Synthesis, Nicotinic Acetylcholine Receptor Binding, and in Vitro and in Vivo Pharmacological Properties of 2′-Fluoro-(substituted thiophenyl)deschloroepibatidine Analogues

Article abstract of DOI:10.1021/acschemneuro.6b00252

The synthesis, nAChR in vitro and in vivo pharmacological properties of 2′-fluoro-3′-(substituted thiophenyl)deschloroepibatidine analogues (5a-f, 6a-d, and 7a-c) are presented herein. All had subnanomolar affinity at α4β2*-nAChRs. Contrary to lead structure epibatidine, a potent nAChR agonist, all were potent α4β2- and α3β4-AChR antagonists in an in vitro functional assay. In vivo, the compounds were also nAChR antagonists with various degrees of agonist activity. Compounds 5e, 5f, 6a, 6c, 6d, and 7c had no agonist effects in the tail-flick, hot-plate, hypothermia, or spontaneous activity tests, whereas 5a-d, 7a and 7b did not have agonist activity in the tail-flick and hot-plate tests but, like varenicline, were agonists in the hypothermia and spontaneous activity tests. Compound 6b had agonist activity in all four in vivo tests. All the compounds were antagonists of nicotine-induced antinociception in the tail-flick test, and all except 5c, 5d, 5f, and 6b were antagonists of nicotine-induced antinociception in the hot-plate test. Compound 7c, which had a K_i = 0.86 nM in the binding assay similar potency at α4β2/α3β4 with selectivity relative to α7 nAChRs, had an AD₅₀ value of 0.001 μg/kg in the tail-flick test with no agonist activity in the in vitro or in vivo test had one of the more interesting profiles.

Full text of DOI:10.1021/acschemneuro.6b00252

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Article
Synthesis, Nicotinic Acetylcholine Receptor Binding, In
Vitro and In Vivo Pharmacological Properties of 2#-Fluoro-
(substituted thiophenyl)deschloroepibatidine Analogs
Pauline W. Ondachi, Ana H. Castro, Benjamin Sherman, Charles W. Luetje, M.
Imad Damaj, Samuel Wayne Mascarella, Hernán A Navarro, and Frank Ivy Carroll
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00252 • Publication Date (Web): 11 Oct 2016
Downloaded from http://pubs.acs.org on October 12, 2016
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Synthesis, Nicotinic Acetylcholine Receptor Binding, In Vitro and In Vivo
Pharmacological Properties of 2′ꢀFluoroꢀ(substituted thiophenyl)deschloroepibatidine
Analogs
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Pauline W. Ondachi†, Ana H. Castro‡, Benjamin Sherman‡, Charles W. Luetje‡, M. Imad
Damaj§, S. Wayne Mascarella†, Hernán A. Navarro†, and F. I. Carroll*†
†Research Triangle Institute, P.O. Box 12194, Research Triangle Park, NC 27709 United States
‡Department of Molecular and Cellular Pharmacology, Miller School of Medicine, University of
Miami, Miami, FL 33101, United States
§Department of Pharmacology, Virginia Commonwealth University Medical Campus, P.O. Box
980615, Richmond Virginia 23298ꢀ0613, United States
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Abstract
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The synthesis, nAChR in vitro and in vivo pharmacological properties of 2′ꢀfluoroꢀ3′ꢀ
(substituted thiophenyl)deschloroepibatidine analogues (5a–f, 6a–d, and 7a–c) are presented
herein. All had subnanomolar affinity at α4β2*ꢀnAChRs. Contrary to lead structure epibatidine, a
potent nAChR agonist, all were potent α4β2ꢀ and α3β4ꢀAChR antagonists in an in vitro
functional assay. In vivo, the compounds were also nAChR antagonists with various degrees of
agonist activity. Compounds 5e, 5f, 6a, 6c, 6d, and 7c had no agonist effects in the tailꢀflick,
hotꢀplate, hypothermia, or spontaneous activity tests whereas 5a–d, 7a and 7b did not have
agonist activity in the tailꢀflick and hotꢀplate tests but like varenicline, were agonists in the
hypothermia and spontaneous activity tests. Compound 6b had agonist activity in all four in vivo
tests. All the compounds were antagonists of nicotineꢀinduced antinociception in the tailꢀflick
test and all except 5c, 5d, 5f, and 6b were antagonists of nicotineꢀinduced antinociception in the
hotꢀplate test. Compound 7c which had a Ki = 0.86 nM in the binding assay similar potency at
α4β2/α3β4 with selectivity relative to α7 nAChRs, AD50 value of 0.001 ꢁg/kg in the tailꢀflick test
with no agonist activity in the in vitro or in vivo test had one of the more interesting profiles.
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Keywords: nAChR antagonist, nicotine receptors, in vitro/in vivo studies, α4β2ꢀ and α3β4ꢀ
nAChR
Introduction
Neuronal nicotinic acetylcholine receptors (nAChRs) are ligandꢀgated ion channels that
are widely expressed throughout the peripheral and central nervous system.1, 2 The major
subtypes found in the mammalian brain are the α4β2ꢀ and α7ꢀnAChRs.3 The α4β2ꢀnAChR can
contain other subunits and are named α4β2*ꢀnAChRs. The α4β2ꢀnAChRs have been implicated
to play a vital role in nicotine (1) (Figure 1) addiction,4 as well as pain, depression, and anxiety.5,
6 Studies have shown that neuronal α3β4ꢀnAChR can also play a significant role in nicotine
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addiction.7-9 α3β4ꢀnAChR antagonists and partial agonists have been shown to reduce nicotine
withdrawal, reward, and selfꢀadministration.10-13 On the other hand, we also reported that
varenicline (2) was an agonist at α3β4ꢀnAChR which may account for several observed
peripheral side effects including nausea and gastrointestinal symptoms.14
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O
Cl
NH
CH3
H
N
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NHC(CH3)3
CH3
N
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Cl
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5a, X = H
5b, X = Cl
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5d, X = OMe
5e, X = CONH2
6a, X = H
6b, X = CONH2
6c, X = F
7a, X = Me
7b, X = CONH2
7c, X = Cl
6d, X = Cl
5f, X = SO2NH2
Figure 1. Structure of Compounds 1, 2, 3, 4, 5a–f, 6a–d, and 7a–c
Over the past several years much less effort has been directed toward the development of
nAChR antagonists than has been directed toward nAChR agonists and partial agonists.
However, recently nAChR antagonists have recently been suggested as potential
pharmacotherapies for depression and nicotine abuse.15, 16 At present, the major drugs used for
the treatment of nicotine dependence are nicotine (1) replacement therapies (NRT), bupropion
(3),17, 18 which acts as weak dopamine and norepinephrine uptake inhibitor as well as a nicotine
antagonist of α3β4* and α4β2*ꢀnAChRs,19 and varenicline (2), an α4β2ꢀnAChR partial agonist.20,
21
Over the last few years we have designed and developed novel nicotinic antagonists as
pharmacological tools and as potential pharmacotherapies for treating nicotine addiction as well
as other CNS disorders using the epibatidine (exoꢀ2ꢀ(2′ꢀchloroꢀ5′ꢀpyridinyl)ꢀ7ꢀ
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azabicyclo[2.2.1]heptane) (4) as the lead structure.22-28 In this study, we report the synthesis,
nAChR binding, and pharmacological properties of 2′ꢀfluoroꢀ3′ꢀ(substituted
thiophenyl)deschloroepibatidine compounds 5a–f, 6a–d, and 7a–c.
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Results and Discussion
The epibatidine analogs 5a–f, 6a–d, and 7a–c were designed to provide a set of
compounds that allowed further characterization of the structural requirements for inhibition of
[3H]epibatidine α4b2*ꢀnAChR binding affinity, in vitro nAChR subtype selectivity and agonist and
antagonist activities functional nicotinic pharmacological and in vivo nAChR agonists and
antagonists nAChR properties using a tailꢀflick, hotꢀplate, hypothermia, and spontaneous activity
test. A comparison of the results from these studies to the results previously obtained with
varenicline provided information concerning the potential of the compounds from the present
study as pharmacological tools for further studying nAChRs and as lead structures and possibly
pharmacotherapies for treating nicotine addiction.
Chemistry. Scheme 1 outlines the synthesis of analogs 5a, 5b and 6a. A Suzuki crossꢀ
coupling of 7ꢀtertꢀbutoxycarbonylꢀ2ꢀexoꢀ(2′ꢀaminoꢀ3′ꢀbromoꢀ5′ꢀpyridinyl)ꢀ7ꢀ
azabicyclo[2.2.1]heptane22 (8) with thiopheneꢀ2ꢀboronic acid in the presence of sodium
carbonate, tetrakis(triphenylphosphine) palladium(0), (Pd(PPh3)4), in N,Nꢀdimethylformamide
and water, heated at 100 °C overnight provided compound 9a.
Scheme 1a
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X
S
X
S
Br
NH2
NH2
Boc
Boc
F
H
N
a or b
N
N
c
N
N
N
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5a, X = H
5b, X = Cl
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9a, X = H
9b, X = Cl
c
S
Br
a
F
H
N
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H
N
N
N
6a
10
a Reagents and Conditions: (a) Thiopheneꢀ2ꢀ or 3ꢀboronic acid, Pd(PPh3)4, Na2CO3, DMF or 1,4ꢀdioxane,
H2O, 100 °C, 18 h; (b) 5ꢀChlorothiopheneꢀ2ꢀboronic acid, Pd2(dba)3 (3 mol %), PPh3 (10 mol %), DME,
H2O, microwave, 120 °C, 1 h (c) 70% HFꢀpyridine, NaNO2, 0 °C to rt, 1 h.
Furthermore, a microwave assisted Suzuki crossꢀcoupling reaction of 8 with 5ꢀchlorothiopheneꢀ
2ꢀboronic acid, in the presence of tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3) and
triphenylphosphine (PPh3) as the catalytic system, sodium carbonate, in 1,2ꢀdimethoxyethane
(DME) and water, irradiated at 120 °C for 1 h furnished a higher yield of compound 9b.
Diazotization of compounds 9a–b in the presence of 70% hydrogen fluoride in pyridine provided
analogs 5a and 5b. Likewise, the 2'ꢀfluoroꢀ3'ꢀbromo compound 10 prepared as previously
reported23 and 3ꢀthiopheneboronic acid were crossꢀcoupled in the presence of
tetrakis(triphenylphosphine) palladium(0) and sodium carbonate in 1,4ꢀdioxane and water
heated at 110 °C overnight provided analog 6a. The synthesis of 5c as outlined in Scheme 2,
commenced with the crossꢀcoupling of the 2'ꢀfluoroꢀ3'ꢀbromo Bocꢀprotected compound 1126 with
thiopheneꢀ2ꢀboronic acid in the presence of tetrakis(triphenylphosphine) palladium(0) and
potassium carbonate in 1,4ꢀdioxane and water, heated at 100 °C overnight provided compound
12. Compound 12 in anhydrous tetrahydrofuran (THF), was subjected to a lithiation using nꢀ
butyllithium at –78 °C. The resulting lithio anion was quenched with Nꢀfluorobenzenesulfonimide
followed by removal of the Bocꢀprotecting group using trifluoroacetic acid in methylene chloride
to provide analog 5c. The methoxythiophene analog 5d (Scheme 2), was synthesized by crossꢀ
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coupling the boronic ester 1326 with 2ꢀbromoꢀ5ꢀmethoxythiophene (14) [prepared in quantitative
yields through a radical bromination reaction of 2ꢀmethoxythiophene (15) using Nꢀ
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bromosuccinimide] in the presence of tetrakis(triphenylphosphine) palladium(0) and potassium
carbonate in 1,4ꢀdioxane and water heated at reflux overnight which provided the Boc protected
product. A trifluoroacetic acid removal of the Bocꢀprotecting group provided the methoxy
substituted analog 5d. Similarly, crossꢀcoupling of boronic ester 13 with 5ꢀbromothiopheneꢀ2ꢀ
carboxamide in the presence of potassium carbonate, tetrakis(triphenylphosphine)palladium(0)
in 1,2ꢀdimethoxyethane and water heated at 85 °C overnight provided the Bocꢀprotected
thiopheneꢀ2ꢀcarboxamide analog and subsequent removal of the Boc group furnished analog
5e. The reaction of compound 13 with 5ꢀbromothiopheneꢀ2ꢀsulfonamide in the presence, 1,1'ꢀ
bis(diphenylphosphino)ferroceneꢀpalladium(II)dichloride and potassium phosphate in 1,4ꢀ
dioxane, dimethylformamide and water, heated at 85 °C overnight, furnished the thiopheneꢀ2ꢀ
sulfonamide Bocꢀprotected analog which on removal of the Bocꢀprotected group using
hydrochloric acid in methanol overnight gave the analog 5f. Following a similar protocol, the
synthesis of analog 6b was accomplished using compound 11 and 4ꢀ(4,4,5,5ꢀteramethylꢀ1,3,2ꢀ
dioxaborolanꢀ2)thiopheneꢀ2ꢀcarboxamide [prepared from 4ꢀbromoꢀ2ꢀcarboxamide under similar
conditions as those used to prepare compound 13] as the starting materials heated at 85 °C in
the presence of 1,1'ꢀbis(diphenylphosphino)ferroceneꢀpalladium(II)dichloride, potassium
phosphate, 1,4ꢀdioxane, dimethylformamide and water to provide the Bocꢀprotected product
which on removal of the Bocꢀprotecting group using hydrochloric acid in methanol furnished 6b.
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Scheme 2a
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F
S
S
N
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F
b,c
Boc
H
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N
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CONH2
F
X
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12
5c
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a
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N
N
i,c
6b
e,c or f,g
or h,g, k
5d, X = OMe
5e, X = CONH2
5f, X = SO2NH2
Br
F
Boc
N
N
d
O
j,c
O
N
B
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S
Boc
N
F
H
N
13
N
7a
l and h,g
H2NOC
S
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H
N
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a Reagents and Conditions: (a) Thiopheneꢀ2ꢀboronic acid, Pd(PPh3)4, K2CO3, 1,4ꢀdioxane, H2O, 100 °C,
overnight; (b) (i) nꢀBuLi, THF, at –78 °C, 40 min to 0 °C (10 min), (ii) Nꢀfluorobenzenesulfonimide, THF, –
78 °C, 1 h to rt. (c) TFA, CH2Cl2, rt, overnight; (d) bis(pinacolato)diboron, KOAc, PdCl2dppf, 1,4ꢀdioxane,
microwave 140 °C, 20 min; (e) 2ꢀbromoꢀ5ꢀmethoxythiophene (14) [prepared from 2ꢀmethoxythiophene
(15) using NBS], Pd(PPh3)4, K2CO3, 1,4ꢀdioxane, H2O, reflux, overnight; (f) 5ꢀbromothiopheneꢀ2ꢀ
carboxamide, Pd(PPh3)4, K2CO3, DME, H2O, 85 °C, overnight; (g) HCl in Et2O/ MeOH, rt, overnight; (h)
PdCl2(dppf) (3 mol %), K3PO4, or Na2CO3, 1,4ꢀdioxane, DMF, H2O, 80 °C, overnight; (i) 4ꢀ(4,4,5,5ꢀ
tetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)thiopheneꢀ2ꢀcarboxamide, [prepared from 4ꢀbromothiopheneꢀ2ꢀ
carboxamide using reagents and conditions in (d)], Pd(PPh3)4, Na2CO3. 1,4ꢀdioxane, DMF, H2O, 100 °C,
overnight; (j) Pd(PPh3)4, K2CO3, 1,4ꢀdioxane, H2O, microwave, 150 °C, 1 h; (k) 5ꢀBromothiopheneꢀ2ꢀ
sulfonamide (l) 5ꢀbromothiopheneꢀ3ꢀcarboxamide (16) [prepared in three steps from thiopheneꢀ3ꢀ
carboxylic acid].
The key step towards the synthesis of analog 7a was accomplished through a microwave
assisted Suzuki crossꢀcoupling. Compound 1126 and 4ꢀmethylthiopheneꢀ2ꢀboronic acid were
crossꢀcoupled in a microwave reactor in the presence of tetrakis(triphenylphosphine)
palladium(0) and potassium carbonate in 1,4ꢀdioxane and water, irradiated for 1 h at 150 °C to
provide the Bocꢀprotected product. A trifluoroacetic acid removal of the Boc group in methylene
chloride provided analog 7a. The coupling partner needed for the synthesis of analog 7b, 5ꢀ
bromothiopheneꢀ3ꢀcarboxamide (16) was prepared from thiopheneꢀ3ꢀcarboxylic acid in three
steps i.e., bromination using Nꢀbromosuccinimide provided 5ꢀbromothiopheneꢀ3ꢀcarboxylic acid
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which was subsequently converted to the acyl chloride using thionyl chloride and finally
amination of the carboxylate was accomplished using an aqueous ammonium hydroxide
solution to furnish compound 16. A 1,1'ꢀbis(diphenylphosphino)ferroceneꢀpalladium(II)dichloride
catalyzed reaction of 16 with boronic ester 13 in the presence of sodium carbonate in
dimethylformamide and water heated at 100 °C provided the Boc protected 7b which gave 7b
upon removal of the Boc group using hydrochloric acid in methanol.
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Scheme 3 outlines the route followed for the synthesis of analogues 6c, 6d and 7c. 3ꢀ
Bromothiopheneꢀ2ꢀtrimethylsilane (18a) and 3ꢀchlorothiopheneꢀ2ꢀtrimethylsilane (18b) were
prepared from 3ꢀbromothiophene (17a) and 3ꢀchlorothiophene (17b), respectively, via 2ꢀ
lithiation using lithium bis(trimethylsilyl)amide and subsequent quenching with
trimethylchlorosilane. Crossꢀcoupling of compound 13 with 18a in the presence of 1,1'ꢀ
bis(diphenylphosphino)ferroceneꢀpalladium(II)dichloride and 2M aqueous solution of sodium
carbonate in 1,2ꢀdimethoxyethane, degassed through bubbling nitrogen and irradiated in a
microwave reactor at 110 °C for 30 min provided compound 19. Lithiation of 19 with nꢀ
butyllithium at –78 °C and subsequent quenching with a solution of hexachloroethane in
tetrahydrofuran stirred at –78 °C for 1 h introduced the chlorine at the desired Cꢀ2 position.
Simultaneous removal of the TMS and Boc groups using trifluoroacetic acid in methylene
chloride furnished the desired analogue 6d. Similarly, 2ꢀlithiated of compound 19 was quenched
with a solution of Nꢀfluorobenzenesulfonimide in tetrahydrofuran, added at –78 °C and allowed
to warm to room temperature overnight provided the difluorinated product. Complete removal of
the Boc group in the difluorinated analogue using either trifluoroacetic or hydrochloric acid
required heating for at least 24 h to provide analogue 6c. Compound 18b was iodinated at the α
position using nꢀbutyllithium at –78 °C and quenching with iodine to provide compound 20,
which was subsequently crossꢀcoupled with 13 in a microwave assisted reaction under similar
conditions to provide compound 21. Concurrent removal of the TMS and Boc groups using
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trifluoroacetic acid in methylene chloride stirred at room temperature overnight provided
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analogue 7c.
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Scheme 3a
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18a, X= Br
18b, X= Cl
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Cl
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+ 13
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Cl
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7c
a Reagents and Conditions: (a) (i) LiHMDS, THF, 0 °C, 1 h, (ii) TMSCl, 0 °C, 1 h; (b) (i) nꢀBuLi (in
hexanes), THF, –78 °C, 1 h, (ii) I2, THF, –78 °C, 30 min; (c) Pd(dppf)Cl2, 2M Na2CO3, DME, µw 110 °C,
30 min; (d) (i) nꢀBuLi, THF, –78 °C, for 40 min then 0 °C (10 min), (ii) either NFSI, THF, –78 °C, 1 h (for
6c) or C6Cl6, THF, –78 °C (for 6d) ; (e) TFA in CH2Cl2, rt, overnight (in the case of 6c, heating was
required for the removal of the Boc group).
α4β2*ꢀnAChR Binding. The nAChR binding affinities of 2′ꢀfluoroꢀ3′ꢀ(substituted
thiophenyl)deschloroepibatidine analogs 5a–f, 6a–d, and 7a–c were determined in rat cerebral
cortex homogenates using [3H]epibatidine and compared to binding affinity values for reference
compounds natꢀepibatidine, DHβE, and varenicline. It should be noted that although other
nAChR subtypes are present in cerebral cortex, the α4β2 nAChR predominates, thus the
reported affinities closely approximate those for this subtype. Compounds 5a–f, 6a–d, and 7a–c
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all had subnanomolar Ki values for inhibition of [3H]epibatidine binding for α4β2*ꢀnAChRs with
values ranging from 0.11 to 0.86 nM (Table 1).
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Table 1. Radioligand Binding and Antinociception Data for 2′ꢀfluoroꢀ3′ꢀ(substituted thiophenyl)deschloroepibatidine Analogs
Antagonist Activity
(% EC50 ACh response
remaining
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4
5
6
7
8
9
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Agonist Activity
(% of max ACh response
at 100 µM)
mg/kg
AD50 (ꢁg/kg)
αβ
[3H]Epibatidine
Compda
X
at 100 µM)
(K , nM)b
i
ED50
ED50
TailꢀFlick
ED50
HotꢀPlate
ED50
Hypothermia
Hotꢀ
Plate
Spontaneous TailꢀFlick
Activity
α4β2
α3β4
37 ± 3
97 ± 4
α7
α4β2
α3β4
nd
α7
0.65
(0.25–
0.85)
0.004
(0.001–
0.008)
1.3
(0.5–1.8)
1.0
(0.6–2.1)
0.5
(0.15–0.78)
nicotine
nat
1.5 ± 0.30
40 ± 1
43 ± 5
150 ± 8
nd
nd
nd
nd
0.004
(0.002–
0.008)
0.001
(0.0005–
0.005)
0.006
0.001–0.01
0.026 ± 0.002
131 ± 13
nd
epibatidine
DHβE
varenicline
39.5 ± 4.9
0.12 ± 0.02
10% @ 10
5% @ 10
10% @ 10
3% @ 10
2.8
2.1
0.2
470
330
(70–
1400)
257
(183–
551)
13 ± 0.4
0
66 ± 4
0
74 ± 5
0
38 ± 2
9 ± 1
nd
nd
9.5
(5.3–14.9)
4
0.52
(0.02–13)
5a
5b
5c
5d
H
F
0.49 ± 0.18
0.20 ± 0.03
0.36 ± 0.05
0.49 ± 0.06
6 ± 1
33 ± 4
(1.1–14)
0.04
(0.01–
0.31)
4.5
(3.3–6.15)
3
7% @ 10
4% @ 10
6% @ 10
14% @ 10
15% @ 10
11% @ 10
0
0
0
0
0
0
0
0
0
14 ± 2
19 ± 2
9 ± 1
4 ± 1
6 ± 1
8 ± 3
12 ± 4
21 ± 2
20 ± 2
(0.5–17)
3710
10
(2–53)
2
25% @
10000
Cl
(1152–
119500
2075
(1145–
3758)
(0.3–13.5)
14.6
(6.7–32)
2.2
(1.6–3)
2% @
10000
CH3O
9900
(5700–
12300)
9% @
10000
196
5e
5f
CONH2
SO2NH2
H
0.71 ± 0.13
0.25 ± 0.1
0.41 ± 0.2
6% @ 10
7% @ 10
1% @ 10
7% @ 10
0% @ 10
9% @ 10
0%@10
0% @ 10
10% @ 10
0%@10
0% @ 10
15% @ 10
1.0 ± 0.2
0
0
0
0
1.2 ± 0.5
0
18 ± 3
14 ± 2
5 ± 1
18 ± 2
15 ± 2
59 ± 9
68 ± 12
16 ± 3
(75–345)
1.01
(0.11–3.5)
0.075
(0.04–
0.12)
0
0
113
(27–475)
6a
3.8 ± 0.4
0.03
(0.007–
0.14)
4.7
(3.4–6.6)
2.8
(1.8–4.9)
1.2
(0.12–12.4)
0.34
(0.17–0.7)
30%@10
00
6b
6c
F
0.17 ± 0.04
0.21 ± 0.04
2.6 ± 0.2
0
1.1 ± 0.1
0
1.2 ± 0.1
1.7 ± 0.5
14 ± 1
9 ± 1
17 ± 5
8 ± 1
22 ± 2
21 ± 2
0.05
(0.01–
0.24)
25%
increase @
10
35
(0.5–
252)
Cl
2%@10
10%@10
0%@10
0%@10
435
(131–
1436)
832
(136–
5051)
4045
(1204–
13583)
2008
1
6d
7a
7b
7c
CONH2
Cl
0.11 ± 0.01
0.22 ± 0.04
0.81 ± 0.2
0.86 ± 0.1
1%@10
1%@10
9% @ 10
4%@10
10%10
5%@10
0
0
0
0
0
0
11 ± 3
13 ± 2
9 ± 2
8 ± 2
8 ± 2
5 ± 1
13 ± 4
62 ± 4
35 ± 2
43 ± 4
53 ± 7
(0.3–10)
9
3.6
(0.8–16)
0.32
(0.03–2.7)
20%@10
13% @ 10
18%@10
0
0
1.9 ± 0.6
(5–16)
1349
(520–
3501)
5.7
(1.3–25)
3.2
(0.2–49)
CH3
0
0
0.001
CONH2
0%@10
25%@10
(0.0008–
0.03)
(977–
4429)
1.5 ± 0.2
6 ± 1
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a All compounds were tested as their a) (±)ꢀisomers; b) [3H]epibatidine Kd is 0.02 nM
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For comparison the reference nAChR agonist natꢀepibatidine, the reference nAChR antagonist
DHβE, and the reference α4β2ꢀnAChR partial agonist varenicline had Ki values for inhibition of
[3H]epibatidine of 0.026, 39.5, and 0.12 nM, respectively. Thus, the 0.11 nM Ki value of 6d was
essentially identical to that of varenicline and was 359 times better than the 39.5 nM Ki value for
DHβE.
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From a structure activity relationship (SAR) perspective it is interesting to note that the
position of the thiophenyl group relative to its attachment to the epibatidine pyridine ring or the
character of the substituent on the thiophene ring had little effect on Ki value for the inhibition of
[3H]epibatidine binding. Compounds with large sulfonamide or carboxamide substituents differed
very little from unsubstituted analogs and electron releasing methoxy or withdrawing chloro
substituents.
In vitro nAChR subtype selectivity. Compounds 5a–f, 6a–d, and 7a–c were tested for
receptor subtype selectivity in an electrophysiology assay. α4β2ꢀ, α3β4, and α7ꢀnAChRs were
expressed in Xenopus oocytes and functional effects of the compounds were determined under
twoꢀelectrode voltage clamp. Values obtained for compounds 5a–f, 6a–d, and 7a–c are
compared to previously determined values for nicotine, natꢀepibatidine and varenicline (Table
1). To assess potential agonist activity, current responses to 100 µM of each compound were
compared to the maximum response that can be achieved with acetylcholine. Compounds 5a–f,
6a–d, and 7a–c showed little or no agonist activity at the α4β2ꢀ, α3β4ꢀ, and α7ꢀnAChRs. This
contrasted with nicotine, natꢀepibatidine and varenicline, which are full or partial agonists at
these nAChR subtypes.
We also tested compounds 5a–f, 6a–d, and 7a–c for antagonist activity. The current
response to an EC50 concentration of acetylcholine in the presence of 100 ꢁM of each
compound was compared to a preceding current response to acetylcholine alone (Table 1). All
compounds displayed antagonist activity at each of the three receptor subtypes. Compound 6b
achieved a similar level of block at all three subtypes. Compounds 5b and 5c were more
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effective antagonists at α3β4 receptors, than at α4β2ꢀ or α7ꢀnAChRs. Compounds 5a, 5d–f, 6a,
6c–d and 7a–c had a similar selectivity profile of α4β2 ≥ α3β4 > α7. We examined the
antagonist properties of representative compound 7c in more detail by constructing
concentrationꢀinhibition curves and obtaining IC50 values. 7c displayed a selectivity of α4β2 >
α3β4 >> α7, with IC50 values of 1.6 ± 0.2 µM, 3.3 ± 0.4 µM and 101 ± 10 µM, respectively. While
7c was only 2ꢀfold more potent at α4β2ꢀ versus α3β4ꢀnAChRs, this compound was 63ꢀfold more
potent at α4β2ꢀ versus α7ꢀnAChRs.
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In vivo properties. Compounds 5a–f, 6a–d, and 7a–c were evaluated for their in vivo
nAChR properties in mice and compared to the nAChR properties of DHβE and varenicline
(Table 1). Similar to varenicline none of the new compounds except 6b showed any agonist
activity in the tailꢀflick or hotꢀplate test after acute administration in the mouse. Also like
varenicline, compounds 5a–d, 6b, and 7a–b had agonist activity in the hypothermia and
spontaneous activity assays. In the hypothermia test the ED50 values range from 1.2 mg/kg for
6b to 14.6 mg/kg for 5d compared to 2.8 mg/kg for varenicline. In the spontaneous activity test,
the ED50 values ranged from 0.34 mg/kg for 6b to 4 mg/kg for 5a compared to 2.1 mg/kg for
varenicline. The lack of activity in the tailꢀflick and hotꢀplate tests for all of these analogues
except 6b combined with the fact the 5e, 5f, 6a, 6d, and 7c did not show any agonist activity in
any of the four tests and their high affinity for inhibition of [3H]epibatidine binding suggested that
these compounds might act as functional nAChR antagonists in the two pain tests. Indeed, all of
the analogues were antagonists in the tailꢀflick test with some of the compounds being highly
potent antagonists in this test. For example, 5b, 6a, 6b, 6c, and 7c had AD50 values of 0.04,
0.075, 0.03, 0.05, and 0.001 ꢁg/kg, respectively. For comparison DHβE had an AD50 value of
450 ꢁg/kg. Compounds 5a and 7a have AD50 values of 0.52 and 0.32 ꢁg/kg in the tailꢀflick test
and thus are similar to the 0.2 ꢁg/kg for varenicline. Compound 6c with an AD50 = 35 ꢁg/kg was
the most potent analog in the hotꢀplate test. Compounds 5a, 5b, 6a, and 6d had AD50 values of
330, 257, 113, and 435 ꢁg/kg, respectively, which is similar to the AD50 = 470 ꢁg/kg for
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varenicline. It is interesting to note that analogue 5a has an in vitro and in vivo profile that
closely resembles that for varenicline. Compound 5b with a Ki = 0.20 nM for inhibition of
[3H]epibatidine binding, ED50 values of 4.5 and 3 mg/kg in the hypothermia and spontaneous
activity test and AD50 values of 0.04 and 257 ꢁg/kg in the tailꢀflick and hotꢀplate are very
comparable to the varenicline values of 0.12 nM for inhibition of [3H]epibatidine binding, ED50 =
2.8 and 2.1 mg/kg in the hypothermia and spontaneous activity test and AD50 values of 0.2 and
470 ꢁg/kg in the tailꢀflick and hotꢀplate tests.
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In order to gain information concerning the predicted ability of compounds 5a–d, 6a–d and
7a–c to cross the blood/brain barrier, their topological polar surface area (TPSA), clogP and
derived logBB values were calculated and compared to varenicline (Table 2). Compounds that
have TPSA values less than 76Å2,29 clogP values in the range 2–4,30 and derived logBB values
greater than ꢀ131 are predicted to cross the blood/brain barrier. All of the compounds except 5f
have TPSA values less than 76Å2. Compounds 5aꢀc, 6a, 6c–d and 7c had TPSA values of
24.92Å2 compared to 37.81Å2 for varenicline. Compound 5d has a TPSA = 34.15Å2, 5e, 6b and
7b have a TPSA = 68.01Å2 and 5f has a TPSA = 85.01Å2. Compounds 5a–d, 6a, 6d, 7a and 7c
all have clogP values between 2–4 compared to a clogP value of 1.01 for varenicline.
Compounds 5e–f, 6b and 7b have clogP values of 1.96, 0.75, 1.97 and 1.83, respectively. All of
the compounds except 5f have logBB values greater than ꢀ1 and thus would be predicted to
penetrate the brain. All of the compounds except 5e–f, 6b and 7b have more positive logBB
values than the logBB value of ꢀ0.27 for varenicline. Wager and coꢀworkers conducted a study
of market CNS drugs and determined that 74% of CNS drugs displayed a CNS MPO
(Multiparameter Optimization) score greater than or equal to four.32 Compounds 5a, 5d–f, 6a–b
and 7b all had a CNS MPO value of four or greater compared to a value of 4.86 for varenicline.
All of the other compounds had CNS MPO values of greater than 3.7.
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Table 2. Calculated Physiochemical Properties of 5a–f, 6a–d, and 7a–c, and Varenicline
5
6
7
8
Compd ID
LogPa
1.01
2.98
3.75
3.29
2.99
1.96
0.75
2.98
1.97
3.29
3.75
3.49
1.83
3.58
TPSAa
37.81
24.92
24.92
24.92
34.15
68.01
85.08
24.92
68.01
24.92
24.92
24.92
68.01
24.92
logBBb
–0.27
0.22
CNS MPO
4.86
4.08
3.70
3.93
4.54
4.50
4.50
4.08
4.50
3.93
3.70
3.83
4.50
3.79
varenicline
5a
5b
5c
5d
5e
5f
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0.34
0.27
0.09
–0.57
–1.01
0.22
6a
6b
6c
6d
7a
7b
7c
–0.57
0.27
0.34
0.30
–0.59
0.32
a ChemAxon Calculator Plugins, Marvin 6.1.0, 2013. b logBB = –0.0148 × TPSA + 0.152 × logP
+ 0.139 (from ref. 33).
In summary several epibatidine analogs (5e, 5f, 6a, 6c, and 7c) had high affinity in the
[3H]epibatidine binding assay, had no agonist activity in in vivo tailꢀflick, hotꢀplate, hyperthermia,
and spontaneous activity tests but were potent antagonists in both the in vitro assay and the in
vivo tailꢀflick test. Several other of the epibatidine analogs (5a–d, 7a, and 7b) did not show
agonist activity in the tailꢀflick and hotꢀplate test but like varenicline were agonist in the
hyporthermia and spontaneous activity test and were potent antagonists in the in vitro assay
and the tailꢀflick test. Compound 6b was an agonist in all four of the in vivo tests and was a
potent antagonist in the in vitro assays and the in vivo tailꢀflick tests. Compound 6c with a Ki of
0.21 nM in the binding assay, an AD50 of 0.05 and 35 ꢁg/kg in the tailꢀflick and hotꢀplate tests,
but no agonist activity, compound 7c with a Ki of 0.86 nM in the binding assay, similar potencies
as an antagonist at α4β2ꢀ and α3β4ꢀnAChRs with good selectivity versus α7ꢀnAChRs and an
AD50 of 0.001 ꢁg/kg in the tailꢀflick test with no agonist activity and compound 5a with a nAChR
profile similar to varenicline in the four in vivo tests but no agonist activity at the α3β4ꢀ and α7ꢀ
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nAChRs like varenicline have properties that suggest utilities as pharmacological tools for
further characterization of nAChRs as well as lead compounds and possible pharmacotherapies
for treating nicotine addiction.
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Methods
Melting points were determined on a Melꢀtemp (Laboratory Devices, Inc.) capillary tube
apparatus. NMR spectra were recorded on a Brunker Avance 300 or AMX 500 Spectrometer
using tetramethylsilane as internal standard. Mass spectra were determined on a PerkinꢀElmer
Sciex API 150EX mass spectrometer outfitted with an APCI and ESI sources. Elemental
analyses were carried out by Atlantic Microlab, Inc., Norcross GA. Analytical thinꢀlayer
chromatography (TLC) was carried out on plates preꢀcoated with silica gel (60 F254). TLC
visualization was accomplished with a UV lamp or in an iodine chamber. Purifications by flash
chromatography were performed on a Combiflash® Teledyne ISCO instrument.
General procedure A for the Synthesis of compounds 5a and 5b. The 2'ꢀaminoꢀ3'ꢀ
(substituted thiophenyl) compound 9a or 9b was stirred in a solution of HFꢀpyridine (70%) at 0
°C for 30 min and was then treated with NaNO2 (10 equiv.). The mixture was stirred at 0 °C for
an additional 20 min then at room temperature for 1 h, after which it was quenched with a 3:1
aqueous solution of NH4OH and extracted with CHCl3. The combined organic layers were dried
(Na2SO4), filtered and concentrated in vacuo. The resultant residue was purified by flash
chromatography on a silica gel column, eluted with CHCl3/MeOH (9/1), to furnish 5a and 5b.
General Procedure B. A solution of 13 (1 equiv.), 5ꢀbromothiopheneꢀ2ꢀcarboxamide or 5ꢀ
bromothiopheneꢀ2ꢀsulfonamide (1.5 equiv.), Pd(PPh3)4 (10 mol %) and K2CO3 (2.5 equiv.) in
DME (10 mL) and H2O (2 mL) was placed in a resealable pressure vessel, degassed through
bubbling nitrogen for 20 min, sealed and heated at 95 °C overnight. After cooling to room
temperature, NH4Cl (20 mL) was added and the organic product extracted with EtOAc (3 × 30
mL). The combined organic layers were dried (Na2SO4), filtered through Celite, concentrated in
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vacuo and the residue was purified by flash chromatography on silica gel columns, eluted with
hexanesꢀethyl acetate, to provide the Bocꢀprotected product of either 5e or 5f, respectively.
2ꢀexo-[2'ꢀFluoroꢀ3'ꢀ(thiophenꢀ2ꢀyl)ꢀ5'ꢀpyridnyl]ꢀ7ꢀazabicyclo[2.2.1]heptane (5a)
Fumarate. Prepared from compound 9a (175 mg, 0.471 mmol) as described in the General
Procedure A to provide 58 mg (45% yield) of 5a free base after purification. (1H NMR (300 MHz,
CDCl3) δ 1.50–1.80 (m, 6H), 1.94–2.01 (m, 1H), 2.83–2.88 (m, 1H), 3.38–3.68 (br s, 1H) 3.68 (s,
1H), 3.89 (s 1H), 7.10 (dd, J = 3.9, 4.7 Hz, 1H), 7.38 (dd, J = 0.9, 5.1 Hz, 1H), 7.55 (t, J = 3.6
Hz, 1H), 8.0 (s, 1H), 8.14 (dd, J = 2.2, 9.5 Hz, 1H); 13C NMR (CDCl3) δ 29.6, 31.1, 40.1, 44.3,
56.6, 62.8, 116.7, 126.6, 127.2, 127.9, 135.5, 137.4, 139.9 144.0, 156.3, 159.4; MS (ESI) m/z
372.2 (M+H)+.
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Compound 5a (125 mg, 0.454 mmol) was dissolved in chloroform (2 mL) in a vial and
treated with 1.3 equiv of fumaric acid (0.65 M) in MeOH and allowed to stand in a refrigerator
overnight. The excess solvent was then removed under reduced pressure and the residue salt
was redissolved in minimal amount of MeOH. The fumarate salts were recrystallized from
MeOH using diethyl ether to provide 111 mg (63% yield) of 5a•C4H4O4 as a brownish solid: mp.
202–204 °C; 1H NMR (300 MHz, MeOHꢀd4) δ 1.84–2.20 (m, 5H), 2.43–2.51 (m, 1H), 3.46–3.51
(m, 1H), 4.33 (br s, 1H) 4.56 (d, J = 3.3 Hz, 1H), 6.67 (s 2H), 7.61 (d, J = 5.1 Hz, 1H), 7.69 (d, J
= 3.8 Hz, 1H), 8.08 (s, 1H), 8.0 (s, 1H), 8.20 (dd, J = 2.4, 9.2 Hz, 1H); 13C NMR (300 MHz,
MeOHꢀd4) δ 27.1, 29.1, 37.9, 43.6, 60.4, 64.3, 127.3, 128.9, 129.1, 136.3, 137.4, 138.8 (JCF
4.5 Hz), 144.9, 145.1 (JCF = 14.6 Hz), 171.5; MS (ESI) m/z 275.3 [(Mꢀfumarate)+,
=
M=C15H15FN2S•C4H4O4]. Anal. calcd for C19H19FN2O4S: C 58.45; H 4.91; N 7.18; found: C 58.22;
H 4.97; N 7.18.
2ꢀexo-[2'ꢀFluoroꢀ3′ꢀ(5ꢀchlorothiophenꢀ2ꢀyl)ꢀ5′ꢀpyridinyl]ꢀ7ꢀazabicyclo[2.2.1]heptane
(5b). Prepared from compound 9b (299 mg, 0.738 mmol) as described in General Procedure A
to furnish 160 mg (70%) of 5b as a yellow oil after purification. 1H NMR (300 MHz, CDCl3) δ
1.51–1.68 (m, 6H), 1.89–1.93 (dd, J = 9.0, 12.4 Hz, 1H), 2.76–2.80 (dd, J = 4.8, 8.9 Hz, 1H),
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3.58 (s, 1H), 3.80 (s, 1H), 4.79 (s, 2H), 6.92 (d, J = 3.81 Hz, 1H), 7.30 (dd, J = 3.96, 0.9 Hz, 1H),
7.99 (d, J = 2.1 Hz, 1H), 8.07 (dd, J =9.6, 2.3 Hz, 1H); 13C NMR (CDCl3) δ 30.1, 31.3, 40.4,
44.2, 56.2, 62.6, 115.7 (JCF = 28.4 Hz), 126.0, 126.8, 131.1 (JCF = 225 Hz), 134.0, 136.6, 140.7,
144.1, 155.8, 159.0; MS (ESI) m/z 309.0 (M+H)+.
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Compound 5b (309 mg, 0.519 mmol) in a vial was dissolved in minimal amount of
chloroform and treated with 1.3 equiv of fumaric acid (0.65 M in MeOH) and allowed to stand in
a refrigerator overnight. The excess solvent was then removed under reduced pressure and the
residue salt was redissolved in minimal amount of MeOH. The fumarate salts were
recrystallized from MeOH using diethyl ether to provide 150 mg (67% yield) of 5b•C4H4O4 as a
yellowish solid: mp. 133–137 °C; 1H NMR (300 MHz, MeOHꢀd4) δ (ppm) 1.88–2.16 (m, 5H),
2.42–2.50 (dd, J = 9.0, 12.4 Hz, 1H), 3.45–3.50 (dd, J = 4.8, 8.9 Hz, 1H), 4.33 (s, 1H), 4.53 (s,
1H), 6.65 (s, 2H), 7.07 (d, J = 3.2 Hz, 1H), 7.54 (d, J = 3.7 Hz, 1H), 8.09 (s, 1H), 8.19 (d, J =9.2
Hz, 1H); 13C NMR (300 MHz, MeOHꢀd4) δ 26.9, 29.0, 37.7, 43.4, 60.2, 64.1, 117.6, 128.5,
131.3, 134.6, 136.2, 137.4, 138.1 (JCF = 4.5 Hz), 145.6 (JCF = 14.6 Hz), 159.7, 171.4; MS (ESI)
m/z 309.1 [(Mꢀfumarate)+, M=C15H14ClFN2S•C4H4O4]. Anal. calcd for C19H18ClFN2O4S•0.5H2O: C
53.15; H 4.34; N 6.52; found: C 53.04; H 4.44; N 6.55.
2ꢀexo-[2'ꢀFluoroꢀ3′ꢀ(5ꢀfluorothiophenꢀ2ꢀyl)ꢀ5′ꢀpyridinyl]ꢀ7ꢀazabicyclo[2.2.1]heptane
(5c) Hydrochloride. A solution of compound 12 (dried over molecular sieves) (346 mg, 0.925
mmol) in anhydrous THF (6 mL) in a flame dried flask was cooled to –78 °C and treated with nꢀ
BuLi (730 ꢁL, 1.6 M in hexanes, 1.16 mmol) dropwise. The mixture was stirred at –78 °C for 40
min then allowed to warm up to 0 °C for 10 min and then cooled back down to –78 °C. Nꢀ
Fluorobenzenesulfonimide (NFSI) (380 mg, 1.203 mmol) was added to the mixture and the
reaction was stirred at –78 °C for 1 h then allowed to warm up to room temperature before being
quenched with a 20ꢀmL aqueous solution of NH4Cl. The organic product was extracted with
EtOAc (3 × 30 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered
through Celite and concentrated in vacuo. The residue was purified on silica gel, eluted with
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EtOAc/hexanes to furnish the Boc protected compound, 7ꢀtertꢀbutoxycarbonylꢀ2ꢀexoꢀ[2'ꢀfluoroꢀ
3′ꢀ(5ꢀfluorothiophenꢀ2ꢀyl)ꢀ5′ꢀpyridinyl]ꢀ7ꢀazabicyclo[2.2.1]heptane; 1H NMR (300 MHz, CDCl3) δ
1.45 (s, 9H), 1.52–1.62 (m, 2H), 1.80–1.86 (m, 3H), 2.00–2.07 (dd, J = 12.4, 9.0 Hz, 1H), 2.88–
2.93 (dd, J = 9.0, 4.7 Hz, 1H), 4.22 (br s, 1H) 4.40 (s, 1H), 3.89 (s 1H), 6.51 (dd, J = 4.1, 2.2 Hz,
1H), 7.16 (t, J = 4.1 Hz, 1H), 7.89 (dd, J = 2.0, 9.6 Hz, 1H), 7.94 (d, J = 1.9 Hz, 1H); 13C NMR
(CDCl3) δ 28.3, 28.8, 29.7, 40.7, 44.7, 56.6, 80.0, 108.2, 123.7, 128.0, 135.8, 139.8, 139.9
143.9, 155.1, 159.4.
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The Bocꢀprotected product was dissolved in anhydrous CH2Cl2 (5 mL) and treated with
TFA (3 mL) and was stirred at room temperature overnight. The mixture was poured into a cold
solution of NH4OH in water (1:1) and was then extracted with CH2Cl2. The organic phase was
washed with brine, dried (Na2SO4), and concentrated in vacuo. Purification on a silica gel
column, eluted with CH2Cl2–MeOH, provided 128 mg of 5c as a yellowish oil: 1H NMR (300
MHz, CDCl3) δ (ppm) 1.50–1.73 (m, 6H), 1.89–1.96 (dd, J = 12.4, 9.0 Hz, 1H), 2.76–2.81 (dd, J
= 9.0, 4.7 Hz, 1H), 3.59 (br s, 1H) 3.80 (s, 1H), 6.51 (dd, J = 4.1, 2.2 Hz, 1H), 7.16 (t, J = 4.1 Hz,
1H), 7.97 (s, 1H), 8.06 (dd, J = 9.6, 2.2 Hz, 1H); 13C NMR (CDCl3) δ 30.3, 31.4, 40.6, 44.4, 56.4,
62.8, 108.2, 116.3, 123.5, 136.6, 140.8, 144.0, 159.3, 164.4, 168.2. MS (ESI) m/z 293.2 (M+H)+.
A solution of 5c (134 mg, 0.458 mmol) in MeOH was treated dropwise with HCl (1.2 equiv)
in diethyl ether to provide 146 mg (93%) of 5c•HCl as a offꢀwhite solid: mp. 86–90 °C; 1H NMR
(MeOHꢀd4) δ (ppm) 1.91–2.16 (m, 5H), 2.44–2.52 (dd, J = 13.1, 9.6 Hz, 1H), 3.47–3.52 (dd, J =
7.5, 4.0 Hz, 1H), 4.35 (br s, 1H) 4.57 (s, 1H), 6.68 (dd, J = 4.1, 2.2 Hz, 1H), 7.40 (t, J = 3.8 Hz,
1H), 8.08 (s, 1H), 8.16 (dd, J = 9.2, 2.2 Hz, 1H); 13C NMR (MeOHꢀd4) δ (ppm) 26.8, 28.9, 37.5,
43.3, 60.5, 64.2, 109.5 (JCF = 10.7 Hz), 117.8, 125.8 (JCF = 4.1 Hz), 137.2, 138.0, 144.8 (JCF
14.7 Hz), 157.6, 160.8; MS (ESI) m/z 293.3 [(M–HCl))+, M = C15H14F2N2S]. Anal. calcd for
C15H15ClF2N2S•0.75H2O: C 52.63; H 4.86; N 8.18; found C 52.49; H 4.94; N 7.93.
=
2ꢀexo-[2'ꢀFluoroꢀ3′ꢀ(5ꢀmethoxythiophenꢀ2ꢀyl)ꢀ5′ꢀpyridinyl]ꢀ7ꢀazabicyclo[2.2.1]heptane
(5d) Fumarate. A solution of the boronic ester 13 (424 mg, 1.01 mmol), 2ꢀbromoꢀ5ꢀ
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methoxythiophene (635 mg, 3.28 mmol), [ which was prepared from 2ꢀmethoxythiophene (3.0
mmol) treated with NBS (3.01 mmol) in CCl4 (2 mL) heated at reflux for 20 min then quenched
with an aqueous solution of NaHCO3, extracted with CH2Cl2, dried, concentrated in vacuo and
used without purification], Pd(PPh3)4 (117 mg, 10 mol %) and K2CO3 (420 mg, 3.04 mmol) in
1,4ꢀdioxane (9 mL) and H2O (1.8 mL) was placed in a resealable pressure vessel, degassed
through bubbling nitrogen for 20 min, sealed and heated at 80 °C for 18 h. After cooling to room
temperature, water (20 mL) was added and the organic product extracted with EtOAc (3 × 30
mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through Celite
and concentrated in vacuo. The residue was purified on silica gel eluted with ethyl acetate –
hexanes to provide 299 mg (73%) of the Bocꢀprotected 5d.
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A solution of Bocꢀprotected 5d (299 mg, 0.74 mmol) in CH2Cl2 (4 mL) was treated with
TFA (1.5 mL) and stirred at room temperature for 1 h. The solvent was removed under reduced
pressure and the residue was treated with a 20ꢀmL NH4OH: H2O solution (3:1) then extracted
with CH2Cl2. The organic portion was dried, filtered and concentrated down then purified on
silica gel, eluted with CHCl3/MeOH (9:1) to provide 119 mg (53%) of 5d as a brownish oil. 1H
NMR (300 MHz, CDCl3) δ (ppm) 1.50–1.73 (m, 6H), 1.78 (s, NꢀH proton), 1.89–1.96 (dd, J =
12.4, 9.0 Hz, 1H), 2.77–2.81 (dd, J = 9.0, 4.7 Hz, 1H), 3.60 (br s, 1H) 3.82 (s, 1H), 3.93 (s, 3H),
6.21 (d, J = 4.0 Hz, 1H), 7.21 (d, J = 4.0 Hz, 1H), 7.90 (s, 1H), 7.97 (dd, J = 9.6, 2.1 Hz, 1H); 13C
NMR (CDCl3) δ 30.2, 31.4, 40.5, 44.5, 56.5, 60.3, 62.8, 104.9, 117.1, 121.5, 125.4, 136.0,
140.4, 142.6, 156.1, 159.2, 167.5. MS (ESI) m/z 305.2 (M+H)+.
A solution of 5d (185 mg, 0.61 mmol) in CH2Cl2 in a vial was treated with a 1.2 equiv of
fumaric acid (0.65 M) in MeOH and the vial was allowed to stand in a refrigerator overnight. The
excess solvent was then removed in vacuo from the salt and then redissolved in minimal
amount of MeOH and the fumarate salt was recrystallized from MeOH using diethyl ether to
provide 136 mg (53%) of the salt 5d•C4H4O4 as a white crystalline solid: mp 166–169 °C.; 1H
NMR (MeOHꢀd4) δ (ppm) 1.87–2.09 (m, 5H), 2.44 –2.52 (dd, J = 13.1, 9.6 Hz, 1H), 3.47–3.52
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(dd, J = 7.5, 4.0 Hz, 1H), 3.94 (s, 3H), 4.33 (br s, 1H) 4.52 (s, 1H), 6.33 (d, J = 3.8 Hz, 1H), 6.66
(s, 2H), 7.34 (d, J = 3.8 Hz, 1H), 7.97 (s, 1H), 8.06 (d, J = 8.3 Hz, 1H); 13C NMR (MeOHꢀd4) δ
(ppm) 26.9, 29.0, 37.6, 43.4, 60.2, 60.8, 64.1, 105.8, 119.4, 127.4 (JCF = 5.6 Hz), 136.2, 137.2,
143.3 (JCF = 14.7 Hz), 160.7, 171.4; MS (ESI) m/z 305.4 [(M–C4H4O4)+, M = C16H17FN2OS].
Anal. calcd for C20H21F2N2O5S: C 57.13; H 5.03; N 6.66; found: C 57.23; H 4.88; N 6.71.
5ꢀ5ꢀ(7ꢀAzabicyclo[2.2.1]heptꢀ2ꢀyl)ꢀ2ꢀfluoropyridinꢀ3ꢀyl)thiopheneꢀ2ꢀcarboxamide (5e)
Hydrochloride. The Bocꢀprotected 5e was prepared from the crossꢀcoupling of compound 13
(399 mg, 0.954 mmol) with 5ꢀbromothiopheneꢀ2ꢀcarboxamide (295 mg, 1.43 mmol) following the
described General Procedure B to provide 179 mg (41%) of the Bocꢀprotected product. 1H NMR
(300 MHz, CDCl3) δ (ppm) 1.42 (s, 9H), 1.55–1.65 (m, 2H), 1.82–1.86 (m, 3H), 2.00–2.07 (dd, J
= 12.4, 9.4 Hz, 1H), 2.92–2.94 (dd, J = 9.0, 5.0 Hz, 1H) 2.97 (br s, NꢀH, 2H), 4.24, 4.39, 7.82 (s,
1H), 7.95–8.06 (m, 4H); 13C NMR δ (ppm) 28.3, 28.8, 29.7, 36.5, 40.6, 44.7, 56.0, 80.0, 117.1
(JCF = 28.4 Hz), 128.8, 129.3, 132.0, 134.7, 137.6, 139.1, 144.5, 155.0, 157.1, 160.3, 163.7. MS
(ESI) m/z 418.7 (M+H)+.
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A solution of the Bocꢀprotected 5e (248 mg, 0.594 mmol) in MeOH (3 mL) was treated with
HCl in diethyl ether (1 mL) and was stirred at room temperature overnight. The salt obtained
was hydrolyzed with NH4OH, extracted with CHCl3/MeOH, dried (Na2SO4), filtered and purified
on silica gel, eluted with CHCl3/MeOH (9:1) to provide 5e (164 mg, 83% yield). Compound 5e
(164 mg, 0.518 mmol) was then treated with 1.1 equivalents of HCl in diethyl ether to furnish
169 mg (86% yield) of 5e•HCl as light yellow solid: sublimes at 175 °C; 1H NMR (MeOHꢀd4) δ
(ppm) 1.90–2.19 (m, 5H), 2.41–2.54 (dd, J = 13.1, 9.6 Hz, 1H), 3.44–3.53 (dd, J = 7.5, 4.0 Hz,
1H), 4.36 (br s, 1H) 4.60 (s, 1H), 7.64–7.78 (m, 3H), 8.10 (d, J = 9.8 Hz, 1H), 8.15 (s, 1H), 8.27
(dd, J = 9.1, 2.4 Hz, 1H); 13C NMR (MeOHꢀd4) δ (ppm) 26.8, 28.9, 37.5, 43.3, 60.5, 64.2, 117.8,
118.0 (JCF = 23.2 Hz), 129.4 (JCF = 4.9 Hz), 130.9, 137.3, 139.0, 146.1 (JCF = 14.8 Hz), 159.9,
161.1, 166.1; MS (ESI) m/z 317.9 [(M–HCl))+, M = C16H16FN3OS]. Anal. calcd for
C16H17ClFN3OS•1.5H2O: C 50.46; H 5.29; N 11.03; found: C; 50.43; H 5.36; N 10.73.
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5ꢀ5ꢀ(7ꢀAzabicyclo[2.2.1]heptꢀ2ꢀyl)ꢀ2ꢀfluoropyridinꢀ3ꢀyl)thiopheneꢀ2ꢀsulfonamide (5f)
Hydrochloride. The Bocꢀprotected 5f was prepared from compound 13 (360 mg, 0.861 mmol)
and 5ꢀbromothiopheneꢀ2ꢀsulfonamide (271 mg, 1.12 mmol) following the described General
Procedure B, to provide 184 mg (47%) of the Bocꢀprotected product. 1H NMR (300 MHz, CDCl3)
δ (ppm) 1.44 (s, 9H), 1.54–1.66 (m, 2H), 1.82–1.87 (m, 3H), 2.02–2.09 (dd, J = 12.6, 9.4 Hz,
1H), 2.93–2.98 (dd, J = 8.8, 5.0 Hz, 1H), 4.24, 4.41, 7.41 (d, J = 3.9 Hz, 1H), 7.62 (d, J = 3.5 Hz,
1H), 8.01–8.05 (m, 2H); 13C NMR δ (ppm) 28.3, 28.8, 29.6, 40.6, 44.6, 56.1, 65.8, 80.3, 115.3
(JCF = 28.4 Hz), 126.5, 131.8, 137.1, 140.1, 141.4, 143.8, 145.9, 155.1, 156.3, 159.4. MS (ESI)
m/z 454.3 (M+H)+.
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A solution of the Bocꢀprotected 5f (184 mg, 0.406 mmol) in MeOH (3 mL) was treated with
HCl in diethyl ether (1 mL) and was stirred at room temperature overnight. The salt obtained
was hydrolyzed with NH4OH, extracted with CHCl3/MeOH, dried (Na2SO4), filtered and purified
on silica gel, eluted with CHCl3/MeOH (9:1), to provide 5f (117 mg, 82% yield). Compound 5f
(191 mg, 0.541 mmol) was then treated with 1.1 equivalents of HCl in diethyl ether to furnish
180 mg (83% yield) of 5f•HCl as white solid: mp. at 160–165 °C; 1H NMR (MeOHꢀd4) δ (ppm)
1.89–2.19 (m, 5H), 2.43–2.51 (dd, J = 13.1, 9.6 Hz, 1H), 3.47–3.53 (dd, J = 7.5, 4.0 Hz, 1H),
4.33 (br s, 1H) 4.53 (s, 1H), 7.62 (d, J = 3.4 Hz, 1H), 7.68 (d, J = 3.5 Hz, 1H), 8.17 (s, 1H), 8.27
(dd, J = 9.2, 2.0 Hz, 1H); 13C NMR (MeOHꢀd4) δ (ppm) 27.0, 29.1, 37.8, 43.4, 60.3, 64.2, 117.4
(JCF = 28.9 Hz), 128.4 (JCF = 4.0 Hz), 131.8, 137.7, 139.1, 140.6, 146.5 (JCF = 14.1 Hz), 147.9,
157.8, 161.0.; MS (ESI) m/z 354.3 [(M–HCl))+, M = C15H16FN3O2S2]. Anal. calcd for
C15H17ClFN3O2S2•0.5H2O: C 45.16; H 4.55; N 10.53; found: C 45.01; H 4.79; N 10.32.
2ꢀexo-[2'ꢀFluoroꢀ3′ꢀ(thiophenꢀ3ꢀyl)ꢀ5′ꢀpyridinyl]ꢀ7ꢀazabicyclo[2.2.1]heptane (6a)
Fumarate. To a resealable reaction pressure vessel under nitrogen was added 10 (151 mg,
0.556 mmol), thiopheneꢀ3ꢀboronic acid (92.5 mg, 0.723 mmol), Pd(PPh3)4 (32 mg, 5 mol %),
sodium carbonate (118 mg, 1.11 mmol), 1,4ꢀdioxane (10 mL) and water (1 mL). The mixture
was degassed through bubbling nitrogen for 40 min and reaction vessel was sealed and heated
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at 110 °C for 24 h. After cooling to room temperature, the mixture was poured into a 30 mL
aqueous solution of NaHCO3 and extracted with EtOAc (3 × 30 mL). The combined organic
layers were dried (Na2SO4), filtered through Celite and the solvent removed in vacuo. The
resultant residue was purified on silica gel, eluted with CHCl3/MeOH (9/1), to furnish 108 mg
(70% yield) of 6a. 1H NMR (300 MHz, CDCl3) δ (ppm) 1.50–1.80 (m, 6H), 1.90–1.97 (m, 1H),
2.79–2.84 (m, 1H), 3.60 (s, 1H) 3.81 (br s, 1H), 3.89 (s 1H), 7.40 (dd, J = 2.9, 5.1 Hz, 1H), 7.47
(dt, J = 1.4, 4.7 Hz, 1H), 7.68–7.70 (m, 1H), 8.0 (t, J = 1.60 Hz,1H), 8.10 (dd, J = 2.4, 9.6 Hz,
1H); 13C NMR (CDCl3) δ 30.2, 31.4, 40.6, 44.5, 56.4, 62.8, 102.3, 116.7, 126.6, 127.2, 127.9,
135.5, 137.4, 139.9 144.0, 156.3, 159.4; MS (ESI) m/z 275.3 (M+H)+.
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Compound 6a (92 mg, 0.334 mmol) was dissolved in chloroform (2 mL) in a vial and
treated with 1.3 equiv of fumaric acid (0.65 M) in MeOH and allowed to stand in a refrigerator
overnight. The excess solvent was then removed under reduced pressure and the residue salt
was redissolved in minimal amount of MeOH. The fumarate salts were recrystallized from
MeOH using diethyl ether to provide 88 mg (67 % yield) of 6a•C4H4O4 as white solid: mp. 175–
177 °C; 1H NMR (300 MHz, MEOHꢀd4) δ 1.85–2.20 (m, 5H), 2.43–2.50 (m, 1H), 3.46–3.51 (m,
1H), 4.34 (s, 1H) 4.55 (d, J = 3.4 Hz,1H), 6.66 (s, 2H), 7.55–7.60 (m, 2H), 7.88–7.90 (m, 1H),
8.08 (s, 1H), 8.22 (dd, J = 2.5, 9.3 Hz, 1H); 13C NMR (300 MHz, MEOHꢀd4) δ 27.1, 29.1, 37.9,
43.6, 60.4, 64.3, 126.4, 127.6, 128.4, 134.9, 137.3, 139.9 (JCF = 4.9 Hz), 144.9, 145.1 (JCF
=
14.7 Hz), 171.5; MS (ESI) m/z 275.3 [(M–fumarate)+, M=C15H15FN2S•C4H4O4]. Anal. calcd for
C19H19FN2O4S: C 56.45; H 4.91; N 7.18; found C 58.21; H 4.87; N 7.14.
4ꢀ5ꢀ(7ꢀAzabicyclo[2.2.1]heptꢀ2ꢀyl)ꢀ2ꢀfluoropyridinꢀ3ꢀyl)thiopheneꢀ2ꢀcarboxamide (6b)
Hydrochloride. A solution of 4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)thiopheneꢀ2ꢀ
carboxamide, (408 mg, 1.61 mmol, 1.5 equiv) [prepared from 4ꢀbromothiopheneꢀ2ꢀcarboxamide
in 63% yield following our previously reported protocol as was used for the synthesis of 13],26
compound 11 (399 mg, 1.07 mmol, 1.0 equiv), Pd(PPh3)4 (62 mg, 0.054 mmol, 5 mol %), and
Na2CO3 (228 mg, 2.15 mmol) in 1,4ꢀdioxane (6 mL), DMF (3 mL) and H2O (1 mL) was degassed
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through bubbling N2, sealed then heated at 100 °C overnight. After cooling to room temperature
the solvent was removed in vacuo. The residue was reꢀdissolved in CHCl3 and a 20ꢀmL
saturated aqueous solution of Na2CO3 was added. The organic product was extracted using
CHCl3 (3 × 30 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered
through Celite and concentrated under reduced pressure. The residue was purified on silica gel,
eluted with EtOAc/hexanes, to provide 308 mg (69% yield) of the Bocꢀprotected 6b. The Boc
protected compound (308 mg, 0.738 mmol) was dissolved in methylene (5 mL) and TFA (1.5
mL) was added and the mixture was stirred at room temperature overnight. The solvent was
then removed under reduced pressure and a solution of NH4OH/H2O (20 mL, 3:1) was added.
The organic product was extracted using CHCl3/ MeOH (10%) (3 × 30 mL). The combined
organic layers were dried (Na2SO4), filtered through Celite and concentrated in vacuo. The
residue was purified by flash chromatography to provide 190 mg (81%) of 6b as a white foamy
compound. 1H NMR (300 MHz, CDCl3) δ1.53–1.71 (m, 5H), 1.91–1.98 (dd, J = 9.0, 12.4 Hz,
1H), 2.22 (br s, 2H), 2.80–2.84 (dd, J = 9.0, 5.0 Hz, 1H), 3.63 (s, 1H), 3.83 (s, 1H), 7.84 (s, 1H),
7.98–8.00 (m, 2 H), 8.13–8.17 (dd, J = 9.6, 2.3 Hz, 1 H); 13C NMR δ (ppm) 30.1, 31.5, 40.5,
44.2, 56.5, 62.9, 117.3 (JCF = 28.3 Hz), 129.1, 129.3, 132.0, 134.8, 138.0, 138.6, 144.7, 157.1,
160.3, 163.7. MS (ESI) m/z 318.1 (M+H)+.
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A solution of the amine 6b (190 mg, 0.60 mmol) in minimal MeOH was treated dropwise
with HCl in diethyl ether. The salt was recrystallized from MeOH using CH2Cl2 to provide 199
mg of 6b•HCl as a white solid: mp > 230 °C; 1H NMR (MeOHꢀd4) δ (ppm) 1.92–2.20 (m, 5H),
2.46–2.53 (dd, J = 9.0, 12.4 Hz, 1H), 3.49–3.54 (dd, J = 9.0, 5.0 Hz, 1H), 4.37 (s, 1H), 4.59 (s,
1H), 8.12–8.25 (m, 4 H); 13C NMR (MeOHꢀd4) δ (ppm) 26.8, 28.9, 37.6, 43.3, 60.5, 64.3, 119.3
(JCF = 27.4 Hz), 121.1 (JCF = 4.6 Hz), 130.6, 131.3, 135.4, 139.8, 144.4 (JCF = 14.8 Hz) ; MS
(ESI) m/z 318.2 [(M–HCl))+, M = C16H17FN2S]. Anal. calcd for C16H17ClFN3OS•1.5H2O: C 50.46;
H 5.29; N 11.03; found: C 50.43; H 5.36; N 10.73.
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2ꢀexo-2ꢀ[6ꢀFluoroꢀ5ꢀ(5ꢀfluorothiophenꢀ3ꢀyl)pyridinꢀ3ꢀyl]ꢀ7ꢀazabicyclo[2.2.1]heptanes
(6c) Hydrochloride. A solution of compound 19 (350 mg, 0.783 mmol, 1.0 equiv, dried over
molecular sieves) in dry THF (15 mL) was placed in flameꢀdried flask and cooled in a dry
ice/acetone bath at –78 °C. The solution was treated dropwise with nꢀBuLi (640 µL, 1.6 M in
hexanes, 1.02 mmol, 1.3 equiv) and stirred at –78 °C for 1 h. A solution of Nꢀ
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fluorobenzenesulfonimide (NFSI) (370 mg, 1.17 mmol, 1.5 equiv, dried over molecular sieves) in
dry THF (5 mL) was then added and the mixture was stirred at –78 °C and slowly allowed to
warmꢀup to room temperature overnight. The reaction mixture was quenched with an aqueous
solution of NH4Cl (20 mL) and the organic product extracted with EtOAc (3 x 30 mL). The
combined organic layers were dried (anhydrous Na2SO4), filtered and concentrated in vacuo.
The residue was purified on silica gel eluted with EtOAc/hexanes, to provide 213 mg (58%) of
the silylatedꢀBocꢀprotected 6c (tertꢀbutylꢀ2ꢀ{5ꢀ[5ꢀfluoroꢀ2ꢀ(trimethylsilyl)thiophenꢀ3ꢀyl]ꢀ6ꢀ
fluoropyridinꢀ3ꢀyl}ꢀ7ꢀazabicyclo[2.2.1]hepaneꢀ7ꢀcarboxylate) as a brownish oil. 1H NMR (300
MHz, CDCl3) δ (ppm) 0.0 (s, 9H), 1.29 (s, 9H),1.40–1.76 (m, 5H), 1.87–1.94 (dd, J = 12.4, 9.0
Hz, 1H), 2.78–2.83 (dd, J = 9.0, 4.7 Hz, 1H), 4.07 (br s, 1H), 4.26 (s, 1H), 6.44 (s, 1H), 7.59 (d,
J = 2.8 Hz, 1H), 7.95 (s, 1H); 19F NMR (CDCl3) δ (ppm) –72.27, –128.36; 13C NMR (CDCl3) δ
0.0, 27.9, 28.4, 29.3, 40.1, 44.4, 55.8, 61.7, 79.5, 111.4, 120.3, 125.7, 129.6, 130.6, 145.2,
154.9, 157.4, 160.5, 166.5, 170.4. MS (ESI) m/z 465.7 (M+H)+.
A solution of silylatedꢀBocꢀprotected product (213 mg, 0.46 mmol) in MeOH (5 mL) was
treated with HCl in diethyl ether (1 mL) and stirred at room temperature overnight. The solvent
was removed under reduced pressure and the residue was treated with a NH4OH:H2O (3:1)
solution then extracted with CH2Cl2. The organic portion was dried, filtered and concentrated
down then purified on silica gel eluted with CHCl3/MeOH (9:1) to provide 105 mg (78%) of 6c as
a colorless oil.
A solution of the amine compound 6c (88 mg, 0.300 mmol) was treated dropwise with HCl
in diethyl ether to provide 88 mg (86%) of 6c•HCl as a brownish solid: mp 60 °C; 1H NMR
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(MeOHꢀd4) δ (ppm) 1.88–2.18 (m, 5H), 2.44–2.52 (dd, J = 13.1, 9.6 Hz, 1H), 3.45–3.52 (dd, J =
7.5, 4.0 Hz, 1H), 4.35 (br s, 1H) 4.57 (s, 1H), 7.12–7.14 (d, J = 1.6 Hz, 1H), 7.32–34 (m, 1H),
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8.10–8.15 (m, 2H); 13C NMR (MeOHꢀd4) δ (ppm) 26.8, 28.8, 37.6, 43.3, 60.5, 64.3, 108.7 (JCF
=
9
27.4 Hz), 114.9, 139.5 (JCF = 4.6 Hz), 145.2, 158.8, 165.3; MS (ESI) m/z 293.3 [(M–HCl))+, M =
C15H14F2N2S]. Anal. calcd for C15H15ClF2N2S•0.75H2O: C 52.63; H 4.86; N 8.18; found: C 52.81;
H 4.95; N 7.97.
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2ꢀ[5ꢀ(5ꢀChlorothiophenꢀ3ꢀyl)ꢀ6ꢀfluoropyridinꢀ3ꢀyl]ꢀ7ꢀazabicyclo[2.2.1]heptane (6d)
Dihydrochloride. A solution of compound 19 (339 mg, 0.769 mmol, 1.0 equiv, dried over
molecular sieves) in dry THF (10 mL) was placed in flameꢀdried flask then cooled to –78 °C
using a dry iceꢀacetone bath. The solution was treated dropwise with nꢀBuLi (570 µL, 1.6 M in
hexanes, 0.911 mmol, 1.2 equiv) and stirred at the bath temperature for 1 h. A solution of
hexachloroethane (234 mg, 0.986 mmol, 1.3 equiv, dried over molecular sieves) in dry THF (5
mL) was then added and the mixture was stirred at –78 °C for an additional 1 h then quenched
with an aqueous solution of NH4Cl (20 mL) and the organic product extracted with EtOAc (3 ×
30 mL). The combined organic layers were dried (anhydrous Na2SO4), filtered and concentrated
in vacuo. The residue was purified on silica gel, eluted with EtOAc/hexanes to provide 302 mg
(83%) of the silylatedꢀBocꢀprotected 6d (tertꢀbutylꢀ2ꢀ{5ꢀ[5ꢀchloroꢀ2ꢀ(trimethylsilyl)thiophenꢀ3ꢀyl]ꢀ
6ꢀfluoropyridinꢀ3ꢀyl}ꢀ7ꢀazabicyclo[2.2.1]hepaneꢀ7ꢀcarboxylate) as a clear oil. 1H NMR (300 MHz,
CDCl3) δ (ppm) 0.0 (s, 9H), 1.27 (s, 9H),1.39–1.73 (m, 5H), 1.87–1.94 (dd, J = 12.4, 9.0 Hz,
1H), 2.77–2.81 (dd, J = 9.0, 4.7 Hz, 1H), 4.05 (br s, 1H), 4.24 (s, 1H), 6.81 (s, 1H), 7.54–7.58
(dd, J = 9.0, 2.5 Hz, 1H), 7.94 (s, 1H); 13C NMR (CDCl3) δ 0.0, 28.0, 28.5, 29.4, 40.2, 44.5, 55.9,
61.8, 79.7, 119.6, 129.6, 133.7, 137.8, 138.5, 140.0, 140.3, 145.2, 155.0, 157.5, 160.6, (ESI)
m/z 481.4 (M+H)+.
A solution of silylatedꢀBocꢀprotected product (302 mg, 0.628 mmol) in CH2Cl2 (10 mL) was
treated with TFA (1 mL) and stirred at room temperature overnight. The solvent was removed
under reduced pressure and the residue was treated with a NH4OH:H2O (3:1) solution then
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extracted with CH2Cl2. The organic portion was dried, filtered and concentrated down then
purified on silica gel, eluted with CHCl3/MeOH (9:1) to provide 85 mg (44%) of 6d as a colorless
oil.
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A solution of the amine 6d (83 mg, 0.267 mmol) was treated dropwise with HCl in diethyl
ether to provide 102 mg (98%) of 6d•2HCl as a brownish solid: mp 60 °C; 1H NMR (MeOHꢀd4) δ
(ppm) 1.88–2.17 (m, 5H), 2.44–2.52 (dd, J = 13.1, 9.6 Hz, 1H), 3.45–3.52 (dd, J = 7.5, 4.0 Hz,
1H), 4.35 (br s, 1H) 4.57 (s, 1H), 7.52 (s, 1H), 7.76 (s, 1H), 8.11 (s, 1H), 8.15–8.19 (dd, J = 9.2,
2.1 Hz, 1H); 13C NMR (MeOHꢀd4) δ (ppm) 26.8, 28.9, 37.6, 43.3, 60.5, 64.3, 117.0 (JCF = 28.2
Hz), 122.8, 127.6, 130.7, 133.7, 139.6 (JCF = 4.6 Hz), 140.7, 145.5, 158.0, 160.2; MS (ESI) m/z
309.0 [(M–HCl))+, M = C15H15Cl2FN2S]. Anal. calcd for C15H16Cl3FN2S•0.25H2O: C 46.65; H 4.31;
N 7.25; found: C 46.56; H 4.41; N 6.96.
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2ꢀexo-[2'ꢀFluoroꢀ3′ꢀ(4ꢀmethylthiophenꢀ2ꢀyl)ꢀ5′ꢀpyridinyl]ꢀ7ꢀazabicyclo[2.2.1]heptane
(7a) Hydrochloride. In a microwave vial was placed 11 (233 mg, 0.626 mmol), 4ꢀ
methylthiopheneꢀ2ꢀboronic acid (115 mg, 0.814 mmol), Pd(PPh3)4 (72.3 mg, 10 mol %), K2CO3
(173 mg, 1.25 mmol), 1,4ꢀdioxane (3 mL) and H2O (0.9 mL). The mixture was degassed through
bubbling nitrogen for 20 min sealed and irradiated in a CEM microwave reactor at 150 °C for 90
min. After cooling, the mixture was filtered through a plug of Celite and anhydrous Na2SO4, and
concentrated in vacuo. The resultant residue was purified on silica gel, eluted with EtOAc–
hexanes to provide 138 mg (57%) of Bocꢀprotected 7a as a colorless oil.
A solution of Bocꢀprotected 7a (308 mg, 0.79 mmol) in CH2Cl2 (3 mL) was treated with
TFA (1 mL) and stirred at room temperature for 2 h. The solvent was removed under reduced
pressure and the residue was treated with a NH4OH:H2O (3:1) solution then extracted with
CH2Cl2. The organic portion was dried, filtered and concentrated down then purified by flash
chromatography to provide 202 mg (88%) of 7a as a colorless oil. 1H NMR (300 MHz, CDCl3) δ
(ppm) 1.50–1.68 (m, 6H), 1.76 (s, NꢀH proton), 1.89–1.96 (dd, J = 12.4, 9.0 Hz, 1H), 2.77–2.81
(dd, J = 9.0, 4.7 Hz, 1H), 2.29 (s, 3H), 3.59 (br s, 1H) 3.81 (s, 1H), 6.96 (s, 1H), 7.35 (s, 1H),
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7.98 (d, J = 1.5 Hz, 1H), 8.07 (dd, J = 9.6, 2.3 Hz, 1H); 13C NMR (CDCl3) δ 15.7, 30.2, 31.4,
40.5, 44.5, 56.2, 62.8, 116.8, 121.9, 129.4, 135.3, 137.2, 138.6, 140.6, 143.8, 156.2, 159.4. MS
(ESI) m/z 289.2 (M+H)+.
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A solution of the amine 7a (179 mg, 0.622) was treated dropwise with HCl (1.2 equiv) in
diethyl ether to provide 173 mg (93%) of 7a•HCl as a brownish solid: mp 192–194 °C; 1H NMR
(MeOHꢀd4) δ (ppm) 1.87–2.17 (m, 5H), 2.31 (s, 3H), 2.44–2.52 (dd, J = 13.1, 9.6 Hz, 1H), 3.47–
3.52 (dd, J = 7.5, 4.0 Hz, 1H), 4.36 (br s, 1H) 4.57 (s, 1H), 7.17 (s, 1H), 7.52 (s, 1H), 8.06 (s,
1H), 8.15 (d, J = 9.3 Hz, 1H); 13C NMR (MeOHꢀd4) δ (ppm) 15.6, 26.8, 28.9, 37.5, 43.3, 56.2,
60.5, 64.2, 118.7 (JCF = 27.4 Hz), 124.3 (JCF = 4.6 Hz), 135.3, 131.1, 135.3, 137.1, 138.4, 139.9,
144.5 (JCF = 14.8 Hz), 157.8, 161.0 ; MS (ESI) m/z 289.3 [(M–HCl))+, M = C16H17FN2S]. Anal.
calcd for C16H18ClFN2S: C 59.16; H 5.59; N 8.62; found: C 58.91; H 5.67; N 8.40.
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5ꢀ(7ꢀAzabicyclo[2.2.1]heptꢀ2ꢀyl)ꢀ3ꢀ(4ꢀcarbamoylthiophenꢀ2ꢀyl)pyridnꢀ2ꢀyl (7b)
Hydrochloride. A solution of 13 (757 mg, 1.81 mmol), 2ꢀbromothiopheneꢀ4ꢀcarboxamide (410
mg, 1.99 mmol, 1.1 equiv), Pd(PPh3)4 (66.2 mg, 5 mol %) and Na2CO3 ( 384 mg, 3.62 mmol) in
DMF (10 mL) and H2O (2 mL) was placed in a resealable pressure vessel, degassed through
bubbling nitrogen for 20 min, sealed and heated at 100 °C overnight. After cooling to room
temperature, NaHCO3 (20 mL) was added and the organic product extracted with CHCl3/MeOH
(10%) (3 × 30 mL). The combined organic layers were dried (Na2SO4), filtered through Celite,
concentrated in vacuo and the residue was purified by on silica gel, eluted with EtOAc/hexanes
to provide the Bocꢀprotected product. A solution of the Bocꢀprotected 7b (370 mg, 0.885 mmol)
in MeOH (3 mL) was treated with HCl in diethyl ether (1 mL) and was stirred at room
temperature overnight. The salt obtained was hydrolyzed with NH4OH, extracted with
CHCl3/MeOH, dried (Na2SO4), filtered and purified on silica gel, eluted with CHCl3/MeOH (9:1),
to provide 7b (147 mg, 53% yield). 1H NMR (300 MHz, CDCl3) δ1.48–1.73 (m, 5H), 1.91–1.98
(dd, J = 9.0, 12.4 Hz, 1H), 2.79–2.84 (dd, J = 9.0, 5.0 Hz, 1H), 3.14 (br s, 2H), 3.65 (s, 1H), 3.84
(s, 1H), 7.83 (s, 1H), 7.93–8.03 (m, 2 H), 8.15–8.19 (dd, J = 9.6, 2.3 Hz, 1 H); 13C NMR δ (ppm)
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30.0, 31.3, 40.4, 44.3, 56.5, 62.8, 115.8 (JCF = 28.3 Hz), 125.9, 130.3, 134.7, 136.5, 137.1,
140.6, 145.0, 156.1, 159.3, 164.9. MS (ESI) m/z 318.4 (M+H)+.
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Compound 7b (170 mg, 0.534 mmol) was then treated with 1.1 equivalents of HCl in
diethyl ether to furnish 100 mg of 7b•HCl as light yellow solid: mp at 160 °C; 1H NMR (MeOHꢀd4)
δ (ppm) 1.90–2.20 (m, 5H), 2.47–2.54 (dd, J = 13.1, 9.6 Hz, 1H), 3.44–3.52 (dd, J = 7.5, 4.0 Hz,
1H), 4.37 (br s, 1H) 4.60 (s, 1H), 7.9–8.27 (m, 4H); 13C NMR (MeOHꢀd4) δ (ppm) 26.8, 28.9,
37.4, 43.3, 60.4, 64.2, 117.9, 128.3 (JCF = 4.9 Hz), 132.2, 136.5, 138.7, 145.9 (JCF = 14.8 Hz),
157.8, 161.1, 167.2; MS (ESI) m/z 317.9 [(M–HCl))+, M = C16H16FN3OS]. Anal. calcd for
C16H17ClFN3OS•1.5H2O: C 50.65; H 5.07; N 10.56; found: C 50.46; H 5.29; N 11.03.
2ꢀ[5ꢀ(4ꢀChlorothiophenꢀ2ꢀyl)ꢀ6ꢀfluoropyridinꢀ3ꢀyl]ꢀ7ꢀazabicyclo[2.2.1]heptane (7c)
Dihydrochloride. A solution of compound 21 (345 mg, 0.718 mmol) in CH2Cl2 (10 mL) was
treated with TFA and stirred at room temperature overnight. The solvent was removed under
reduced pressure and the residue was treated with a NH4OH:H2O (3:1) solution then extracted
with CH2Cl2. The organic portion was dried, filtered and concentrated down then purified by on
silica gel, eluted with CHCl3/MeOH (9:1), to provide 183 mg (82%) of 7c as a colorless oil.
A solution of the amine 7c (180 mg, 0.582 mmol) in CH2Cl2 (2 mL) was treated with HCl in
diethyl ether to furnish 206 mg of 7c•2HCl as yellow solid: mp at 125 °C; 1H NMR (MeOHꢀd4) δ
(ppm) 1.88–2.16 (m, 5H), 2.45–2.53 (dd, J = 13.1, 9.6 Hz, 1H), 3.49–3.54 (dd, J = 7.5, 4.0 Hz,
1H), 4.36 (br s, 1H) 4.58 (s, 1H), 7.49 (d, J = 1.5 Hz, 1H) 7.63 (s, 4H), 8.13 (s, 1H), 8.21–8.25
(dd, J = 8.9, 2.1 Hz, 1H); 13C NMR (MeOHꢀd4) δ (ppm) 26.8, 28.9, 37.6, 43.3, 60.5, 64.2, 117.8,
123.5 (JCF = 4.9 Hz), 127.1, 128.7, 132.2, 136.5, 138.7, 145.9 (JCF = 14.8 Hz), 146.0, 157.8,
161.0; MS (ESI) m/z 309.0 [(M–HCl))+, M = C16H15Cl2FN3OS]. Anal. calcd for
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C15H16Cl3FN2S•0.25H2O: C 46.65; H 4.31; N 7.25; found: C 46.62; H 4.43; N 7.17.
7ꢀtertꢀButoxycarbonylꢀ2ꢀexo-[2'ꢀaminoꢀ3′ꢀ(thiophenꢀ2ꢀyl)ꢀ5′ꢀpyridinyl]ꢀ7ꢀ
azabicyclo[2.2.1]heptane (9a). A solution of compound 8 (301 mg, 0.817 mmol), thiopheneꢀ2ꢀ
boronic acid (136 mg, 1.06 mmol), Pd(PPh3)4 (47.2 mg, 5 mol %), Na2CO3 (173 mg, 1.63 mmol)
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