Discovery and Development of 3-(6-Chloropyridine-3-yloxymethyl)-2-azabicyclo[3.1.0]hexane Hydrochloride (SUVN-911): A Novel, Potent, Selective, and Orally Active Neuronal Nicotinic Acetylcholine α4β2 Receptor Antagonist for the Treatment of Depression

Article abstract of DOI:10.1021/acs.jmedchem.9b00790

A series of chemical optimizations guided by in vitro affinity at the α4β2 receptor in combination with selectivity against the α3β4 receptor, pharmacokinetic evaluation, and in vivo efficacy in a forced swim test resulted in identification of 3-(6-chloropyridine-3-yloxymethyl)-2-azabicyclo[3.1.0]hexane hydrochloride (9h, SUVN-911) as a clinical candidate. Compound 9h is a potent α4β2 receptor ligand with a K_i value of 1.5 nM. It showed >10 μM binding affinity toward the ganglionic α3β4 receptor apart from showing selectivity over 70 other targets. It is orally bioavailable and showed good brain penetration in rats. Marked antidepressant activity and dose-dependent receptor occupancy in rats support its potential therapeutic utility in the treatment of depression. It does not affect the locomotor activity at doses several folds higher than its efficacy dose. It is devoid of cardiovascular and gastrointestinal side effects. Successful long-term safety studies in animals and phase-1 evaluation in healthy humans for safety, tolerability, and pharmacokinetics paved the way for its further development.

Full text of DOI:10.1021/acs.jmedchem.9b00790

Subscriber access provided by Gothenburg University Library
Article
Discovery and Development of 3-(6-Chloropyridine-3-yloxymethyl)-2-
azabicyclo[3.1.0]hexane hydrochloride (SUVN-911): A Novel, Potent,
Selective and Orally Active Neuronal Nicotinic Acetylcholine
#4#2 Receptor Antagonist for the Treatment of Depression
Ramakrishna Nirogi, Abdul Rasheed Mohammed, Anil K. Shinde, Srinivasa Rao Ravella, Narsimha
Bogaraju, Ramkumar Subramanian, Venkat Reddy Mekala, Raghava Choudary Palacharla, Nageswararao
Muddana, Jagadeesh Babu Thentu, Gopinadh Bhyrapuneni, Renny Abraham, and Venkat Jasti
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00790 • Publication Date (Web): 06 Feb 2020
Downloaded from pubs.acs.org on February 6, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Discovery and Development of 3-(6-Chloropyridine-
3
-yloxymethyl)-2-azabicyclo[3.1.0]hexane
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
hydrochloride (SUVN-911): A Novel, Potent,
Selective and Orally Active Neuronal Nicotinic
Acetylcholine α4β2 Receptor Antagonist for the
Treatment of Depression
Ramakrishna Nirogi,* Abdul Rasheed Mohammed, Anil K. Shinde, Srinivasa Rao Ravella,
Narsimha Bogaraju, Ramkumar Subramanian, Venkat Reddy Mekala, Raghava Choudary
Palacharla, Nageswararao Muddana, Jagadeesh Babu Thentu, Gopinadh Bhyrapuneni, Renny
Abraham, Venkat Jasti.
Discovery Research, Suven Life Sciences Ltd, Serene Chambers, Road-5, Avenue-7, Banjara
Hills, Hyderabad 500 034, India
Tel.: +91-40-23556038; Fax: +91-40-23541152; E-mail: nvsrk@suven.com
Keywords-α4β2 antagonist, Structure-activity relationship, receptor occupancy, forced swim test,
antidepressant, depression
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ABSTRACT. A series of chemical optimizations guided by in vitro affinity at α4β2 receptor in
combination with selectivity against α3β4 receptor, pharmacokinetic evaluation and in vivo
efficacy in forced swim test resulted in identification of 3-(6-chloropyridine-3-yloxymethyl)-2-
azabicyclo[3.1.0]hexane hydrochloride (9h, SUVN-911) as a clinical candidate. Compound 9h is
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a potent α4β2 receptor ligand with K value of 1.5 nM. It showed >10 µM binding affinity
i
towards ganglionic α3β4 receptor apart from showing selectivity over 70 other targets. It is orally
bioavailable and showed good brain penetration in rats. Marked antidepressant activity and dose
dependent receptor occupancy in rats supports its potential therapeutic utility in the treatment of
depression. It does not affect the locomotor activity at doses several folds higher than its efficacy
dose. It is devoid of cardiovascular and gastrointestinal side effects. Successful long term safety
studies in animals and Phase-1 evaluation in healthy humans for safety, tolerability and
pharmacokinetics paved the way for its further development.
INTRODUCTION
Depression is a common and severe psychological condition with changes in mood, affecting
1
approximately 151 million people worldwide. It is characterized by loss of interest, pleasure,
2
feelings of guilt, low self-worth, low confidence, disturbed sleep or appetite, poor concentration,
3
,4
and is associated with suicidal thoughts. Most of the medications used to treat depression target
either serotonergic and noradrenergic transmitter systems or inhibit monoamine oxidase to
reduce the degradation of serotonin (5-HT) and norepinephrine (NE).
Pitfalls of existing therapy with antidepressants are slow onset of action accompanied by
serious side effects like cognitive impairment, loss of interest in sex and higher non responders to
5
the treatment. Therefore, there is an unmet and urgent medical need for potent, safe and viable
pharmacotherapy with differentiated mechanism of action. Literature evidence implicates a role
2
ACS Paragon Plus Environment

Page 3 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
of cholinergic signaling in depressive symptoms. Physostigmine is an acetyl cholinesterase
inhibitor, potentiates cholinergic transmission and produces depressive-like symptoms in
humans. Conversely, α4β2 nicotinic acetylcholine receptor (nAChR) antagonists, or compounds
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
that induce receptor desensitization have shown to reduce depressive symptoms.6,7,8 Presynaptic
neuronal nAChR desensitization induces monoamines release including NE, dopamine (DA),
9
and 5-HT. Some of the classical antidepressants including tricyclic antidepressants and selective
serotonin reuptake inhibitors (SSRIs) inhibit nAChRs.10,11 This supports the hypothesis that
modulation of the cholinergic system through desensitization/inactivation of nAChR could hold
promise for the development of new antidepressants. Further, nAChRs have been investigated
for developing the compounds/modulators that may potentially treat various central nervous
system disorders including depression.12,13
Mecamylamine, a non-selective and non-competitive antagonist of nAChRs originally
developed for treating hypertension has been investigated extensively for antidepressant
activity.14,15 The pharmacological studies suggested that the S-enantiomer of mecamylamine
(TC-5214) is more efficacious antagonist at α4β2 nAChRs than mecamylamine itself and could
act as allosteric modulator at α4β2 nAChRs.16 TC-5214 exhibited robust anxiolytic- and
antidepressant-like effects in animal models. This behavioral activity was attributed to its
1
7
antagonist effects at α4β2 nAChRs. In Phase 2 clinical study, TC-5214 as an augmentation
strategy to antidepressants in patients who were inadequate responders to SSRI citalopram
showed an average of 6 point improvement on primary efficacy endpoint, the Hamilton
Depression Rating Scale (HAM-D). It also showed improvement on other secondary measures.18
However, in Phase 3 clinical trial, TC-5214 as an adjunctive therapy in patients with major
depressive disorder (MDD) who were inadequate responders to SSRI or serotonin-
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
norepinephrine reuptake inhibitors (SNRI), failed to meet the primary efficacy endpoint.19
Possible reasons for failure were reported as higher placebo effect, combination treatment and
non-selectivity of TC-5214. Apart from TC-5214, other reported α4β2 nAChR ligands such as
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
20
21
22
23
TC-2216, varenicline, sazetidine-A, sazetidine-A-isoxazole analogue (I), and cyclopropyl
2
4
sazetidine analogue (II) showed antidepressant activity in pre-clinical models of depression
(Figure 1) showing the potential of α4β2 nAChR ligands in treating depression.
Ganglionic type nAChR α3β4 mediates the effects on temperature, locomotor activity and
seizures elicitation and is also believed to be partially responsible for the cardiovascular and
gastrointestinal liabilities of nicotine.25,26 Hence it is important to achieve selectivity for central
α4β2 nAChRs over ganglionic α3β4 nAChRs. The potent α4β2 nAChR ligands with high
27
selectivity over α3β4 may offer promising efficacy with fewer side effects. It is reported that in
nAChR ligand ABT-089, the 2-methyl substituent on the pyridine ring (Figure 1) contributed for
2
8
its reduced propensity to activate peripheral ganglionic-like nAChRs. It is also reported that,
the constrained compounds such as TC-2216 (Figure 1) shows high potency for α4β2 nAChR
and weaker potency towards α3β4 nAChR as they possess shorter distance between secondary
2
9
30
amine and pyridinyl nitrogen. Hanessian et al reported that, introduction of cyclopropyl group
at 4,5-position of L-proline introduces conformational constraint in the molecule and causes
considerable “flattening” of the pyrrolidine ring relative to L-proline. We hypothesized that the
flattening of the pyrrolidine ring may have effect on the distance between secondary amine and
pyridinyl nitrogen and may offer selectivity against ganglionic α3β4 nAChR. Based on this
hypothesis, constraining of 3-pyridinyl ether class of nAChR ligands were conceptualized. The
cyclopropyl group was introduced on all available places in 5-memebered pyrrolidine central
core and at 5,6-position in 6-membered piperidine central core to evaluate its impact on the
4
ACS Paragon Plus Environment

Page 5 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
affinity for at both central α4β2 and ganglionic α3β4 nAChRs. Thus, 4,5-methanopyrrolidinol
derivatives30,31,32 (Series 1, 2 and 3), 3,4-methanopyrrolidinol derivatives (Series 4), 2,3-
methanopyrrolidinol derivatives (Series 5), 5,6-methanopiperidinol derivatives (Series 6) and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
,5-methanopyrrolidinol derivatives with extended distance between pyrrolidine and pyridinyl
ring (Series 7) were synthesized (Figure 1) and screened for their in vitro affinities towards α4β2
and α3β4 nAChRs. The focused structure-activity relationships around these series covering in
vitro affinity, selectivity over α3β4 nAChR, metabolic stability, pharmacokinetics, efficacy in
forced swim test (FST) model of depression, target engagement and safety assessments afforded
a potent, selective and safe α4β2 nAChR clinical candidate 9h (SUVN-911).
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N
N
H CHN
O
N
3
HN
N
NH
N
N
CH3
O
NHCH3
N
H
N
CH3
CH3
Nicotine
Physostigmine
TC-5214
TC-2216
Varenicline
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OH
N
O
HN
O
HN
HN
O
O
OH
N
OH
N
N
Sazetidine-A
I
II
R
R
R
O
O
O
R
O
O
N
N
N
N
N
H
N
N
N
N
N
H
R1
H
ABT-089
Series 1
Series 2
Series 3
Series 4
R
N
R
O
O
O
Cl
O
N
N
H
N
N
H
R
N
N
HCl
H
H
N
Series 5
Series 6
Series 7
9h, SUVN-911
R = H, CH3
R = H, alkyl, halo, CN,
alkyne, heterocyclic
1
Figure 1. Reported α4β2 nAChR ligands along with current series of compounds.
Chemistry.
The general synthetic strategy used for the synthesis of the new compounds reported here is
summarized in Schemes 1-7. The starting materials L-pyroglutaminol (1), L-pyroglutamic acid
and substituted hydroxy pyridines were sourced from commercial vendors whereas starting
materials 3833 and 4334 were synthesized as reported in the literature. The synthesis of
compounds 9 commenced with reaction of (S)-5-(hydroxymethyl)-2-pyrrolidinone 1 (Scheme 1)
6
ACS Paragon Plus Environment

Page 7 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
with TBDPS-Cl which afforded compound 2. Amino group protection in compound 2 with Boc
anhydride afforded compound 3. The super hydride reduction of compound 3 yielded compound
4 which on treatment with TFAA and Hunig’s base afforded olefin 5. Olefin 5 on treatment with
diethyl zinc and diiodomethane gave a mixture of trans isomer 6a and cis isomer 6b in ~95:5
ratio, respectively. The diastereomers 6a and 6b were separated by silica gel column
chromatography. The assigned stereochemistry of 6a has been compared and confirmed by
synthesizing it as per earlier reported protocol.30 Compound 6a after TBDPS deprotection
afforded compound 7 which was on further reaction with substituted 3-hydroxypyridines under
Mitsunobu reaction conditions afforded compounds 8. The selection of different substituted 3-
hydroxypyridines was based on SAR, their use in the prior art compounds, their commercial
availability and ease of their synthesis. Boc group deprotection in compounds 8 afforded
compounds 9. Treatment of compounds 9 with formic acid and formaldehyde mixture resulted in
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
respective N-methyl derivatives 10. Treatment of compound 9h (R = H, R = 6-Cl) with acetic
1
anhydride and triethylamine had resulted in compound 11.
Scheme 1. Synthesis of trans 4,5-methanopyrrolidinol derivatives 9, 10 and 11.a
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
b
OH
c
OTBDPS
OTBDPS
OTBDPS
O
N
H
O
O
N
HO
N
H
N
Boc
Boc
1
2
3
4
d
OTBDPS
e
OTBDPS
OTBDPS
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
N
Boc
Boc
Boc
6
a
6b
5
%6a : 6b = 95 : 5
HO
f
OH
g
O
R
h
O
R
N
R
6a
+
N
N
N
Boc
N
Boc
N
R1
j
i
7
8
1
1
9, R = H
1
R = 6-Cl,
R = -COCH
1
0, R = CH
1 3
1
3
aReagents and conditions: (a) Imidazole, DMAP, TBDPS-Cl, DCM,0 ºC-r.t. 12.0 h (b)
Boc O, DMAP, acetonitrile, r.t., 16.0 h, 71% for two steps (c) LiEt BH, THF, 0 ºC, 1.0 h, 95%
(d) Pr EtN, TFAA, toluene, 0 ºC-r.t., 1.0 h, 110 ºC, 12.0 h, 70% (e) Et Zn, CH I , DCM, 0 ºC-
r.t., 3.0 h, 78% (f) TBAF, THF, 0 ºC-r.t., 12.0 h, 88% (g) PPh , DEAD, THF, 0 ºC-r.t., 16.0 h (h)
Dry HCl, IPA, 0 ºC-r.t., 6.0 h (i) HCOOH, 40% aq. HCHO, 80 ºC 3.0 h (j) Et N, Ac O, DCM, 0
2
i
3
2
2
2 2
3
3
2
ºC-r.t., 3.0 h, 91%.
Stereo selective synthesis of compounds 19 with cis stereo chemistry commenced with L-
3
2
pyroglutamic acid (Scheme 2) as reported earlier. L-Pyroglutamic acid was converted to its
ethyl ester 12. Ethyl ester 12 was then protected with Boc group to obtain compound 13.
Compound 13 on reduction with LiEt BH afforded ~1:1 mixture of aminols 14. The aminols 14
3
on dehydration reaction yielded olefin 15. Olefin 15 was cyclopropanated using diethyl zinc and
diiodomethane to obtain a mixture of diastereomers 16a and 16b in ~1:4 ratio respectively.
Diastereomers 16a and 16b were separated by silica gel column purification. Reduction of ester
group in 16b with LiAlH afforded alcohol 17 which on Mitsunobu reaction with substituted 3-
4
hydroxypyridines afforded compounds 18. The Boc deprotection in compounds 18 yielded
compounds 19.
Scheme 2. Synthesis of cis 4,5-methanopyrrolidinol derivatives 19.a
8
ACS Paragon Plus Environment

Page 9 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a
b
c
OH
OEt
OEt
OEt
O
N
O
O
N
HO
N
H
N
H
O
O
Boc
O
Boc
O
L-Pyroglutamic acid
12
13
14
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
d
OEt
e
g
OEt
OEt
+
N
N
N
Boc
Boc
O
Boc
O
O
1
5
16a
16b
%
16a : 16b = 1 : 4
OH HO
+
O
h
O
f
R
R
16b
N
R
N
N
Boc
Boc
N
HCl
N
H
N
1
7
18
19
a
Reagents and conditions: (a) K CO , EtI, DCM, 0 ºC-r.t. 6.0 h. 92% (b) Boc O, Et N,
2
3
2
3
i
acetonitrile, 16.0 h, 85% (c) LiEt BH, THF, 78 ºC, 1.0 h, 90% (d) Pr EtN, TFAA, toluene, 0
ºC-r.t., 1.0 h, 110 ºC, 12.0 h, 70% (e) Et Zn, CH I , DCM, 0 ºC-r.t., 3.0 h, 64% (f) LiAlH , THF,
ºC, 1.0 h, 88% (g) PPh , DEAD, THF, 0 ºC-r.t., 16.0 h (h) Dry HCl, IPA, 0 ºC-r.t., 6.0 h.
3
2
2
2 2
4
0
3
Synthesis of compounds 31 commenced with reaction of benzylamine with allyl bromide
Scheme 3) which afforded compound 22. Compound 22 on treatment with acetyl chloride
afforded acetylated compound 23 which on cyclization using cyclopentylmagnesium chloride
(
3
5
and titaniumisopropoxide yielded compound 24. N-oxidation of compound 24 afforded N-oxide
5 which on debenzylation followed by Boc protection afforded compound 26. Compound 26 on
2
treatment with sec-butyllithium and carbondioxide36 yielded diastereomeric mixture of
methanoproline 27. The diastereomers were not separated. The racemic compound 27 was on
BH -DMS reduction afforded racemic alcohol 28. The racemic alcohol 28 on mesylation
3
followed by displacement with potassium salt of substituted 3-hydroxypyridines afforded
compounds 30. The Boc deprotection yielded targeted compounds 31 as a diastereomeric
mixture.
Scheme 3. Synthesis of 5-methyl, 4,5-methanopyrrolidinol derivatives 31.a
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
NH2
N
b
c
N
+
Br
H
O
2
1
22
23
20
OH
O
d
e
f
g
OH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
N
N
O
N
H C
H3C
H3C Boc
H C Boc
3
3
H C Boc
3
Ph
N
25 Ph
26
27
2
4
28
O
O
R
OMs
HO
R
j
h
i
+
R
N
N
N
N
HCl
N
H C
3
H
H C Boc
H C Boc 29
3
30
31
3
a
Reagents and conditions: (a) Cs CO , CH CN, reflux, 3.0 h, 33%. (b) CH COCl,
NaHCO , EtOAc : H O, 0 ºC-r.t., 0.5 h, 72%. (c) Titanium (IV) isopropoxide, cyclopentyl-MgCl,
2
3
3
3
3
2
THF, 0 ºC-r.t., 1.0 h. 50%. (d) H O , MeOH, 0 ºC-r.t., 24 h, 100%. (e) BOC O, DCM, 0 ºC-r.t.,
2
2
2
1
6.0 h, 50%. (f) sec-BuLi, THF, CO , 78 ºC-r.t., 3.0 h, 51%. (g) BH -DMS, THF, 0 ºC-r.t., 3.0
h, 64%. (h) CH SO Cl, Et N, DCM, 0 ºC-r.t., 0.5 h (i) KOH, DMF, 75 ºC, 5.0 h (j) Dry HCl,
2
3
3
2
3
IPA, 0 ºC-r.t., 6.0 h.
3
7
Compounds 37 were prepared by following the protocol as reported earlier. Reaction of an
intermediate compound 3 with NaHMDS and PhSeCl followed by treatment with hydrogen
peroxide afforded olefin 32. Olefin 32 on cyclopropanation with diazomethane in presence of
palladium acetate afforded a mixture of diastereomers 33a and 33b in 9:1 ratio, respectively. The
trans isomer 33a which was isolated by silica gel column purification, on subjecting to borane
reduction afforded compound 34. Deprotection of TBDPS group in compound 34 afforded
compound 35. Alcohol 35 on treatment with substituted 3-hydroxypyridines using Mitsunobu
conditions afforded compounds 36. Deprotection of Boc in 36 yielded compounds 37 (Scheme
4
).
Scheme 4. Synthesis of 3,4-methanopyrrolidinol derivatives 37.a
1
0
ACS Paragon Plus Environment

Page 11 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a
b
OTBDPS
OTBDPS
OTBDPS
3a
OTBDPS
33b
O
O
N
N
O
+
O
N
N
Boc
Boc
Boc
Boc
3
32
3
%33a : 33b = 9 : 1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HO
OH +
R
e
c
d
f
R
R
OTBDPS
O
O
3
3a
N
N
N
N
N
Boc
Boc
Boc
H
HCl
3
4
N
N
3
5
36
3
7
a
Reagents and conditions: (a) i. NaHMDS, PhSeCl, THF, 78 ºC, 2.0 h. ii. H O THF, 0
ºC, 2.0 h, 65% (b) CH N , Pd(OAc) , ether, 0 ºC, 24.0 h, 87% (c) BH -THF, THF, r.t., 16.0 h,
5% (d) TBAF, THF, 0 ºC-r.t., 12.0 h, 69% (e) PPh , DEAD, THF, 0 ºC-r.t., 16.0 h (f) Dry HCl,
2
2,
2
2
2
3
7
3
IPA, 0 ºC-r.t., 6.0 h.
3
3
Synthesis of compounds 42 commenced with Boc protection of key intermediate 38 which
afforded compound 39. The ester group reduction in compound 39 with LiAlH yielded alcohol
4
4
0. Alcohol 40 on reaction with substituted 3-hydroxypyridines under Mitsunobu conditions
yielded compounds 41. Boc deprotection in 41 afforded compounds 42 (Scheme 5).
Scheme 5. Synthesis of 2,3-methanopyrrolidinol derivatives 42. a
OCH3
O
OCH3
O
OH
HO
O
a
c
b
+
R
R
N
N
N
N
N
N
H
Boc
Boc
R
1
38
39
40
4
1, R = Boc
1
d
4
2, R = H
1
a
Reagents and conditions: (a) Boc O, Et N, DCM, 0 ºC-r.t. 16.0 h, 62% (b) LiAlH , THF,
ºC, 1.0 h, 91%. (c) PPh , DEAD, THF, 0 ºC-r.t., 16.0 h (d) Dry HCl, IPA, 0 ºC-r.t., 6.0 h.
2
3
4
0
3
Compounds 51 were prepared (Scheme 6) starting with key intermediate 43 which in turn was
3
4
prepared by following earlier reported procedures. Compound 43 was protected with TBDPS
group to obtain compound 44 which was on Boc protection afforded compound 45. Compound
4
5 on reduction with LiEt BH yielded diastereomeric mixture of compounds 46. Compound 46
3
1
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
upon subjecting to dehydration reaction conditions using TFAA and Hunig’s base, yielded olefin
4
7. Olefin 47 on cyclopropanation with diethyl zinc and diiodomethane yielded diastereomeric
mixture of compounds 48a and 48b which were separated by silica gel column purification.
Major diastereomer 48a on TBDPS group deprotection afforded alcohol 49. Alcohol 49 was
reacted with substituted 3-hydroxypyridines under Mitsunobu reaction conditions to yield
compounds 50. Boc deprotection in compound 50 afforded targeted compounds 51.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 6. Synthesis of 5,6-methanopiperidinol derivatives 51.a
a
b
OH
c
OTBDPS
OTBDPS
OTBDPS
O
N
O
O
HO
N
H
N
N
H
Boc
Boc
4
3
4
4
45
4
6
d
OTBDPS
e
OTBDPS
OTBDPS
+
N
N
N
Boc
Boc
Boc
48a
48b
4
7
%
48a : 48b = 95 : 5
HO
f
OH
g
O
R
h
O
R
N
R
48a
+
N
N
N
Boc
N
Boc
N
H
HCl
51
5
0
4
9
a
Reagents and conditions: (a) Imidazole, DMAP, TBDPS-Cl, 0 ºC-r.t., 12.0 h, 94% (b)
i
Boc O, Et N, DCM, 0 ºC-r.t. 16.0 h, 81% (c) LiEt BH, H O THF, 0 ºC, 1.0 h, 81% (d) Pr EtN,
2
3
3
2
2
2
TFAA, toluene, 0 ºC-r.t., 1.0 h, 110 ºC, 12.0 h, 75% (e) Et Zn, CH I , DCM, 0 ºC-r.t., 3.0 h, 87%
2
2 2
(f) TBAF, THF, 0 ºC-r.t., 12.0 h, 78% (g) PPh , DEAD, THF, 0 ºC-r.t., 16.0 h (h) Dry HCl, IPA,
3
0 ºC-r.t., 6.0 h.
Compounds 59a and 59b were prepared as shown in scheme 7. Compound 7, already described
in scheme 1, was mesylated and reacted with KCN to get compound 53. Cyano group hydrolysis
in compound 53 with a mixture of HCl and acetic acid afforded compound 54. Acid 54 was
esterified with methanol to obtain methyl ester 55. Compound 55 on Boc protection yielded
compound 56. The ester 56 was reduced with LiAlH to obtain compound 57. Compound 57 on
4
1
2
ACS Paragon Plus Environment

Page 13 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
reaction with 5-chloro-3-hyroxypyridine or 6-chloro-3-hyroxypyridine under Mitsunobu reaction
conditions afforded compounds 58a and 58b, respectively. Boc deprotection afforded 5-chloro
derivative 59a and 6-chloro derivative 59b, respectively as hydrochloride salts.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 7. Synthesis of 4,5-methanopyrrolidinol derivatives 59a and 59b.a
O
O
CN
c
d
e
OH a
b
OMs
OH
OCH3
N
N
N
N
N
HCl
H
Boc
Boc
Boc
H
HCl
7
52
53
54
5
5
O
HO
OH +
g
h
f
OCH3
O
R
O
N
N
N
N
Boc
N
H
Boc
Boc
HCl
N
N
5
9
5
6
57
R
R
58a, R = 5-Chloro
59a, R = 5-Chloro
59b, R = 6-Chloro
5
8b, R = 6-Chloro
a
Reagents and conditions: (a) Et N, CH SO Cl, DCM, 0 ºC, 1.0 h, 98% (b) KCN,
3
3
2
DMSO, 90 C, 3.0 h, 28% (c) Con. HCl, glacial AcOH, reflux, 6.0 h, 100% (d) SOCl , MeOH,
2
r.t., 16.0 h, 100% (e) Boc O, Et N, DCM, 0 C-r.t., 16.0 h, 65% (f) LiAlH , THF, 0 C, 1.0 h,
2
3
4
7
3% (g) PPh , DEAD, THF, 0 C-r.t. 16.0 h (h) Dry HCl, IPA, 0 C, 6.0 h.
3
RESULTS AND DISCUSSION
The in vitro affinity of each compound at α4β2 nAChRs was determined by measuring the
3
displacement of [ H]-()-cytisine from a membrane preparation of cloned human α4β2 receptors
stably expressed in CHO-K1 cells and mean ± S.E.M K values were presented in Table 1 and
i
3
8
28
Table 2. The observed in vitro values of known reference standards nicotine, ABT-089, TC-
2
9
39
2
216, and varenicline in the current assay were in agreement with respective reported values
(Table 1). The first trans 4,5-methanoprolinol compound 9a (Series 1) with no substitution on
1
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
pyridine showed a K value of 7.9 nM. The reported compound, 3-(pyrrolidin-2-ylmethoxy)-
i
pyridine which is structurally similar to the compound 9a showed a K value of 0.16 nM. The K
i
i
value of this reported compound was determined using a membrane preparation from whole rat
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
40
brain. The N-methyl derivative 10a showed a K value of 5.5 nM which is in concurrence with
i
the reported observation that desmethyl derivatives and N-methyl derivatives show similar in
4
0
vitro affinities. The 2-methyl pyridine derivative 9b was surprisingly inactive whereas its N-
methyl derivative 10b showed a modest affinity with a K value of 231 nM. However,
i
corresponding reported 2-methylpyridine derivative ABT-089 (Figure 1) in the current assay
2
8
showed a K value of 26.0 nM (reported value; 16.7 nM). The minor difference in the binding
i
affinity of ABT-089 could be because of a difference in the assay conditions used.
Having seen huge difference in in vitro affinities between pyridine derivative (9a) and 2-
methylpyridine derivative (9b), the effect of diverse substitutions on various positions of
pyridine was explored. The 6-methyl pyridine derivatives 9c and 10c, 2-chloropyridine
derivatives 9d and 10d, 2-fluoropyridine derivatives 9e and 10e showed potent in vitro affinities
in the range of 17.4 to 31.9 nM (Table 1). The 5-chloropyridine derivatives 9f and 10f showed
sub-nanomolar in vitro affinities with K values of 0.19 and 0.37 nM respectively suggesting
i
chloro substitution at 5-position is favorable. However, 5-bromopyridine derivatives 9g and 10g
showed modest affinity with K values of 6.4 and 4.2 nM respectively. The reason for lower
i
affinity of 5-bromopyridine derivatives in comparison to 5-chloropyridine derivatives could be
due to bulky size of bromine. The 6-chloropyridine derivatives 9h and 10h showed K values of
i
1
.5 nM and 3.4 nM respectively, suggesting chloro substitution at 6-position also favorable.
These compounds were more potent than un-substituted pyridine analogues (9a and 10a) and
less potent than 5-chloropyridine derivatives (9f and 10f). The 6-fluoropyridine derivatives 9i
1
4
ACS Paragon Plus Environment

Page 15 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
and 10i followed the suit and showed in vitro affinities of 3.9 nM and 1.1 nM similar to 6-
chloropyridine derivatives. The 6-bromopyridine analogue 9j was moderately potent with a Ki
value of 46.5 nM in sync with earlier observation that bulky size of bromine has negative impact
on in vitro affinity. Additional compounds with bulky substitutions like 6-
trifluoromethylpyridine derivative 9k and 6-methoxy derivative 9l were turned out to be inactive
further confirming that bulky groups are not well tolerated. Multiple halo substitutions on
pyridine ring had resulted in 5,6-dichloropyridine derivative 9m and 2,5,6-trichloropyridine
derivative 9n. Both of these compounds showed weak in vitro affinities suggesting more than
one halo substitution may not be well tolerated. Replacement of halogen at 5-position of pyridine
with heterocyclic rings resulted in compounds 9o, 9p and 9q. The 1,3,4-oxadiazole derivative 9o,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
-methyl-1,3,4-oxadiazole derivative 9p and 5-vinyl-1,3,4-oxadiazole derivative 9q showed very
weak in vitro affinities suggesting heterocyclic rings on pyridine may not be tolerated. However,
5-(hex-5-yn-1-ol) pyridine derivative 9r showed potent in vitro affinity with a K value of 2.7 nM
i
suggesting alkynyl groups are well tolerated. In the next set of modifications, the effect of
replacing 3-hydroxypyridinyl group with 2-hydroxypyridinyl, 4-hydroxypyridinyl and with
different heteroaromatic groups was evaluated. However, this modification was found to be futile
as replacing 3-hydroxypyridinyl ring with 2-hydroxypyridinyl (9s), 4-hydroxypyridinyl (9t), 3-
hydroxyquinolinyl (9u), 2-hydroxypyrazine (9v), 2-chloro-3-hydroxypyrazine (9w) and 6-
chloro-3-hydroxypyridazine (9x) had resulted in complete loss of in vitro affinity showing the
importance of 3-hydroxypyridinyl ring for the affinity. To explore the effect of enantiomers on
affinity, R-enantiomer (9y) of compound 9h (S-enantiomer) was synthesized and tested for its in
vitro affinity. The R-isomer 9y with a K value of 39.0 was about 26 folds less potent than S-
i
1
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
isomer 9h. Masking of basic nitrogen of pyrrolidine ring with acetyl group resulted in in vitro
inactive compound (11).
Table 1. hα4β2 nAChR radioligand binding values of compounds 9, 10 and 11.a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
6
ACS Paragon Plus Environment

Page 17 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
OAr
N
R1
Series 1
Compound
No.
hα4β2 K i nM ± Compound
hα4β2 K i nM ±
Ar
R1
Ar
R1
H
S.E.M
No.
S.E.M
N
N
S-Nicotine
Varenicline
N.A
N.A
N.A
N.A
2.2 ± 0.3
9q
O
2657.0 ± 121.9
2.7 ± 0.1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
0.04 ± 0.01
26.0 ± 1.3
9r
H
(
)4
N
HO
ABT-089
TC-2216
N.A
N.A
N.A
N.A
H
9s
H
H
>10000
>10000
N
18.6 ± 0.7
7.9 ± 0.4
9t
9u
N
9
9
9
9
a
b
c
d
H
600.0 ± 15.8
>10000
N
N
N
N
H
>10000
9v
H
N
N
N
N
H
26.2 ± 1.3
17.5 ± 0.6
17.4 ± 1.3
0.19 ± 0.02
6.4 ± 0.2
9w
9x
H
5112.0 ± 496.2
4736.0 ± 464.3
39.0 ± 3.1
5.5 ± 0.2
Cl
N
H
H
N
Cl
N
Cl
Enantiomer of
9e
H
9y
H
9h
F
N
Cl
Br
9
f
H
10a
10b
10c
10d
10e
10f
10g
10h
10i
11
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
COCH3
N
N
N
N
N
9g
H
231.0 ± 7.9
20.0 ± 1.2
28.1 ± 0.3
31.9 ± 1.8
0.37 ± 0.01
4.2 ± 0.6
N
9
h
H
1.5 ± 0.2
Cl
F
N
N
9
i
j
H
3.9 ± 0.4
Cl
9
H
46.5 ± 5.9
768.0 ± 58.0
>10000
N
Br
F
N
Cl
Br
9
k
H
N
CF3
N
N
N
N
9l
H
N
OMe
Cl
9
m
H
149.2 ± 5.2
265.4 ± 8.4
131.1 ± 2.8
407.3 ± 18.2
3.4 ± 0.2
N
Cl
Cl
Cl
F
9
9
9
n
o
p
H
1.1 ± 0.1
Cl
N
Cl
N
N
O
H
>10000
N
N
N
Cl
N
N
O
H
1
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
3
Displacement of [ H]-()-cytisine binding to cloned human α4β2 receptors stably
expressed in CHO-K1 cells. K values were determined from average of two independent
experiments tested in duplicate. Compounds 9q, 10a, 10c, 10f, 10j are free bases; 9e, 9g are
tartrates; 9o, 9p, 9r are oxalates and all other compounds are HCl salts.
i
In the next set of modification, the effect of diastereomers on in vitro affinity was evaluated.
Several cis 4,5-methanoproline derivatives which are diastereomers of compounds 9 were
synthesized and tested for in vitro affinities (Table 2). Compound 19a having no substitution on
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
pyridine showed a K value of 1.0 nM as against 7.9 nM for its trans counterpart 9a. However, 2-
i
chloropyridine derivative 19b with a K of 327 nM, 5-chloropyridine derivative 19c with a K of
i
i
2
.9 nM were about 19 and 15 folds less potent than trans isomers 9d and 9f, respectively. The 5-
bromopyridine derivative 19d with a K of 1.38 nM and 6-methylpyridine derivative 19e with a
i
K of 6.4 nM showed four folds more affinity than trans isomers 9g and 9c, respectively. The 6-
i
chloropyridine derivative 19f with a K of 1.2 nM and 6-fluoropyridine derivative 19g with a K
i
i
of 4.7 nM showed similar affinities as that of trans isomers 9h and 9i, respectively. The 6-
methoxypyridine derivative 19h showed moderate affinity with a K value of 329 nM, whereas
i
its trans counterpart 9l was found to be inactive. The R-enantiomer 19i with K value of 3.6 nM
i
was ~3 fold less potent than corresponding S-enantiomer 19f.
In order to evaluate the effect of further substitution on 4,5-methanopyrrolidine ring, a methyl
group was introduced at 5-position of the ring which resulted in racemic compounds 31.
Compound with no substitution on pyridine (31a) and chloro substitution at different positions of
3
-hydroxypyridine (31b, 31c and 31d) were made as only the chloro substitution was found to be
optimal in foregoing scaffolds. However, these compounds were found to be either less potent
or inactive. As these compounds showed modest in vitro affinities, no attempt was made to
separate the individual isomers. In the next set of modification, cyclopropyl ring was moved
from 4,5 to 3,4-position to get 3,4-methanopyrrolidinol derivatives 37a, 37b and 37c. This move
1
8
ACS Paragon Plus Environment

Page 19 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
had resulted in potent in vitro compounds with K values ranging between 0.14 to 0.2 nM. In an
i
extended SAR, the cyclopropyl ring was moved to 2,3-position to get 2,3-methanopyrrolidinol
derivatives 42a, 42b and 42c. These compounds showed very weak in vitro affinities in the range
of 200 to 900 nM suggesting this modification is not tolerated.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
After exploring the effect of cyclopropyl group on 5-membered pyrrolidine, effect of it on 6-
membered piperidine ring was studied. The piperidine derivatives 51a, 51c and 51d showed
affinity akin to 4,5-methanopyrrolidine derivatives and showed in vitro affinities in the range of
1
1.0 to 31.0 nM. However, 2-chloropyridine derivative 51b was found to be inactive. In the next
modification, the distance between central pyrrolidine ring and pyridine nitrogen was increased
by introduction of an additional methylene group in compounds 59a and 59b. This modification
was done to check the effect of distance between central pyrrolidine and pyridine nitrogen. As
2
9
expected, compounds 59a and 59b showed reduced in vitro potencies with K values of 394.7
i
and 327.3 respectively. Replacement of the ether linkage with some other hetero atoms/groups
such as S, NH or NR was not explored as they may have resulted in increase in distance between
central core and pyridine nitrogen. Balboni et al had reported that replacement of the oxygen
linker with NH or NR in pyrrolidinol derivatives resulted in less potent compounds.41,42
Table 2. hα4β2 nAChR radioligand binding values of compounds 19, 31, 37, 42, 51,and 59.a
1
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
OAr
OAr
OAr
OAr
OAr
OAr
N
H
N
H
N
H
N
H
N
H
N
H
Series 2
Series 3
Series 4
Series 5
Series 6
Series 7
Compound
hα4β2 Ki nM ± Compound
hα4β2 Ki nM ±
Ar
Series
N.A
Ar
Series
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
No.
SEM
No.
SEM
Cl
S-Nicotine
N.A
N.A
2.2 ± 0.3
31c
3
3
577.4 ± 5.5
N
N
Varenicline
ABT-089
N.A
0.04 ± 0.01
26.0 ± 1.3
31d
37a
6881.3 ± 363.4
Cl
Cl
N.A
N.A
N.A
N.A
2
4
4
4
5
5
5
6
6
6
6
7
7
0.14 ± 0.02
0.2 ± 0.0
N
TC-2216
18.6 ± 0.7
1.0 ± 0.1
37b
37c
42a
42b
42c
51a
51b
51c
51d
59a
59b
N
19a
19b
19c
19d
19e
0.2 ± 0.0
N
N
Cl
Cl
2
327.1 ± 6.6
2.9 ± 0.2
241.9 ± 13.5
827.2 ± 16.7
340.7 ± 12.0
11.4 ± 1.0
>10000
Cl
N
N
Cl
Br
2
N
N
N
2
1.38 ± 0.05
6.4 ± 0.2
N
Cl
2
N
N
N
19f
2
1.2 ± 0.1
Cl
Cl
N
Cl
19g
19h
19i
2
4.7 ± 0.6
31.3 ± 2.9
20.5 ± 0.8
394.7 ± 21.0
327.3 ± 6.2
N
F
N
2
329.3 ± 15.9
3.6 ± 0.2
N
OMe
N
Cl
Cl
Enantiomer of
9f
2
1
N
31a
31b
3
>10000
N
N
Cl
3
1341.2 ± 11.4
Cl
N
2
0
ACS Paragon Plus Environment

Page 21 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a
3
Displacement of [ H]-()-cytisine binding to cloned human α4β2 receptors stably
expressed in CHO-K1 cells. K values were determined from average of two independent
experiments tested in duplicate. Compound 37c is free base and all other compounds are HCl
i
salts.
Compounds 9a, 9d, 9f, 9h, 9i, 9y from Series 1, 19a, 19f, 19i from Series 2, 37a, 37b from
Series 4, and 51a, 51d from Series 6 which represent major SAR modifications were evaluated
for α3β4 affinity, metabolic stability in human and rat microsomes, interaction with CYP 3A4,
CYP 2D6 enzymes, pharmacokinetics and brain penetration in Wistar rats (Table 3). It is evident
from the in vitro values obtained for these compounds at α3β4 nAChR that, the compounds with
trans configuration (Series1 and Series 6) showed high selectivity against ganglionic α3β4
nAChR. The cis 4,5-methanopyrrolidinol derivatives 19a, 19f and 19i, (Series 2) showed
moderate affinity between 500-1000 nM while 3,4-methanopyrrolidinol analogues 37a and 37b
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
Series 4) showed potent in vitro affinity (<50 nM) towards α3β4 nAChR.
Table 3. In vitro affinity values for α3β4 nAChR, metabolic stability, CYP inhibition and
pharmacokinetics in rat for selected compounds.
a
2
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CYP
Inhibition
CYP
Inhibition
Human liver
Rat liver
Bio-
b
α3β4 K i nM
SEM
Cmax ng/mL AUC ng*hr/mL
Cbrain/Cplasma
Compound
microsomal
microsomal availability
T1/2 ±S.D
±
S.D
±S.D
IC50 (µM)
2D6
IC50 (µM)
3A4
±
c
c
d
±S.D
stability ±S.D stability ±S.D %F ±S.D
S-Nicotine 1744.4 ± 22.3
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Varenicline
ABT-089
TC-2216
9a
39.6 ± 2.2
>10000
NA
NA
NA
NA
NA
NA
NA
NA
>10000
NA
NA
NA
NA
>10000
12.1 ± 3.6
29.5 ± 12.6
40.7 ± 7.5
33.4 ± 8.3
10.7 ± 3.8
42.2 ± 5.6
2.6 ± 2.3
13.6 ± 4.8
18.3 ± 8.6
6.7 ± 0.6
6.04 ± 1.9
68.4 ± 9.3
17.9 ± 2.4
14.9 ± 19.0
40.4 ± 6.0
32.2 ± 0.6
5.1 ± 8.4
26.2 ± 5.7
3.9 ± 5.8
36.0 ± 7.7
69.6 ± 9.4
6.2 ± 4.5
12.1 ± 6.1
53.1 ± 3.9
72.8 ± 0.7
92 ± 16 7611 ± 1660 10925 ± 646 4.19 ± 1.53 3.80 ± 0.29
26.2
> 42
> 45
38.1
> 45
17.1
30.2
> 45
32.1
> 45
> 45
> 45
7.9
> 45
> 45
> 45
> 45
> 45
> 45
31.9
> 45
> 45
21.1
> 45
> 45
> 45
9
d
>10000
54 ± 23
50 ± 13
66 ± 12
96 ± 7
35 ± 2
87 ± 11
66 ± 4
NT
921 ± 268
949 ± 200
2158 ± 658 2.15 ± 0.51 3.60 ± 0.99
1188 ± 201 1.07 ± 0.04 3.79 ± 0.66
9
f
>10000
9
h
>10000
1906 ± 675 3507 ± 657 3.34 ± 0.62 2.34 ± 0.25
3757 ± 952 15569 ± 3501 4.02 ± 2.26 2.38 ± 0.41
9
i
>10000
9y
9a
19f
9i
7a
7b
1a
51d
6014 ± 431
558.3 ± 19.8
1019 ± 39
525.2 ± 13.4
26.4 ± 2.5
8.2 ± 1.0
>10000
1192 ± 206 1767 ± 204
1.6 ± 0.1
3.83 ± 0.57
1
1503 ± 29
638 ± 38
NT
2707 ± 219 1.08 ± 0.26 3.07 ± 0.21
1616 ± 100 2.73 ± 0.23 2.14 ± 0.52
1
NT
NT
NT
NT
NT
NT
NT
NT
3
NT
NT
3
NT
NT
NT
5
17 ± 7
48 ± 18
853 ± 293
665 ± 270
0.22 ± 0.01 0.32 ± 0.09
>10000
98.9 ± 0.4
3343 ± 795 4478 ± 1185 0.68 ± 0.16 0.16 ± 0.05
a
b
Data represents, K values for α3β4 nAChR were determined K values were determined
from average of two independent experiments tested in duplicate; results are expressed as
%metabolized at 30 minutes post incubation; Fasted male Wistar rats were used, Vehicle used is
water for injection for both oral and i.v. routes. Dosing volumes: 10 mL/kg for p.o. and 2 mL/kg
for i.v.; NA, not available; NT, not tested. Values are mean ± S.D, n=3 animals/time point.
i
i
c
d
Compounds 9a, 9d, 9f, 9h, 9i, 9y from Series 1, compounds 19a, 19f and 19i from Series 2
and compounds 37a, 37b from Series 4 have shown low-to-moderate metabolism both in rat and
human liver microsomes. Compounds 51a and 51d which represent six membered piperidine
series, showed moderate to high metabolism. The increased metabolism of 51a and 51d may be
because of an increase in metabolic soft spots on the ring. All the compounds as mentioned
above were tested for metabolizing enzymes CYP 2D6 and CYP 3A4 inhibition potential in
2
2
ACS Paragon Plus Environment

Page 23 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
pooled human liver microsomes using specific CYP isoform marker activities. These compounds
showed very weak inhibition (>17 µM) towards CYP 2D6 and CYP3A4 enzymes barring
compound 51d which showed IC value of 7.9 µM for CYP 2D6. The low inhibitory potential
5
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
values suggest the drug-drug interaction liability in clinic for these compounds is remote. As
evident from rat PK and brain penetration data, compounds from Series 1 (9a, 9d, 9f, 9h, 9i and
9
y) have shown high oral exposures, longer half-lives and adequate brain penetration in Wistar
rats (Table 3). Compound 51d from Series 6 showed high oral exposures and moderate half-life
with plasma to brain penetration ratio of 0.16.
On the basis of good plasma exposures and adequate brain penetration, compounds 9a, 9d, 9f,
4
3
9h, 9i, 9y and 51d were evaluated in depression model, the forced swim test (FST) in Wistar
rats. The FST is considered as one of the most commonly used assays to study depressive-like
behavior in rodents and based on an assumption that immobility reflects a measure of behavioral
despair. Additionally, the FST has been shown to share some factors that are influenced or
altered by the depression in humans. The FST experiment outcomes of selected compounds are
presented
in
Table
4.
Table 4: Forced swim test of compounds 9a, 9d, 9f, 9h, 9i, 9y, and 51d in Wistar rats.a
Immobility time in seconds
(Mean± SEM)
Compound
Vehicle
3.0 mg/k.g p.o.
10.0 mg/kg p.o.
183.9±12.1**
210.4±18.1*
198.8±19.6*
241.7±9.8
TC-5214
271.9±7.2
241.9±2.7
262.5±10.6
255.3±3.4
242.9±4.7
249.1±6.4
9
9
a
231.0±2.5
d
204.8±15.9*
249.9±15.2
155.9±21.7**
9f
9
h
81.5±13.6**
2
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
9
9
i
253.1±13.2
254.8±9.0
281.6±3.9
259.6±9.6
256.8±9.0
238.6±10.5*
263.5±7.7
279.0±4.9
264.9±9.4
y
51d
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Imipramine
45 mg/kg, i.p.)
2
71.9±7.2
176.5±6.8**
(
aNumber of animals used in each group n = 10, evaluated using one way ANOVA
followed by Dunnett's test. Data represents Mean ± SEM of immobility time in seconds.
p<0.05,**p<0.01.
*
The positive control imipramine showed significant antidepressant effects at a tested dose of
45 mg/kg, i.p. (**p<0.01). TC-5214 showed potent antidepressant effects at tested dose of 10
mg/kg p.o. Compound 9d and 9h showed significant effects at 3.0 and 10.0 mg/kg p.o., however
the magnitude of effect was pronounced in case of 9h. Compound 51d showed activity at 3.0
mg/kg but not at 10.0 mg/kg p.o. On the other hand compound 9a showed activity only at higher
dose that is, 10 mg/kg p.o. Compounds 9f, 9i and 9y were inactive at tested doses (Table 4). As
compounds 9d and 9h showed significant antidepressant effects in FST at both tested doses,
compounds were further evaluated at 1.0, 3.0 and 10.0 mg/kg to check dose dependency. The
dosing was done once a day for three days. Both of the compounds showed antidepressant like
activity with no signs of tachyphylaxis. Compound 9h and compound 9d at per oral doses of 1.0,
3
.0 and 10.0 mg/kg reduced immobility time to ~29%, 36%, 66% and ~10%, 25%, 16%
respectively. Once again, the effect of compound 9h in FST was robust as compared to
compound 9d. Compound 9h showed significant activity at all tested doses whereas, compound
9
d showed significant activity only at 3 mg/kg p. o. Both experiments were done on the same
day and thus kept the common positive control duloxetine. Duloxetine showed ~33% reduction
in immobility time at 30 mg/kg, i.p. (Figure 2). As compound 9h showed robust efficacy in
2
4
ACS Paragon Plus Environment

Page 25 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
preliminary non-clinical model of depression, it was evaluated for its functional activity at α4β2
nAChR, selectivity against other receptors, PK in dog, target engagement and safety.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
Cl
O
N
N
N
N
Cl
d
9h
9
H
H
300
200
100
0
300
200
100
0
15.8%
1
0%
2
4.7%
*
3
2.67%
*
2
9.35%
*
3
5.81%
*
*
6
6.4%
**
Vehicle
mL/kg, p.o.
1 mg/kg
3 mg/kg
10 mg/kg
Vehicle
mL/kg, p.o.
1 mg/kg
3 mg/kg
10 mg/kg
Duloxetine
30 mg/kg, i.p.
2
2
Compound 9h
Compound 9d
aData represents Mean ± SEM of immobility time in seconds. One way ANOVA
followed by Dunnett’s post hoc test was used to assess the efficacy (F4,34 = 2.99 for compound
h and F4,37 = 3.55 for compound 9d); n = 8-10; *p<0.05,**p<0.01.
9
Figure 2. Effect of compounds 9h and 9d in rat FST on repeated treatment for 3 days.a
The functional activity of 9h at α4β2 nAChR was tested at commercial service provider B’Sys
GmbH, a European ion channel contract research organization. The whole-cell patch-clamp
technique was used to investigate the effect of 9h on acetylcholine-induced currents recorded
from HEK293 cells stably expressing the human nAChR α4β2 receptor. Compound 9h was
tested at five concentrations (n=3 cells). Compound 9h exhibited antagonism of acetylcholine-
induced currents with IC values of 7.31 μM with a Hill coefficient of 0.94 for the steady state
5
0
current amplitudes and 6.39 μM with a Hill coefficient of 0.69 for the peak current amplitudes
Figure 3).
(
2
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
00
75
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
2
0
5
0
Steady State
Peak current
1
3
10
30
100
Compound 9h (µM)
aData represents Mean ± SEM of triplicates, compound 9h tested at five different
concentrations in presence of 10 µM of acetylcholine a nAChR α4β2 receptor agonist.
Figure 3. Effect of compounds 9h in human nAChR α4β2 receptor whole cell patch clamp
a
assay.
Compound 9h showed good selectivity against over 70 receptors which included GPCRs, ion
channels, enzymes, peptides, steroids, second messengers, growth factors and prostaglandins.
The selectivity has been evaluated at Caliper Life Sciences Inc. (Supporting information).
To assess target engagement of compound 9h, receptor occupancy (RO) study was carried out
in rat brain. Compound 9h showed dose dependent RO in dose ranges of 0.3-30 mg/kg with an
ED of 1.61 mg/kg, p.o. exhibiting good correlation between plasma exposures and receptor
5
0
occupancy (Figure 4). Nicotine was a positive control in this experiment.
2
6
ACS Paragon Plus Environment

Page 27 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
% Receptor occupancy (ED : 1.61 mg/kg)
50
Brain (ng/g) (EC : 333.7 ng/g)
50
Plasma (ng/mL) (EC : 259.1 ng/mL)
50
1
00
100000
10000
1000
100
8
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
60
4
0
0
0
2
10
1
100
0.1
1
10
Compound 9h (mg/kg, p.o.)
aTracer: ZW-104, 1 µg/kg, i.v. 45 min, male Wistar rats (fasted, n = 4 animals for each
dose)
Figure 4. Compound 9h exposure correlation with α4β2 receptor occupancy in Wistar rats.a
-6
In Caco-2 permeability study, compound 9h showed good permeability (68.8 x 10 cm/sec)
with an efflux ratio [Papp (B-A)/Papp (A-B)] of 0.7 suggesting it is not a substrate of P-gp. In
protein binding studies compound 9h showed unbound fraction of ~31%, 31% and 21% in rat,
dog and human plasma protein, respectively.
Compound 9h showed ideal lead like characteristics as evident from the above results; it was
further evaluated in studies like dog PK, CYP inhibition in other isoforms and safety
assessments. To support compound requirement (with less than 0.15% single known impurity as
per ICH guidelines) for these studies, the synthetic scheme was optimized from lab scale to
production scale. In order to avoid cumbersome silica gel column purification on a large scale,
the diastereomeric mixture (6a and 6b, Scheme 1) was used in the next step of synthesis without
separation. However, in the end of the synthesis, undesired isomer (19f, ~2-5%) was separated
2
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
from desired isomer 9h by chiral resolution process. The free base of compound 9h (with ~2-5%
of diastereomer 19f) was treated with (+)-camphor-10-sulfonic acid (CSA) to form a 9h-CSA
salt. The 9h-CSA salt was recrystallized from isopropanol to get 9h-CSA with <0.1% undesired
diastereomer. The pure 9h-CSA salt was converted to free base and then to HCl salt to isolate 9h
as mono-hydrochloride salt with >99.8% diastereomeric excess (de). The relative
stereochemistry of compound 9h was established by converting well-established minor isomer
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
5
1
6a (Scheme 2) to 9h. Compound 16a on ester reduction to alcohol followed by Mitsunobu
reaction with 2-chloro-5-hydroxypyridine then Boc deprotection with dry HCl yielded desired
isomer 9h. Alternatively, the relative stereochemistry of 9h was confirmed by synthesizing
3
0
compound 6a (Scheme 1) by well-established route as reported earlier. The formation of mono-
1
hydrochloride salt was confirmed by H-NMR in DMSO-d which showed only two
6
exchangeable protons at  10.02 (Experimental section). The HCl content in 9h was estimated as
13.85%, by argentometric titration method using silver nitrate as the titer with the confidence
interval of 0.78 to +0.37. The estimated HCl content had matched with theoretical HCl content
(13.96%) for mono-hydrochloride salt. Estimation of acid dissociation constant (pKa) gave
single pKa value of 8.9 (Supporting information). Compound 9h is a non-hygroscopic and
crystalline mono-hydrochloride salt with excellent water solubility (452.0 mg/mL, BCS Class I
category).
Dog PK of compound 9h was assessed in Beagle dogs at 3 mg/kg, p.o. and 1 mg/kg, i.v. dose.
Compound 9h showed rapid absorption (Tmax 0.30 h) with high oral exposures (AUC0−t = 4349 ±
2
370 ng*h/mL) and showed a favorable terminal i.v. half-life (1.9 ± 0.4 h). Intravenous clearance
(10 ± 3 mL/min/kg) and volume of distribution (1.3 ± 0.1 L/kg) were moderate. The observed
absolute oral bioavailability (%F) was 83 ± 22%.
2
8
ACS Paragon Plus Environment

Page 29 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Compound 9h was tested in assays predictive of cardiovascular toxicity like human hERG
patch clamp assay and dog telemetry. Drug-induced inhibition of hERG (I ) current can delay
Kr
repolarization; the in vitro electrophysiology hERG (I ) study provides valuable information in
Kr
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
assessing the test substance potential for QT interval prolongation and elucidating cellular
mechanisms affecting repolarization which leads to cardio-toxicity. Compound 9h was tested in
whole cell hERG patch clamp assay on Nanion automated Patch clamp system using human
recombinant hERG channel expressed in HEK293 cells. Compound 9h showed a negligible
affinity towards hERG channel with IC >10 µM. In dog telemetry study, compound 9h
5
0
administered orally to male dogs did not show any adverse effect on body weight, body
temperature, blood pressure, heart rate, or the ECG at up to tested doses of 25 mg/kg p.o.
Compound 9h was well differentiated from amphetamine like stimulants as it did not show any
effect on behavior related with autonomic, neuromuscular, sensorimotor, central nervous system
and / or other physiological measures in modified Irwin’s test at highest tested dose of 10 mg/kg
p.o. Compound 9h did not show behavioral sensitization up to the highest tested dose of 10
mg/kg p.o., whereas the positive control nicotine caused behavioral sensitization at a tested dose
of 3 µmol/kg s.c. Compound 9h did not show any effect on latency to fall at a dose of 30 mg/kg,
p.o. when assessed in rotarod test suggesting it does not cause motor in coordination in animal
models.
Compound 9h is non-mutagenic in all four strains of Salmonella typhimurium, TA1537,
TA1535, TA98, and TA100, and Escherichia coli WP2 uvrA both in the absence and presence of
metabolic activation. It is nonclastogenic in in vitro chromosomal aberration tests in human
peripheral blood lymphocytes or in vivo micronucleus assay in mice bone marrow. It does not
show drug-drug interaction liability as IC values were found to be >45 µM for all major CYPs.
5
0
2
9
ACS Paragon Plus Environment