptor agonist/antagonist at the peripheral and dorsal
ganglion/spinal cord participate in the development and mainte-
nance of secondary mechanical allodynia and hyperalgesia in rat.6
Since the first 5-HT6 antagonists, Ro 04-6790 (1) and Ro 63-
0563 (2) having arylsulfonyl amide moieties, were discovered ser-
endipitously in random screening by scientists at Roche in 1998,
arylsulfonyl-related scaffolds still have common structural
features.7 Currently, several 5-HT6 antagonists possessing arylsufo-
nyl moieties such as AVN-211(3), SB-737050A, SB-742457,
BVT-74316, and SAM-532 (Fig. 1) are in clinical trials (Phase I or
Phase II).
Several researchers have reported that the basic pharmaco-
phore model of 5-HT6 ligands have two aromatic sites, which could
be involved in secondary binding such as
p-stacking and the pri-
mary binding site at the aspartate residue (Fig. 2).8 Based on the
explored structure and pharmacophore model, we have designed,
synthesized, and evaluated the biological activity of a novel series
of 5,6-dihydro-1H-pyrazolo[3,4-c]pyridine-7-ones having various
arylsulfonylamino groups at the 3-position, and introduced aryl
groups such as p-toluyl, p-methoxy phenyl, and cyclic amines such
as piperidine at the 6-position (Fig. 3).
Synthesis of the final compounds was carried out according to
Schemes 1 and 2. The 3-arylsulfonylamino-5,6-dihydro-pyrazolo
[3,4-c]pyridine-7-one derivatives having
a p-toluyl (5) or p-
methoxyphenyl (6) group were prepared from the corresponding
6-amino-5,6-dihydro-pyrazolo[3,4-c]pyridine-7-one intermediate
4 through a coupling reaction with various corresponding aryl-
sulfonyl chlorides in good yield. Alkylation of aniline compound
1 followed by reaction with 1-bromo-3-cyano propane provided
cyanopropylated amine 2. Next, the reaction with diethylacetoacetate
⇑
Corresponding authors. Tel.: +82 2 958 5166; fax: +82 2 958 5189.
E-mail addresses: hrhim@kist.re.kr (H. Rhim), gsnam@kist.re.kr (G. Nam).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.100
Please cite this article in press as: Devegowda, V. N.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.05.100

2
V. N. Devegowda et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
H3C
N
H3C
N
CH3
N
NH
N
NH
N
O
SCH3
O
O
O
S
S
H3C
N
HN
CH3
N
H2N
N
S
O
H
H
O
NH2
NH2
2
1
3
AVN-211
Ro 63-0563
Ro-04-6790
H
N
NH
OCH3
O
H3C
N
N
N
O
OCH3
CH3
NH
S
O
H
N
O
S
S
Cl
O
O
S
O
O
4
5
6
SB-737050A
SB-742457
BVT-74316
H
N
H3C
N
I
CH3
N
H
N
N
N
S
H3C
O
S
O
O
O
O
H3CO
7
8
SAM-532
SB-258585
Figure 1. Structure of select 5-HT6 antagonists in clinical trials (Phase I and Phase II).
Hydrophobic
site
transformed to cyanopropylated derivative 8, followed by
a
condensation reaction with diethylacetoacetate to give 4-cyano-
2,3-diketo piperazine 9 in moderate yield. To prepare 3-arylsulf-
onylated-5,6-dihydro-pyrazolo[3,4-c]pyridine-7-ones 11 and 12,
the intermediate 10 was synthesized by treatment with hydrazine
hydrate and glacial acetic acid in ethanol. A subsequent nucleophilic
substitution reaction was employed as the amino intermediate 10,
and a similar synthetic sequence was followed by Boc-deprotection
with TFA, resulting in the desired final compounds (11a, b, 12a–g).
All of the 3-arylsulfonylamino-5,6-dihydro-pyrazolo[3,4-]pyri-
dine-7-one derivatives having an aromatic group (5a–j, 6a–d) or
piperidinyl group (11a, b, 12a–g) at the 6-position were evaluated
for 5-HT6 receptor antagonistic activity in a functional cyclic aden-
osine monophosphate (cAMP) assay using HeLa cells stably
expressing the human 5-HT6 receptor.9 The results are summa-
rized in Table 1.
According to the results, we examined the structure–activity
relationship of the dihydro-pyrazolo[3,4-c]pyridine-7-one
analogs 5 and 6 having toluyl and p-methoxy phenyl groups at
the 6-position depending on various arylsulfonylamine func-
tionalities. Among the compounds bearing substituted phenyl
groups at the 6-position of the core skeleton, compound 6b
(R1 = OCH3, X = 3-FPh) displayed the most potent activity
Double acceptor
Proton
donor
O
O
S
N
Ar
H
Hydrophobic
site
Figure 2. Structural model for 5-HT6 pharmacophore.
O
H
6
N
1 N
R
2
7
N
O
S
5
3
O
4
N
H
Ar
CH3
OCH3
NH
NH
R
=
Ar = Phenyl, Substituted Phenyl, Naphthyl
(IC50 = 0.58 0.15 lM) and was superior to the corresponding
Figure 3. Designed 3-arylsulfonylamino-5,6-dihydro-6-substituted-pyrazolo[3,4-c]
5c (R1 = CH3, X = 3-FPh). Other compounds (5a, 5b, 5f, and 6c)
bearing fluoro or chloro phenyl groups displayed lower activity.
However, compound 5j, bearing a toluyl group, and a 2-methyl-
3-chlorophenyl sulfonyl group, also showed good activity
(IC50 = 1.66 0.25). The introduction of the methoxy group
retained similar potency between 5g and 6d, whereas the
dimethoxy compound 5h showed poor activity.
From the results of six-membered exocyclic amine piperidine
derivatives 11 (n = 0) and 12 (n = 1), the 1-naphthalene sulfonyl
derivatives 11b (IC50 = 0.77 0.9) and 12b (IC50 = 0.31 0.1)
showed good activities. However, the 2-naphthyl compound 12c
showed lower activity than the 1-napthyl compound 12b. This
means that the geometry of the aromatic ring might play an impor-
tant role in 5-HT6 inhibitory activity.
pyridine-7-ones.
under basic conditions gave cyclic 4-cyano-2,3-diketopiperidine 3,
followed by reductive cyclization with hydrazine hydrate to give
3-amino-5,6-dihydro-pyrazolo[3,4-c]pyridinone 4 almost quantita-
tively. The coupling reaction of amine 4 with commercially available
substituted phenylsulfonyl chlorides, including fluorophenyl sulfonyl
chloride, chlorophenyl sulfonyl chloride, methoxyphenyl chloride,
(2-methyl-5-methyl)phenyl sulfonyl chloride, (2-methyl-5-chloro)-
phenyl sulfonyl chloride, and trifluoromethylphenyl sulfonyl chlo-
ride, provided 3-arylsulfonylamino-5,6-dihydro-6-arylsubstituted-
pyrazolo[3,4-c]pyridine-7-ones (5a–j, 6a–d).
Analogs with piperidine or piperidynylmethyl introduced to the
6-position (11, 12) instead of an aromatic group were synthesized
according to Scheme 2. Boc-protected piperidinyl amine 7 was
Monosubstituted phenyl derivatives such as the methyoxyphe-
nyl (11a, 12a), chlorophenyl (12e), and t-butylphenyl (12g)
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V. N. Devegowda et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
3
O
O
H
N
CN
a
R1
NH2
R1
N
CN
b
R1
O
2
1
3
R1
R1
O
H
N
H
N
N
N
c
d
N
N
O
S
O
NH
NH2
5, R1 CH
4
=
X
3
6, R1 OCH
=
3
Scheme 1. Reagents and conditions: (a) Br(CH2)3CN, K2CO3, CH3CN, reflux, 2 days, yield 80–85%; (b) diethylacetoacetate, NaOEt, EtOH, reflux, overnight, yield 60–65%; (c)
hydrazine hydrate, glacial AcOH, EtOH, 65 °C, yield 95–99%; (d) arylsulfonyl chloride, pyridine, DMAP, DCM, 0 °C to room temperature, yield 81–95%.
O
NH CN
n
NH2
a
n
O
b
N
N
N
N
Boc
n
Boc
N
Boc
O
CN
8
7
9
O
H
H
N
N
n
N
N
c
d, e
n
N
N
HN
Boc
O
S
NH2
O
Ar
NH
10
11, n = 0
12, n = 1
Scheme 2. Reagents and conditions: (a) Br(CH2)3CN, K2CO3, CH3CN, reflux, 2 days, yield quantitative; (b) diethylacetoacetate, NaOEt, EtOH, reflux, overnight, yield 60–65%;
(c) hydrazine hydrate, glacial AcOH, EtOH, 65 °C, yield 85–89%; (d) arylsulfonyl chloride, pyridine, DMAP, DCM, 0 °C to room temperature (RT), yield 81–95%; (e) TFA/CH2Cl2,
0 °C to RT.
Table 1
Percent inhibition and IC50 values of the 6-substituted 3-arylsulfonylamino-5,6-dihydro-pyrazolo[3,4-c]pyridine-7-one derivatives 5a–j, 6a–e, 11a, b and 12a–g against the 5-HT6
receptor
R1
O
O
H
N
H
N
N
N
n
N
N
O
S
HN
O
S
O
Ar
O
N
H
NH
5, R1 = CH3
X
11, n = 0
12, n = 1
6, R1 OCH3
=
Compd
X
Inhibition
(%, 10 M)
5-HT6 functional cAMP
Compd
Ar
Inhibition
(%, 10 M)
5-HT6 functional cAMP
assay IC50 M)
l
assay IC50
(lM)
l
(l
5a
5b
5c
5d
5e
5f
5g
5h
5i
H
2-F
3-F
4-F
3-CF3
3-Cl
3-OMe
2,5-(OMe)2
2-F, 5-Me
2-Me, 3-Cl
H
3-F
4-F
3-OMe
23.5 12.2
29.6 10.6
18.1 6.9
67.9 5.9
69.0 4.3
5.4 5.1
11a
11b
12a
12b
12c
12d
12e
12f
p-Methoxyphenyl
1-Naphthyl
p-Methoxyphenyl
1-Naphthyl
2-Naphthyl
3-Chloro-2-methylphenyl
3-Chlorophenyl
2-Fluoro-5-methylphenyl
4-(t-Butyl)phenyl
p-Methoxyphenyl
26.1 6.9
89.5 7.7
15.1 6.4
94.1 3.0
40.9 6.8
51.7 9.7
31.0 13.9
30.6 4.8
11.5 5.1
97.7 1.9
0.77 0.9
0.31 0.1
5.22 1.76
3.28 0.83
63.2 4.8
3.8 3.6
6.77 1.29
25.9 11.8
92.2 8.8
30.6 9.2
95.2 7.0
30.6 9.2
63.3 3.2
12g
SB-258585
5j
1.66 0.25
0.58 0.15
7.08 0.80
0.019 0.01
6a
6b
6c
6d
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4
V. N. Devegowda et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
Table 2
surgical manipulation, the effect of 12b on neuropathic pain
including cold and mechanical allodynia was evaluated.
Inhibitory effects of selected compounds on specific CYP enzyme activities in cDNA-
expressing CYP microsomes
Oral administration of compound 12b exhibited a good allevia-
tion effect on cold allodynia. The effect of 12b on cold allodynia
was significant at 1 h after administration and was sustained for
5 h. We used gabapentin as a reference, which is the most potent
neuropathic pain drug in clinical use. Compound 12b showed five
fold stronger potency than gabapentin (Fig. 4A, B). In mechanical
allodynia, compound 12b also showed a pain inhibitory effect,
but the efficacy after 5 h had almost disappeared (Fig. 4C, D).
In summary, we identified a novel series of 6-N-aryl and 6-N-
piperidyl amine substituted 3-arylsulfonylamino-5,6-dihydro-1H-
pyrazolo[3,4-c]pyridine-7-ones as 5-HT6 ligands. The synthesis
and SAR of this series compounds was reported in the current
study. The 6-N substituent, whether phenyl or cyclic amine, exhib-
ited little effect on activity, as demonstrated by the low activity of
various phenyl substituted derivatives at the 4-position, except for
compounds 5j (3-Cl-2-MePh) and 6b (3-FPh). However, larger aro-
matic groups like naphthalene at the 4-position play an important
role in the inhibition of the 5-HT6 receptor. The best combination
of substituents was 6-N-4-methylpiperidine with 1-naphthalene
sulfonamide at the 4-position, as identified by the best compound
12b (IC50 = 310 nM). The lead compound 12b exhibited good met-
abolic stability, CYP450 liability, and in vivo neuropathic pain alle-
viation effect. Based on these results, we will conduct further
optimization studies with this class of compounds for the develop-
ment of neuropathic pain treatments.
Compd HLMa stability
remaining %
% Control of CYP-450b (10
1A2c 2D6d 2C9e 2C19f
l
M)
3A4g
6b
11b
12b
96.32
100.88
90.85
63.49 87.85 78.56
67.66 95.13 94.54
81.81 19.52
99.82 16.74
79.14 97.19 98.42 103.62 17.78
a
HLM, human liver metabolic.
Values represent the remaining % activities and the mean SD from triplicate
experiments.
b
c
a-Naphthoflavon.
d
e
f
Quinidine.
Sulfaphenazol.
Miconazole.
Ketoconazole.
g
displayed lower activity. The 3-chloro-2-methylphenyl sulfonyl
derivative 12d presented twofold lower activity compared to the
corresponding aromatic substituted derivative 5j.
Some of the selected potent compounds were profiled for their
metabolic stability10 and human P450 enzyme inhibitory activity11
(Table 2). Compounds136b, 11b, and 12b showed good stability,
with 96.32%, 100.88%, and 90.85% concentration, respectively,
remaining with human hepatic microsomes at 30 min. Also, they
also show high remaining activities for isozymes of human CYP
450, except for 3A4, indicating that it is not involved in drug
metabolism and drug interaction.
Acknowledgments
The selected three compounds exhibited similar properties for
stability and CYP inhibition; therefore, the most potent compound
in vitro (12b) was selected and evaluated for in vivo neuropathic
pain relief activity in a rat model (Fig. 4). Behavioral testing for
neuropathic pain according to the spinal nerve ligation model
(SNL model)12 was performed with rats (n = 25). After 14 days of
This research was supported by the Basic Science Research Pro-
gram through the National Research Foundation of Korea (NRF)
funded by the Ministry of Education, Science and Technology
(KRF-2010-0021233) and the KIST Institutional program
(2E23770 and 2E23870).
A. Cold allodynia
B. Cold allodynia (%MPE)
12b 100 mg/kg (n=4)
Gabapentin 100 mg/kg (n=4)
120
100
80
60
40
20
0
100
80
*
60
*
*
40
*
*
20
0
*
Base
1h
3h
5h
Pre N14D 1h
3h
5h
Experimental time (hour)
Experimental time (day or hour)
C. Mechanical allodynia
D. Mechanical allodynia (%MPE)
100
15
12
9
*
*
*
80
60
40
20
0
6
3
0
Base
1h
3h
5h
Pre N14D 1h
3h
5h
Experimental time (hour)
Experimental time (day or hour)
Figure 4. Effect on cold allodynia (A and B) and mechanical allodynia (C and D) after oral administration of gabapentin (s, 100 mg/kg, n = 4) and 12b (d, 100 mg/kg, n = 4) on
neuropathic pain-induced rats. Experimental time is expressed as D for days after neuropathic injury (N) and h for hours after gabapentin or 12b administration. ⁄P < 0.05
(gabapentin), ⁄P < 0.05 (12b) versus pre-administration value (paired t-test), |P < 0.05 gabapentin vs 12b (unpaired t-test).
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V. N. Devegowda et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
5
5-HT6 receptor. To analyze cAMP levels, the cAMP dynamic 2 HTRF kit (Cisbio,
Avignon, France), which provides a homogeneous high-throughput assay, was
used. 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. The
plates were incubated at room temperature for 1 h. The fluorescence intensity
of accumulated cAMP level was measured at 314 nm excitation, and 668 and
620 nm emission using a FlexstationÒ 3 microplate reader (Molecular Devices,
Downingtown, PA).
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13. Representative spectroscopic data of selected compounds that were evaluated for
biological activity: 6b 1H NMR (DMSO, 300 MHz): d 13.53 (s, 1H, NH), 10.39 (s,
1H, NH), 7.68–7.49 (m, 4H), 7.26 (d, J = 8.8 Hz, 2H), 6.95 (d, J = 8.8 Hz, 2H), 3.86
(t, J = 6.6 Hz, 2H), 3.76 (s, 3H), 2.74 (t, J = 6.6 Hz, 2H) 13C NMR (DMSO, 75 MHz):
d 157.9, 157.8, 141.2, 135.4, 134.4, 132.0, 131.9, 127.6, 123.5, 120.5, 120.2,
116.0, 114.4, 114.0, 55.7, 52.1, 20.0; 11b 1H NMR (DMSO, 300 MHz): d 10.32 (s,
1H), 8.82 (br, 1H, NH), 8.74 (br, 1H, NH), 8.32 (s, 1H), 8.10 (d, J = 8.4 Hz, 2H),
8.02 (d, J = 7.9 Hz, 1H), 7.77 (d, J = 8.6 Hz, 1H), 7.72–7.61 (m, 2H), 4.57 (t,
J = 12.0 Hz, 1H), 3.34–3.30 (m, 4H), 3.00 (q, J = 11.0 Hz, 2H), 2.54 (t, J = 6.2 Hz,
2H), 1.96 (q, J = 11.9 Hz, 2H), 1.67 (d, J = 11.7 Hz, 2H) 13C NMR (DMSO,
75 MHz): d 158.2, 140.5, 137.8, 134.9, 134.7, 132.0, 129.6, 129.5, 129.3, 128.2,
128.0, 128.0, 122.9, 115.3, 47.3, 43.2, 42.5, 25.8, 19.8; 12b 1H NMR (DMSO,
300 MHz): d 10.30 (s, 1H), 8.96 (br, 1H, NH), 8.72 (br, 1H, NH), 8.32 (s, 1H), 8.10
(d, J = 8.7 Hz, 2H), 8.02 (d, J = 7.9 Hz, 1H), 7.77 (dd, J = 1.7, 8.6 Hz, 1H), 7.72–
7.62 (m, 2H), 3.43 (t, J = 6.7 Hz, 2H), 3.28–3.19 (m, 4H), 2.77 (q, J = 9.9 Hz, 2H),
2.52 (t, J = 6.5 Hz, 2H), 1.87 (br, 1H), 1.69 (d, J = 12.4 Hz, 2H), 1.34 (q,
J = 11.4 Hz, 2H) 13C NMR (DMSO, 75 MHz):
132.0, 129.6, 129.5, 129.3, 128.2, 128.0, 122.9, 115.4, 50.2, 48.4, 43.1, 32.5,
d
158.6, 140.5, 137.8, 134.6,
26.4, 19.5. HR-FABMS calcd for
440.17547.
C
22H25N5O3S [M+H]+: 440.17509. found:
9. Functional cAMP assay: All of the synthesized compounds were evaluated
against adenylate cyclase activity using HeLa cells stably expressing the human
Please cite this article in press as: Devegowda, V. N.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.05.100