Synthesis, Nicotinic Acetylcholine Receptor Binding, and in Vitro and

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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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ACS Chemical Neuroscience

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§, 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 23298-0613, United States

ACS Paragon Plus Environment


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Abstract 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 Ki = 0.86 nM in the binding assay similar potency at α4β2/α3β4 with selectivity relative to α7 nAChRs, 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.

Keywords: nAChR antagonist, nicotine receptors, in vitro/in vivo studies, α4β2- and α3β4nAChR

Introduction Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated 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 α4β2- and α7-nAChRs.3 The α4β2-nAChR can contain other subunits and are named α4β2*-nAChRs. The α4β2-nAChRs 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 α3β4-nAChR can also play a significant role in nicotine


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addiction.7-9 α3β4-nAChR antagonists and partial agonists have been shown to reduce nicotine withdrawal, reward, and self-administration.10-13 On the other hand, we also reported that varenicline (2) was an agonist at α3β4-nAChR which may account for several observed peripheral side effects including nausea and gastrointestinal symptoms.14 O NH N CH3

Cl

2

X

3

4

X

S

S

F H

N N

5a, X = H 5b, X = Cl 5c, X = F 5d, X = OMe 5e, X = CONH 2 5f, X = SO 2NH 2

F N

H

N

6a, 6b, 6c, 6d,

Cl N N

X S

H

H

NHC(CH 3) 3

N

N

1

CH3

N

X=H X = CONH 2 X=F X = Cl

F N N

7a, X = Me 7b, X = CONH 2 7c, X = Cl

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 α4β2-nAChR 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 (exo-2-(2′-chloro-5′-pyridinyl)-7-



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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′-fluoro-3′-(substituted thiophenyl)deschloroepibatidine compounds 5a–f, 6a–d, and 7a–c.

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 tail-flick, hot-plate, 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 crosscoupling of 7-tert-butoxycarbonyl-2-exo-(2′-amino-3′-bromo-5′-pyridinyl)-7azabicyclo[2.2.1]heptane22 (8) with thiophene-2-boronic acid in the presence of sodium carbonate, tetrakis(triphenylphosphine) palladium(0), (Pd(PPh3)4), in N,N-dimethylformamide and water, heated at 100 °C overnight provided compound 9a. Scheme 1a


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X S

Br NH 2

Boc N

X

NH 2

Boc N

a or b

N

S

8

N

N 5a, X = H 5b, X = Cl

9a, X = H 9b, X = Cl

c

F

H N

c

S Br F

H N

N 10

a

F

H N

N 6a

a

Reagents and Conditions: (a) Thiophene-2- or 3-boronic acid, Pd(PPh3)4, Na2CO3, DMF or 1,4-dioxane, H2O, 100 °C, 18 h; (b) 5-Chlorothiophene-2-boronic acid, Pd2(dba)3 (3 mol %), PPh3 (10 mol %), DME, H2O, microwave, 120 °C, 1 h (c) 70% HF-pyridine, NaNO2, 0 °C to rt, 1 h.

Furthermore, a microwave assisted Suzuki cross-coupling reaction of 8 with 5-chlorothiophene2-boronic acid, in the presence of tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3) and triphenylphosphine (PPh3) as the catalytic system, sodium carbonate, in 1,2-dimethoxyethane (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'-fluoro-3'-bromo compound 10 prepared as previously reported23 and 3-thiopheneboronic acid were cross-coupled in the presence of tetrakis(triphenylphosphine) palladium(0) and sodium carbonate in 1,4-dioxane and water heated at 110 °C overnight provided analog 6a. The synthesis of 5c as outlined in Scheme 2, commenced with the cross-coupling of the 2'-fluoro-3'-bromo Boc-protected compound 1126 with thiophene-2-boronic acid in the presence of tetrakis(triphenylphosphine) palladium(0) and potassium carbonate in 1,4-dioxane and water, heated at 100 °C overnight provided compound 12. Compound 12 in anhydrous tetrahydrofuran (THF), was subjected to a lithiation using nbutyllithium at –78 °C. The resulting lithio anion was quenched with N-fluorobenzenesulfonimide followed by removal of the Boc-protecting group using trifluoroacetic acid in methylene chloride to provide analog 5c. The methoxythiophene analog 5d (Scheme 2), was synthesized by cross-



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coupling the boronic ester 1326 with 2-bromo-5-methoxythiophene (14) [prepared in quantitative yields through a radical bromination reaction of 2-methoxythiophene (15) using Nbromosuccinimide] in the presence of tetrakis(triphenylphosphine) palladium(0) and potassium carbonate in 1,4-dioxane and water heated at reflux overnight which provided the Boc protected product. A trifluoroacetic acid removal of the Boc-protecting group provided the methoxy substituted analog 5d. Similarly, cross-coupling of boronic ester 13 with 5-bromothiophene-2carboxamide in the presence of potassium carbonate, tetrakis(triphenylphosphine)palladium(0) in 1,2-dimethoxyethane and water heated at 85 °C overnight provided the Boc-protected thiophene-2-carboxamide analog and subsequent removal of the Boc group furnished analog 5e. The reaction of compound 13 with 5-bromothiophene-2-sulfonamide in the presence, 1,1'bis(diphenylphosphino)ferrocene-palladium(II)dichloride and potassium phosphate in 1,4dioxane, dimethylformamide and water, heated at 85 °C overnight, furnished the thiophene-2sulfonamide Boc-protected analog which on removal of the Boc-protected 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 4-(4,4,5,5-teramethyl-1,3,2dioxaborolan-2)thiophene-2-carboxamide [prepared from 4-bromo-2-carboxamide 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)ferrocene-palladium(II)dichloride, potassium phosphate, 1,4-dioxane, dimethylformamide and water to provide the Boc-protected product which on removal of the Boc-protecting group using hydrochloric acid in methanol furnished 6b. Scheme 2a


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F S

S F

Boc N

b,c

N

X

F

H N

N 5c

S F

H N

CONH 2

S 12

F

H N

a

N

N i,c

e,c or f,g or h,g, k 5d, X = OMe 5e, X = CONH 2 5f, X = SO2NH 2

Br F

Boc N

O B

6b

N

d

O

j,c 11

F

Boc N

N 13 l and h,g

S F

H N

N 7a

H 2NOC S F

H N

N 7b

a

Reagents and Conditions: (a) Thiophene-2-boronic acid, Pd(PPh3)4, K2CO3, 1,4-dioxane, H2O, 100 °C, overnight; (b) (i) n-BuLi, THF, at –78 °C, 40 min to 0 °C (10 min), (ii) N-fluorobenzenesulfonimide, THF, – 78 °C, 1 h to rt. (c) TFA, CH2Cl2, rt, overnight; (d) bis(pinacolato)diboron, KOAc, PdCl2dppf, 1,4-dioxane, microwave 140 °C, 20 min; (e) 2-bromo-5-methoxythiophene (14) [prepared from 2-methoxythiophene (15) using NBS], Pd(PPh3)4, K2CO3, 1,4-dioxane, H2O, reflux, overnight; (f) 5-bromothiophene-2carboxamide, Pd(PPh3)4, K2CO3, DME, H2O, 85 °C, overnight; (g) HCl in Et2O/ MeOH, rt, overnight; (h) PdCl2(dppf) (3 mol %), K3PO4, or Na2CO3, 1,4-dioxane, DMF, H2O, 80 °C, overnight; (i) 4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)thiophene-2-carboxamide, [prepared from 4-bromothiophene-2carboxamide using reagents and conditions in (d)], Pd(PPh3)4, Na2CO3. 1,4-dioxane, DMF, H2O, 100 °C, ; overnight (j) Pd(PPh3)4, K2CO3, 1,4-dioxane, H2O, microwave, 150 °C, 1 h; (k) 5-Bromothiophene-2sulfonamide (l) 5-bromothiophene-3-carboxamide (16) [prepared in three steps from thiophene-3carboxylic acid].

The key step towards the synthesis of analog 7a was accomplished through a microwave assisted Suzuki cross-coupling. Compound 1126 and 4-methylthiophene-2-boronic acid were cross-coupled in a microwave reactor in the presence of tetrakis(triphenylphosphine) palladium(0) and potassium carbonate in 1,4-dioxane and water, irradiated for 1 h at 150 °C to provide the Boc-protected 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, 5bromothiophene-3-carboxamide (16) was prepared from thiophene-3-carboxylic acid in three steps i.e., bromination using N-bromosuccinimide provided 5-bromothiophene-3-carboxylic 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)ferrocene-palladium(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. Scheme 3 outlines the route followed for the synthesis of analogues 6c, 6d and 7c. 3Bromothiophene-2-trimethylsilane (18a) and 3-chlorothiophene-2-trimethylsilane (18b) were prepared from 3-bromothiophene (17a) and 3-chlorothiophene (17b), respectively, via 2lithiation using lithium bis(trimethylsilyl)amide and subsequent quenching with trimethylchlorosilane. Cross-coupling of compound 13 with 18a in the presence of 1,1'bis(diphenylphosphino)ferrocene-palladium(II)dichloride and 2M aqueous solution of sodium carbonate in 1,2-dimethoxyethane, degassed through bubbling nitrogen and irradiated in a microwave reactor at 110 °C for 30 min provided compound 19. Lithiation of 19 with nbutyllithium 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 C-2 position. Simultaneous removal of the TMS and Boc groups using trifluoroacetic acid in methylene chloride furnished the desired analogue 6d. Similarly, 2-lithiated of compound 19 was quenched with a solution of N-fluorobenzenesulfonimide 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 n-butyllithium at –78 °C and quenching with iodine to provide compound 20, which was subsequently cross-coupled 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 analogue 7c. Scheme 3a S

S TMS

a X 17a, X= Br 17b, X= Cl

X 18a, X= Br 18b, X= Cl b I

S TMS 20 Cl

X S

S TMS 18a + 13

c F

Boc N

d,e

TMS

6c, X= F 6d, X=Cl

e F

Boc N

N 21

Cl

Cl

S

c

N

N 19

20 + 13

F

H N

S F

H N

N 7c

a

Reagents and Conditions: (a) (i) LiHMDS, THF, 0 °C, 1 h, (ii) TMSCl, 0 °C, 1 h; (b) (i) n-BuLi (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) n-BuLi, 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′-fluoro-3′-(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 nat-epibatidine, 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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1 Table 1. Radioligand Binding and Antinociception Data for 2′-fluoro-3′-(substituted thiophenyl)deschloroepibatidine Analogs 2 Antagonist Activity 3 Agonist Activity (% EC50 ACh response 4 mg/kg AD (% of max ACh response 50 (µg/kg) αβ remaining at 100 µM) 5 Compda [3H]Epibatidine at 100 µM) X 6 (Ki, nM)b ED50 ED50 ED50 ED50 HotSpontaneous Tail-Flick α4β2 α3β4 α7 α4β2 α3β4 α7 7 Tail-Flick Hot-Plate Hypothermia Plate Activity 8 0.65 1.3 1.0 0.5 9 nicotine 1.5 ± 0.30 (0.25– 40 ± 1 37 ± 3 43 ± 5 nd nd nd (0.5–1.8) (0.6–2.1) (0.15–0.78) 10 0.85) 0.004 0.004 0.001 11 nat 0.006 0.026 ± 0.002 (0.001– (0.002– (0.0005– 131 ± 13 97 ± 4 150 ± 8 nd nd nd 12epibatidine 0.001–0.01 0.008) 0.008) 0.005) 13 DHβE 39.5 ± 4.9 14varenicline 0.12 ± 0.02 10% @ 10 10% @ 10 2.8 2.1 0.2 470 13 ± 0.4 66 ± 4 74 ± 5 38 ± 2 nd nd 330 15 9.5 4 0.52 H 0.49 ± 0.18 5% @ 10 3% @ 10 (70– 0 0 0 9±1 6±1 33 ± 4 16 5a (5.3–14.9) (1.1–14) (0.02–13) 1400) 17 0.04 257 4.5 3 18 5b (0.01– (183– 0 0 0 14 ± 2 4±1 12 ± 4 F 0.20 ± 0.03 7% @ 10 14% @ 10 (3.3–6.15) (0.5–17) 0.31) 551) 19 3710 20 5c 10 2 25% @ Cl 0.36 ± 0.05 4% @ 10 15% @ 10 (1152– 0 0 0 19 ± 2 6±1 21 ± 2 (2–53) (0.3–13.5) 10000 21 119500 2075 22 14.6 2.2 2% @ CH3O 0.49 ± 0.06 6% @ 10 11% @ 10 (1145– 0 0 0 9±1 8±3 20 ± 2 23 5d (6.7–32) (1.6–3) 10000 3758) 24 9900 196 25 5e CONH2 0.71 ± 0.13 6% @ 10 7% @ 10 0%@10 0%@10 (5700– 1.0 ± 0.2 0 0 18 ± 3 18 ± 2 59 ± 9 (75–345) 12300) 26 1.01 9% @ 27 5f SO2NH2 0.25 ± 0.1 7% @ 10 0% @ 10 0% @ 10 0% @ 10 0 0 1.2 ± 0.5 14 ± 2 15 ± 2 68 ± 12 (0.11–3.5) 10000 28 0.075 113 29 6a H 0.41 ± 0.2 1% @ 10 9% @ 10 10% @ 10 15% @ 10 (0.04– 0 0 0 5±1 3.8 ± 0.4 16 ± 3 (27–475) 0.12) 30 0.03 31 4.7 2.8 1.2 0.34 30%@10 6b F 0.17 ± 0.04 (0.007– 2.6 ± 0.2 1.1 ± 0.1 1.2 ± 0.1 14 ± 1 17 ± 5 22 ± 2 (3.4–6.6) (1.8–4.9) (0.12–12.4) (0.17–0.7) 00 32 0.14) 33 0.05 35 25% (0.01– 34 6c (0.5– 0 0 1.7 ± 0.5 9±1 8±1 21 ± 2 Cl 0.21 ± 0.04 2%@10 10%@10 0%@10 increase @ 0.24) 10 252) 35 36 435 1 CONH2 0.11 ± 0.01 1%@10 10%10 0%@10 5%@10 (131– 0 0 0 11 ± 3 8±2 62 ± 4 37 6d (0.3–10) 1436) 38 832 9 3.6 0.32 39 7a Cl 0.22 ± 0.04 1%@10 20%@10 (136– 0 0 1.9 ± 0.6 13 ± 2 8±2 35 ± 2 (5–16) (0.8–16) (0.03–2.7) 40 5051) 1349 4045 41 5.7 3.2 0.81 ± 0.2 9% @ 10 13% @ 10 (520– (1204– 0 0 0 9±2 5±1 43 ± 4 7b CH 3 42 (1.3–25) (0.2–49) 3501) 13583) 43 0.001 2008 44 7c CONH2 0.86 ± 0.1 4%@10 18%@10 0%@10 25%@10 (0.0008– (977– 0 1.5 ± 0.2 0 6±1 13 ± 4 53 ± 7 0.03) 4429) 45 46a ACS Paragon Plus Environment All compounds were tested as their a) (±)-isomers; b) [3H]epibatidine Kd is 0.02 nM 47 48


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For comparison the reference nAChR agonist nat-epibatidine, the reference nAChR antagonist DHβE, and the reference α4β2-nAChR 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. 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. α4β2-, α3β4, and α7-nAChRs were expressed in Xenopus oocytes and functional effects of the compounds were determined under two-electrode voltage clamp. Values obtained for compounds 5a–f, 6a–d, and 7a–c are compared to previously determined values for nicotine, nat-epibatidine 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 α4β2-, α3β4-, and α7-nAChRs. This contrasted with nicotine, nat-epibatidine 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 µM 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 α4β2- or α7-nAChRs. 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 concentration-inhibition 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 2-fold more potent at α4β2- versus α3β4-nAChRs, this compound was 63-fold more potent at α4β2- versus α7-nAChRs. 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 tail-flick or hot-plate 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 tail-flick and hot-plate 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 tail-flick 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 µg/kg, respectively. For comparison DHβE had an AD50 value of 450 µg/kg. Compounds 5a and 7a have AD50 values of 0.52 and 0.32 µg/kg in the tail-flick test and thus are similar to the 0.2 µg/kg for varenicline. Compound 6c with an AD50 = 35 µg/kg was the most potent analog in the hot-plate test. Compounds 5a, 5b, 6a, and 6d had AD50 values of 330, 257, 113, and 435 µg/kg, respectively, which is similar to the AD50 = 470 µg/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 µg/kg in the tail-flick and hot-plate 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 µg/kg in the tail-flick and hot-plate tests. 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 5a-c, 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 co-workers 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

a

Compd ID

LogPa

TPSAa

logBBb

CNS MPO

varenicline

1.01

37.81

–0.27

4.86

5a

2.98

24.92

0.22

4.08

5b

3.75

24.92

0.34

3.70

5c

3.29

24.92

0.27

3.93

5d

2.99

34.15

0.09

4.54

5e 5f

1.96 0.75

68.01 85.08

–0.57 –1.01

4.50 4.50

6a

2.98

24.92

0.22

4.08

6b

1.97

68.01

–0.57

4.50

6c 6d

3.29 3.75

24.92 24.92

0.27 0.34

3.93 3.70

7a

3.49

24.92

0.30

3.83

7b

1.83

68.01

–0.59

4.50

7c

3.58

24.92

0.32

3.79

b

ChemAxon Calculator Plugins, Marvin 6.1.0, 2013. 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 tail-flick, hot-plate, hyperthermia, and spontaneous activity tests but were potent antagonists in both the in vitro assay and the in vivo tail-flick test. Several other of the epibatidine analogs (5a–d, 7a, and 7b) did not show agonist activity in the tail-flick and hot-plate test but like varenicline were agonist in the hyporthermia and spontaneous activity test and were potent antagonists in the in vitro assay and the tail-flick 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 tail-flick tests. Compound 6c with a Ki of 0.21 nM in the binding assay, an AD50 of 0.05 and 35 µg/kg in the tail-flick and hot-plate tests, but no agonist activity, compound 7c with a Ki of 0.86 nM in the binding assay, similar potencies as an antagonist at α4β2- and α3β4-nAChRs with good selectivity versus α7-nAChRs and an AD50 of 0.001 µg/kg in the tail-flick 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 α3β4- and α7-



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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.

Methods Melting points were determined on a Mel-temp (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 Perkin-Elmer Sciex API 150EX mass spectrometer outfitted with an APCI and ESI sources. Elemental analyses were carried out by Atlantic Microlab, Inc., Norcross GA. Analytical thin-layer chromatography (TLC) was carried out on plates pre-coated 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'-amino-3'(substituted thiophenyl) compound 9a or 9b was stirred in a solution of HF-pyridine (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.), 5-bromothiophene-2-carboxamide or 5bromothiophene-2-sulfonamide (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 hexanes-ethyl acetate, to provide the Boc-protected product of either 5e or 5f, respectively. 2-exo-[2'-Fluoro-3'-(thiophen-2-yl)-5'-pyridnyl]-7-azabicyclo[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)+. 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, MeOH-d4) 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, MeOH-d4) 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 [(M-fumarate)+, 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. 2-exo-[2'-Fluoro-3′-(5-chlorothiophen-2-yl)-5′-pyridinyl]-7-azabicyclo[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)+. 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, MeOH-d4) 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, MeOH-d4) 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 [(M-fumarate)+, 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. 2-exo-[2'-Fluoro-3′-(5-fluorothiophen-2-yl)-5′-pyridinyl]-7-azabicyclo[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 nBuLi (730 µL, 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. NFluorobenzenesulfonimide (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 20-mL 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, 7-tert-butoxycarbonyl-2-exo-[2'-fluoro3′-(5-fluorothiophen-2-yl)-5′-pyridinyl]-7-azabicyclo[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. The Boc-protected 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 off-white solid: mp. 86–90 °C; 1H NMR (MeOH-d4) 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 (MeOH-d4) 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. 2-exo-[2'-Fluoro-3′-(5-methoxythiophen-2-yl)-5′-pyridinyl]-7-azabicyclo[2.2.1]heptane (5d) Fumarate. A solution of the boronic ester 13 (424 mg, 1.01 mmol), 2-bromo-5-



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methoxythiophene (635 mg, 3.28 mmol), [ which was prepared from 2-methoxythiophene (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 1,4-dioxane (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 Boc-protected 5d. A solution of Boc-protected 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 20-mL 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, N-H 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 (MeOH-d4) 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 (MeOH-d4) 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. 5-5-(7-Azabicyclo[2.2.1]hept-2-yl)-2-fluoropyridin-3-yl)thiophene-2-carboxamide (5e) Hydrochloride. The Boc-protected 5e was prepared from the cross-coupling of compound 13 (399 mg, 0.954 mmol) with 5-bromothiophene-2-carboxamide (295 mg, 1.43 mmol) following the described General Procedure B to provide 179 mg (41%) of the Boc-protected 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, N-H, 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)+. A solution of the Boc-protected 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 (MeOH-d4) 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 (MeOH-d4) 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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5-5-(7-Azabicyclo[2.2.1]hept-2-yl)-2-fluoropyridin-3-yl)thiophene-2-sulfonamide (5f) Hydrochloride. The Boc-protected 5f was prepared from compound 13 (360 mg, 0.861 mmol) and 5-bromothiophene-2-sulfonamide (271 mg, 1.12 mmol) following the described General Procedure B, to provide 184 mg (47%) of the Boc-protected 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)+. A solution of the Boc-protected 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 (MeOH-d4) 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 (MeOH-d4) 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. 2-exo-[2'-Fluoro-3′-(thiophen-3-yl)-5′-pyridinyl]-7-azabicyclo[2.2.1]heptane (6a) Fumarate. To a resealable reaction pressure vessel under nitrogen was added 10 (151 mg, 0.556 mmol), thiophene-3-boronic acid (92.5 mg, 0.723 mmol), Pd(PPh3)4 (32 mg, 5 mol %), sodium carbonate (118 mg, 1.11 mmol), 1,4-dioxane (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)+. 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, MEOH-d4) 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, MEOH-d4) 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. 4-5-(7-Azabicyclo[2.2.1]hept-2-yl)-2-fluoropyridin-3-yl)thiophene-2-carboxamide (6b) Hydrochloride. A solution of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thiophene-2carboxamide, (408 mg, 1.61 mmol, 1.5 equiv) [prepared from 4-bromothiophene-2-carboxamide 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 1,4-dioxane (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 re-dissolved in CHCl3 and a 20-mL 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 Boc-protected 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)+. 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 (MeOH-d4) 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 (MeOH-d4) 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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2-exo-2-[6-Fluoro-5-(5-fluorothiophen-3-yl)pyridin-3-yl]-7-azabicyclo[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 flame-dried flask and cooled in a dry ice/acetone bath at –78 °C. The solution was treated dropwise with n-BuLi (640 µL, 1.6 M in hexanes, 1.02 mmol, 1.3 equiv) and stirred at –78 °C for 1 h. A solution of Nfluorobenzenesulfonimide (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 warm-up 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 silylated-Boc-protected 6c (tert-butyl-2-{5-[5-fluoro-2-(trimethylsilyl)thiophen-3-yl]-6fluoropyridin-3-yl}-7-azabicyclo[2.2.1]hepane-7-carboxylate) 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 silylated-Boc-protected 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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(MeOH-d4) 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), 8.10–8.15 (m, 2H); 13C NMR (MeOH-d4) δ (ppm) 26.8, 28.8, 37.6, 43.3, 60.5, 64.3, 108.7 (JCF = 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. 2-[5-(5-Chlorothiophen-3-yl)-6-fluoropyridin-3-yl]-7-azabicyclo[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 flame-dried flask then cooled to –78 °C using a dry ice-acetone bath. The solution was treated dropwise with n-BuLi (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 silylated-Boc-protected 6d (tert-butyl-2-{5-[5-chloro-2-(trimethylsilyl)thiophen-3-yl]6-fluoropyridin-3-yl}-7-azabicyclo[2.2.1]hepane-7-carboxylate) 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 silylated-Boc-protected 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. 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 (MeOH-d4) 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 (MeOH-d4) 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. 2-exo-[2'-Fluoro-3′-(4-methylthiophen-2-yl)-5′-pyridinyl]-7-azabicyclo[2.2.1]heptane (7a) Hydrochloride. In a microwave vial was placed 11 (233 mg, 0.626 mmol), 4methylthiophene-2-boronic acid (115 mg, 0.814 mmol), Pd(PPh3)4 (72.3 mg, 10 mol %), K2CO3 (173 mg, 1.25 mmol), 1,4-dioxane (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 Boc-protected 7a as a colorless oil. A solution of Boc-protected 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, N-H 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)+. 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 (MeOH-d4) 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 (MeOH-d4) 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. 5-(7-Azabicyclo[2.2.1]hept-2-yl)-3-(4-carbamoylthiophen-2-yl)pyridn-2-yl (7b) Hydrochloride. A solution of 13 (757 mg, 1.81 mmol), 2-bromothiophene-4-carboxamide (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 Boc-protected product. A solution of the Boc-protected 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)+. 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 (MeOH-d4) 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 (MeOH-d4) 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. 2-[5-(4-Chlorothiophen-2-yl)-6-fluoropyridin-3-yl]-7-azabicyclo[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 (MeOH-d4) 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 (MeOH-d4) 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 C15H16Cl3FN2S•0.25H2O: C 46.65; H 4.31; N 7.25; found: C 46.62; H 4.43; N 7.17. 7-tert-Butoxycarbonyl-2-exo-[2'-amino-3′-(thiophen-2-yl)-5′-pyridinyl]-7azabicyclo[2.2.1]heptane (9a). A solution of compound 8 (301 mg, 0.817 mmol), thiophene-2boronic acid (136 mg, 1.06 mmol), Pd(PPh3)4 (47.2 mg, 5 mol %), Na2CO3 (173 mg, 1.63 mmol)



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in DMF (10 mL) and H2O (1 mL) was placed in a resealable pressure vessel, degassed through bubbling nitrogen for 40 min, sealed and heated at 100 °C overnight. After cooling, the solvent was removed in vacuo, and a 10-mL aqueous solution of NaHCO3 was added. The organic product was extracted using dichloromethane (3 × 30 mL), dried over MgSO4, filtered through Celite and solvent removed under reduced pressure. The residue was purified by on a silica gel column, eluted with EtOAc-hexanes, to furnish 177 mg (58%) of 9a. 1H NMR (300 MHz, CDCl3) δ 1.40 (s, 9H), 1.47–1.57 (m, 2H), 1.74–1.84 (m, 3H), 1.91–195 (dd, J = 9.0, 12.4 Hz, 1H), 2.76–2.80 (dd, J = 4.8, 8.9 Hz, 1H), 4.13 (s, 1H), 4.36 (s, 1H), 4.76 (s, 2H), 7.11 (t, J = 4.6 Hz, 1H), 7.22 (d, J = 3.5 Hz, 1H), 7.35 (d, J = 4.1 Hz, 1H), 7.49 (d, J = 2.2 Hz, 1H), 7.93 (d, J = 2.2 Hz, 1H); 13C NMR (CDCl3) δ 28.3, 28.8, 29.7, 40.3, 44.9, 62.2, 79.5, 114.6, 125.6, 126.0, 127.7 131.8, 137.0, 139.8, 146.2, 154.5, 155.1; MS (ESI) m/z 372.2 (M+H)+. 7-tert-Butoxycarbonyl-2-exo-[2'-amino-3′-(5-chlorothiophen-2-yl)-5′-pyridinyl]-7azabicyclo[2.2.1]heptane (9b). A solution of compound 8 (235 mg. 0.638 mmol), 5chlorothiophenyl-2-boronic acid (124 mg, 0.766 mmol), Pd2(dba)3 (16.5 mg, 3 mol %), PPh3 (16.7 mg, 10 mol %) and sodium carbonate (176 mg, 1.66 mmol) was placed in a microwave vial and degassed through bubbling nitrogen. The mixture was then irradiated in a microwave reactor at 120 °C for 1 h. After cooling, the solvent was removed in vacuo, and 10 mL of water were added to the residue. The organic product was extracted using dichloromethane (3 × 30 mL), dried over MgSO4, filtered through Celite and solvent removed under reduced pressure. The residue was purified on silica gel, eluted with EtOAc/hexanes, to furnish 225 mg (86%) of 9b. 1H NMR (300 MHz, CDCl3) δ 1.41(s, 9H), 1.47–1.57 (m, 2H), 1.74–1.84 (m, 3H), 1.91–1.95 (dd, J = 9.0, 12.4 Hz, 1H), 2.75–2.79 (dd, J = 4.8, 8.9 Hz, 1H), 4.14 (s, 1H), 4.36 (s, 1H), 4.79 (s, 2H), 6.92 (d, J = 3.8 Hz, 1H), 6.98 (d, J = 3.8 Hz, 1H), 7.43 (d, J = 2.1 Hz, 1H), 7.93 (d, J = 2.1 Hz, 1H); 13C NMR (CDCl3) δ 28.3, 28.8, 29.7, 40.3, 44.8, 62.2, 113.9, 125.5, 126.7, 127.2, 130.0, 132.0, 137.0, 138.5 146.5, 154.3, 155.1; MS (ESI) m/z 406.4 (M+H)+.


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7-tert-Butoxycarbonyl-2-exo-[2'-fluoro-3′-(thiophen-3-yl)-5′-pyridinyl]-7azabicyclo[2.2.1]heptane (12). A solution of compound 11 (462 mg, 1.24 mmol, 1.0 equiv.), thiophene-2-boronic (207 mg, 1.62 mmol, 1.3 equiv.), Pd(PPh3)4 (144 mg, 10 mol %), K2CO3 (430 mg, 3.11 mmol, 2.5 equiv) in DMF (10 mL) and water (1 mL) was degassed through bubbling nitrogen for 20 min, sealed and heated at 80 °C overnight. After cooling to room temperature the mixture was diluted with H2O (20 mL) and organic layer was extracted with EtOAc (3 × 30 mL). The combined organic layer were dried over anhydrous Na2SO4, filtered through Celite and concentrated in vacuo. Purification on silica gel, eluted with EtOAc-hexanes, provided 364 mg (78%) of 12 as a clear oil. 1H NMR (300 MHz, CDCl3) δ 1.45 (s, 9H), 1.52– 1.65 (m, 2H), 1.81–1.88 (m, 3H), 2.02–2.06 (dd, J = 9.0, 12.4 Hz, 1H), 2.89–2.94 (dd, J = 4.8, 8.9 Hz, 1H), 4.23 (s, 1H), 4.40 (s, 1H), 7.11–7.14 (dd, J = 5.1, 1.4 Hz, 1H), 7.39–7.41 (dd, J = 5.3, 1.2 Hz, 1H), 7.53–7.55 (dt, J = 3.8, 1.3 Hz, 1H), 7.96 (t, J = 2.2 Hz, 1H), 8.03 (dd, J = 2.4, 9.5 Hz, 1H); 13C NMR (CDCl3) δ 28.3, 28.8, 29.7, 40.6, 44.7, 56.0, 62.0, 116.9, 126.7, 127.1, 128.0, 135.4, 136.8, 139.7, 143.8, 155.1, 156.3, 159.5; MS (ESI) m/z 375.4 (M+H)+. tert-Butyl-2-{6-fluoro-5-[2-(trimethylsilyl)thiophen-3-yl]pyridin-3-yl}-7azabicyclo[2.2.1]heptane-7-carboxylate (19). Preparation of 3-bromothiophene-2-trimethylsilane (18a): A solution of 3bromothiophene (4.0 g, 24.6 mmol) in dry THF was placed in a flame-dried flask at 0 °C and was treated with LHMDS (31.9 mL, 1.0 M in THF, 1.2 equiv). After stirring for 1 h at 0 °C, TMSCl (2.6 mL, 31.94 mmol, 1.3 equiv) was added and the mixture was stirred for an additional 1 h at 0 °C then quenched with an aqueous solution of NH4Cl. The organic layer was extracted with EtOAc (3 × 50 mL), dried over Na2SO4, filtered and concentrated in vacuo. Purification on silica gel, eluted with ethyl acetate–hexanes, provided 5.36g (93%) of 18a as yellowish oil. A clear oil was obtained upon distillation. bp. 219 °C at atmospheric pressure; 1H NMR (300 MHz, CDCl3) δ 0.22 (s, 9H), 6.92 (d, J = 4.6 Hz, 1H), 7.25 (d, J = 4.6 Hz, 1H); 13C NMR (CDCl3) δ 0.0, 118.0, 131.4, 133.3, 135.1.



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A solution of compound 13 (561 mg, 1.34 mmol, 1.0 equiv), compound 18a (631 mg, 2.68 mmol, 2.0 equiv), Pd(dppf)Cl2 (49 mg, 0.067 mmol, 5 mol %) and 2M Na2CO3 ( 1.5 mL) in DME (6 mL) was placed in a microwave vial and degassed for 10 min then was irradiated in a microwave reactor at 110 °C for 30 min. After cooling to room temperature, an aqueous solution of NH4Cl was added and the organic product extracted with EtOAc. The combined organic layers were dried (Na2SO4), filtered and concentrated in vacuo. Purification on silica gel, eluted with EtOAc/hexanes, provided 424 mg (71%) of compound 19 as an oil. 1H NMR (300 MHz, CDCl3) δ 0.0 (s, 9H), 1.23 (s, 9H), 1.39–1.49 (m, 2H), 1.67–1.71 (m, 3H), 1.83–1.90 (dd, J = 9.0, 12.4 Hz, 1H), 2.74–2.79 (dd, J = 4.8, 8.9 Hz, 1H), 4.04 (s, 1H), 4.22 (s, 1H), 6.97 (d, J = 4.6 Hz, 1H), 7.44 (d, J = 4.7 Hz, 1H), 7.54 (dd, J = 9.3, 4.7 Hz, 1H), 7.91 (s, 1H); 13C NMR (CDCl3) δ 0.0, 27.8, 28.3, 29.3, 40.0, 44.4, 55.8, 61.7, 79.4, 120.3 (JCF = 32.1 Hz), 129.5, 130.5, 137.7, 138.2, 140.3 (JCF = 4.2 Hz), 144.5 (JCF = 14.0 Hz), 154.9, 157.5, 160.7. MS (ESI) m/z 447.7 (M+H)+. tert-Butyl-2-{5-[4-chloro-5-(trimethylsilyl)thiophen-2-yl]-6-fluoropyridin-3-yl}-7azabicyclo[2.2.1]heptane-7-carboxylate (22) Preparation of 5-iodo-3-chlorothiophene-2-trimethylsilane (20). A solution of 3chlorothiophene (2.43 g, 20.5 mmol) in dry THF was placed in a flame-dried flask at 0 °C and was treated with LHMDS (24.6 mL, 1.2 equiv., 1.0 M in THF). After stirring for 1 h, TMSCl (3.3 mL, 26.6 mmol) was added and the mixture was stirred for an additional 1 h at 0 °C then quenched with an aqueous solution of NH4Cl. The organic layer was extracted with EtOAc (3 × 50 mL), dried over Na2SO4, filtered and concentrated in vacuo. Purification on silica gel, eluted with EtOAc/hexanes, provided 3.2 g (81%) of 18b as an oil which was distilled before the next step. bp. 202 °C at atmospheric pressure; 1H NMR (300 MHz, CDCl3) δ 0.22 (s, 9H), 6.95 (d, J = 5.5 Hz, 1H), 7.25 (d, J = 4.8 Hz, 1H); 13C NMR (CDCl3) δ 0.0, 130.6, 130.8, 132.7, 132.8. A solution of 18b (1.28 g, 6.73 mmol, 1.0 equiv. (dried over molecular sieves)) in dry THF was placed in a flame-dried flask and cooled to –78 °C. The reaction was treated with a


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dropwise addition of n-BuLi (5.15 mL, 1.6 M in hexanes, 8.08 mmoL, 1.2 equiv) and stirred for 1 h at –78 °C. A solution of I2 (2.22 g, 8.75 mmol, 1.3 equiv. (dried over molecular sieves)) in dry THF was added and the reaction was stirred for an additional 0.5 h then quenched with an aqueous solution of NH4Cl (20 mL). The organic product was extracted with EtOAc (3 × 30 mL). The combined organic layers were washed with sodium thiosulfate, dried (Na2SO4), filtered and concentrated in vacuo. Purification of the residue on silica gel, eluted with EtOAc/hexanes, provided 1.13 g (53%) of 20 as a dark brown oil. 1H NMR (300 MHz, CDCl3) δ 0.17 (s, 9H), 6.92 (s, 1H); 13C NMR (CDCl3) δ 0.0, 78.3, 132.8, 139.3, 140.8. A solution of compound 13 (620 mg, 1.48 mmol, 1.0 equiv), compound 20 (703 mg, 2.22 mmol, 1.5 equiv), Pd(dppf)Cl2 (53 mg, 0.074 mmol, 5 mol %) and 2M Na2CO3 ( 1.5 mL) in DME (6 mL) was placed in a microwave vial and degassed for 10 min then was irradiated in a microwave reactor at 110 °C for 30 min. After cooling to room temperature, an aqueous solution of NH4Cl was added and the organic product extracted with EtOAc. The combined organic layers were dried (Na2SO4), filtered and concentrated in vacuo. Purification on silica gel, eluted with EtOAc/hexanes, provided 301 mg (42%) of compound 21 as an oil. 1H NMR (300 MHz, CDCl3) δ 0.0 (s, 9H), 1.27 (s, 9H), 1.32–1.45 (m, 2H), 1.63–1.70 (m, 3H), 1.79–1.87 (dd, J = 9.0, 12.4 Hz, 1H), 2.69–2.73 (dd, J = 4.8, 8.9 Hz, 1H), 4.03 (s, 1H), 4.20 (s, 1H), 7.24 (d, J = 1.4 Hz, 1H), 7.76 (s, 1H), 7.80 (dd, J = 9.5, 2.4 Hz, 1H); 13C NMR (CDCl3) δ 0.0, 29.2, 29.6, 30.5, 41.6, 45.4, 56.8, 62.8, 80.8, 116.9 (JCF = 28.6 Hz), 130.1, 133.2, 134.6, 137.2, 140.4 (JCF = 4.2 Hz), 145.5 (JCF = 14.0 Hz), 155.9, 157.0, 160.2. MS (ESI) m/z 481.5 (M+H)+. [3H]Epibatidine Binding Assay. The inhibition of [3H] binding at rat brain α4β2*-nAChRs was conducted as previously reported.28 Electrophysiology. Compounds 5a–f, 6a–d, and 7a–c were tested for agonist and antagonist activity using rat α4β2, α3β4 and α7 nAChRs expressed in Xenopus laevis oocytes and assayed by two-electrode voltage clamp electrophysiology. The care and use of X. laevis frogs in this study was approved by the University of Miami Animal Research Committee and



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meet the guidelines of the National Institutes of Health. X. laevis oocytes were surgically obtained and follicle cells removed by treatment with collagenase B for 2 h at room temperature. cRNA encoding the rat α3, α4, α7, β2, and β4 neuronal nAChR subunits was synthesized using mMessage mMachine kits (Ambion). Oocytes were injected with 10–40 ng of cRNA in 25–50 nL of water and incubated at 19 °C in modified Barth's saline (in mM: 88 NaCl, 1 KCl, 2.4 NaHCO3, 0.3 Ca(NO3)2 0.41 CaCl2, 0.82 MgSO4, 15 mM HEPES, pH 7.6 and 2.5 µg/mL ciprofloxacin). For α4β2 and α3β4 receptors, cRNA transcripts encoding each subunit were injected into oocytes at a molar ratio of 1:1. Acetylcholine-induced current responses were measured 2–6 days after cRNA injection using two-electrode voltage clamp in an automated parallel electrophysiology system (OpusExpress 6000A, Molecular Devices). Micropipettes were filled with 3M KCl and had resistances of 0.2–2.0 MΩ. For α4β2 and α3β4 receptors, current responses were recorded at a holding potential of -70 mV, filtered (4-pole, Bessel, low pass) at 20 Hz (-3db) and sampled at 100 Hz. For α7 receptors, current responses were recorded at a holding potential of -40 mV (to minimize the contribution of Ca2+-activated Cl- channels), filtered (4-pole, Bessel, low pass) at 100 Hz (-3db) and sampled at 500 Hz. Current responses were captured and stored using OpusXpress 1.1 software (Molecular Devices). Oocytes were perfused at room temperature (25 °C) with ND96 (in mM: 96 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 5 HEPES, pH 7.5). Compounds 5a–f, 6a–d, and 7a–c were diluted in ND96 and applied for 15 sec (α4β2 and α3β4) or 5 sec (α7) at a flow rate of 4 mL/min, with 3-min washes between applications. Agonist activity was assessed (Table 1) by comparing the current response to 100 µM of each compound to the mean current response of three preceding applications of acetylcholine, applied at an EC20 concentration (20 µM for α4β2, 110 µM for α3β4) or an EC50 concentration (300 µM for α7). Agonist activity of each compound is expressed as a percentage of the maximal response to acetylcholine. Antagonist activity was assessed (Table 1) by comparing the current response to an EC50 concentration of acetylcholine (70 µM for α4β2, 200


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µM for α3β4, 300 µM for α7) in the presence of 100 µM of each compound to the mean current response of three preceding applications of acetylcholine (EC50 concentration). The antagonist activity of compound 7c was assessed in more detail by generating concentration-inhibition curves. Data were fit (Prism 7, Graphpad) to the equation: I=Imax/[1+(IC50/X)n], where I is the current response at agonist concentration (X), Imax is the maximum current, IC50 is the ligand concentration producing half-maximal inhibition of the current response, and n is the Hill coefficient. In vivo Methods. The epibatidine analogues were tested for their effects on body temperature and two pain models (tail-flick and hot-plate assays) and locomotor activity after acute s.c. administration as previously described.33 Antinociception was assessed by the tailflick and hot plate methods. A control response (2–4 sec for tail-flick and 8–19 sec for hot-plate) in male adult ICR mice was determined for each mouse before treatment, and a test latency was determined after drug administration. In order to minimize tissue damage, a maximum latency of 10 sec for tail flick and 40 sec for hot-plate was imposed. Antinociceptive response was calculated as percent maximum possible effect (% MPE), where %MPE = [(testcontrol)/(10(or 40 for hot-plate)-control)] × 100. Groups of eight to twelve animals were used for each dose and for each treatment. The mice were tested 5 min after either vehicle or analogs injection s.c. for the dose-response evaluation. The animals were tested 5 min after administration of the analogs. Right after the antinociceptive testing, mice were evaluated for their locomotor activity. Mice were placed into individual Omnitech photocell activity cages (28 × 16.5 cm). Interruptions of the photocell beams (two banks of eight cells each) were then recorded for the next 10 min. Data were expressed as number of photocell interruptions. Finally, 20 min after injection of vehicle or epibatidine analogs, their rectal temperature was measured by a thermistor probe (inserted 24 mm) and digital thermometer (Yellow Springs Instrument Co., Yellow Springs, OH).



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Readings were taken just before injection and 20 min post drug. The difference in rectal temperature before and after treatment was calculated for each mouse. The ambient temperature of the laboratory varied from 21–24 °C from day to day. For the antagonist experiments, male ICR adult mice were pretreated s.c. with either saline or epibatidine analogues 10 min before nicotine as previously described.34 Nicotine was administered at a dose of 2.5 mg/kg, s.c. (an ED84 dose), and mice were tested 5 min later. ED50 and AD50 values with 95% confidence limits were determined. Data was analyzed statistically by an analysis of variance followed by the Fisher PLSD multiple comparison test. The null hypothesis was rejected at the 0.05 level. ED50 values with 95% CL for behavioral data were calculated by unweighted least-squares linear regression as described by Tallarida and Murray.35 Calculated Pharmacokinetic Properties. The topological polar surface area (TPSA) and calculated lipophilicity (clogP) values were calculated using the ChemAxon Instant JChem package. Predictions of the logarithm of the in vivo blood-brain ratio (logBB) were based on the Clark and Pickett model (Eq.3).31, 36

Author Information Corresponding Author Telephone: 919 541-6679 Fax: 919 541-8868 Email: [email protected]

Author Contributions P. W. O. performed synthesis of compounds. A. H. C., B. S. and M. I. D. performed the pharmacological studies. P. W. O., A. H. C., B. S., C. W. L., M. I. D., S. W. M., H. A. N., and F. I. C. designed the studies, performed analysis of the data, and wrote the manuscript.


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Funding Source This research was supported by the National Institute on Drug Abuse Grant DA12001.

Acknowledgment We thank Mr. Keith Warner for conducting the [3H]epibatidine binding studies. Abbreviations NRT, nicotine replacement therapy; nAChR, nicotinic acetylcholine receptor; DHβE, dihydrobeta-erythroidine; DD, drug discrimination; SA, self-administration; PdCl2(dppf), 1,1'bis(diphenylphosphino)ferrocene-palladium(II) dichloride; MPE, maximum potential effect; cRNA, complimentary RNA.

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Figure 1. Structure of Compounds 1, 2, 3, 4, 5a–f, 6a–d, and 7a–c 169x98mm (288 x 289 DPI)


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a Reagents and Conditions: (a) Thiophene-2- or 3-boronic acid, Pd(PPh3)4, Na2CO3, DMF or 1,4-dioxane, H2O, 100 °C, 18 h; (b) 5-Chlorothiophene-2-boronic acid, Pd2(dba)3 (3 mol %), PPh3 (10 mol %), DME, H2O, microwave, 120 °C, 1 h (c) 70% HF-pyridine, NaNO2, 0 °C to rt, 1 h. 151x76mm (288 x 289 DPI)



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a Reagents and Conditions: (a) Thiophene-2-boronic acid, Pd(PPh3)4, K2CO3, 1,4-dioxane, H2O, 100 °C, overnight; (b) (i) n-BuLi, THF, at –78 °C, 40 min to 0 °C (10 min), (ii) N-fluorobenzenesulfonimide, THF, – 78 °C, 1 h to rt. (c) TFA, CH2Cl2, rt, overnight; (d) bis(pinacolato)diboron, KOAc, PdCl2dppf, 1,4-dioxane, microwave 140 °C, 20 min; (e) 2-bromo-5-methoxythiophene (14) [prepared from 2-methoxythiophene (15) using NBS], Pd(PPh3)4, K2CO3, 1,4-dioxane, H2O, reflux, overnight; (f) 5-bromothiophene-2carboxamide, Pd(PPh3)4, K2CO3, DME, H2O, 85 °C, overnight; (g) HCl in Et2O/ MeOH, rt, overnight; (h) PdCl2(dppf) (3 mol %), K3PO4, or Na2CO3, 1,4-dioxane, DMF, H2O, 80 °C, overnight; (i) 4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)thiophene-2-carboxamide, [prepared from 4-bromothiophene-2carboxamide using reagents and conditions in (d)], Pd(PPh3)4, Na2CO3. 1,4-dioxane, DMF, H2O, 100 °C, overnight; (j) Pd(PPh3)4, K2CO3, 1,4-dioxane, H2O, microwave, 150 °C, 1 h; (k) 5-Bromothiophene-2sulfonamide (l) 5-bromothiophene-3-carboxamide (16) [prepared in three steps from thiophene-3-carboxylic acid]. 184x164mm (288 x 288 DPI)


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a Reagents and Conditions: (a) (i) LiHMDS, THF, 0 °C, 1 h, (ii) TMSCl, 0 °C, 1 h; (b) (i) n-BuLi (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) n-BuLi, 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). 132x139mm (289 x 289 DPI)



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ToC Graphic 147x34mm (288 x 290 DPI)


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Synthesis, Nicotinic Acetylcholine Receptor Binding, and in Vitro and

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