Synthesis and Pharmacology of (Pyridin-2-yl)methanol Derivatives as

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Synthesis and Pharmacology of (Pyridin-2-yl)methanol Derivatives as Novel and Selective Transient Receptor Potential Vanilloid 3 (TRPV3) Antagonists Arthur R Gomtsyan, Robert G Schmidt, Erol K Bayburt, Gregory A. Gfesser, Eric A. Voight, Jerome F Daanen, Diana L Schmidt, Marlon D. Cowart, Huaqing Liu, Robert J. Altenbach, Michael E. Kort, Bruce Clapham, Phil B. Cox, Anurupa Shrestha, Rodger Henry, David N. Whittern, Regina M Reilly, Pamela S Puttfarcken, Jill-Desiree Brederson, Ping Song, Bin Li, Susan M Huang, Heath A McDonald, Torben R Neelands, Steve P. McGaraughty, Donna M Gauvin, Shailen K Joshi, Patricia N Banfor, Jason A Segreti, Mohamad Shebley, Connie R. Faltynek, Michael J. Dart, and Philip R. Kym J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00287 • Publication Date (Web): 14 Apr 2016 Downloaded from http://pubs.acs.org on April 15, 2016

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Journal of Medicinal Chemistry

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Synthesis and Pharmacology of (Pyridin-2-yl)methanol Derivatives as Novel and Selective Transient Receptor Potential Vanilloid 3 (TRPV3) Antagonists Arthur Gomtsyan,* Robert G. Schmidt, Erol K. Bayburt, Gregory A. Gfesser, Eric A. Voight, Jerome F. Daanen, Diana L. Schmidt, Marlon D. Cowart, Huaqing Liu, Robert J. Altenbach, Michael E. Kort, Bruce Clapham, Phil B. Cox, Anurupa Shrestha, Rodger Henry, David N. Whittern, Regina M. Reilly, Pamela S. Puttfarcken, Jill-Desiree Brederson, Ping Song, Bin Li, Susan M. Huang, Heath A. McDonald, Torben R. Neelands, Steve P. McGaraughty, Donna M. Gauvin, Shailen K. Joshi, Patricia N. Banfor, Jason A. Segreti, Mohamad Shebley, Connie R. Faltynek, Michael J. Dart, Philip R. Kym Research & Development, AbbVie Inc., 1 North Waukegan Road, North Chicago, Illinois 60064, United States

Abstract Transient receptor potential vanilloid 3 (TRPV3) is a Ca2+- and Na+- permeable channel with a unique expression pattern. TRPV3 is found in both neuronal and non-neuronal tissues, including dorsal root ganglia, spinal cord and keratinocytes. Recent studies suggest that TRPV3 may play a role in inflammation, pain sensation and skin disorders. TRPV3 studies have been challenging, in part due to a lack of research tools such as selective antagonists. Herein we provide the first detailed report on the development of potent and selective TRPV3 antagonists featuring a pyridinyl methanol moiety. Systematic optimization of pharmacological, physicochemical and ADME properties of original lead 5a resulted in identification of a novel and selective TRPV3 antagonist 74a, which demonstrated a favorable preclinical profile in two different models of neuropathic pain as well as in a reserpine model of central pain.

Introduction

ACS Paragon Plus Environment

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Transient receptor potential channel, subfamily V, member 3 (TRPV3) is a nonselective cation channel on neuronal and non-neuronal cells that responds to both thermal (31-37°C) and chemical stimuli.1,2 Although its physiological functions have yet to be fully elucidated, unique attributes of TRPV3 distinguish it from other vanilloid family members. Unlike other vanilloid TRPs, TRPV3 is sensitized dramatically by repeated stimulation.1,3 Predominant expression is observed in human spinal cord, dorsal root ganglia, and keratinocytes.2,4,5 While numerous plantderived natural products have been shown to activate TRPV3, they show little selectivity. 6 The synthetic small molecule, 2-aminoethyl diphenylborinate (2-APB) is most often used to characterize TRPV3 function.7 Activation of TRPV3 elicits release of multiple molecules implicated in barrier formation, hair growth, itch, and pain perception, suggesting the potential for neuronal and non-neuronal functions of this channel.8-10 In addition, multiple TRPV3 gain-offunction mutations have been linked to Olmsted syndrome, a rare but debilitating skin disease.11,12 Its distinctive pharmacology and tissue distribution suggest that pursuit of TRPV3 antagonists for the treatment of chronic pain and skin disease represents an attractive opportunity.13,14 Nevertheless, TRPV3 is one of the least studied TRP subtypes, a consequence of lack of efficient research tools such as selective antagonists. We have identified a small molecule lead series that combines the favorable attributes of high potency and selectivity for TRPV3, desirable drug-like physiochemical properties, and efficacy in models of pathological pain. Here we report on identification and characterization of a novel class of pyridinol antagonists of TRPV3.



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Results and Discussion As a starting point, the project team identified compound 5a originating from medicinal chemistry efforts following a high throughput screen (HTS) of the AbbVie compound collection. Although a relatively lipophilic compound (cLogP 3.9), this (S)-enantiomer of the racemic hit was singled out for its potency at TRPV3 (human Kb = 0.08 µM; IC50 = 0.16 µM), structural compactness, relatively low molecular weight of 308 and, therefore, the potential to access desirable drug-like space. Chirality was found to be critically important, since the enantiomeric counterpart (R)-5a was virtually inactive. The first set of investigations we undertook was aimed at reducing the high unbound clearance of 5a in both rat (rClint u = 150 L/h/kg) and human liver microsomes (hClint u = 40 L/h/kg) (Table 1). Table 1. SAR and microsomal stability of 5a and its analogs modified at aromatic sitesa, b

OH N

R1

X R2

Compound

R1

R2

X

5a 5b 5c (±)-5d (±)-5e (±)-5f (±)-5g 5h (±)-5i

3,4-di-Cl 3-OCF3 4-OCF3 2-OCF3 3,4-di-Cl 3,4-di-Cl 3,4-di-Cl 3,4-di-Cl 4-OCF3

H H H H 3-F 4-F 5-F H H

CH CH CH CH CH CH CH N N

hTRPV3Kb (µM) 0.08 0.08 0.14 0.55 0.61 0.64 0.91 0.37 1.54


RLM / HLM Clint u (L/h/kg) 151/41 103/133 63/20 110/74 180/50 231/32 190/46 50/16 29/6

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OH N

Cl Cl

3.4

380/100

(±)-5j a All experiments to determine Kb values were performed with at least duplicates at each compound dilution and all Kb values were averaged when determined in two or more independent experiments. bIn vitro microsomal stability of compounds was determined using a high-throughput automated 384-well plate assay using published protocols.15 Microsomal intrinsic clearance (Clint) values were corrected for non-specific microsomal binding using experimentally measured unbound fraction (fumic) according to published methods,16 and results were reported as unbound Clint (Clint u).

Compound 5a was incubated in rat liver microsomes for 1 h at 37°C, metabolite distribution was analyzed by HPLC (Figure 1) and the structures of the metabolites were elucidated by 1H NMR. Both pyridine and benzene rings as well as alcohol and cyclobutane moieties were determined to be sites of metabolic oxidation resulting in approximately equal ratio of corresponding metabolites. Blocking different positions of the pyridine fragment with fluorine [(±)-5e-g] did not offer any gains in metabolic stability, but replacement of the pyridine ring with pyrimidine produced compound 5h with 2.5-3-fold diminished microsomal clearance in rats (Table 1). Alternatively, the 4-position on the other aromatic site, phenyl ring, was found to be slightly favored over the 3- and 2-positions in regard to stability (5bc, (±)-5d). Taking advantage of these observations, we synthesized (±)-5i that combined a pyrimidine fragment on its left-hand side with a 4-trifluoromethoxyphenyl group on the right-hand side. Compound (±)-5i did exhibit improved metabolic stability over 5a (rClint u 29 L/h/kg vs. 151 L/h/kg) but also substantially weaker TRPV3 potency (1.54 µM for (±)-5i vs 0.08 µM for 5a, 0.14 µM for 5c and 0.37 µM for 5h) even factoring in its racemic nature.



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mA 4

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Cl

OH N

Cl

3

Oxidation at 2

Oxidation of

5a

cyclobutane

alcohol

Oxidation at

1

aromatic sites 0

-

-

2

2

3

3

4

4

5 mi

Figure 1. Composition of metabolites of 5a (HPLC UV trace)

Exploration of SAR and structure-stability relationship around the alcohol functionality in 5a indicated that TRPV3 potency is highly sensitive to modifications in this region. Structural manipulations such as O-alkylation, homologation to hydroxymethyl group, removal of OH or its replacement with an amine led to a significant reduction in potency. In order to block the oxidation of the alcohol to the ketone, a methyl group was incorporated at the carbon bearing an OH group. However, the resulting tertiary alcohol (±)-5j was over 40-fold less potent than the parent 5a (Table 1). A tertiary OH group was better tolerated in molecules with modified topology of the central moiety such as cyclopentanes 7-10 (Table 2). Unlike the compounds from the original series, these new analogs showed no prohibitive dependency on the stereochemical disposition at the benzylic carbon as all four diastereomers displayed comparable TRPV3 potency in the range of 0.1-0.6 µM. However, the two most potent diastereomers 7 and 8 were also the ones that


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displayed the highest values of intrinsic unbound clearance in microsomes (rClint u 151 and 460 L/h/kg, respectively). By introducing polarity into the cyclopentane ring we expected to reduce the number of potential sites of oxidative metabolism and improve Table 2. SAR and microsomal stability of tertiary alcohols 7-10 and 15-18 with cyclopentane and tetrahydrofuran corea, b

HO N

HO

1 3

O 3 5

N OCF3

CF3

7-10

15-18

Compound

Chirality

hTRPV3 Kb (µM)

7 8 9 10 15 16 17 18

1S, 3R 1R, 3R 1R, 3S 1S, 3S 3S, 5R 3R, 5S 3S, 5S 3R, 5R

0.24 0.13 0.54 0.61 0.42 1.12 1.71 2.46

RLM/HLM Clint u (L/h/kg) 151/9 460/23 86/20 21/24 10/7 20/4 23/13 9/7

a

All experiments to determine Kb values were performed with at least duplicates at each compound dilution and all Kb values were averaged when determined in two or more independent experiments. bIn vitro microsomal stability of compounds was determined using a high-throughput automated 384-well plate assay using published protocols.15 Microsomal intrinsic clearance (Clint) values were corrected for non-specific microsomal binding using experimentally measured unbound fraction (fumic) according to published methods,16 and results were reported as unbound Clint (Clint u).

drug-like properties. Indeed, the tetrahydrofuran moiety in 15-18 reduced cLogP by about 0.7 and resulted in up to 50-fold improvement in metabolic stability compared with cyclopentane analogues 7-10 (Table 2). Although tetrahydrofuran 15 exhibited a significant advantage over the



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carbon analog 7 with respect to metabolic stability, we were concerned about the low TRPV3 potency of this compound when its free fraction in plasma was factored in. According to the free drug hypothesis, the pharmacological effects in vivo are elicited by free (unbound) drug concentration.17 On that measure, 15 exhibited plasma adjusted potency of 12 µM calculated from its total potency in FLIPR assay (Kb = 0.42 µM) and free fraction of 0.03 in plasma. Such a high concentration of the compound required for pharmacological effects was undesirable based on pharmacokinetic and toxicological considerations. In addition to the two aromatic rings and an alcohol moiety in 5a, the cyclobutane ring was also found to undergo oxidation in rat liver microsomes, representing another major metabolic pathway for 5a. The initial strategy to mitigate that liability was to replace the cyclobutane ring with heterocycles. The 3-oxetane replacement improved the metabolic stability as expected,18-20 (compound 24), but at the same time it led to a complete erosion in TRPV3 potency (Table 3). Replacing the ether oxygen with Table 3. SAR and microsomal stability of oxetanes 24 and 37ab, azetidines 29ab and tetrahydrofurans 40 and 46aba, b OH

R

1 2

N

n X

Y

Compound

n

R

X

Y

Chirality

24 29a 29b 37a 37b 40

0 0 0 0 0 1

3,4-di-Cl 4-CF3 4-CF3 3,4-di-Cl 3,4-di-Cl 3,4-di-Cl

CH2 CH2 CH2 O O CH2

O NH NMe CH2 CH2 O

racemate racemate racemate 1,2-trans 1,2-cis racemate


hTRPV3 Kb (µM) 7.37 6.68 6.63 0.38 0.15 0.63

RLMHLM Clint u (L/h/kg) 25/8 3/2 7/3 60/13 3/4 59/25

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1 3,4-di-Cl O CH2 1,2-trans 0.043 179/23 46a 1 3,4-di-Cl O CH2 1,2-cis 0.47 not determined 46b a All experiments to determine Kb values were performed with at least duplicates at each compound dilution and all Kb values were averaged when determined in two or more independent experiments. bIn vitro microsomal stability of compounds was determined using a high-throughput automated 384-well plate assay using published protocols.15 Microsomal intrinsic clearance (Clint) values were corrected for non-specific microsomal binding using experimentally measured unbound fraction (fumic) according to published methods,16 and results were reported as unbound Clint (Clint u).

nitrogen resulted in azetidine analogs 29a and 29b with profiles similar to that of 3-oxetanes. TRPV3 potency rebounded for 2-oxetanes 37ab. The tetrahydrofurans 40 and 46ab also had improved potency for TRPV3, but lower microsomal stability. Replacement of C-H bonds in the cyclobutane ring with C-F bonds yielded compounds with good potency and moderate to good stability, as shown by gem-difluoro analogs 55a and 55c (Table 4). However, further exploration of gem-difluoro-cyclobutane Table 4. SAR and microsomal stability of gem-difluorocyclobutane and diol analogsa, b OH

X

Y

N R3 R1

R2

Compound

R1

R2

R3

X

Y

55a 55c 60b 60e 60f 61b 61e 61f 62b 62c

F F H H H OH OH OH OH OH

F F OH OH OH H H H Me Me

3,4-di-Cl 4-OCF3 3-OCF3 3-CF3 4-CF3 3-OCF3 3-CF3 4-CF3 3-OCF3 4-OCF3

CH CH CH CH CH CH CH CH CH CH

CH CH CH CH CH CH CH CH CH CH

hTRPV3 (µM) 0.08 0.04 0.04 0.09 2.24 0.08 0.21 1.87 0.28 0.33


Kb RLM/HLM Clint u (L/h/kg) 51/15 12/11 7/9 11/8 3/3 13/7 6/3 3/2 12/10 8/4


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

OH OH Me Me Me Me

62e 62f 63b 63c 63e 63f

Me Me OH OH OH OH

3-CF3 4-CF3 3-OCF3 4-OCF3 3-CF3 4-CF3

CH CH CH CH CH CH

CH CH CH CH CH CH

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0.30 0.84 1.31 1.51 1.55 4.79

19/9 9/3 3/7 6/3 4/2 6/2

>10.4

6/3

0.27 0.06 0.32 0.53 0.56 0.50 0.73 4.27

3/2 3/2 3/2 12/5 6/2 5/4 7/2 3/4

>9.1

6/2

OCF3

OH N

OH

64 H H OH OH OH OH Me Me

72a 72b 73a 73b 74a 74b 75a 75b OH N

OH OH H H Me Me OH OH

3-OCF3 3-OCF3 3-OCF3 3-OCF3 3-OCF3 3-OCF3 3-OCF3 3-OCF3

N CH N CH N CH N CH

CH N CH N CH N CH N

N CF3

HO

76 a

All experiments to determine Kb values were performed with at least duplicates at each compound dilution and all Kb values were averaged when determined in two or more independent experiments. bIn vitro microsomal stability of compounds was determined using a high-throughput automated 384-well plate assay using published protocols.15 Microsomal intrinsic clearance (Clint) values were corrected for non-specific microsomal binding using experimentally measured unbound fraction (fumic) according to published methods,16 and results were reported as unbound Clint (Clint u).

analogs was plagued by consistent cardiovascular side-effects in rat studies. Thus, 55c at plasma concentration of 4.7 µg/mL produced a statistically significant increase in cardiac contractility (dP/dt50) to a maximum of 29% above vehicle in anesthetized rats. This effect was also accompanied by a 16% increase in mean arterial pressure above vehicle. Additionally, compound 55c was found to be strong activator of the human pregnan X receptor (PXR) (EC50 = 5.7 µM), a nuclear receptor playing a key role in regulating the expression of metabolizing enzymes.21 One of the most significant implications of PXR activation could be drug-drug-interaction (DDI) due to the increased expression of CYP450 enzymes.22 Therefore, the gem-difluoro-cyclobutane


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series was abandoned and SAR design strategy shifted towards targeting diols such as 60b and 61b where the introduction of the second hydroxyl group at the cyclobutane would preempt the formation of possible products of metabolic oxidation. Having reached favorable in vitro unbound clearance for the first two diols 60b and 61b (rClint u = 7 and 13 L/h/kg respectively), combined with excellent TRPV3 potency, we began a rigorous examination of this series (Table 4). Three key structural elements that influenced the activity of compounds against TRPV3 in the diol series were: 1) stereochemistry at the benzylic carbon adjacent to the pyridine ring, 2) syn/anti-isomerism at the cyclobutane ring and 3) the nature of secondary versus tertiary hydroxyl group at the cyclobutane ring. Analogous to 5a, TRPV3 potency in diols is highly sensitive to the stereo-configuration of the benzylic carbon with (S)isomers being significantly more potent than their (R)-counterparts (for example, compare 74a vs. 76 and 63c vs. 64). A syn-relationship between cyclobutane hydroxyl and pyridinyl-methanol fragment is favored over anti-configuration, although the potency difference is less pronounced in the case of sec-cyclobutanols (60b vs. 61b or 73a vs. 72a) than in the case of tertcyclobutanols (74b vs. 75b or 62f vs. 63f). Finally, a sec-OH group on the cyclobutane ring generally provides higher potency than a tert- OH group, however, the magnitude of the difference is dependent on the identity of the right-hand side fragment and, as mentioned above, syn/anti isomerism. Thus, sec-alcohol 72b in the anti-series featuring a 4-pyridyl fragment was 70-fold more potent than the corresponding tert-alcohol 75b (Kb = 0.06 µM vs. 4.3 µM), yet synisomeric analogs 73b and 74b with the same 4-pyridyl fragment were virtually equipotent with Kb value about 0.5 µM. These effects were substantially muted for other pairs of sec/tert-OH cyclobutanol analogs. For example, with a 2-pyridyl instead of a 4-pyridyl group, there was only a modest 2.7- and 1.5-fold reduction in potency going from secondary to tertiary alcohols in both



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anti- (72a vs 75a) and syn-series (73a vs. 74a). Importantly, unlike the gem-difluoro series, the compounds in the diol series were largely devoid of cardiovascular side-effects and did not activate PXR. Based on the most balanced combination of pharmacological, ADME and physicochemical properties, compound 74a was chosen for more advanced characterization. Despite its modest potency in the FLIPR assay (human Kb = 0.56 µM; IC50 = 0.93 µM), compound 74a was calculated to exhibit Kb = 0.62 µM when free fraction of 0.89 in human plasma protein was factored in. Based on this measure, 74a was superior to a number of its analogs and represented one of the most potent TRPV3 antagonists in the series. This lead compound was also potent in a patch-clamp electrophysiological assay, where it blocked TRPV3 agonist 2-APB-activated currents in a stable recombinant HEK293 cell line expressing human TRPV3 with IC50 = 0.38 µM. In addition, 74a was highly selective for TRPV3 as screening at 10 µM in CEREP and AbbVie internal panels of biological targets (including TRPV1, Nav1.5, Nav1.7 Nav1.8 Cav1.2 Cav2.2 Cav3.2, hERG) showed no evidence of binding >50% (see Supporting Information). A number of molecular and structural parameters define compound 74a as more drug-like than the parent compound 5a and lead 15 from the tetrahydrofuran series (Figure 2). For example, the advantages of 74a over 5a and 15 were manifested in lower lipophilicity, higher fraction of sp3hybridized carbon atoms and higher lipophilic efficiency parameter LLE. Additionally, 74a exhibits an attractive CNS MPO of 4.5 (out of maximal desirable score of 6) based on six fundamental molecular properties.23

OH N

Cl

HO

O

OH N

N

CF3

Cl OCF3


N

HO

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Properties MW logD (calcd) TPSA LLE (pKb – LogD LLE (pKb – LogD (based on plasma adjusted pKb) fSP3 Ar - SP3 PFI (LogD – NAR) Human Clint u (L/h/kg) hep Human Fu plasma CNS MPO

5a 308 4.2 33 2.9

15 339 3.7 52 2.68

74a 338 2.0 66 4.25

0.7

1.41

4.2

0.31 6 6.2 14 0.007 4

0.35 5 5.7 16 0.06 5.0

0.41 3 4.0 2 0.89 4.5

Figure 2. Properties of initial lead 5a and more advanced leads 15 and 74aa. forecast index; NAR, number of aromatic rings

a

PFI, property

The improved unbound microsomal stability of 74a in vitro (rClint u = 6 L/h/kg) translated well to the in vivo setting where unbound clearance of rClp u = 2.5 L/h/kg (iv) was recorded in rat PK studies (Table 5). For comparison, the unbound clearance for tetrahydrofuran analog 15 after iv administration in rats was 7-fold higher, correlating with its higher microsomal clearance in vitro. Table 5. Pharmacokinetic parameters of 15 and 74a in ratsa Compound 15 74a

Total Clp (iv)b (L/h/kg) 0.6 1.6

Unbound Clp (iv) (L/h/kg) 17.6 2.5

Vss (L/kg) 3.2 1.4

T1/2 (iv) (h) 3.8 0.5

Cmax (µg/mL) 0.13 0.19

AUC0-8 (po) (µg*h/mL) 0.93 0.38

a

Unbound AUC0-8 (po) (µg*h/mL) 0.03 0.24

F (po) (%) 56 26

iv doses were 1 mg/kg for both compounds and po doses were 1 mg/kg for 15 and 2 mg/kg for 74a. bAs a reference, rat hepatic blood flow is 3.3 L/h/kg and human hepatic blood flow is 1.2 L/h/kg



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Following ADME and PK characterization, compound 74a was assessed in rat behavioral models of neuropathic pain (Figure 3). Dose-dependent efficacy with an estimated EC50 value around 11-12 µmol was observed in both the chronic constriction injury (CCI) and sciatic nerve ligation (SNL) models of neuropathic pain. A

B

Figure 3. Compound 74a (10, 30,100 mg/kg po) was anti-allodynic in both the CCI (A) and SNL (B) models of neuropathic pain. In both models, the compound dose-dependently attenuated mechanical allodynia induced by von Frey hair stimulation to the ipsilateral (injured) hind paw. A response threshold of 15 g is considered to be the maximal possible effect (MPE). The blue symbols represent %MPE in the neuropathic models, and the red symbols represent the plasma concentrations at the specific doses for these studies. For comparison, gabapentin (Gaba) was also administered at 150 mg/kg. Veh = vehicle (10% DMSO in Polyethylene glycol), n = 6 per group. **p95% ee. Carbononitrile 51 was used as a key intermediate for the preparation of 4trifluoromethoxyphenyl analog 55c as opposed to ester 49 for the synthesis of dichlorophenyl analog 55a. One pot two-step alkylation of 4-trifluoromethoxyphenylacetonitrile 1c with epibromohydrin by stepwise use of methyl lithium and methylmagnesium bromide as bases afforded 51c in excellent yield. Oxidation of alcohol with Dess-Martin periodinane and subsequent fluorination of the resulting ketone 52c with DAST yielded carbononitrile intermediate 53. Similar to the synthesis of 55a from 49, compound 53 was converted to the target 55c through installation of the pyridine moiety followed by asymmetric reduction of the ketone 54. Diol analogs 60-64 were prepared starting from the cyclobutanones 52 (Scheme 8). Intermediates 52 were carried through five steps starting with ketal formation and installation of the pyridine moiety to give 57. It should be noted that the formation of the ketal 56 using 1,2bis(trimethylsilyloxy)ethane and catalytic trimethylsilyl trifluoromethanesulfonate35 was significantly superior to more traditional use of ethyleneglycol resulting in 97% yield of compound 56. Ketone reduction in 57 followed by hydrolysis of the ketal and chiral separation gave enantiopure ketoalcohols 59. The latter were reduced with sodium borohydride to provide diols 60 and 61 with secondary alcohol on the cyclobutane ring Scheme 9. Synthesis of bis-pyridyl diolsa


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CN

O

O

66

X

OH

X

O

e

O

O

69ab

CF3 O

70ab

71ab

h

g

X

OH

Y

N

CF3 + H

Y

Y

N

O

OH

X

CF3

68ab

OH f

O N

CF3 d

O

67

CF3 O

O

Y

N

Y

NC

c

b

a

65

X

CN

CN

O

CF3 + HO

OH h

OH

Y

18

74a X=N, Y=CH 74b X=CH, Y=N


CF3

X

N

H

N

(R) 71a

OH CF3

HO


N

Y

N

17 OH

OH

X

N

X

N

Y CF3

OH


N CF3

HO

76

a

Reagents and conditions: (a) ruthenium (III) chloride trihydrate, NaIO4, CH2Cl2-MeCN-H2O, 0°C – room temperature, 89%; (b) HOCH2CH2OH, TsOH.H2O, toluene, reflux with Dean-Stark trap, 1 h, 73%; (c) 2-fluoro-4(trifluoromethyl)pyridine, KHMDS, toluene, room temperature to 60°C, 2.5 h, 84% for 68a; (d) 2-bromopyridine, Et2O, n-butyl lithium, -78°C, then 1 N HCl, 0°C – room temperature, 0.75 h, 79% for 69a; (e) NaBH4, MeOH CH2Cl2, room temperature, 1 h, 84% for 70a; (f) HCl, acetone, H2O , room temperature, 16 h, 97%, then chiral chromatography on Chiralpac AD-H, 43% for 71a; (g) NaBH4, MeOH - CH2Cl2, room temperature, 16 h, 81%, then SFC on Chiralpac AD-H, 24% for 72a and 39% for 73a; (h) MeLi (1.6 M in Et2O), THF, -78°C – 0°C, 16 h, 40% for 74a and 14% for 75a

or treated with methyl lithium to yield diols 62 and 63 containing tertiary alcohol on the cyclobutane ring.



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Diols 72-76 flanked by pyridine fragments in each side of the molecule were prepared in largely similar way as their mono-pyridine analogs with most notable difference being the synthesis of the key intermediate 68 (Scheme 9). Oxidation of 3-methylenecyclobutanecarbonitrile (65) with sodium periodate in the presence of ruthenium (III) chloride afforded ketone 66 in good yield. Ketalization followed by nucleophilic addition of 2-fluoro-4-(trifluoromethyl)- pyridine or other pyridyl halides afforded 68. The latter were elaborated to the target compounds 72-76 by a similar set of reactions described above for the conversion of 56 to 60-64.

Conclusions This manuscript represents the first detailed report on the development of potent and selective TRPV3 antagonists. Initial lead compound 5a was a potent TRPV3 antagonist, but it also exhibited poor metabolic stability in liver microsomes. In an effort to address this primary deficiency, we systematically modified the structural fragments identified as sites of metabolic activation. Blockade of the metabolic hot spots with fluorine atoms or replacement of the oxidation-prone carbon atoms in the cyclobutane moiety with oxygen or nitrogen atoms either did not lead to the desired outcome or resulted in loss of TRPV3 potency. However, the introduction of an hydroxyl group into the cyclobutane ring proved to be critical for increased metabolic stability and improved plasma adjusted TRPV3 potency. This approach, in conjunction with some manipulations of the right hand side aromatic group, afforded 74a, a molecule with improved logD, LLE and other druglike properties that demonstrated antinociceptive activity in rat models of pathological pain.

Experimental Section


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General methods . Unless otherwise noted, all materials were obtained from commercial suppliers and used without further purification. Anhydrous solvents were obtained from Aldrich (Milwaukee, WI) and used directly. 1H NMR data were recorded using Varian Mercury 300 Mz, Varian Inova 500 Mz or Agilent 400 MR spectrometers. The data are reported as follows: chemical shift in ppm from internal tetramethylsilane standard on the δ scale, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, qn = quintet, m = multiplet), coupling constants (Hz), and integration. Mass spectral data were collected using Finnigan DCI/MS SSQ7000, Thermo Scientific DSQ II (DCI) or Thermo Finnigan LCQ Deca (ESI) spectrometers. Analytical LC-MS was performed on a Thermo MSQ-Plus mass spectrometer and Agilent 1100/1200 HPLC system running Xcalibur 2.0.7, Open-Access 1.4, and custom login software. The mass spectrometer was operated under positive APCI or ESI ionization conditions. The HPLC system comprised an Agilent Binary pump, degasser, column compartment, autosampler and diode-array detector, with a Polymer Labs ELS-2100 evaporative light-scattering detector. The column used was a Phenomenex Kinetex C8, 2.6 μm 100Å (2.1mm × 30mm), at a temperature of 65 °C. A gradient of 5-100% acetonitrile (A) and 0.1% trifluoroacetic acid in H2O (B) was used, at a flow rate of 1.5 mL/min (0-0.05 min 5% A, 0.05-1.2 min 5-100% A, 1.2-1.4 min 100% A, 1.4-1.5 min 1005% A. 0.25 min post-run delay). Preparative reverse phase HPLC purification was done on a Phenomenex Luna C8(2) 5 µm 100Å AXIA column (30 mm × 75 mm) by using a gradient of acetonitrile and 0.1% trifluoroacetic acid in H2O. A Biotage initiator was used for microwave reactions. HRMS data was collected using an Agilent 6550 Q-TOF using an electrospray ion source. The purity for all compounds tested in bioassays was 95+%.



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1-(3,4-Dichlorophenyl)cyclobutanecarbonitrile (2a). To a suspension of sodium hydride (6.45 g, 269 mmol) in DMSO (250 mL) was added a solution of 2-(3,4-dichlorophenyl)acetonitrile (1a) (10 g, 53.8 mmol) and 1,3-dibromopropane (11.94 g, 59.1 mmol) in diethyl ether (60 mL) over 30 min at < 30° C.The reaction mixture was stirred at 15°C for 16 h and then diluted with 250 mL of ether and 150 mL of H2O. The organic layer was separated and washed with H2O and brine. A fter drying (Na2 SO 4 ), filtering, and concentrating, the residue was purified by column chromatography on silica gel (petroleum ether: EtOA c= 10:1) to give 2a (10.2 g, 84 % yield).

1

H NMR (300 MHz, DMSO-d6) δ 7.51 (m, 2H), 7.22 (m, 2H), 2.89 – 2.02 (m, 6H).

MS (ESI) m/z 227 (M + H)+.

1-(4-(Trifluoromethoxy)phenyl)cyclobutanecarbaldehyde (3b). To a solution of 2b (4.82 g, 0.02 mol) in dry CH2Cl2 (100 mL) at -78 oC under an argon atmosphere was added diisobutylaluminum hydride (24 mL, 1 M solution in toluene). The reaction mixture was stirred at –78 oC for 1 h and then quenched by dropwise addition of potassium sodium tartrate (10% solution in H2O). The resulting mixture was warmed to room temperature, stirred vigorously for 40 min and then diluted with CH2Cl2. The organic phase was separated and the aqueous phase extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether:EtOAc = 100:0 to 30:1) to give 3b as a viscous oil (2.7 g, yield 55.3%). MS (ESI) m/z 245 (M + H)+.


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(1-(3,4-Dichlorophenyl)cyclobutyl)(pyridin-2-yl)methanone (4a). To a cooled to -78°C solution of 2-bromopyridine (0.968 mL, 9.95 mmol) and THF (15 mL) was added n-butyllithium (4.0 mL, 2.5 M solution, 9.95 mmol). After 10 min, a solution of 2a (1.5 g, 6.63 mmol) in THF (10 mL) was added dropwise and after another 10 min 10 mL 2 N H2SO4 was added. The reaction mixture was stirred at 50-60 °C for 30 min, after which the aqueous phase was separated and extracted with EtOAc. The combined organic phases were washed with H2O, brine, dried over Na2SO4, and filtered. After concentration in vacuo, the crude product was purified by column chromatography on silica gel (EtOAc:hexane = 0:20) to give 4a (1.12 g, 55.5 % yield). 1

H NMR (400 MHz, CD3CN) δ 8.47 (ddd, J = 4.8, 1.6, 0.9 Hz, 1H), 7.93 (dt, J = 7.9, 1.0 Hz,

1H), 7.83 (td, J = 7.7, 1.7 Hz, 1H), 7.64 (d, J = 2.1 Hz, 1H), 7.43 - 7.32 (m, 3H), 3.02 - 2.91 (m, 2H), 2.66 - 2.57 (m, 2H), 2.02 - 1.95 (m, 1H), 1.91 - 1.79 (m, 1H). MS (ESI) m/z 307 (M + H)+.

(S)-(1-(3,4-Dichlorophenyl)cyclobutyl)(pyridin-2-yl)methanol (5a) (by chiral separation of racemic mixture (±)-5a). To solution of 4a (1.19 g, 3.9 mmol) in methanol (50 mL) was added NaBH4 (0.45 g, 11.8 mmol) in portions, and the mixture was stirred overnight at room temperature. After removal of the solvent, the pH of the remainder was adjusted to 7-8 by addition of 1 N HCl and then extracted with EtOAc. The organic phase was dried over Na2SO4 and after concentration in vacuo, the residue was purified by column chromatography on silica gel (petroleum ether: EtOAc=10: 1) to give racemic alcohol (±)-5a (0.54 g, 45% yield). MS (ESI) m/z 309 (M + H)+. The latter (88 mg) was separated on chiral column Chiralcel OJ-H (isopropanol : hexane = 2:98) to obtain 5a (34 mg, 35% yield, 98% ee), [α]D = -57.4° (c 0.5, MeOH) and (R)-5a) (40 mg, 41%, 94% ee), [α]D = 51.8° (c 0.5, MeOH). Analytical data for 5a: 1

H NMR (300 MHz, DMSO-d6) δ ppm 8.42 (ddd, J = 4.8, 1.8, 0.9 Hz, 1H), 7.55 (d, J = 7.7, 1.8



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Hz, 1H), 7.36 (d, J = 8.3 Hz, 1H), 7.18 (ddd, J = 7.5, 4.8, 1.2 Hz, 1H), 6.90 (d, J = 2.1 Hz, 1H), 6.77 (d, J = 7.9 Hz, 1H), 6.70 (dd, J = 8.3, 2.1 Hz, 1H), 5.65 (d, J = 4.4 Hz, 1H), 4.92 (d, J = 4.3 Hz, 1H), 2.62-2.82 (m, 2H), 2.07-2.27 (m, 2H), 1.99 (s, 1H), 1.64-1.78 (m, 1H). MS (ESI) m/z 309 (M + H)+. Calculated for C16H15Cl2NO: C 62.35%, H 4.91%, N 4.54%; Found: C 62.51%, H 5.04%, N 4.46%. (S)-(1-(3,4-Dichlorophenyl)cyclobutyl)(pyridin-2-yl)methanol (5a) (by asymmetric synthesis). 4a (3.44 g, 11.23 mmol) and formic acid (1.85 mL, 48.3 mmol) were cooled in an ice bath and triethylamine (3.91 mL, 28.1 mmol) was added. The white slurry was warmed to room temperature and (S,S)-N-(p-touenesulfonyl)-1,2-diphenylethanediamine(chloro)(pcumene)ruthenium (II) (0.072 g, 0.112 mmol) added. The reaction mixture was warmed to 35°C, stirred for 18 h, diluted with dichloromethane and saturated aqueous NaHCO3 and extracted twice with dichloromethane. The organic layers were dried (Na2SO4), filtered, concentrated in vacuo and the residue was chromatographed on silica gel (0-75% EtOAc : hexanes) to give 5a (3.38g, 98 % yield), 96% ee according to chiral HPLC on Chiralcel OJ-H column (2% isopropanol/hexanes). 1H NMR (300 MHz, DMSO-d6) δ 8.42 (ddd, J = 4.8, 1.8, 0.9 Hz, 1H), 7.55 (d, J = 7.7, 1.8 Hz, 1H), 7.36 (d, J = 8.3 Hz, 1H), 7.18 (ddd, J = 7.5, 4.8, 1.2 Hz, 1H), 6.90 (d, J = 2.1 Hz, 1H), 6.77 (d, J = 7.9 Hz, 1H), 6.70 (dd, J = 8.3, 2.1 Hz, 1H), 5.65 (d, J = 4.4 Hz, 1H), 4.92 (d, J = 4.3 Hz, 1H), 2.62-2.82 (m, 2H), 2.07-2.27 (m, 2H), 1.99 (s, 1H), 1.64-1.78 (m, 1H). MS (ESI) m/z 309 (M + H)+.

The following two enantiomerically pure compounds 5b and 5c and one racemic compound (±)5d were prepared by a 3-step procedure described for 5a by replacing 2-(3,4dichlorophenyl)acetonitrile (1a) with the corresponding phenylacetonitriles 1b-d.


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(S)-Pyridin-2-yl(1-(3-(trifluoromethoxy)phenyl)cyclobutyl)methanol (5b). Chiral separation was performed on Chiralpak IA column (EtOH : Hexane =3:97 with 0.1% diethyl amine), >99% ee, [α]D = -0.93 (c 1.0, MeOH). 1H NMR (400 MHz, CDCl3): δ 8.32 (d, J=4Hz, 1H), 7.55 (t, J=8Hz, 1H), 7.14 (t, J=8Hz, 1H), 7.08-7.04 (m, 1H), 6.98 (t, J=8Hz, 1H), 6.88 (d, J=8Hz, 1H), 6.85 (d, J=8Hz, 1H), 4.95 (s, 1H), 4.39 (br, 1H), 2.77-2.66 (m, 2H), 2.35-2.26 (m,2H), 2.14-2.03 (m, 1H), 1.91-1.81 (m,1H). MS (ESI) m/z 324 (M + H)+. HRMS m/z: [M + H]+ calcd for C17H17F3NO2, 324.12059; found, 324.12112.

(S)-Pyridin-2-yl(1-(4-(trifluoromethoxy)phenyl)cyclobutyl)methanol (5c). Chiral separation was performed on Chiralpak IA column (EtOH : Hexane =5:95 with 0.1% diethyl amine), 99% ee, [α]D = -0.97 (c 1.0, MeOH). 1H NMR (400 MHz, CDCl3): δ 8.34 (d, J=8.0 Hz, 1H), 7.47 (t, J=8.0 Hz, 1H), 7.11 (t, J=8.0 Hz, H), 7.00 (d, J=8.0 Hz, 2H), 6.85 (d, J=8.0 Hz, 2H), 6.70 (d, J=8.0 Hz, 1H), 4.90 (s, 1H), 4.44 (br, 1H), 2.73 (m, 2H), 2.29 (m,2H), 2.04 (m, 1H), 1.83 (m,1H). MS (ESI) m/z 324 (M + H)+. Calculated for C17H16F3NO2: C 63.15%, H 4.99%, N 4.33%; Found: C 63.25%, H 4.90%, N 4.30%.

Pyridin-2-yl(1-(2-(trifluoromethoxy)phenyl)cyclobutyl)methanol ((±)-5d). This racemic compound was prepared by a 3-step procedure described for 1C by replacing 2-(3,4dichlorophenyl)acetonitrile with 2-(2-trifluoromethoxyphenyl)acetonitrile. 1H-NMR (400 MHz, CD3OD): δ 8.26 (d, J=4.0Hz, 1H), 7.44-7.40 (m, 1H), 7.12-7.08 (m, 2H), 7.01-6.92 (m, 2H), 6.83 (d, J=7.6Hz, 2H), 7.70 (d, J=7.6Hz, 1H), 5.01 (s, 1H), 2.72-2.61 (m, 2H), 2.31-2.21 (m,



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2H), 1.98-1.96 (m, 1H), 1.73-1.67 (m, 1H). MS (ESI) m/z 324 (M + H)+. HRMS m/z: [M + H]+ calcd for C17H17F3NO2, 324.12059; found, 324.12107.

Compounds (±)-5e-g were prepared by a 2-step procedure described for 4a by replacing 2bromopyridine with fluorinated 2-bromopyriidnes.

(1-(3,4-Dichlorophenyl)cyclobutyl)(3-fluoropyridin-2-yl)methanol ((±)-5e). 1H NMR (300 MHz, DMSO) δ 8.33 (dt, J = 4.6, 1.5, 1H), 7.49 (ddd, J = 10.4, 8.4, 1.3, 1H), 7.39 (d, J = 8.4, 1H), 7.32 (dt, J = 8.5, 4.3, 1H), 7.06 (d, J = 2.2, 1H), 6.86 (dd, J = 8.4, 2.2, 1H), 5.50 (d, J = 6.4, 1H), 4.95 (d, J = 5.7, 1H), 2.93 – 2.78 (m, 1H), 2.78 – 2.63 (m, 1H), 2.24 – 2.10 (m, 2H), 1.91 – 1.61 (m, 2H). MS (ESI) m/z 326 (M+H)+. Calculated for C16H14Cl2FNO: C 58.91%, H 4.33%, N 4.29%; Found: C 58.87%, H 4.10%, N 4.22%.

(1-(3,4-Dichlorophenyl)cyclobutyl)(4-fluoropyridin-2-yl)methanol ((±)-5f). 1H NMR (300 MHz, DMSO) δ 8.42 (d, J = 3.0, 1H), 7.49 (td, J = 8.9, 3.0, 1H), 7.37 (d, J = 8.4, 1H), 6.92 (d, J = 2.1, 1H), 6.78 (dd, J = 8.8, 4.7, 1H), 6.67 (dd, J = 8.3, 2.1, 1H), 5.77 (d, J = 4.3, 1H), 4.94 (d, J = 4.1, 1H), 2.78 – 2.59 (m, 2H), 2.33 – 2.01 (m, 2H), 1.97 – 1.84 (m, 1H), 1.80 – 1.63 (m, 1H). MS (ESI) m/z 326 (M+H)+. Calculated for C16H14Cl2FNO: C 58.91%, H 4.33%, N 4.29%; Found: C 59.17%, H 4.13%, N 4.22%

(1-(3,4-Dichlorophenyl)cyclobutyl)(5-fluoropyridin-2-yl)methanol ((±)-5g). 1H NMR (300


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MHz, DMSO) δ 8.42 (d, J = 3.0, 1H), 7.49 (td, J = 8.9, 3.0, 1H), 7.37 (d, J = 8.4, 1H), 6.92 (d, J = 2.1, 1H), 6.78 (dd, J = 8.8, 4.7, 1H), 6.67 (dd, J = 8.3, 2.1, 1H), 5.77 (d, J = 4.2, 1H), 4.94 (d, J = 3.9, 1H), 2.78 – 2.60 (m, 2H), 2.24 – 2.04 (m, 2H), 1.97 – 1.84 (m, 1H), 1.80 – 1.64 (m, 1H). MS (ESI) m/z 326 (M+H)+. Calculated for C16H14Cl2FNO: C 58.91%, H 4.33%, N 4.29%; Found: C 58.80%, H 4.19%, N 4.27%

(S)-(1-(3,4-dichlorophenyl)cyclobutyl)(pyrimidin-2-yl)methanol (5h). Racemic (±)-5h was prepared by a 2-step procedure described for (±)-5i by replacing 2b with 2a. 1H NMR (300 MHz, DMSO-d6) δ 8.66-8.69 (m, 2H), 7.33-7.37 (m, 2H), 6.98 (d, J = 2.1 Hz, 1H), 6.74 (dd, J = 8.3, 2.1 Hz, 1H), 5.17-5.20 (m, 1H), 4.88-4.91 (m, 1H), 2.68-2.93 (m, 2H), 2.15-2.28 (m, 2H), 1.66-1.92 (m, 2H). MS (DCI) m/z 309 (M+H)+. Chiral separation of using supercritical fluid chromatography (SFC) with Chiralcel AD column (5% MeOH/CO2 with 0.1% diethylamine) gave 5h, [α]D=-23.2° (c=0.42 CH3OH). 1H NMR (300 MHz, DMSO-d6) δ 8.66-8.69 (m, 2H), 7.33-7.37 (m, 2H), 6.97 (d, J = 2.1 Hz, 1H), 6.74 (dd, J = 8.3, 2.1 Hz, 1H), 5.18 (d, J = 6.3 Hz, 1H), 4.91 (s, 1H), 2.74-2.92 (m, 2H), 2.15-2.28 (m, 2H), 1.79-1.92 (m, 1H), 1.64-1.79 (m, 1H). MS (DCI) m/z 309 (M+H)+. Also was isolated (R)-5h, [α]D=+27.5° (c=0.46 CH3OH). 1H NMR (300 MHz, DMSO-d6) δ 8.87 (d, J = 4.8 Hz, 1H), 7.55-7.57 (m, 2H), 7.29 (dd, J = 8.4, 2.2 Hz, 1H), 2.89-2.97 (m, 1H), 2.54-2.74 (m, 2H), 1.23-2.00 (m, 5H). MS (DCI) m/z 309 (M+H)+. HRMS m/z: [M + H]+ calcd for C15H15Cl2N2O, 309.05559; found, 309.05589.



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Pyrimidin-2-yl(1-(4-(trifluoromethoxy)phenyl)cyclobutyl)methanol ((±)-5i). To a solution of n-butyllithium (0.48 mL, 1.2 mmol, 2.5 M in hexanes) was added 2-(tributylstannyl)pyrimidine (0.37 g, 1.0 mmol) in THF (6.0 mL) under nitrogen atmosphere at -95°C to -100°C. After 45 min, 64A (0.24 g, 1.0 mmol) was added at -95oC, and the resulting mixture was stirred for an additional 30 min and then warmed to room temperature for 10 min. Saturated aq. NH4Cl was added and the mixture was extracted with CH2Cl2 (30 mL), concentrated and purified by PrepTLC (petroleum ether:EtOAc = 15:1 to 10:1) to give (±)-5i (0.03 g, yield 9.3%). 1H NMR (400 MHz, CDCl3): δ 8.58 (d, J=8Hz, 2H), 7.17 (t, J=4Hz, 1H), 6.95 (d, J=8Hz, 2H), 6.81 (d, J=8Hz, 2H), 5.24 (s, 1H), 3.53 (br, 1H), 3.05-2.97 (m, 1H), 2.86-2.79 (m, 1H), 2.46-2.35 (m,2H), 2.232.11 (m, 1H), 1.96-1.86 (m,1H). MS (ESI) m/z 325 (M + H)+. HRMS m/z: [M + H]+ calcd for C16H16F3N2O2, 325.11584; found, 325.11627.

1-(1-(3,4-Dichlorophenyl)cyclobutyl)-1-(pyridin-2-yl)ethanol (5j). To the solution of 4a (0.014 g, 0.046 mmol) in THF (1 mL) was added methylmagnesium bromide (0.023 mL, 0.069 mmol) and the mixture stirred at room temperature for 0.5 h. Reaction mixture was diluted with MTBE, washed with saturated aqueous NH4Cl, dried (Na2SO4), concentrated under reduced pressure and chromatographed on silica gel (EtOAc:hexanes = 0:100 to 1:4)) to give 5j (0.01 g, 68 % yield). 1H NMR (300 MHz, DMSO-d6) δ 8.40 (ddd, J = 4.8, 1.8, 0.9 Hz, 1H), 7.62 (m, 1H), 7.38-7.17 (m, 3H), 6.85 (d, J = 2.1 Hz, 1H), 6.77 (m, 1H), 5.39 (s, 1H), 2.86 (m, 2H), 2.382.00 (m, 2H), 1.58 (m, 2H), 1.50 (s, 3H). MS (DCI m/z 322 (M+H)+.


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(1S,3R)-1-(Pyridin-2-yl)-3-(4-(trifluoromethyl)phenyl)cyclopentanol (7) and (1R,3R)-1(pyridin-2-yl)-3-(4-(trifluoromethyl)phenyl)cyclopentanol (8). To a solution of 2bromopyridine (2.57 mL, 26.95 mmol) in anhydrous THF (50 mL) at -78°C was added n- BuLi in hexane (11.0 mL, 2.5 M, 27.5 mmol). After 0.5 h, a solution of (R)-3-(4(trifluoromethyl)phenyl)cyclopentanone (6a) (5.48 g, 24.0 mmol) in THF (25 mL) was slowly added over 15 min. The reaction mixture was allowed to warm to 0°C over 1.5 h and was then stirred at room temperature for 1 h before being quenched with 1 M aqueous citric acid (10 mL). The aqueous phase was removed and the organic phase was washed with brine, dried (Na2SO4), concentrated under reduced pressure and chromatographed on silica gel (1 to 5% MeCN in 1:1 CH2Cl2 / heptane) to give 7 (2.75 g, 37% yield) and 8 (2.21 g, 30% yield). Analytical data for 7: [α]D = -72.3° (c=1.0, MeOH). 1H NMR (300 MHz, CDCl3) δ 8.56 - 8.52 (m, 1H), 7.74 (ddd, J = 8.0, 7.5, 1.6 Hz, 1H), 7.57 (d, J = 8.6 Hz, 2H), 7.53 (d, J = 8.6 Hz, 2H), 7.46 (d, J = 8.0 Hz, 1H), 7.25 - 7.19 (m, 1H), 5.16 (bs, 1H), 3.52 - 3.38 (m, 1H), 2.67 (dd, J = 14.3, 10.1 Hz, 1H), 2.37 - 2.27 (m, 1H), 2.27 - 2.09 (m, 4H). MS (ESI) m/z 308 (M+H)+. HRMS m/z: [M + H]+ calcd for C17H17F3NO, 308.12568; found, 308.12606. Analytical data for 8: [α]D = -12.8° (c=1.0, MeOH). 1H NMR (300 MHz, CDCl3) δ 8.54 (ddd, J = 4.9, 1.7, 1.0 Hz, 1H), 7.74 (ddd, J = 7.9, 7.5, 1.8 Hz 1H), 7.56 (d, J = 8.5 Hz, 2H), 7.48 - 7.40 (m, 3H), 7.22 (ddd, J = 7.5, 4.9, 1.1 Hz, 1H), 5.22 (bs, 1H), 3.83 - 3.70 (m, 1H), 2.60 - 2.46 (m, 1H), 2.46 - 2.28 (m, 2H), 2.23 - 2.07 (m, 2H), 2.03 - 1.89 (m, 1H). MS (ESI) m/z 308 (M+H)+. HRMS m/z: [M + H]+ calcd for C17H17F3NO, 308.12568; found, 308.12598.



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(1R,3S)-1-(pyridin-2-yl)-3-(4-(trifluoromethyl)phenyl)cyclopentanol (9) and (1S,3S)-1(pyridin-2-yl)-3-(4-(trifluoromethyl)phenyl)cyclopentanol (10). These compounds were prepared according to the procedure described for the synthesis of 7 and 8 replacing (R)-3-(4(trifluoromethyl)phenyl)cyclopentanone (6a) with (S)-3(4(trifluoromethyl)phenyl)cyclopentanone (6b). Analytical data for 9: [a]D =+77.1 o (c 1.50, MeOH). 1H NMR: (400 MHz, CDCl3): δ 8.55-8.54 (m, 1H), 7.76-7.72 (m, 1H), 7.58-7.52 (m, 4H), 7.46 (d, J = 8.0 Hz, 1H), 7.24-7.21 (m, 1H), 5.19 (broad s, 1H), 3.49-3.40 (m, 1H), 2.702.64 (m, 1H), 2.35-2.29 (m, 1H), 2.27-2.12 (m, 4H). MS (ESI) m/z 308 (M+H)+. HRMS m/z: [M + H]+ calcd for C17H17F3NO, 308.12568; found, 308.12606. Analytical data for 10: [α]D =+28.0 o (c 0.75, MeOH). 1H NMR: (400 MHz, CDCl3): δ 8.55-8.54 (m, 1H), 7.76-7.72 (m, 1H), 7.56 (d, J = 8.4 Hz, 2H), 7.47-7.41 (m, 3H), 7.24-7.21 (m, 1H), 5.25 (broad s, 1H), 3.81-3.72 (m, 1H), 2.57-2.49 (m, 1H), 2.44-2.30 (m, 2H), 2.22-2.09 (m, 2H), 2.01-1.91 (m, 1H). MS (ESI) m/z 308 (M+H)+. HRMS m/z: [M + H]+ calcd for C17H17F3NO, 308.12568; found, 308.12591.

2-(4-(Trifluoromethoxy)phenyl)pent-4-yn-2-ol (12). To a solution of n-BuLi (21.60 mL, 1.6 M, 34.6 mmol) in diethyl ether (20 mL) at 98% ee by chiral SFC on ChiralPak AD-H)). 1H NMR (500 MHz, CD3CN) δ 7.58 (d, J = 8.8 Hz, 2H), 7.26 (d, J = 8.2 Hz, 2H), 3.45 (s, 1H), 2.65 (dd, J = 2.5, 1.1 Hz, 2H), 2.18 (t, J = 2.7 Hz, 1H), 1.57 (s, 3H).

5-methyl-5-(4-(trifluoromethoxy)phenyl)dihydrofuran-3(2H)-one (13). To a solution of 12 (1.06 g, 4.34 mmol) in CH2Cl2 (45 mL) at 0°C, 3,5-dichloropyridine 1-oxide (1.42 g, 8.68 mmol) was added, followed by a solution of methanesulfonic acid (0.51 mL, 7.81 mmol)) in CH2Cl2 (5 mL). Then triphenylphosphinegold(I) bis(trifluoromethanesulfonyl)imidate (0.064 g, 0.087 mmol) was added, and the yellow solution was stirred at 0°C for 3h and at room temperature for 16 h. Reaction mixture was diluted with H2O (50 mL) and CH2Cl2 (50 mL), organic layer was separated, washed with brine (10 mL), dried (Na2SO4), concentrated under reduced pressure, and chromatographed the residue on silica gel (0-25% EtOAc/heptanes) to obtain (0.6 g, 52.8% yield). 1H NMR (400 MHz, DMSO) δ 7.58 (d, J = 8.7 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H), 4.18 (d, J = 17.2 Hz, 1H), 3.94 (d, J = 17.2 Hz, 1H), 2.88 (s, 2H), 1.58 (s, 3H).



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Trans-(3R,5S)-5-methyl-3-(pyridin-2-yl)-5-(4-(trifluoromethoxy)phenyl)tetrahydrofuran-3ol (trans-14) and cis-(3S,5S)-5-methyl-3-(pyridin-2-yl)-5-(4-(trifluoromethoxy)phenyl) tetrahydrofuran-3-ol (cis-14). A solution of 52C 13 (0.59 g, 2.26 mmol) and lanthanum trichloride lithium chloride complex (3.77 mL, 2.26 mmol) stirred at room temperature for 1 h was added to a -70°C solution of pyridyl lithium prepared by addition of a solution of 2bromopyridine (0.33 mL, 3.40 mmol) in THF (6 mL) to n-BuLi (1.36 mL, 2.5 M, 3.40 mmol) After stirring 1 h, AcOH (0.26 mL, 4.53 mmol) was added dropwise at -70°C, then reaction mixture was allowed to warm to room temperature. Reaction mixture was diluted with MTBE (50 mL) and H2O (10 mL), layers were separated, and the organic layer was washed with brine (10 mL), then dried (Na2SO4), concentrated under reduced pressure and chromatographed on silica gel (0-10% MTBE/DCM) to obtain trans-14 (0.43 g, 55.3 % yield) and cis-14 (0.21 g, 27.1 % yield). Relative stereochemistry was determined by 2D NMR. Analytical data for trans14 : 1H NMR (400 MHz, CDCl3) δ 8.52 (ddd, J = 4.9, 1.7, 0.9 Hz, 1H), 7.77 (ddd, J = 8.0, 7.5, 1.8 Hz, 1H), 7.60 (dt, J = 8.0, 1.1 Hz, 1H), 7.57 – 7.53 (m, 2H), 7.23 (dddd, J = 8.9, 7.8, 2.4, 1.1 Hz, 3H), 4.79 (s, 1H), 4.27 (d, J = 9.6 Hz, 1H), 4.05 (dd, J = 9.6, 0.8 Hz, 1H), 2.78 (dd, J = 13.6, 0.8 Hz, 1H), 2.64 (d, J = 13.5 Hz, 1H), 1.65 (s, 3H). MS (ESI) m/z 340 (M+H)+. Analytical data for cis-14: 1H NMR (400 MHz, CDCl3) δ 8.50 (ddd, J = 4.9, 1.7, 1.0 Hz, 1H), 7.64 (td, J = 7.7, 1.7 Hz, 1H), 7.51 – 7.44 (m, 2H), 7.24 – 7.16 (m, 4H), 5.59 (s, 1H), 4.20 (d, J = 9.5 Hz, 1H), 4.08 (d, J = 9.6 Hz, 1H), 2.66 (s, 2H), 1.77 (s, 3H). MS (ESI) m/z 340 (M+H)+.


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(3R,5S)-5-Methyl-3-(pyridin-2-yl)-5-(4-(trifluoromethoxy)phenyl)tetrahydrofuran-3-ol (15) and (3S,5R)-5-methyl-3-(pyridin-2-yl)-5-(4-(trifluoromethoxy)phenyl)tetrahydrofuran-3-ol (16). Racemic mixture of trans-14 (0.42 g, 1.24 mmol) was subjected to chiral separation by SFC on ChiralPak IA column (5-50% MeOH :CO2) to obtain 15 (0.14 g, 33%, >99% ee) and 16 (0.18 g, 42%, 98.5% ee). Analytical data for 15 : 1H NMR (400 MHz, CDCl3) δ 8.52 (ddd, J = 4.9, 1.7, 0.9 Hz, 1H), 7.77 (ddd, J = 8.0, 7.5, 1.8 Hz, 1H), 7.60 (dt, J = 8.0, 1.1 Hz, 1H), 7.57 – 7.53 (m, 2H), 7.23 (dddd, J = 8.9, 7.8, 2.4, 1.1 Hz, 3H), 4.79 (s, 1H), 4.27 (d, J = 9.6 Hz, 1H), 4.05 (dd, J = 9.6, 0.8 Hz, 1H), 2.78 (dd, J = 13.6, 0.8 Hz, 1H), 2.64 (d, J = 13.5 Hz, 1H), 1.65 (s, 3H). MS (ESI) m/z 340 (M+H)+. HRMS m/z: [M + H]+ calcd for C17H17F3NO3, 340.11550; found, 340.11518. Analytical data for 16: 1H NMR (400 MHz, CDCl3) δ 8.52 (ddd, J = 4.9, 1.7, 0.9 Hz, 1H), 7.77 (ddd, J = 8.0, 7.5, 1.8 Hz, 1H), 7.60 (dt, J = 8.0, 1.1 Hz, 1H), 7.57 – 7.53 (m, 2H), 7.23 (dddd, J = 8.9, 7.8, 2.4, 1.1 Hz, 3H), 4.79 (s, 1H), 4.27 (d, J = 9.6 Hz, 1H), 4.05 (dd, J = 9.6, 0.8 Hz, 1H), 2.78 (dd, J = 13.6, 0.8 Hz, 1H), 2.64 (d, J = 13.5 Hz, 1H), 1.65 (s, 3H). MS (ESI) m/z 340 (M+H)+. HRMS m/z: [M + H]+ calcd for C17H17F3NO3, 340.11550; found, 340.11614.

(3S,5S)-5-methyl-3-(pyridin-2-yl)-5-(4-(trifluoromethoxy)phenyl)tetrahydrofuran-3-ol (17) and (3R,5R)-5-methyl-3-(pyridin-2-yl)-5-(4-(trifluoromethoxy)phenyl)tetrahydrofuran-3-ol (18). Racemic mixture of cis-14 (0.20 g, 0.59 mmol) was subjected to chiral separation by SFC on ChiralPak IA column (5-50% MeOH:CO2) to obtain 17 (0.061 g, 31.5%, 99% ee) and 18 (0.068 g, 34%, 97% ee). Analytical data for 17 : 1H NMR (400 MHz, CDCl3) δ 8.50 (ddd, J = 4.9, 1.7, 1.0 Hz, 1H), 7.64 (td, J = 7.7, 1.7 Hz, 1H), 7.51 – 7.44 (m, 2H), 7.24 – 7.16 (m, 4H), 5.59 (s, 1H), 4.20 (d, J = 9.5 Hz, 1H), 4.08 (d, J = 9.6 Hz, 1H), 2.66 (s, 2H), 1.77 (s, 3H). MS



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(ESI) m/z 340 (M+H)+. HRMS m/z: [M + H]+ calcd for C17H17F3NO3, 340.11550; found, 340.11598. Analytical data for 18 : 1H NMR (400 MHz, CDCl3) δ 8.50 (ddd, J = 4.9, 1.7, 1.0 Hz, 1H), 7.64 (td, J = 7.7, 1.7 Hz, 1H), 7.51 – 7.44 (m, 2H), 7.24 – 7.16 (m, 4H), 5.59 (s, 1H), 4.20 (d, J = 9.5 Hz, 1H), 4.08 (d, J = 9.6 Hz, 1H), 2.66 (s, 2H), 1.77 (s, 3H). MS (ESI) m/z 340 (M+H)+. HRMS m/z: [M + H]+ calcd for C17H17F3NO3, 340.11550; found, 340.11614.

2-(3,4-Dichlorophenyl)acetaldehyde (20). The mixture of 2-(3-chlorophenyl)ethanol (19) (6.9 g, 36.5 mmol) and Dess-Martin periodinane (18.6 g, 43.8 mmol) in 200 mL of CH2Cl2 was stirred under nitrogen atmosphere for 4 h at room temperature. Then saturated NaHCO3 (500 mL) and Na2S2O3 (100 mL) was added and the stirring continued for another 30 min. The resulting mixture was extracted by CH2Cl2 (3 × 300 mL), the organic layers were combined, dried over Na2SO4 and concentrated to obtain crude 20 (4.18 g), which was used directly in the next step.

2-(3,4-Dichlorophenyl)-2-(hydroxymethyl)propane-1,3-diol (21). A solution 20 (4.18 g, 22.09 mmol), paraformaldehyde (5.3 g, 176.7 mmol) and Ca(OH)2 (16.3 g, 220.9 mmol) in THF (200 mL) was stirred at 60 oC for 5 days. After cooling to room temperature, the mixture was filtered through a celite pad and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel (100% EtOAc) to obtain crude 21 (1.28 g), which was used directly in the next step.


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(3-(3,4-Dichlorophenyl)oxetan-3-yl)methanol (22). A mixture of 21 (1.28 g, 5.10 mmol), diethyl carbonate (722 mg, 6.10 mmol) and KOH (15 mg) was heated to 80 oC allowing the triol to melt and the mixture to become homogeneous. The mixture was then heated at 95 oC for 4 h, during which time EtOH distilled off from the mixture. Distillation was continued until the pot temperature was 190 oC, and then the pressure was reduced to 50 mm, maintaining the pot temperature at 190oC for 1 h. The residue was purified by preparative TLC on silica gel (with Petroleum ether:EtOAc=1:1) to obtain 22 (0.6 g), which was used directly in the next step.

3-(3,4-Dichlorophenyl)oxetane-3-carbaldehyde (23). The mixture of 22 (0.6 g, 2.58 mmol) and Dess-Martin periodinane (1.3 g, 3.09 mmol) in 30 mL of CH2Cl2 was stirred under nitrogen atmosphere for 4h at room temperature. Then saturated NaHCO3 (120 mL) and Na2S2O3 (30 mL) was added with stirring for another 30 min. The mixture was extracted by CH2Cl2 (3 × 100 mL), organic layers were combined, dried over Na2SO4 and concentrated to give crude 23 (0.6 g), which was directly used in the next step without further purification.

(3-(3,4-Dichlorophenyl)oxetan-3-yl)(pyridin-2-yl)methanol (24). To a mixture of 2bromopyridine (0.49 g, 3.1 mmol) in 8 mL of THF was added n-BuLi (3.35 mmol, 1.6 M, 2.1 mL) dropwise at -78 oC under argon atmosphere. After stirring for 20 min at the same temperature, 23 (0.6 g, 2.58 mmol) in 4 mL of THF was added dropwise at -78 oC. The resulting mixture was stirred for another 1 h, after which EtOH (15 mL) was added, the solvent was concentrated and the residue purified by preparative TLC on silica gel (petroleum ether:EtOAc=1:1.5) to give 24 (0.37 g, 46% yield). 1HNMR (400 MHz, CD3OD): δ 4.72-4.80



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(m, 2H) ,5.13 (d, J = 6.0 Hz, 1 H), 5.23 (t, J = 8.8 Hz, 2H), 6.56 (m, 1H), 6.78-6.85 (m, 2H), 7.10-7.20 (m, 2H), 7.47-7.51 (m, 1H), 8.31(d, J = 7.8 Hz, 1H). MS (ESI) m/z 310 (M+H)+. HRMS m/z: [M + H]+ calcd for C15H14Cl2NO2, 310.03961; found, 310.04003.

tert-Butyl 3-cyano-3-(4-(trifluoromethyl)phenyl)azetidine-1-carboxylate (26). To a solution of 1-fluoro-4-(trifluoromethyl)benzene (25) (1.64 g, 10mmol) in toluene (25 mL) was added tertbutyl 3-cyanoazetidine-1-carboxylate (1.8 g, 10 mmol) and KHMDS (0.5 M in toluene) (2.99 g, 0.5 M, 15.00 mmol). The reaction mixture was stirred at 60°C for 16 h and cooled to room temperature. After the addition of 1 N HCl (25 mL), the reaction mixture was extracted with EtOAc (3 × 20mL)m the combined organic extracts were washed with brine (100 mL), dried over MgSO4, filtered and concentrated. The residue was purified by preparative TLC on silica gel (EtOAc: petroleum ether = 1 : 5) to give 26 (1.03 g, 32% yield). MS (ESI) m/z 327 (M+H)+.

tert-Butyl 3-picolinoyl-3-(4-(trifluoromethyl)phenyl)azetidine-1-carboxylate (27). To a solution of 2-bromopyridine (0.768 g, 4.86 mmol) in dry THF (20 mL) was added n-butyllithium (2.5 M in hexane, 0.6 mL, 4.86 mmol) at -78oC. After stirring for 30 min, the solution of 26 (1.0 g, 3.24 mmol) in THF (5 mL) was added. The mixture was stirred at -78oC for 30 min, diluted with sat NH4Cl (2 × 20 mL) and extracted with EtOAc (2 × 20mL). The combined organic layers were dried over Na2SO4, filtered and concentrated. The resulting residue was purified by column chromatography on silica gel (petroleum ether/EtOAc =10/1) to give 27 (0.45 g, 34% yield). MS (ESI) m/z 407 (M+H)+.


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tert-Butyl 3-(hydroxy(pyridin-2-yl)methyl)-3-(4-(trifluoromethyl)phenyl)azetidine-1carboxylate (28). To a solution of 27 (500 mg, 1.11 mmol) in dry MeOH (5 mL) was added NaBH4 (84 mg, 2.22 mmol) at 0oC. The mixture was stirred at 25oC for 1 h and H2O (10 mL) was added slowly. The aqueous phase was separated and extracted with CH2Cl2 (2 × 20mL). The combined organic phases were washed with H2O, brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by column chromatography on silica gel (petroleum ether/EtOAc =1/1) to give 28 (0.35 g, 78% yield). 1H NMR: (400 MHz, CD3OD): δ 8.31 (s, 1H), 7.41-7.37 (m, 3H), 7.10-7.07 (m, 1H), 6.81 (d, J = 8.0 Hz, 2H), 6.60 (s, 1H), 4.94 (s, 1H), 4.55-4.45 (m, 3H), 4.06-4.02 (m, 2H), 1.36 (s, 9H). MS (ESI) m/z 409 (M+H)+.

Pyridin-2-yl(3-(4-(trifluoromethyl)phenyl)azetidin-3-yl)methanol (29a). To a solution of 28 (390 mg, 0.87 mmol) in MeOH (2 mL) was added HCl\ MeOH (2 M, 2 mL, 4 mmol) at room temperature. The reaction mixture was stirred at ambient temperature for 1 h, solvent was removed under reduced pressure and the resulting residue was partitioned between EtOAc (20 mL) and aq. sat NaHCO3 (20 mL). The organic layer was separated, dried over Na2SO4, filtered, concentrated, and the residue was purified by column chromatography on silica gel (petroleum ether/EtOAc =1/1) to give 29a (0.08 g, 32% yield). 1H NMR: (400 MHz, CD3OD): δ 8.49 (s, 1H), 7.94 (s, 1H), 7.53 (m, 3H), 7.05-7.15 (m, 3H), 5.35 (s, 1H), 4.70-4.75 (m, 2H), 4.19-4.27 (m, 2H). MS (ESI) m/z 309 (M+H)+. HRMS m/z: [M + H]+ calcd for C16H16F3N2O, 309.12092; found, 309.12135.



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(1-Methyl-3-(4-(trifluoromethyl)phenyl)azetidin-3-yl)(pyridin-2-yl)methanol (29b). The mixture of 29a (0.14 g, 0.440 mmol), formaldehyde (0.013 mL, 0.484 mmol) and formic acid (0.042 mL, 1.10 mmol) were heated at 90 °C for 18 h. After addition of concentrated HCl (1 mL), the volatiles were removed under reduced pressure and the resulting residue was dissolved in H2O (5 mL). The aqueous solution was adjusted to pH = 8 with 30% NaOH and extracted with Et2O (3 × 10 mL). The combined organic phases were dried (Na2SO4), filtered, concentrated, and the residue was purified by column chromatography on silica gel (petroleum ether/EtOAc =1/1) to give 29b (0.018 g, 13% yield). 1H NMR: (400 MHz, CDCl3): δ 8.30 (d, J = 4.8 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.38 (d, J = 8.0 Hz, 2H), 7.11 (d, J = 4.2 Hz, 1H), 6.92 (d, J = 7.6 Hz, 1H), 6.79 (d, J = 8 Hz, 2H), 5.18 (s, 1H), 4.09-4.01 (m, 2H), 3.38-3.32 (m, 2H), 2.37 (s, 3H). MS (ESI) m/z 323 (M+H)+. HRMS m/z: [M + H]+ calcd for C17H18F3N2O, 323.13657; found, 323.13664.

Ethyl 2-(3,4-dichlorophenyl)-2-hydroxypent-4-enoate (31). To the solution of ethyl 2-(3,4dichlorophenyl)-2-oxoacetate (30) (100 g, 404 mmol) in CH2C12 at 0 °C was added allyltributyltin (126 g, 485 mmol) and TiC14 neat (92.2 g, 485 mmol) and the mixture was stirred for 40 h from 0°C to room temperature. Purification by chromatography on silica gel provided 31 (90 g, 77%). MS (ESI) m/z 290 (M+H)+.

Ethyl 2-(3,4-dichlorophenyl)-2-hydroxy-4-oxobutanoate (32). Ozonolysis of 31 (25 g, 86.5 mmol) in CH2C12 at -78° C. for 4 h followed by treatment with DMSO and CH2C12 at -78°C to room temperature for 9 h provided 32 (33.0 g) that was used in the next step without further purification.


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Ethyl 2-(3,4-dichlorophenyl)-2,4-dihydroxybutanoate (33). A solution of 32 (33.0 g, 113 mmol) in MeCN and acetic acid with tetramethylammonium triacetoxyborohydride at 40° C for 40 h provided 33 (24.0 g) that was used in the next step without further purification.

Ethyl 2-(3,4-dichlorophenyl)-2-hydroxy-4-(tosyloxy)butanoate (34). To the solution 33 (8.8 g, 30 mmol) in CH2C12 ( 300 mL) was added tosyl chloride (11.46 g, 60 mmol)) and 1,8diazabicyclo[5.4.0]undec-7-ene (11.4 g, 75 mmol) and the mixture was stirred at room temperature for 16 h. Purification by column chromatography on silica gel provided 34 (3.3 g, 25% for 3 steps).

Ethyl 2-(3,4-dichlorophenyl)oxetane-2-carboxylate (35). Treatment of 34 (5.4 g, 12 mmol) in THF with potassium tert-butoxide and 18-crown-6 at room temperature for 2 h provided 35 (2.1 g) that was used in the next step without further purification.

(2-(3,4-Dichlorophenyl)oxetan-2-yl)(pyridin-2-yl)methanone (36). 2.5 M n-Butyllithium (8.72 mL, 21.81 mmol) in hexanes was added to diethyl ether (30 mL) and cooled to -78ºC. 2Bromopyridine (2.22 mL, 22.7 mmol) was added and after 30 min 35 (5.0 g, 18.2 mmol) in diethyl ether (25 mL) was added dropwise. The reaction was warmed to 0ºC over a period of 2.5 h, and then quenched with saturated NH4Cl solution. The mixture was extracted twice with diethyl ether, and the combined organic layers washed with brine, dried with MgSO4, and concentrated. The residue was chromatographed on silica gel column (20% EtOAc:hexanes) to give 36 (3.66 g, 65.4% yield). 1H NMR (300 MHz, d6-DMSO) δ 8.66 – 8.60 (m, 1H), 8.01 –



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7.96 (m, 2H), 7.79 (d, J = 2.1 Hz, 1H), 7.66 – 7.55 (m, 2H), 7.47 (dd, J = 8.4, 2.1 Hz, 1H), 4.57 – 4.39 (m, 2H), 3.55 – 3.42 (m, 1H), 3.26 – 3.14 (m, 1H). MS (DCI) m/z 308 (M+H)+.

(R)-((S)-2-(3,4-Dichlorophenyl)oxetan-2-yl)(pyridin-2-yl)methanol (37a) and (R)-((R)-2-(3,4dichlorophenyl)oxetan-2-yl)(pyridin-2-yl)methanol (37b). To a solution of 36 (3.66 g, 11.9 mmol) in triethylamine (4.14 mL, 29.7 mmol) and formic acid (1.96 mL, 51.1 mmol) was added (S,S)-N-(p-toluenesulfonyl)-1,2-diphenylethanediamine(chloro)(p-cymene)ruthenium(II) (0.076 g, 0.119 mmol) and the reaction mixture heated at 35ºC for 16 h. The reaction was cooled to room temperature, diluted with CH2C12 and washed with saturated NaHCO3 solution. The organic layer was dried with MgSO4 and concentrated. The residue was chromatographed on silica gel column (35% EtOAc:hexanes) to give 37a (1.19 g, 32.3% yield) and 37b (1.46 g, 40% yield) which were dissolved in 2 N hydrogen chloride in methanol and concentrated to give 37a HCl salt (1.14 g, 85.7% yield) and 37b HCl salt (0.76 g, 47% yield). Analytical data for 37a HCl salt: 1H NMR (300 MHz, d6-DMSO) δ 8.67 (d, J = 5.1 Hz, 1H), 8.27 (t, J = 7.3 Hz, 1H), 7.78 (t, J = 6.1 Hz, 1H), 7.57 (s, 1H), 7.54 (s, 1H), 7.35 (d, J = 2.0 Hz, 1H), 7.05 (dd, J = 8.3, 2.0 Hz, 1H), 5.21 (s, 1H), 4.42 – 4.27 (m, 2H), 3.40 – 3.27 (m, 1H), 2.69 – 2.57 (m, 1H). MS (DCI) m/z 310 (M+H)+. [〈]D = +43.7º (c 0.60, MeOH). Calculated for C15H13Cl2NO2 •HCl: C 51.97%, H 4.07%, N 4.04%; Found: C 51.98%, H 3.82%, N 3.94%. Analytical data for 37b HCl salt: 1H NMR (300 MHz, d6-DMSO) δ 8.63 (d, J = 5.4 Hz, 1H), 8.37 (t, J = 7.5 Hz, 1H), 7.82 (t, J = 6.4 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.51 (d, J = 8.3 Hz, 1H), 7.24 (d, J = 2.0 Hz, 1H), 6.95 (dd, J = 8.4, 2.0 Hz, 1H), 5.51 (s, 1H), 4.55 – 4.33 (m, 2H), 3.22 – 3.10 (m, 1H), 2.78 – 2.66 (m, 1H). MS (DCI) m/z 310 (M+H)+. [α]D = -22.3º (c 0.705, MeOH). Calculated for C15H13Cl2NO2 •HCl: C 51.97%, H 4.07%, N 4.04%; Found: C 52.02%, H 3.87%, N 4.11%.


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3-(3,4-Dichlorophenyl)tetrahydrofuran-3-carbonitrile (38). To a suspension of NaH (60% by weight, 1.2 g, 30 mmol) in 1-methylpyrrolidin-2-one (20 mL) was added a solution of 2-(3,4dichlorophenyl)acetonitrile (1a) (1.86 g, 10 mmol) and 1-chloro-2-(chloromethoxy)ethane (1.29 g, 10 mmol) in THF (10 mL) at -20 ºC. The mixture was allowed to warm to room temperature after completion of addition and stirred for 24 h. The reaction was quenched by ice water and extracted with EtOAc (3 ×30 mL). The combined organic layers were washed with water (3×50 mL), brine (50 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by preparative TLC on silica gel (EtOAc / hexane = 1:5) to give 38 (0.41 g, 17.4% yield). MS (DCI) m/z 215 (M-CN)+.

(3-(3,4-Dichlorophenyl)tetrahydrofuran-3-yl)(pyridin-2-yl)methanone (39). To a solution of 2-bromopyridine (0.412 g, 2.6 mmol) in dry THF was added n-BuLi (1.05 mL, 2.5 M solution in n-hexane) at -78°C. After stirring for 15min, the solution of 38 (0.42 g, 1.74 mmol) in THF (2 mL) was added. The mixture was stirred at -78°C for 15min, then 5 mL of 1 M H2SO4 solution was added slowly. The mixture was heated to 50°C-60°C for 30min. The aqueous phase was separated and extracted with EtOAc (3×50 mL). The combined organic phase was washed with water (2 × 50 mL), brine (50 mL), dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether: EtOAc=3:1) to give 39 (0.073 g, 13.4 % yield). MS (DCI) m/z 323 (M+H)+.



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(3-(3,4-Dichlorophenyl)tetrahydrofuran-3-yl)(pyridin-2-yl)methanol (40). To a solution of 39 (0.074 g, 0.23 mmol) in methanol (25 mL) was added NaBH4 (0.027 g, 0.70 mmol) in portions, and the mixture was stirred overnight at room temperature. After removal of the solvent under reduced pressure, the pH of solution was adjusted to 7-8 by addition of 1 N HCl and then extracted with EtOAc (3×50 mL). The combined organic phase was dried over Na2SO4, filtered and concentrated to afford the crude product which was purified by preparative TLC on silica gel (petroleum ether: EtOAc=1:1) to give 40 (0.034 g, 46% yield). 1H-NMR(400 MHz, CDCl3): δ 8.39 (d, J=4.0Hz, 1H), 7.62-7.54 (m, 1H), 7.27-7.15 (m, 2H), 6.98-6.93 (m, 2H), 6.85-6.65 (m, 1H), 4.87-4.85 (m, 1H), 4.52-4.38 (m, 1H), 4.11-4.01 (m, 1H), 3.93-3.80 (m, 2H), 2.87-2.58 (m, 1H), 2.18-2.08 (m, 1H). MS (DCI) m/z 324 (M+H)+.

Cyclopropyl(3,4-dichlorophenyl)methanone (42). To a solution of 3,4-dichloro-N-methoxy-Nmethylbenzamide (41) (3.74 g, 16 mmol) in THF (50 mL) was added cyclopropanecarbonylmagnesium bromide (2 N in THF, 20 mL, 40 mmol) dropwise at 0°C. Reaction mixture was stirred for 4 h at room temperature and quenched with 30 mL of aq. NH4Cl. The aqueous phase was separated and extracted with EtOAc (50 mL×2). The combined organic phase was washed with water (50 mL), brine (50 mL), dried over Na2SO4 and concentrated to give crude 42 (3.4 g), which was used in the next step without further purification. MS (DCI) m/z 215 (M+H)+.

4-Chloro-1-(3,4-dichlorophenyl)butan-1-one (43). A solution of 42 (3.63 g, 16.9 mmol), TsOH.H2O (3.2 g, 16.9 mmol) and pyridine hydrochloride (3.88 g, 33.8 mmol) in CH3CN (60


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mL) was heated to 160°C for 3 h under microwave irradiation. Volatiles were removed under reduced pressure to give a residue, which was re-dissolved by EtOAc (100 mL). The mixture was washed with NaHCO3 (aq.) (50 mL), brine (50 mL), dried over MgSO4, and concentrated to give a residue, which was purified by chromatography on silica gel column (petroleum ether: EA =20:1) to give 43 (2.12 g, 50%). MS (DCI) m/z 253 (M+H)+.

2-(3,4-Dichlorophenyl)tetrahydrofuran-2-carbonitrile (44). To a solution of 43 (2.0 g, 8.0 mmol) in MeOH (15 mL) was carefully added KCN (0.8 g, 12.3 mmol) and the mixture was stirred at 35°C for 48 h. The mixture was diluted with EtOAc (50 mL), washed with sat. aq. NaHCO3 (20 mL) and brine (10 mL). The aq. solution was oxidized with excessive amount of NaClO. The organic phase was dried over MgSO4, concentrated to give crude 44 (1.74 g), which was used directly in the next step without further purification. MS (DCI) m/z 242 (M+H)+.

(2-(3,4-Dichlorophenyl)tetrahydrofuran-2-yl)(pyridin-2-yl)methanone (45). To a solution of 2-bromopyridine (0.42 g, 2.65 mmol) in THF (10 mL) was added n-BuLi (1.65 mL, 2.65 mmol, 1.6 N in hexane) at -78°C. After 15 min, solution of 44 (0.43 g, 1.76 mmol) in THF (2 mL) was added. The mixture was stirred at -78°C for 15min and 2 mL of 1 M H2SO4 was added slowly, after which the mixture was heated to 50°C-60°C for 30min. After cooling to room temperature, the aqueous phase was separated and extracted with EtOAc (15 mL×3). The combined organic phase was washed with water (50 mL) and brine (50 mL), then dried over Na2SO4 and concentrated under reduced pressure to obtain crude 45 (0.57 g), which was used in the next step without further purification. MS (DCI) m/z 322 (M+H)+.



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trans-(R)-((S)-2-(3,4-Dichlorophenyl)tetrahydrofuran-2-yl)(pyridin-2-yl)methanol (46a) and cis-(R)-((R)-2-(3,4-dichlorophenyl)tetrahydrofuran-2-yl)(pyridin-2-yl)methanol (46b). To a solution of 45 (0.58 g, 1.79 mmol) in methanol (10 mL) was added NaBH4 (135 mg, 3.55 mmol) portion wise, and the mixture was stirred at room temperature for 16 h. After evaporating most of the solvent, the residue was diluted with 10 mL of water and extracted with EtOAc (15 mL×3). The combined organic phase was dried over Na2SO4, concentrated and the residue was purified by preparative TLC on silica gel (petroleum ether: EtOAc =3:1) to give 8 (0.22 g, 38% yield) and 9 (0.19 g, 33% yield). Analytical data for 46a: 1H-NMR (400 MHz, CDCl3): δ 8.23 (d, J=4.4Hz, 1H), 6.98-7.62 (m, 6H), 5.13 (broad s, 1H), 4.79 (s, 1H), 3.87-4.13 (m, 2H), 2.62-2.70 (m, 2H), 2.25-2.32 (m, 1H), 1.98-2.04 (m, 1H), 1.24-1.27 (m, 1H). MS (DCI) m/z 324 (M+H)+. HRMS m/z: [M + H]+ calcd for C16H16Cl2NO2, 324.05526; found, 324.05565. Analytical data for 46b: 1H-NMR (400 MHz, CDCl3): δ ppm 8.55 (t, J=2.4Hz, 1H), 6.83-7.53 (m, 6H), 4.78 (s, 1H), 3.87-4.13 (m, 2H), 3.73-3.91 (m, 2H), 2.52-2.59 (m, 1H), 2.02-2.09 (m, 1H), 1.63-1.72 (m, 2H). MS (DCI) m/z 324 (M+H)+. HRMS m/z: [M + H]+ calcd for C16H16Cl2NO2, 324.05526; found, 324.05562.

1-(3,4-Dichlorophenyl)-3,3-dimethoxycyclobutanecarboxamide (47). A mixture of 3,4dichlorophenylacetonitrile (1a) (9.30 g, 50 mmol), 1,3-dibromo-2,2-dimethoxypropane (13.10 g, 50 mmol) and sodium tert-butoxide (10.57 g, 110 mmol) in DMSO (100 mL) and water (5 mL) was heated to 125ºC for 1 h, then stirred at ambient temperature for 20 h. The reaction mixture was diluted with water and extracted three times with diethyl ether. The organic layers were


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combined, washed with brine, dried with MgSO4, and concentrated. The crude product was triturated with 1:1 diethyl ether / hexanes, and the solid collected by filtration was 47 (7.16 g, 47.1% yield). 1H NMR (300 MHz, d6-DMSO) δ 7.61 – 7.55 (m, 2H), 7.37 (s, 1H), 7.29 (dd, J = 8.4, 2.2 Hz, 1H), 6.95 (s, 1H), 3.05 (s, 3H), 2.99 (s, 3H), 2.97 – 2.89 (m, 2H), 2.46 – 2.37 (m, 2H).

Ethyl 1-(3,4-dichlorophenyl)-3-oxocyclobutanecarboxylate (48). A solution of 47 (9.73 g, 32.0 mmol) in ethanol (160 mL) and concentrated sulfuric acid (40 mL) was heated to reflux for 2 h. The reaction mixture was cooled, diluted with water (100 mL) and stirred for 30 min. The mixture was poured onto a mixture of ice and 10 N sodium hydroxide (75 mL), and extracted twice with diethyl ether. The organic layers were combined, washed with brine, dried with MgSO4 and concentrated to give 48 (5.25 g, 57.2% yield). 1H NMR (300 MHz, d6-DMSO) δ 7.68 – 7.63 (m, 2H), 7.37 (dd, J = 8.4, 2.3 Hz, 1H), 4.10 (q, J = 7.1 Hz, 2H), 3.82 – 3.60 (m, 4H), 1.12 (t, J = 7.1 Hz, 3H). MS (DCI) m/z 304 (M+H)+.

Ethyl 1-(3,4-dichlorophenyl)-3,3-difluorocyclobutanecarboxylate (49). To a 0ºC solution of 48 (3.96 g, 13.79 mmol) in CH2Cl2 (50 mL) was added dropwise a solution of diethylaminosulfur trifluoride (3.64 mL, 27.6 mmol) in CH2Cl2 (10 mL). The reaction mixture was allowed to warm to room temperature and stirred for 16 h, after which it was quenched with saturated solution of NaHCO3. The mixture was extracted twice with CH2Cl2, and the combined organic layers washed with saturated NH4Cl solution, dried with MgSO4, and concentrated. The residue was chromatographed on silica gel column (0-15% EtOAc:hexanes) to give 49 (3.06 g,



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72% yield). 1H NMR (300 MHz, d6-DMSO) δ 7.70 – 7.60 (m, 2H), 7.35 (dd, J = 8.4, 2.3 Hz, 1H), 4.09 (q, J = 7.1 Hz, 2H), 3.47 – 3.34 (m, 2H), 3.29 – 3.11 (m, 2H), 1.11 (t, J = 7.0 Hz, 3H).

[1-(3,4-Dichlorophenyl)-3,3-difluorocyclobutyl](pyridin-2-yl)methanone (50). 2.5 M nButyllithium (9.86 mL, 24.65 mmol) in hexanes was added to diethyl ether (60 mL) and cooled to -78ºC in a dry ice / acetone bath. 2-Bromopyridine (2.48 mL, 25.5 mmol) was added slowly, and the reaction was stirred for 30 min before a solution of 49 (5.08 g, 16.43 mmol) in diethyl ether (15 mL) was added dropwise over 10 min. The reaction mixture was warmed to room temperature by removing the cooling bath, then quenched by the addition of saturated NH4Cl solution, and extracted twice with diethyl ether. The combined organic layers were dried with MgSO4 and concentrated. The residue was chromatographed on silica gel column (20-35% CH2Cl2 : hexanes) to give 50 (5.00 g, 89% yield). MS (DCI+): m/z 342.0 (M+H). 1H NMR (300 MHz, d6-DMSO) δ 8.61 (dt, J = 4.7, 1.2 Hz, 1H), 8.03 – 7.93 (m, 2H), 7.74 (d, J = 2.2 Hz, 1H), 7.62 – 7.53 (m, 2H), 7.44 (dd, J = 8.5, 2.3 Hz, 1H), 3.68 – 3.48 (m, 2H), 3.46 – 3.32 (m, 2H). MS (DCI) m/z 342 (M+H)+.

3-Hydroxy-1-[3-(trifluoromethoxy)phenyl]cyclobutanecarbonitrile (51b). To a -75ºC solution of 2-[3-(trifluoromethoxy)phenyl]acetonitrile (1b) (10.28 g, 51.1 mmol) in anhydrous THF (80 mL) was added methyl lithium (31.9 mL, 1.6 M in diethyl ether, 51.1 mmol) dropwise and the yellow solution was stirred at -75ºC for 1 h. Epibromohydrin (4.23 mL, 51.1 mmol) in THF (15 mL) was added slowly and the reaction was stirred at -75ºC for 1 h. 3.0 M Methyl magnesium bromide (17.04 mL, 51.1 mmol) in diethyl ether was added dropwise, and the


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reaction was allowed to warm to room temperature for 16 h. The reaction was quenched with water (50 mL), added 3 N hydrochloric acid (100 mL), extracted twice with EtOAc (200 mL), washed with brine, and dried over Na2SO4. Organic phase was concentrated under reduced pressure and the residue chromatographed on silica gel (0-100% EtOAc in heptane) to give 51b (11.97 g, 91% yield) as a yellow oil. MS (DCI) m/z 275 (M+NH4)+.

3-Hydroxy-1-[4-(trifluoromethoxy)phenyl]cyclobutanecarbonitrile (51c). 2-[4(Trifluoromethoxy) phenyl]acetonitrile (1c) (5.0 g, 24.86 mmol) was dissolved in THF (40 mL) and chilled to -75ºC. Methyllithium (15.54 mL, 1.6 M solution, 24.86 mmol) in diethyl ether was added dropwise, and the yellow solution was stirred at -75ºC for 1 h. Epibromohydrin (2.1 mL, 24.86 mmol) in THF (10 mL) was added slowly, and the reaction was stirred at -75ºC for 1 h. Methyl magnesium bromide (8.29 mL, 3.M solution, 24.86 mmol) in diethyl ether was added dropwise, and the reaction was warmed to room temperature and stirred for 16 h. The reaction mixture was quenched with water (50 mL), then added 3 N HCl (100 mL), extracted twice with EtOAc (200 mL), washed with brine, and dried over Na2SO4. Column chromatography on silica gel (0-100% EtOAc in heptane) provided 51c (6.38 g, 100% yield). MS (DCI) m/z 275 (M+NH4)+.

3-Hydroxy-1-[3-(trifluoromethyl)phenyl]cyclobutanecarbonitrile (51e). To a -75ºC solution of 2-[3-(trifluoromethyl)phenyl]acetonitrile (1e) (10.25 g, 55.4 mmol) in anhydrous THF (80 mL) was added methyl lithium (34.6 mL, 1.6 M in diethyl ether, 55.4 mmol) dropwise and the yellow solution was stirred at -75ºC for 1 h. Epibromohydrin (4.58 mL, 55.4 mmol) in THF (15



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mL) was added slowly and the reaction was stirred at -75ºC for 1 h. 3.0 M Methyl magnesium bromide (18.45 mL, 55.4 mmol) in diethyl ether was added dropwise, and the reaction was allowed to warm to room temperature for 16 h. The reaction was quenched with water (50 mL), added 3 N hydrochloric acid (100 mL), extracted twice with EtOAc (200 mL), washed with brine, and dried over Na2SO4. Organic phase was concentrated under reduced pressure and the residue chromatographed on silica gel (0-100% EtOAc in heptane) to give 51e (12.21 g, 91% yield) as a yellow oil. MS (DCI) m/z 259 (M+NH4)+.

3-Oxo-1-[3-(trifluoromethoxy)phenyl]cyclobutanecarbonitrile (52b). To a solution of 51b (11.97 g, 46.5 mmol) in CH2Cl2 (200 mL) was added Dess-Martin Periodinane (29.6 g, 69.8 mmol) and the reaction mixture was stirred at room temperature for 16 h. The reaction was filtered through a pad of silica, rinsed with CH2Cl2, and the filtrate was concentrated. The residue was chromatographed on silica gel (100% CH2Cl2) to give 52b (11.32 g, 95% yield) as a yellow oil. 1H NMR (300 MHz, d6-DMSO) δ 7.68 - 7.59 (m, 3H), 7.49 - 7.40 (m, 1H), 4.17 - 4.05 (m, 2H), 3.99 - 3.87 (m, 2H).

3-Oxo-1-[4-(trifluoromethoxy)phenyl]cyclobutanecarbonitrile (52c). To a solution of 51c (6.38 g, 24.79 mmol) in CH2Cl2 (100 mL) was added Dess-Martin periodinane (15.77 g, 37.2 mmol). The yellow solution was stirred for 16 h at room temperature, concentrated and chromatographed on a silica gel column (100% CH2Cl2) to obtain 52c (5.75 g, 91% yield) 1H NMR (300 MHz, d6-DMSO) δ 7.77 - 7.70 (m, 2H), 7.54 - 7.46 (m, 2H), 4.17 - 4.07 (m, 2H), 3.97 - 3.86 (m, 2H).


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3-Oxo-1-[3-(trifluoromethyl)phenyl]cyclobutanecarbonitrile (52e). To a solution of 51e (12.21 g, 50.6 mmol) in CH2Cl2 (200 mL) was added Dess-Martin Periodinane (32.2 g, 76 mmol) and the reaction mixture was stirred at room temperature for 16 h. The reaction was filtered through a pad of silica, rinsed with CH2Cl2, and the filtrate was concentrated. The residue was chromatographed on silica gel (100% CH2Cl2) to give 52e (11.36 g, 94% yield) as a yellow oil. 1H NMR (300 MHz, d6-DMSO) δ 7.98 - 7.89 (m, 2H), 7.85 - 7.70 (m, 2H), 4.18 - 4.06 (m, 2H), 4.05 - 3.93 (m, 2H). MS (DCI) m/z 257 (M+NH4)+.

3,3-Difluoro-1-[4-(trifluoromethoxy)phenyl]cyclobutanecarbonitrile (53). To a 0ºC solution of 52c (5.75 g, 22.54 mmol) in CH2Cl2 (40 mL) was added slowly a solution of diethylaminosulfur trifluoride (7.5 mL, 56.8 mmol) in CH2Cl2 (20 mL). The orange solution was stirred for 48 h at room temperature, quenched with saturated NaHCO3 solution (200 mL), extracted with CH2Cl2 (200 mL), washed with saturated NaHCO3 solution (200 mL), and dried over Na2SO4. Chromatography on a silica gel column (0-30% EtOAc in heptane) gave 53 (5.25 g, 84% yield) 1H NMR (300 MHz, d6-DMSO) δ 7.71 - 7.64 (m, 2H), 7.53 - 7.45 (m, 2H), 3.66 3.50 (m, 2H), 3.50 - 3.34 (m, 2H).

{3,3-Difluoro-1-[4-(trifluoromethoxy)phenyl]cyclobutyl}(pyridin-2-yl)methanone (54). This compound was prepared analogously to the synthesis of 50 using starting material 53 instead 49. 1

H NMR (300 MHz, d6-DMSO) δ 8.64 - 8.57 (m, 1H), 8.02 - 7.91 (m, 2H), 7.66 - 7.59 (m, 2H),

7.59 - 7.53 (m, 1H), 7.30 (dd, J = 8.9, 0.9 Hz, 2H), 3.70 - 3.52 (m, 2H), 3.42 - 3.25 (m, 2H). MS (ESI) m/z 358 (M+H)+.



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(S)-[1-(3,4-Dichlorophenyl)-3,3-difluorocyclobutyl](pyridin-2-yl)methanol hydrochloride (55a). To a solution of 50 (5 g, 14.61 mmol) in formic acid (2.41 mL, 62.8 mmol) and triethylamine (5.09 mL, 36.5 mmol) was added (S,S)-N-(p-toluenesulfonyl)-1,2diphenylethanediamine(chloro)(p-cymene)ruthenium(II) (0.093 g, 0.146 mmol) and the reaction mixture was heated at 35ºC for 16 h. The reaction mixture was cooled, diluted with CH2Cl2, and washed with saturated NaHCO3 solution. The organic layer was dried with MgSO4 and concentrated. The residue was chromatographed on silica gel column with 100% CH2Cl2. The resulting residue was dissolved in 2 N hydrogen chloride in methanol and concentrated to give 55a (4.60 g, 83% yield, >95% ee by chiral HPLC). 1H NMR (300 MHz, d6-DMSO) δ 8.54 (d, J = 5.2 Hz, 1H), 8.13 – 7.95 (m, 1H), 7.65 – 7.51 (m, 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 6.7 Hz, 1H), 7.15 (d, J = 2.0 Hz, 1H), 6.82 (dd, J = 8.4, 2.2 Hz, 1H), 5.10 (s, 1H), 3.62 – 3.24 (m, 2H), 3.04 – 2.78 (m, 2H). MS (DCI) m/z 344 (M+H)+. [α]D = -35.6º (c 0.715, MeOH). Calculated for C16H13Cl2F2NO •HCl: C 50.49%, H 3.71%, N 3.68%; Found: C 50.65%, H 3.69%, N 3.59%.

(S)-{3,3-Difluoro-1-[4-(trifluoromethoxy)phenyl]cyclobutyl}(pyridin-2-yl)methanol hydrochloride (55c). This compound with >95% ee was prepared analogously to the synthesis of 55a using starting material 54 instead 50. 1H NMR (300 MHz, d4-Methanol) δ 8.48 - 8.38 (m, 2H), 7.94 - 7.85 (m, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.14 (dd, J = 8.8, 0.7 Hz, 2H), 7.05 (d, J = 8.8 Hz, 2H), 5.33 (s, 1H), 3.55 - 3.35 (m, 2H), 3.09 - 2.89 (m, 2H). MS (ESI) m/z 360 (M+H)+. [α]D


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= -24.6º (c 1.0, MeOH). Calculated for C17H14F5NO2 •HCl: C 51.59%, H 3.82%, N 3.54%; Found: C 51.55%, H 3.91%, N 3.49%.

2-[3-(Trifluoromethoxy)phenyl]-5,8-dioxaspiro[3.4]octane-2-carbonitrile (56b). To a solution of 52b (11.318 g, 44.4 mmol) and 1,2-bis(trimethylsilyloxy)ethane (17 mL, 69.2 mmol) in CH2Cl2 (100 mL) was added trimethylsilyl trifluoromethanesulfonate (1.0 mL, 5.53 mmol) and the resulting yellow solution was stirred at room temperature for 16 h. The reaction was quenched with triethylamine (1.2 mL, 8.66 mmol), washed twice with saturated NaHCO3 solution, and dried over Na2SO4. Organic layer was concentrated under reduced pressure and the residue chromatographed on silica gel (0-100% EtOAc in heptane) to give 56b (12.85 g, 97% yield) as a yellow liquid. 1H NMR (300 MHz, d6-DMSO) δ 7.65 - 7.52 (m, 2H), 7.48 (s, 1H), 7.44 - 7.36 (m, 1H), 4.00 - 3.92 (m, 2H), 3.90 - 3.81 (m, 2H), 3.23 - 3.13 (m, 2H), 3.03 - 2.92 (m, 2H).

2-[4-(Trifluoromethoxy)phenyl]-5,8-dioxaspiro[3.4]octane-2-carbonitrile (56c). To a solution of 52C (30.0 g, 120 mmol), ethylene glycol (8.4 mL, 150 mmol) and 4-toluenesulfonic acid hydrate (0.69 g, 3.6 mmol) in toluene (240 mL) was heated at reflux with distillation into a Dean-Stark trap for 90 minutes. Trimethyl orthoformate (5.0 mL, 46 mmol) was added and the solution was stirred for 3 h at room temperature and then warmed to 100ºC where it was kept for about 1.5 h before additional ethylene glycol (1.7 mL, 30 mmol) was added. The solution was heated at 100ºC overnight, the flask was removed from the hot bath and allowed to cool, and the reaction mixture was washed three times with 0.5 M aqueous K2HPO4, dried (Na2SO4),



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concentrated, and chromatographed on silica (10 - 25% EtOAc : hexanes) to give 56c (28.6 g, 81% yield), which was used without further purification.

2-[3-(Trifluoromethyl)phenyl]-5,8-dioxaspiro[3.4]octane-2-carbonitrile (56e). To a solution of 52e (11.36 g, 47.5 mmol) and 1,2-bis(trimethylsilyloxy)ethane (18 mL, 73.2 mmol) in CH2Cl2 (100 mL) was added trimethylsilyl trifluoromethanesulfonate (1.0 mL, 5.53 mmol) and the resulting yellow solution was stirred at room temperature for 16 h. The reaction was quenched with triethylamine (1.2 mL, 8.66 mmol), washed twice with saturated NaHCO3 solution, and dried over Na2SO4. Organic layer was concentrated under reduced pressure and the residue chromatographed on silica gel (0-100% EtOAc in heptane) to give 56e (13.32 g, 99% yield) as a yellow liquid. 1H NMR (300 MHz, d6-DMSO) δ 7.87 - 7.79 (m, 2H), 7.79 - 7.66 (m, 2H), 4.04 3.92 (m, 2H), 3.91 - 3.82 (m, 2H), 3.25 - 3.15 (m, 2H), 3.06 - 2.96 (m, 2H).

Pyridin-2-yl{2-[3-(trifluoromethoxy)phenyl]-5,8-dioxaspiro[3.4]octan-2-yl}methanone (57b). A solution of 2-bromopyridine (6.35 mL, 66.6 mmol) in diethyl ether (40 mL) was added dropwise to a -75ºC solution of n-butyl lithium (25.8 mL, 2.5 M in hexanes, 64.4 mmol) in diethyl ether (90 mL) and the resulting orange-red solution was stirred for 1.5 h, after which a solution of 56b (12.85 g, 42.9 mmol) in diethyl ether (40 mL) was added dropwise, and the reaction mixture slowly warmed to 0ºC over the period of 3.5 h. Then the reaction mixture was quenched with 1 N hydrochloric acid (150 mL) and stirred the biphasic mixture for 45 min at room temperature. A solution of 3 N NaOH (150 mL) was added to that mixture, extracted twice with EtOAc (300 mL), washed with brine, and dried over Na2SO4. Organic layer was


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concentrated under reduced pressure and the residue chromatographed on silica gel (0-50% EtOAc in heptane) to give 57b (12.47 g, 77% yield) as a yellow oil. 1H NMR (500 MHz, d3MeCN) δ 8.50 (dd, J = 4.5, 0.4 Hz, 1H), 7.95 (d, J = 7.9 Hz, 1H), 7.84 (td, J = 7.8, 1.7 Hz, 1H), 7.52 (s, 1H), 7.44 - 7.38 (m, 2H), 7.33 (t, J = 8.0 Hz, 1H), 7.10 - 7.05 (m, 1H), 3.87 - 3.78 (m, 4H), 3.37 - 3.30 (m, 2H), 3.01 - 2.94 (m, 2H). MS (ESI) m/z 380 (M+H)+.

Pyridin-2-yl{2-[4-(trifluoromethoxy)phenyl]-5,8-dioxaspiro[3.4]octan-2-yl}methanone (57c). A solution of 2-bromopyridine (14.8 mL, 152 mmol) in diethyl ether (80 mL) was added dropwise to a -75ºC solution of n-butyl lithium (59 mL, 2.5 M in hexanes, 148 mmol) in diethyl ether (10 mL) and the resulting orange-red solution was stirred for 1 h after which a solution of 56c (28.5 g, 95 mmol) in diethyl ether (100 mL) was added dropwise, and the reaction mixture slowly warmed to 0ºC over the period of 16 h. Then the reaction mixture was quenched with 1 N hydrochloric acid (150 mL) and stirred the biphasic mixture for 45 minutes at room temperature. A solution of 3 N NaOH (150 mL) was added to that mixture, extracted twice with EtOAc (300 mL), washed with brine, and dried over Na2SO4. Organic layer was concentrated under reduced pressure and the residue chromatographed on silica gel (0 - 5% EtOAc in 2:3 CH2Cl2 : hexanes) to give 57c (22 g, 60.8% yield) as a yellow oil. ). 1H NMR (300 MHz, d6-DMSO) δ 8.59 - 8.55 (m, 1H), 7.98 - 7.91 (m, 2H), 7.58 - 7.50 (m, 3H), 7.29 - 7.23 (m, 2H), 3.82 (dd, J = 9.9, 3.8 Hz, 2H), 3.77 (dd, J = 9.9, 3.8 Hz, 2H), 3.3 - 3.23 (m, 2H), 2.99 - 2.91 (m, 2H). MS (ESI) m/z 380 (M+H)+.



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Pyridin-2-yl{2-[3-(trifluoromethyl)phenyl]-5,8-dioxaspiro[3.4]octan-2-yl}methanone (57e). A solution of 2-bromopyridine (6.95 mL, 72.9 mmol) in diethyl ether (40 mL) was added dropwise to a -75ºC solution of n-butyl lithium (28.2 mL, 2.5 M in hexanes, 70.6 mmol) in diethyl ether (10 mL) and the resulting orange-red solution was stirred for 1.5 h, after which a solution of 56e (13.32 g, 47.0 mmol) in diethyl ether (40 mL) was added dropwise, and the reaction mixture slowly warmed to 0ºC over the period of 3 h. Then the reaction mixture was quenched with 1 N hydrochloric acid (150 mL) and stirred the biphasic mixture for 45 min at room temperature. A solution of 3 N NaOH (150 mL) was added to that mixture, extracted twice with EtOAc (300 mL), washed with brine, and dried over Na2SO4. Organic layer was concentrated under reduced pressure and the residue chromatographed on silica gel (0-50% EtOAc in heptane) to give 57e (13.02 g, 76% yield) as a yellow oil. 1H NMR (500 MHz, d3MeCN) δ 8.51 (d, J = 4.6 Hz, 1H), 7.96 (d, J = 7.8 Hz, 1H), 7.88 - 7.81 (m, 2H), 7.73 (d, J = 7.3 Hz, 1H), 7.50 - 7.39 (m, 3H), 3.87 - 3.79 (m, 4H), 3.36 (d, J = 13.7 Hz, 2H), 3.02 (d, J = 13.6 Hz, 2H). MS (ESI) m/z 364 (M+H)+.

Pyridin-2-yl{2-[3-(trifluoromethoxy)phenyl]-5,8-dioxaspiro[3.4]oct-2-yl}methanol (58b). To a solution of 57b (12.47 g, 32.9 mmol) in CH2Cl2 (100 mL) and MeOH (10 mL) was added sodium borohydride (1.37 g, 36.2 mmol), and the resulting mixture was stirred at room temperature for 16 h. A solution of 3 N NaOH (200 mL) was added to that mixture, extracted twice with EtOAc (300 mL), washed with brine, and dried over Na2SO4. Organic layer was concentrated under reduced pressure and the residue chromatographed on silica gel (0-100% EtOAc in hexanes) to give 58b (12.36 g, 99% yield) as a yellow oil. 1H NMR (501 MHz, d3MeCN) δ 8.32 (d, J = 4.8 Hz, 1H), 7.50 (td, J = 7.7, 1.7 Hz, 1H), 7.22 (t, J = 8.0 Hz, 1H), 7.14


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(dd, J = 7.5, 4.9 Hz, 1H), 7.06 - 7.02 (m, 1H), 6.91 (d, J = 7.8 Hz, 1H), 6.79 (d, J = 7.9 Hz, 1H), 6.64 (s, 1H), 5.00 (d, J = 6.1 Hz, 1H), 4.18 (d, J = 6.1 Hz, 1H), 3.93 - 3.86 (m, 2H), 3.81 - 3.75 (m, 2H), 3.18 (dd, J = 12.9, 3.2 Hz, 1H), 3.03 (dd, J = 13.0, 3.2 Hz, 1H), 2.58 (dd, J = 13.0, 3.1 Hz, 1H), 2.53 (dd, J = 12.9, 3.1 Hz, 1H). MS (ESI) m/z 382 (M+H)+.

Pyridin-2-yl{2-[4-(trifluoromethoxy)phenyl]-5,8-dioxaspiro[3.4]octan-2-yl}methanol (58c). To a solution of 57c (21.8 g, 57 mmol) in CH2Cl2 (200 mL) and MeOH (20 mL) was added sodium borohydride (2.3 g, 61 mmol), and the resulting mixture was stirred at room temperature for 16 h. A solution of 3 N NaOH (200 mL) was added to that mixture, extracted twice with EtOAc (300 mL), washed with brine, and dried over Na2SO4. Organic layer was concentrated under reduced pressure and the residue chromatographed on silica gel (20 - 40% EtOAc:CH2Cl2) to give 58c (17.1 g, 78% yield) as a white powder. 1H NMR (300 MHz, d6-DMSO) δ 8.40 (ddq, J = 4.8, 1.7, 0.8 Hz, 1H), 7.44 (ddd, J = 7.7, 7.7, 1.8 Hz, 1H), 7.14 (ddd, J = 7.5, 4.8, 1.1 Hz, 1H), 7.10 - 7.04 (m, 2H), 6.85 - 6.79 (m, 2H), 6.55 (d, J = 7.9 Hz, 1H), 5.74 (d, J = 4.2 Hz, 1H), 4.96 (d, J = 4.2 Hz, 1H), 3.90 - 3.83 (m, 2H), 3.78 - 3.71 (m, 2H), 3.22 (dd, J = 12.9, 3.4 Hz, 1H), 3.04 (dd, J = 12.9, 3.4 Hz, 1H), 2.57 - 2.5 (m, 1H), 2.41 (dd, J = 13.0, 2.3 Hz, 1H). MS (ESI) m/z 382 (M+H)+.

Pyridin-2-yl{2-[3-(trifluoromethyl)phenyl]-5,8-dioxaspiro[3.4]oct-2-yl}methanol (58e). To a solution of 57e (13.02 g, 35.8 mmol) in CH2Cl2 (100 mL) and MeOH (11 mL) was added sodium borohydride (1.49 g, 39.4 mmol), and the resulting mixture was stirred at room temperature for 16 h. A solution of 3 N NaOH (200 mL) was added to that mixture, extracted twice with EtOAc



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(300 mL), washed with brine, and dried over Na2SO4. Organic layer was concentrated under reduced pressure and the residue chromatographed on silica gel (0-100% EtOAc in hexanes) to give 58e (12.39 g, 95% yield) as a yellow oil. 1H NMR (400 MHz, d3-MeCN) δ 8.34 - 8.29 (m, 1H), 7.50 (td, J = 7.7, 1.7 Hz, 1H), 7.43 (d, J = 7.7 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 7.18 - 7.10 (m, 2H), 6.97 (s, 1H), 6.79 (d, J = 7.9 Hz, 1H), 5.02 (d, J = 6.0 Hz, 1H), 4.15 (d, J = 6.0 Hz, 1H), 3.94 - 3.87 (m, 2H), 3.82 - 3.75 (m, 2H), 3.20 (dd, J = 12.9, 3.2 Hz, 1H), 3.05 (dd, J = 13.0, 3.3 Hz, 1H), 2.64 (ddd, J = 13.1, 3.2, 0.7 Hz, 1H), 2.55 (dd, J = 12.9, 3.1 Hz, 1H). MS (ESI) m/z 366 (M+H)+.

3-[(S)-Hydroxy(pyridin-2-yl)methyl]-3-[3-(trifluoromethoxy)phenyl]cyclobutanone (59b). A solution of 58b (12.364 g, 32.4 mmol) in acetone (200 mL), H2O (20 mL) and 12 N HCl (25 mL) was stirred overnight at room temperature. Mixture was concentrated to about half of original volume, added 3 N NaOH (150 mL), extracted twice with EtOAc (300 mL), washed with brine, and dried over Na2SO4. Organic layer was concentrated under reduced pressure and the residue chromatographed on silica gel (0-100% EtOAc in heptane) to give a racemic mixture of alcohols (10.82 g, 99% yield) as a colorless oil which solidified upon cooling. This mixture (10.2 g, 30.2 mmol) was separated on a Chiralpak AD-H column (10% EtOH in hexanes) to provide 59b (4.0 g, 39% yield) as a yellow oil. 1H NMR (500 MHz, d3-MeCN) δ 8.43 (d, J = 4.8 Hz, 1H), 7.49 (td, J = 7.7, 1.7 Hz, 1H), 7.34 (t, J = 8.0 Hz, 1H), 7.22 - 7.17 (m, 1H), 7.14 (dd, J = 8.2, 0.9 Hz, 1H), 7.05 (d, J = 7.8 Hz, 1H), 6.79 (s, 1H), 6.60 (d, J = 7.8 Hz, 1H), 4.97 (s, 1H), 4.51 (broad s, 1H), 3.79 (ddd, J = 17.1, 6.0, 2.4 Hz, 1H), 3.64 (ddd, J = 17.4, 6.0, 2.3 Hz, 1H), 3.33 - 3.25 (m, 1H), 3.24 - 3.16 (m, 1H). MS (ESI) m/z 338 (M+H)+. [α]D = -22.9º (c 1.0, MeOH).


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(S)-3-[hydroxy(pyridin-2-yl)methyl]-3-[4-(trifluoromethoxy)phenyl]cyclobutanone (59c) and (R)-3-[hydroxy(pyridin-2-yl)methyl]-3-[4-(trifluoromethoxy)phenyl]cyclobutanone (R59c). A solution of 58c (17.0 g, 44.6 mmol) in acetone (250 mL) and 6 N HCl (25 mL) was stirred overnight at room temperature. Mixture was concentrated to about half of original volume, added 3 N NaOH (60 mL), extracted twice with EtOAc (300 mL), washed with brine, and dried over Na2SO4. Organic layer was concentrated to give a racemic mixture of alcohols (15.06 g, 100% yield) as a white solid. This mixture (15.06 g, 44.6 mmol) was separated on a Chiralpak AD-DAC column (10% EtOH in hexanes) to provide 59c (7.52 g, 50% yield) as a white solid and R-59c. Analytical data for 59c: 1H NMR (400 MHz, d3-MeCN) δ 8.43 (ddd, J = 4.8, 1.4, 0.9 Hz, 1H), 7.49 (td, J = 7.7, 1.8 Hz, 1H), 7.20 (ddd, J = 7.5, 4.9, 1.0 Hz, 1H), 7.13 (d, J = 8.7 Hz, 2H), 7.04 (d, J = 8.8 Hz, 2H), 6.62 (d, J = 7.9 Hz, 1H), 4.97 (d, J = 5.7 Hz, 1H), 4.47 (d, J = 6.0 Hz, 1H), 3.78 (ddd, J = 17.0, 6.0, 2.5 Hz, 1H), 3.63 (ddd, J = 17.3, 6.0, 2.4 Hz, 1H), 3.28 (ddd, J = 17.0, 3.6, 2.4 Hz, 1H), 3.20 (ddd, J = 17.3, 3.5, 2.5 Hz, 1H). MS (ESI) m/z 338 (M+H)+. [α]D = -18.8º (c 1, MeOH). Analytical data for R-59c: 1H NMR (400 MHz, CD3CN) δ 8.43 (ddd, J = 4.8, 1.4, 0.9 Hz, 1H), 7.49 (td, J = 7.7, 1.8 Hz, 1H), 7.20 (ddd, J = 7.5, 4.9, 1.0 Hz, 1H), 7.13 (d, J = 8.7 Hz, 2H), 7.04 (d, J = 8.8 Hz, 2H), 6.62 (d, J = 7.9 Hz, 1H), 4.97 (d, J = 5.7 Hz, 1H), 4.47 (d, J = 6.0 Hz, 1H), 3.78 (ddd, J = 17.0, 6.0, 2.5 Hz, 1H), 3.63 (ddd, J = 17.3, 6.0, 2.4 Hz, 1H), 3.28 (ddd, J = 17.0, 3.6, 2.4 Hz, 1H), 3.20 (ddd, J = 17.3, 3.5, 2.5 Hz, 1H). MS (ESI) m/z 338 (M+H)+. [α]D = +26.0 (c=1, MeOH).

3-[(S)-Hydroxy(pyridin-2-yl)methyl]-3-[3-(trifluoromethyl)phenyl]cyclobutanone 59e). A solution of 58e (12.389 g, 33.9 mmol) in acetone (200 mL), water (20 mL) and 12 N HCl (25 mL) was stirred overnight at room temperature. Mixture was concentrated to about half of



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original volume, added 3 N NaOH (150 mL), extracted twice with EtOAc (300 mL), washed with brine, and dried over Na2SO4. Organic layer was concentrated under reduced pressure and the residue chromatographed on silica gel 0-100% EtOAc in 1:1 heptane: CH2Cl2)) to give a racemic mixture of alcohols (10.04 g, 92% yield) as a white solid. This mixture (9.93 g, 30.9 mmol) was separated on a Chiralpak AD-H column (10% EtOH in hexanes) to provide 59e (4.52 g, 46% yield) as a white solid. 1H NMR (500 MHz, d3-MeCN) δ 8.45 - 8.40 (m, 1H), 7.53 (dd, J = 7.7, 0.5 Hz, 1H), 7.49 (td, J = 7.7, 1.7 Hz, 1H), 7.42 (t, J = 7.8 Hz, 1H), 7.27 (d, J = 7.7 Hz, 1H), 7.20 (ddd, J = 7.5, 4.8, 1.0 Hz, 1H), 7.14 (s, 1H), 6.61 (d, J = 7.8 Hz, 1H), 4.98 (s, 1H), 4.47 (broad s, 1H), 3.81 (ddd, J = 17.1, 6.0, 2.5 Hz, 1H), 3.67 (ddd, J = 17.4, 6.1, 2.4 Hz, 1H), 3.32 (ddd, J = 17.1, 3.6, 2.5 Hz, 1H), 3.25 (ddd, J = 17.4, 3.5, 2.6 Hz, 1H). MS (ESI) m/z 322 (M+H)+. [α]D = -34.3º (c 1.0, MeOH).

trans-3-[(S)-Hydroxy(pyridin-2-yl)methyl]-3-[3-(trifluoromethoxy)phenyl]cyclobutanol (60b) and cis-3-[(S)-hydroxy(pyridin-2-yl)methyl]-3-[3-(trifluoromethoxy)phenyl] cyclobutanol (61b). To a solution of 59b (1.32 g, 3.91 mmol) in CH2Cl2 (30 mL) and MeOH (3 mL), was added sodium borohydride (0.31 g, 8.22 mmol) and the reaction mixture was stirred at room temperature for 16 h. A solution of 3 N NaOH (200 mL) was added to that mixture, extracted twice with EtOAc (200 mL), washed with brine, and dried over Na2SO4. Organic layer was concentrated under reduced pressure and the residue chromatographed on silica gel (0-10% MeOH in EtOAc) to give mixture of geometrical isomers (1.08 g, 3.18 mmol, 81% yield) as a white solid. The latter (0.992 g, 2.92 mmol) was separated by chiral preparative SFC on a Chiralpak AD-H column (10% isopropanol (0.5% isopropylamine) in CO2) to obtain 60b (0.419 g, 42% yield) and 61b (0.242 g, 24% yield). Analytical data for 60b: 1H NMR (500 MHz, d3-


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MeCN) δ 8.37 (d, J = 4.7 Hz, 1H), 7.51 (td, J = 7.7, 1.6 Hz, 1H), 7.25 (t, J = 8.0 Hz, 1H), 7.17 (dd, J = 7.4, 4.9 Hz, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.87 (d, J = 7.8 Hz, 1H), 6.76 (d, J = 7.9 Hz, 1H), 6.60 (s, 1H), 4.79 (d, J = 6.4 Hz, 1H), 4.30 - 4.22 (m, 1H), 4.20 (d, J = 6.4 Hz, 1H), 3.15 (ddd, J = 11.8, 7.3, 4.7 Hz, 1H), 3.09 (ddd, J = 11.9, 7.3, 4.7 Hz, 1H), 3.02 (d, J = 6.9 Hz, 1H), 2.07 (ddd, J = 14.8, 11.8, 7.1 Hz, 2H). MS (ESI) m/z 340 (M+H)+. [α]D = -67.6º (c 1.0, MeOH). HRMS m/z: [M + H]+ calcd for C17H17F3NO3, 340.11550; found, 340.11585. Analytical data for 61b: 1H NMR (500 MHz, d3-MeCN) δ 8.31 (d, J = 4.8 Hz, 1H), 7.51 (td, J = 7.7, 1.7 Hz, 1H), 7.25 (t, J = 8.0 Hz, 1H), 7.16 - 7.12 (m, 1H), 7.05 (dd, J = 7.8, 1.4 Hz, 2H), 6.80 - 6.74 (m, 2H), 4.83 (d, J = 6.2 Hz, 1H), 4.37 (d, J = 6.2 Hz, 1H), 3.92 - 3.83 (m, 1H), 3.24 (d, J = 6.7 Hz, 1H), 2.67 - 2.59 (m, 3H), 2.46 (dd, J = 12.3, 7.5 Hz, 1H). MS (ESI) m/z 340 (M+H)+. [α]D = -39.4º (c 1.0, MeOH). HRMS m/z: [M + H]+ calcd for C17H17F3NO3, 340.11550; found, 340.11572.

The following four compounds were prepared analogously to 60b and 61b by using differently substituted phenylacetonitriles in the first step of the sequence.

trans-3-[(S)-Hydroxy(pyridin-2-yl)methyl]-3-[3-(trifluoromethyl)phenyl]cyclobutanol (60e). 1

H NMR (500 MHz, d3-MeCN) δ 8.16 (d, J = 4.8 Hz, 1H), 7.31 (td, J = 7.7, 1.7 Hz, 1H), 7.23

(d, J = 7.8 Hz, 1H), 7.14 (t, J = 7.8 Hz, 1H), 6.97 (ddd, J = 7.4, 4.9, 0.8 Hz, 1H), 6.89 (d, J = 7.8 Hz, 1H), 6.75 (s, 1H), 6.57 (d, J = 7.9 Hz, 1H), 4.61 (d, J = 3.4 Hz, 1H), 4.05 (p, J = 7.1 Hz, 1H), 3.97 (d, J = 5.5 Hz, 1H), 3.02 - 2.89 (m, 2H), 2.84 (broad s, 1H), 1.90 (ddd, J = 11.6, 7.1, 4.2 Hz, 2H). MS (ESI) m/z 324 (M+H)+. [α]D = -71.8º (c 1.0, MeOH). Calculated for C17H16F3NO2: C63.15%, H 4.99%, N 4.33%; Found: C 63.29%, H 5.14%, N 4.15%.



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trans-3-[(S)-Hydroxy(pyridin-2-yl)methyl]-3-[4-(trifluoromethyl)phenyl]cyclobutanol (60f). 1

H NMR (501 MHz, DMSO-d6) δ 8.41 (d, J = 4.1 Hz, 1H), 7.53 (td, J = 7.7, 1.6 Hz, 1H), 7.47

(d, J = 8.2 Hz, 2H), 7.17 (dd, J = 6.9, 5.3 Hz, 1H), 6.95 (d, J = 8.0 Hz, 2H), 6.81 (d, J = 7.9 Hz, 1H), 5.60 (s, 1H), 4.87 (d, J = 6.5 Hz, 1H), 4.74 (s, 1H), 4.04 - 3.93 (m, 1H), 3.15 - 3.05 (m, 2H), 2.05 - 1.93 (m, 2H). MS (ESI) m/z 324 (M+H)+. [α]D = -68.0º (c 1.0, MeOH). Calculated for C17H16F3NO2·0.27 MeOH: C 62.49%, H 5.19%, N 4.22%; Found: C 62.39%, H 4.82%, N 4.17%.

cis-3-[(S)-Hydroxy(pyridin-2-yl)methyl]-3-[3-(trifluoromethyl)phenyl]cyclobutanol (61e). 1

H NMR (500 MHz, d3-MeCN) δ 8.30 (d, J = 4.8 Hz, 1H), 7.52 (td, J = 7.7, 1.5 Hz, 1H), 7.44

(d, J = 7.7 Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.27 (d, J = 7.9 Hz, 1H), 7.14 (dd, J = 7.4, 4.9 Hz, 1H), 7.11 (s, 1H), 6.79 (d, J = 7.9 Hz, 1H), 4.86 (d, J = 5.5 Hz, 1H), 4.33 (d, J = 6.0 Hz, 1H), 3.92 - 3.82 (m, 1H), 3.26 (d, J = 5.8 Hz, 1H), 2.74 - 2.61 (m, 3H), 2.48 (dd, J = 11.3, 7.6 Hz, 1H). MS (ESI) m/z 324 (M+H)+. [α]D = -45.6º (c 1.0, MeOH). Calculated for C17H16F3NO2: C 63.15%, H 4.99%, N 4.33%; Found: C 63.12%, H 5.47%, N 4.17%.

cis-3-[(S)-Hydroxy(pyridin-2-yl)methyl]-3-[4-(trifluoromethyl)phenyl]cyclobutanol (61f). 1

H NMR (300 MHz, DMSO-d6) δ 8.35 (ddd, J = 4.8, 1.7, 0.8 Hz, 1H), 7.52 (td, J = 7.7, 1.8 Hz,

1H), 7.46 (d, J = 8.1 Hz, 2H), 7.18 - 7.10 (m, 3H), 6.74 (d, J = 7.9 Hz, 1H), 5.64 (s, 1H), 5.01 (d, J = 5.9 Hz, 1H), 4.82 (s, 1H), 3.81 - 3.65 (m, 1H), 2.70 - 2.56 (m, 2H), 2.55 - 2.41 (m, 2H). MS


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(ESI) m/z 324 (M+H)+. [α]D = -41.6º (c 1.0, MeOH). Calculated for C17H16F3NO2·0.18 MeOH: C 62.70%, H 5.12%, N 4.26%; Found: C 62.61%, H 4.76%, N 4.23%.

cis-3-[(S)-Hydroxy(pyridin-2-yl)methyl]-1-methyl-3-[3-(trifluoromethoxy)phenyl] cyclobutanol (62b) and trans-3-[(S)-hydroxy(pyridin-2-yl)methyl]-1-methyl-3-[3(trifluoromethoxy)phenyl]cyclobutanol (63b). To a -78°C solution of 59b (2.0 g, 5.93 mmol) in anhydrous 2-methyltetrahydrofuran (30 mL) was slowly added methyl lithium (9.0 mL, 1.6 M in diethyl ether, 14.4 mmol), and the reaction mixture was allowed to warm to room temperature for 16 h. The reaction was quenched with 1 M aqueous KH2PO4 (50 mL), then water (200 mL) was added, extracted twice with EtOAc (200 mL), washed with brine, and dried over Na2SO4. Organic layers were concentrated under reduced pressure and the residue chromatographed on silica gel (0-10% MeOH in EtOAc) to obtain 62b (0.94 g, 45.0% yield) and 63b (0.17 g, 8.3% yield). Analytical data for 62b: 1H NMR (400 MHz, d3-MeCN) δ 8.31 (ddd, J = 4.9, 1.6, 1.0 Hz, 1H), 7.49 (td, J = 7.7, 1.8 Hz, 1H), 7.24 (t, J = 8.0 Hz, 1H), 7.14 (ddd, J = 7.5, 4.9, 1.1 Hz, 1H), 7.07 - 7.01 (m, 1H), 6.95 (ddd, J = 7.8, 1.5, 1.0 Hz, 1H), 6.71 (d, J = 7.9 Hz, 1H), 6.66 (s, 1H), 4.91 (d, J = 5.7 Hz, 1H), 4.48 (d, J = 6.0 Hz, 1H), 3.40 (s, 1H), 2.94 - 2.86 (m, 1H), 2.78 - 2.71 (m, 1H), 2.44 - 2.36 (m, 2H), 0.99 (s, 3H). MS (ESI) m/z 354 (M+H)+. [α]D = -43.7º (c 1.0, MeOH). Calculated for C18H18F3NO3 •0.34 H2O: C 60.14%, H 5.24%, N 3.90%; Found: C 60.12%, H 4.88%, N 3.77%. Analytical data for 63b: 1H NMR (400 MHz, d3-MeCN) δ 8.32 (ddd, J = 4.9, 1.6, 1.0 Hz, 1H), 7.49 (td, J = 7.7, 1.8 Hz, 1H), 7.23 (t, J = 8.0 Hz, 1H), 7.14 (ddd, J = 7.6, 4.9, 1.1 Hz, 1H), 7.05 - 6.99 (m, 1H), 6.95 - 6.89 (m, 1H), 6.74 (d, J = 7.9 Hz, 1H), 6.64 (s, 1H), 4.87 (d, J = 5.1 Hz, 1H), 4.20 (d, J = 5.7 Hz, 1H), 3.04 - 2.97 (m, 1H), 2.90 - 2.84 (m, 1H), 2.82 (s, 1H), 2.38 - 2.28 (m, 2H), 1.40 (s, 3H). MS (ESI) m/z 354 (M+H)+. [α]D = -60.8º (c



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1.0, MeOH). Calculated for C18H18F3NO3:C 61.19%, H 5.13%, N 3.96%; Found: C 60.12%, H 4.78%, N 3.45%.

cis-3-[(S)-Hydroxy(pyridin-2-yl)methyl]-1-methyl-3-[4-(trifluoromethoxy)phenyl] cyclobutanol hydrochloride (62c) and trans-3-[(S)-hydroxy(pyridin-2-yl)methyl]-1-methyl3-[4-(trifluoromethoxy)phenyl]cyclobutanol hydrochloride (63c). To a -78°C solution of 59c (6.94 g, 20.5 mmol) in anhydrous THF (100 mL) was added slowly methyl lithium (28.3 mL, 1.6 M in diethyl ether, 45 mmol), and the reaction was allowed to warm to room temperature for 16 h. The reaction was quenched with 1 M aqueous KH2PO4 (50 mL), then water (200 mL) was added, extracted twice with EtOAc (200 mL), washed with brine, and dried over Na2SO4. Organic layers were concentrated under reduced pressure and the residue chromatographed on silica gel (30 - 100% EtOAc:hexanes, then 1 - 5% MeOH:EtOAc) to obtain 62c (0.77 g, 9.5% yield) and 63c (3.8 g, 47% yield). Both compounds were converted to their corresponding hydrochloride salts by dissolving them in excess of HCl in MeOH and then removing all the volatiles under reduced pressure. Analytical data for 62c hydrochloride: 1H NMR (300 MHz, d6-DMSO) δ 8.46 (d, J = 4.9 Hz, 1H), 8.21 – 7.95 (m, 1H), 7.74 – 7.50 (m, 1H), 7.37 – 7.15 (m, 1H), 7.11 (d, J = 8.1 Hz, 2H), 6.87 (d, J = 8.7 Hz, 2H), 5.10 (s, 1H), 2.89 (dd, J = 19.8, 12.6 Hz, 2H), 2.31 (dd, J = 19.1, 12.3 Hz, 2H), 1.38 (s, 3H). MS (ESI) m/z 354 (M+H)+. [α]D = -19.2º (c 1.0, MeOH).Analytical data for 63c hydrochloride: MS (ESI+): m/z 354 (M+H). 1H NMR (300 MHz, d6-DMSO) δ 8.44 (d, J = 5.0 Hz, 1H), 8.27 – 8.00 (m, 1H), 7.78 – 7.54 (m, 1H), 7.50 – 7.24 (m, 1H), 7.12 (d, J = 8.2 Hz, 2H), 6.94 (d, J = 8.7 Hz, 2H), 5.21 (s, 1H), 2.82 (dd, J = 37.5, 11.9 Hz, 2H), 2.35 (ddd, J = 24.5, 12.2, 3.5 Hz, 2H), 0.90 (s, 3H). MS (ESI) m/z 354 (M+H)+.


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[α]D = -15.2º (c 1.0, MeOH). Calculated for C18H18F3NO3·HCl: C 55.46%, H 4.91%, N 3.59%; Found: C 55.17%, H 4.55%, N 3.41%.

cis-3-[(S)-Hydroxy(pyridin-2-yl)methyl]-1-methyl-3-[3-(trifluoromethyl)phenyl] cyclobutanol (62e) and trans-3-[(S)-hydroxy(pyridin-2-yl)methyl]-1-methyl-3-[3(trifluoromethyl)phenyl]cyclobutanol (63e). To a -78°C solution of 59e (2.011 g, 6.26 mmol) in anhydrous THF (35 mL) was added slowly methyl lithium (9 mL, 1.6 M in diethyl ether, 14.4 mmol), and the reaction was allowed to warm to room temperature for 16 h. The reaction was quenched with 1 M aqueous KH2PO4 (50 mL), then water (200 mL) was added, extracted twice with EtOAc (200 mL), washed with brine, and dried over Na2SO4. Organic layers were concentrated under reduced pressure and the residue chromatographed on silica gel (0-10% MeOH in EtOAc) to obtain 62e (1.18 g, 56% yield) and 63e (0.31 g, 14.8% yield). Analytical data for 62e: 1H NMR (500 MHz, d3-MeCN) δ 8.29 (d, J = 4.8 Hz, 1H), 7.50 (td, J = 7.7, 1.7 Hz, 1H), 7.42 (d, J = 7.7 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.19 - 7.10 (m, 2H), 6.99 (s, 1H), 6.73 (d, J = 7.9 Hz, 1H), 4.94 (d, J = 3.9 Hz, 1H), 4.43 (d, J = 5.3 Hz, 1H), 3.41 (s, 1H), 2.93 (d, J = 12.0 Hz, 1H), 2.78 (d, J = 12.2 Hz, 1H), 2.50 - 2.40 (m, 2H), 0.99 (s, 3H). MS (ESI) m/z 338 (M+H)+. [α]D = -48.6º (c 1.0, MeOH). HRMS m/z: [M + H]+ calcd for C18H19F3NO2, 338.13624; found, 338.13653. Analytical data for 63e: 1H NMR (500 MHz, d3-MeCN) δ 8.31 (dd, J = 4.8, 0.4 Hz, 1H), 7.49 (td, J = 7.7, 1.7 Hz, 1H), 7.41 (d, J = 7.8 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 7.17 - 7.11 (m, 2H), 6.98 (s, 1H), 6.74 (d, J = 7.9 Hz, 1H), 4.90 (s, 1H), 4.16 (broad s, 1H), 3.06 - 3.00 (m, 1H), 2.92 - 2.86 (m, 1H), 2.83 (t, J = 6.9 Hz, 1H), 2.40 (d, J = 12.9 Hz, 1H), 2.34 (d, J = 12.7 Hz, 1H), 1.41 (s, 3H). MS (ESI) m/z 338 (M+H)+. [α]D = -64.2º (c 1.0, MeOH). HRMS m/z: [M + H]+ calcd for C18H19F3NO2, 338.13624; found, 338.13652.



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Compounds 62f and 63f were prepared analogously to 62c and 63c by using 4trifluoromethylphenylacetonitrile (1f) instead of 4-trifluorometoxylphenylacetonitrile (1c) in the first step of the sequence.

cis-3-[(S)-Hydroxy(pyridin-2-yl)methyl]-1-methyl-3-[4-(trifluoromethyl)phenyl] cyclobutanol (62f) and trans-3-[(S)-hydroxy(pyridin-2-yl)methyl]-1-methyl-3-[4(trifluoromethyl)phenyl]cyclobutanol (63f). Analytical data for 62f: 1H NMR (300 MHz, d6DMSO) δ 8.37 (ddd, J = 4.8, 1.8, 0.9 Hz, 1H), 7.51 - 7.40 (m, 3H), 7.13 (ddd, J = 7.5, 4.8, 1.2 Hz, 1H), 6.99 (d, J = 8.1 Hz, 2H), 6.59 (d, J = 7.9 Hz, 1H), 5.64 (d, J = 4.5 Hz, 1H), 4.98 (d, J = 4.5 Hz, 1H), 4.95 (s, 1H), 2.97 (d, J = 12.2 Hz, 1H), 2.77 (d, J = 12.0 Hz, 1H), 2.39 (dd, J = 12.1, 3.6 Hz, 1H), 2.24 (dd, J = 12.1, 3.5 Hz, 1H), 0.87 (s, 3H). MS (ESI) m/z 338 (M+H)+. [α]D = 45.0º (c 1.0, MeOH). Calculated for C18H18F3NO2·0.12 H20: C 63.68%, H 5.42%, N 4.13%; Found: C 63.69%, H 5.32%, N 4.06%. Analytical data for 63f: 1H NMR (300 MHz, d6-DMSO) δ 8.41 (ddd, J = 4.8, 1.8, 0.9 Hz, 1H), 7.51 - 7.39 (m, 3H), 7.15 (ddd, J = 7.4, 4.9, 1.0 Hz, 1H), 6.93 (d, J = 8.0 Hz, 2H), 6.57 (d, J = 7.9 Hz, 1H), 5.64 (d, J = 4.6 Hz, 1H), 4.88 (d, J = 4.6 Hz, 1H), 4.69 (s, 1H), 3.02 (d, J = 12.2 Hz, 1H), 2.88 (dd, J = 12.5, 1.4 Hz, 1H), 2.34 (d, J = 12.4 Hz, 1H), 2.22 (d, J = 12.4 Hz, 1H), 1.35 (s, 3H). MS (ESI) m/z 338 (M+H)+. [α]D = -61.1º (c 1.0, MeOH). Calculated for C18H18F3NO2: C 64.09%, H 5.38%, N 4.15%; Found: C 63.34%, H 4.86%, N 4.08%.


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cis-3-[(R)-Hydroxy(pyridin-2-yl)methyl]-1-methyl-3-[4-(trifluoromethoxy)phenyl] cyclobutanol hydrochloride (64). This compound was prepared from R-59c by the protocol described above for the synthesis of 24: 1H NMR (300 MHz, d6-DMSO) δ 8.36 (dq, J = 4.9, 0.8 Hz, 1H), 7.45 (ddd, J = 7.7, 7.7, 1.8 Hz, 1H), 7.12 (ddd, J = 7.5, 4.8, 1.1 Hz, 1H), 7.09 - 7.03 (m, 2H), 6.87 (d, J = 8.8 Hz, 2H), 6.56 (d, J = 7.9 Hz, 1H), 5.58 (d, J = 4.6 Hz, 1H), 4.94 (d, J = 4.6 Hz, 1H), 4.92 (s, 1H), 2.92 (d, J = 12.1 Hz, 1H), 2.74 (d, J = 12.1 Hz, 1H), 2.35 (dd, J = 12.1, 3.5 Hz, 1H), 2.20 (dd, J = 12.1, 3.5 Hz, 1H), 0.89 (s, 3H). MS (ESI) m/z 354 (M+H)+. [α]D = +12.7° (c 1.0, MeOH).

3-Oxocyclobutanecarbonitrile (66). To a solution of 3-methylenecyclobutanecarbonitrile (65) (9.2 g, 99 mmol) in a mixture of CH2Cl2 (175 mL), MeCN (175 mL) and water (263 mL) was added ruthenium(III) chloride trihydrate (0.61 g, 2.35 mmol) in one portion at 0°C. Sodium periodate (86 g, 400 mmol) was added portionwise over 90 min keeping the temperature below 10ºC. After the addition was completed, the reaction was diluted with CH2Cl2 (300 mL) and filtered to remove insoluble material. Organic phase was separated, and the aqueous phase was extracted twice with CH2Cl2 (200 mL). The organic layers were combined, filtered and concentrated under reduced pressure to give 66(8.4 g, 88 mmol, 89 % yield) as a white solid. 1H NMR (400 MHz, CDCl3): δ 3.49 (d, J = 8.0 Hz, 4H), 3.17-3.22 (m, 1H).

5,8-Dioxaspiro[3.4]octane-2-carbonitrile (67). To a solution of 66 (5.00 g, 52.6 mmol) in toluene (100 mL) was added ethane-1,2-diol (2.94 mL, 52.6 mmol) and p-toluenesulfonic acid (0.50 g, 2.63 mmol) at room temperature. The reaction was heated to reflux for 2 h under a



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Dean-Stark trap. The reaction mixture was washed with saturated solution of NaHCO3 (2 × 100 mL), dried over Na2SO4, filtered and concentrated to give 67 (5.6 g, 73 % yield) as a yellow oil. 1

H NMR (400 MHz, CDCl3): δ 3.84 (s, 4H), 2.70-2.84 (m, 1H), 2.64-2.68 (m, 4H).

2-(4-(Trifluoromethyl)pyridin-2-yl)-5,8-dioxaspiro[3.4]octane-2-carbonitrile (68a). To a solution of Example 67 (22.0 g, 158 mmol) and 2-fluoro-4-(trifluoromethyl)- pyridine (24.91 g, 151 mmol) in toluene (200 mL) was added potassium hexamethyldisilazide (330 mL, 0.5 M in toluene, 165 mmol), then the reaction was stirred at 60 oC for 4 h. The reaction was quenched with water (500 mL) and extracted with EtOAc (500 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (5-50% EtOAc in heptane) to give 68a (35.8 g, 84% yield). 1

H NMR (500 MHz, CD3CN) δ 8.86 (d, J = 5.1 Hz, 1H), 7.84 (s, 1H), 7.63 (d, J = 5.7 Hz, 1H),

4.00 (t, J = 6.4 Hz, 2H), 3.89 (t, J = 6.4 Hz, 2H), 3.25 - 3.18 (m, 2H), 3.13 - 3.05 (m, 2H). MS (ESI) m/z 285 (M+H)+.

2-(2-(Trifluoromethyl)pyridin-4-yl)-5,8-dioxaspiro[3.4]octane-2-carbonitrile (68b). To a solution of 4-chloro-2-(trifluoromethyl)pyridine (10.0 g, 55.1 mmol) and 67 (11.57 g, 83 mmol) in THF (100 mL) was added lithium hexamethyldisilazide (83 mL, 82.6 mmol) dropwise at 78°C and the reaction was stirred for 16 h. The reaction solution was quenched with saturated NH4Cl solution (30 mL) and the aqueous layer was extracted with EtOAc (100 mL). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether:EtOAc = 3:1) to give 68b


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(12.4 g, 78 % yield) as a yellow oil. 1H NMR (400 MHz, CDCl3): δ 8.79 (d, J = 5.2 Hz, 1H), 7.91 (d, J = 0.8 Hz, 1H), 7.74 (dd, J1 = 1.6 Hz, J2 = 5.2 Hz, 1H), 3.91-4.06 (m, 4H), 3.36 (dd, J1 = 3.2 Hz, J2 = 10.8 Hz, 2H), 2.99 (dd, J1 = 3.2 Hz, J2 = 11.2 Hz, 2H). MS (ESI) m/z 285 (M+H)+.

Pyridin-2-yl(2-(4-(trifluoromethyl)pyridin-2-yl)-5,8-dioxaspiro[3.4]octan-2-yl)methanone (69a). To a solution of 2-bromopyridine (16.22 mL, 170 mmol) in diethyl ether (60 mL) was added n- BuLi (65.5 mL, 2.5 M solution in hexanes, 164 mmol) dropwise at -78°C. after stirring reaction mixture at -78°C for 1 h, A solution of 68a (35.82 g, 126 mmol) in diethyl ether (150 mL) was added at -78°C and the reaction was stirred for 2 h at the same temperature. Aqueous HCl (3 M, 150 mL) was added dropwise slowly at -78°C. The biphasic mixture warmed to room temperature and stirred for 0.5 h, and then the mixture was basified with 3 M aqueous NaOH (200 mL). The aqueous phase was separated and extracted twice with EtOAc (400 mL), and the combined organic phases were washed with brine (400 mL), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (0-50% EtOAc in heptane) to give 69a (36.3 g, 79 % yield). 1H NMR (400 MHz, CD3CN) δ 8.49 (d, J = 5.1 Hz, 1H), 8.31 (ddd, J = 4.7, 1.7, 0.9 Hz, 1H), 8.08 - 8.03 (m, 2H), 7.87 (td, J = 7.7, 1.7 Hz, 1H), 7.41 - 7.35 (m, 2H), 3.94 - 3.82 (m, 4H), 3.28 - 3.21 (m, 2H), 3.04 - 2.96 (m, 2H). MS (DCI) m/z 365 (M+H)+.

Pyridin-2-yl(2-(2-(trifluoromethyl)pyridin-4-yl)-5,8-dioxaspiro[3.4]octan-2-yl)methanone (69b). To a solution of 2-bromopyridine (25.5 g, 161 mmol) in THF (50 mL) was added n-



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butyllithium (95 mL, 1.6 M in hexanes, 152 mmol) dropwise at -78°C and the reaction was stirred for 30 min. Compound 68b (27.8 g, 98 mmol) in diethyl ether (120 mL) was added and the reaction was stirred at -78°C for 4 h. After acidification by the addition of 2 N HCl to pH = 4, the reaction was stirred for 15 min and extracted twice with EtOAc (100 mL). The organic phase was separated, washed with brine (200 mL), dried over MgSO4 and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether:EtOAc = 3:1) to give 69b (34.0 g, 86 % yield) as a yellow oil. MS (ESI) m/z 365 (M+H)+.

Pyridin-2-yl(2-(4-(trifluoromethyl)pyridin-2-yl)-5,8-dioxaspiro[3.4]octan-2-yl)methanol (70a). To a solution of Example 69a (36.33 g, 100 mmol) in CH2Cl2 (300 mL) and MeOH (30 mL) was added NaBH4 (4.15 g, 110 mmol) in one portion, and then the reaction was stirred at room temperature for 3 h. To the reaction mixture was added 3 N NaOH (300 mL) and extracted with EtOAc (450 mL). The combined organic phases were washed twice with brine (450 mL), dried over Na2SO4, filtered and the residue was purified by column chromatography on silica gel (0-100% EtOAc in heptane) to obtain 70a (30.8 g, 84 % yield). 1H NMR (400 MHz, CD3CN) δ 8.57 (d, J = 5.1 Hz, 1H), 8.30 (ddd, J = 4.8, 1.6, 1.0 Hz, 1H), 7.50 (td, J = 7.7, 1.8 Hz, 1H), 7.42 - 7.37 (m, 1H), 7.29 - 7.24 (m, 1H), 7.13 (ddd, J = 7.5, 4.9, 1.1 Hz, 1H), 6.79 (d, J = 7.9 Hz, 1H), 5.04 (d, J = 6.6 Hz, 1H), 4.40 (d, J = 6.6 Hz, 1H), 3.90 - 3.84 (m, 2H), 3.80 - 3.73 (m, 2H), 3.09 (ddd, J = 13.0, 2.8, 0.7 Hz, 1H), 3.01 (ddd, J = 13.1, 2.7, 0.6 Hz, 1H), 2.79 (ddd, J = 13.1, 3.6, 0.8 Hz, 1H), 2.69 (ddd, J = 13.0, 3.6, 0.9 Hz, 1H). MS (ESI) m/z 367 (M+H)+.


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Pyridin-2-yl{2-[2-(trifluoromethyl)pyridin-4-yl]-5,8-dioxaspiro[3.4]oct-2-yl}methanol (70b). To a solution of 69b (34.0 g, 93 mmol) in CH2Cl2 (50 mL) and MeOH (5 mL) was added NaBH4 (5.30 g, 140 mmol) and the reaction was stirred at room temperature for 3 h. After addition of water (35 mL), the reaction mixture was extracted twice with CH2Cl2 (60 mL). The organic phase was separated, washed with brine (200 mL), dried over MgSO4 and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether:EtOAc = 2:1 to 1:1) to give 70b (20.6 g, 60.3 % yield) as a colorless oil. 1H NMR (400 MHz, CDCl3): δ 8.49 (d, J = 5.2 Hz, 1H), 8.26 (d, J = 4.8 Hz, 1H), 7.55-7.61 (m, 1H), 7.13-7.17 (m, 1H), 6.99-7.04 (m, 2H), 6.94 (s, 1H), 5.13 (d, J = 6.4 Hz, 1H), 4.43 (d, J = 7.2 Hz, 1H), 3.974.01 (m, 2H), 3.84-3.89 (m, 2H), 3.09-3.23 (m, 2H), 2.64-2.71 (m, 2H). MS (ESI) m/z 367 (M+H)+.

(S)-3-(Hydroxy(pyridin-2-yl)methyl)-3-(4-(trifluoromethyl)pyridin-2-yl)cyclobutanone (71a) and (R)-3-(Hydroxy(pyridin-2-yl)methyl)-3-(4-(trifluoromethyl)pyridin-2yl)cyclobutanone ((R)-71a). To a solution of 70a (28.6 g, 78 mmol) in acetone (112 mL) was added 6 N HCl (56 mL) and the reaction was stirred at room temperature for 16 h. The reaction mixture was basified with 3 N NaOH (120 mL), extracted twice with EtOAc (200 mL) and the combined organic phases were washed with brine (100 mL), dried over Na2SO4 and concentrated to give an enantiomeric mixture of keto-alcohols (25 g, 76 mmol, 97 % yield) as a white solid. This mixture was separated on a chiral column ChiralPak AD-H, 50 ×250 mm, 5 μm; CO2/isopropanol/diethylamine =85/25/0.1) to give 71a (10.7g, 43% yield) as well as its Renantiomer (R)-71a (10.2g, 40% yield). Analytical data for 71a: 1H NMR (400 MHz, CD3CN) δ 8.47 (d, J = 5.1 Hz, 1H), 8.20 (ddd, J = 4.9, 1.6, 1.0 Hz, 1H), 7.34 (td, J = 7.7, 1.8 Hz, 1H), 7.30



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- 7.26 (m, 1H), 7.24 - 7.19 (m, 1H), 7.00 (ddd, J = 7.5, 4.9, 1.1 Hz, 1H), 6.55 (d, J = 7.9 Hz, 1H), 4.97 (d, J = 6.2 Hz, 1H), 4.39 (d, J = 6.3 Hz, 1H), 3.55 (ddd, J = 17.4, 5.6, 2.5 Hz, 1H), 3.41 (ddd, J = 17.6, 5.6, 2.3 Hz, 1H), 3.29 (ddd, J = 17.5, 3.9, 2.4 Hz, 1H), 3.19 (ddd, J = 17.7, 3.9, 2.5 Hz, 1H). MS (ESI) m/z 323 (M+H)+. [α]D20 = -21.8 º (c = 1.0, MeOH). Analytical data for (R)-71a: 1H NMR (500 MHz, CD3CN) δ 8.47 (d, J = 5.1 Hz, 1H), 8.23 - 8.17 (m, 1H), 7.34 (td, J = 7.7, 1.7 Hz, 1H), 7.29 (dd, J = 5.1, 0.7 Hz, 1H), 7.21 (d, J = 0.5 Hz, 1H), 7.00 (ddd, J = 7.3, 4.9, 0.9 Hz, 1H), 6.55 (d, J = 7.9 Hz, 1H), 4.97 (d, J = 6.1 Hz, 1H), 4.39 (d, J = 6.2 Hz, 1H), 3.55 (ddd, J = 17.4, 5.6, 2.4 Hz, 1H), 3.41 (ddd, J = 17.7, 5.6, 2.3 Hz, 1H), 3.29 (ddd, J = 17.4, 3.9, 2.4 Hz, 1H), 3.19 (ddd, J = 17.7, 3.9, 2.5 Hz, 1H). MS (ESI) m/z 323 (M+H)+. [α]D20 = +21.2 º (c = 1.0, MeOH).

3-[(S)-Hydroxy(pyridin-2-yl)methyl]-3-[2-(trifluoromethyl)pyridin-4-yl]cyclobutanone (71b). To a solution of 70b (20.1 g, 54.9 mmol) in acetone (100 mL) was added 6 N HCl (50.0 mL) and the reaction was stirred for 16 h. The reaction was basified with 3 N NaOH to keep pH = 8 and extracted twice with EtOAc (60 mL). The organic phase was washed by brine (100 mL), dried over MgSO4 and concentrated under reduced pressure to give enantiomeric mixture of alcohols (17.0 g, 92.0% yield) as a yellow solid. Chiral HPLC separation on ChiralPak AD-H column (30 ×250 mm, 5 μm; mobile phase CO2/EtOH/diethylamine =90/10/0.1) gave 71b (5.0 g, 29% yield) as a white solid. 1H NMR: (400 MHz, CDCl3): δ 8.62 (t, J = 3.6 Hz, 1H), 8.47 (d, J = 4.8 Hz, 1H), 7.48-7.53 (m, 1H), 7.14-7.25 (m, 1H), 7.14 (t, J = 1.6 Hz, 2H), 6.51 (d, J = 8.0 Hz, 1H), 4.97 (s, 1H), 4.75 (broad s, 1H), 3.88-3.94 (m, 1H), 3.74-3.81 (m, 1H), 3.30-3.60 (m, 1H), 3.17-3.23 (m, 1H). MS (ESI) m/z 323 (M+H)+. [α]D20 = -42.4 º (c = 1.0 in MeOH).


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trans-3-[(S)-Hydroxy(pyridin-2-yl)methyl]-3-[4-(trifluoromethyl)pyridin-2-yl]cyclobutanol (72a) and cis-3-[(S)-hydroxy(pyridin-2-yl)methyl]-3-[4-(trifluoromethyl)pyridin-2yl]cyclobutanol (73a). To a solution of 71a (4.5 g, 13.96 mmol) in CH2Cl2 (30 mL) and MeOH (3.00 mL) was added NaBH4 (0.63 g, 16.76 mmol) at 0°C, then the reaction was stirred at room temperature for 3 h. To the reaction mixture was added water (10 mL), extracted twice with CH2Cl2 (40 mL), and the combined organic phases were dried over Na2SO4, filtered and concentrated under reduced pressure. The crude mixture was subjected to chiral separation on a chiral column ChiralPak AD-H, 50 ×250 mm, 5 μm; CO2/isopropanol/diethylamine =85/25/0.1) to give 72a (1.05 g, 24% yield, >99% ee) and 73a (1.7 g, 39% yield, >99% ee) as white solids. Analytical data for 72a: 1H NMR: (400 MHz, 400 MHz, CDCl3): δ 8.66 (d, J = 5.2 Hz, 1H), 8.44 (d, J = 4.0 Hz, 1H), 7.42-7.47 (td, J = 2.0 Hz, 1H), 7.30-7.33 (dd, J1 =0.8 Hz, J2 = 4.8 Hz, 1H), 7.10-7.16 (m, 1H), 7.06 (s, 1H), 6.59 (d, J = 8.0 Hz , 1H), 4.98 (d, J = 4.8 Hz, 1H), 4.82 (d, J = 6.4 Hz, 1H), 4.36 (t, J = 6.8 Hz, 1H), 3.12-3.25 (m, 2H), 2.23-2.41 (m, 2H). MS (ESI) m/z 325 (M+H)+. [α]D20 = -53.9 º (c = 1.0, MeOH). HRMS m/z: [M + H]+ calcd for C16H16F3N2O2, 325.11584; found, 325.11500. Analytical data for 73a: 1H NMR: (400 MHz, 400 MHz, CDCl3) : δ 8.68 (d, J = 5.2 Hz, 1H), 8.41 (d, J = 4.8 Hz, 1H), 7.30-7.40 (m, 2H), 7.11 (d, J = 6.4 Hz, 2H), 6.35 (d, J = 8.0 Hz, 1H), 5.23 (d, J = 6.0 Hz, 1H), 5.03 (d, J =6.0 Hz, 1H), 4.15 (d, J = 6.0 Hz, 1H), 2.95-2.98 (m, 1H), 2.75-2.80 (m, 2H), 2.56-2.60 (m, 1H). MS (ESI) m/z 325 (M+H)+. [α]D20 = -32.6 º(c = 1.0, MeOH).



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trans-3-[(S)-hydroxy(pyridin-2-yl)methyl]-3-[5-(trifluoromethyl)pyridin-3-yl]cyclobutanol (72b) and cis-3-[(S)-hydroxy(pyridin-2-yl)methyl]-3-[5-(trifluoromethyl)pyridin-3yl]cyclobutanol (73b). To a solution of 71b (80 mg, 0.248 mmol) in CH2Cl2 (5 mL) and MeOH (0.5 mL) was added NaBH4 (9.39 mg, 0.248 mmol), then the reaction was stirred at room temperature for 3 h. After addition of H2O (5 mL), the mixture was extracted twice with CH2Cl2 (15 mL) and the organic phase was washed with brine (20 mL) and dried over MgSO4. After removal of solvent, the residue (80 mg, 99 % yield) was resolved by chiral HPLC ChiralPak ADH column (30 ×250 mm, 5 μm; mobile phase CO2/EtOH/DEA =85/15/0.1) to give 72b (10 mg, 12.5% yield) and 73b (40 mg, 50% yield) as white solids. Analytical data for 72b: 1H NMR: (400 MHz, 400 MHz, CDCl3) : δ 8.55 (s, 1H), 8.29 (d, J = 4.4 Hz, 1H ), 8.14 (d, J = 1.6 Hz, 1H ), 7.45-7.49 (m, 1H), 7.08-7.13 (m, 2H), 6.68 (d, J = 7.6 Hz, 1H), 4.74 (s, 1H), 4.48 (t, J = 7.2 Hz, 1H), 3.12-3.19 (m, 2H), 2.12-2.21 (m, 2H). MS (ESI) m/z 325 (M+H)+. HRMS m/z: [M + H]+ calcd for C16H16F3N2O2, 325.11584; found, 325.11597. Analytical data for 73b: 1H NMR: (400 MHz, 400 MHz, CDCl3) : δ 8.57 (s, 1H), 8.27 (d, J = 1.6 Hz, 1H ), 8.24 (d, J = 4.8 Hz, 1H ), 7.42-7.47 (m, 1H), 7.22 (s, 1H), 7.07 (dd, J1 = 4.8 Hz, J2 = 2.0 Hz, 1H ), 6.61 (d, J = 8.0 Hz, 1H), 4.87 (s, 1H), 4.03-4.09 (m, 1H), 2.64-2.73 (m, 4H). MS (ESI) m/z 325 (M+H)+. HRMS m/z: [M + H]+ calcd for C16H16F3N2O2, 325.11584; found, 325.11559.

cis-3-[(S)-Hydroxy(pyridin-2-yl)methyl]-1-methyl-3-[4-(trifluoromethyl)pyridin-2yl]cyclobutanol (74a) and trans-3-[(S)-hydroxy(pyridin-2-yl)methyl]-3-[4(trifluoromethyl)pyridin-2-yl]cyclobutanol (75a). To a -78°C solution of 71a (10.7 g, 33.2 mmol) in anhydrous THF (250 mL) was slowly added methyl lithium (46 mL, 1.6 M in diethyl ether, 73.6 mmol), and the reaction mixture was allowed to warm to room temperature for 7 h.


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The reaction was quenched with 1 M aqueous KH2PO4 (200 mL), then water (200 mL) was added, extracted twice with EtOAc (400 mL), washed with brine, and dried over Na2SO4. Organic layers were concentrated under reduced pressure and the residue chromatographed on silica gel (0-10% MeOH in MeCN: CH2Cl2 1:2) to obtain 74a (4.49 g, 40.1% yield) and 75a (1.57 g, 14% yield). Analytical data for 74a: 1H NMR (500 MHz, CD3CN) δ 8.58 (d, J = 5.1 Hz, 1H), 8.28 (d, J = 4.8 Hz, 1H), 7.49 (td, J = 7.7, 1.7 Hz, 1H), 7.37 (d, J = 5.0 Hz, 1H), 7.18 (s, 1H), 7.12 (ddd, J = 7.4, 4.9, 0.7 Hz, 1H), 6.72 (d, J = 7.9 Hz, 1H), 4.98 (s, 1H), 4.61 (s, 1H), 3.43 (s, 1H), 2.85 (d, J = 12.4 Hz, 1H), 2.74 (d, J = 12.6 Hz, 1H), 2.63 (dd, J = 12.5, 3.3 Hz, 1H), 2.55 (dd, J = 12.5, 3.4 Hz, 1H), 0.97 (s, 3H). MS (ESI) m/z 339 (M+H)+. [α]D = -42.5º (c 1.0, MeOH). HRMS m/z: [M + H]+ calcd for C17H18F3N2O2, 339.13149; found, 339.13190. Analytical data for 75a: MS: 339.0 [M+H]+; 1H NMR: (400 MHz, 400 MHz, CDCl3): δ 8.60 (d, J = 4.8 Hz, 1H), 8.43 (d, J = 4.8 Hz, 1H), 7.49-7.50 (m, 1H), 7.25-7.30 (m, 2H), 7.14 (dd, J1 = 4.8 Hz, J2 = 7.2 Hz, 1H), 6.72 (d, J = 8.0 Hz, 1H), 5.06 (s, 1H), 4.85 (br, 1H), 3.47 (s, 1H), 2.99 (d, J = 13.2 Hz, 1H), 2.89 (d, J = 13.2 Hz, 1H), 2.64 (dd, J1 = 2.8 Hz, J2 = 9.2 Hz, 1H), 2.48 (dd, J1 = 2.8 Hz, J2 = 9.2 Hz, 1H), 1.39 (s, 3H). MS (ESI) m/z 339 (M+H)+. [α]D20 = -59.8 º (c = 1.0, MeOH). HRMS m/z: [M + H]+ calcd for C17H18F3N2O2, 339.13149; found, 339.13171.

cis-3-[(S)-Hydroxy(pyridin-2-yl)methyl]-1-methyl-3-[2-(trifluoromethyl)pyridin-4yl]cyclobutanol (74b) and trans-3-[(S)-hydroxy(pyridin-2-yl)methyl]-1-methyl-3-[2(trifluoromethyl)pyridin-4-yl]cyclobutanol (75b). To a solution of Example 71b (2.8g, 8.69 mmol) in THF (50 mL) was added methyllithium (6.37 mL, 3.0 M in diethyl ether, 19.11 mmol) dropwise at -78oC and then the reaction was stirred for 16 h slowly allowing to reach room temperature. The reaction mixture was quenched with saturated KH2PO4 solution (30 mL) and



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extracted twice with EtOAc (50 mL). The combined organic phases were washed by brine (50 mL), dried over MgSO4, filtered and concentrated under reduced pressure. The crude mixture was separated by chiral HPLC on ChiralPak AD-H column (CO2/EtOH/Hexane/DEA =85/7.5/7.5/0.1) to give 74b (0.6 g, 21 % yield) and 75b (0.1 g, 3.4% yield) as white solids. Analytical data for 74b: MS: 1H NMR: (400 MHz, 400 MHz, CDCl3) : δ 8.49 (d, J = 4.8 Hz, 1H), 8.32 (d, J = 4.4 Hz, 1H), 7.35-7.40 (m, 1H), 7.09-7.12 (m, 1H), 6.93-6.97 (m, 2H), 6.31 (d, J = 8.0 Hz, 1H), 4.83 (s,1H), 2.79-2.97 (dd, J1 = 12.8 Hz, J2 = 59.2Hz, 2H), 2.30-2.48 (dd, J1 = 12.8 Hz, J2 = 58.4 Hz, 2H), 1.14 (s, 3H). MS (ESI) m/z 339 (M+H)+. HRMS m/z: [M + H]+ calcd for C17H18F3N2O2, 339.13149; found, 339.13190. Analytical data for 75b: 1H NMR: (400 MHz, 400 MHz, CDCl3):δ 8.44 (d, J = 5.2 Hz, 1H), 8.24 (d, J = 4.8 Hz, 1H), 7.45-7.50 (m, 1H), 7.07-7.11 (m, 1H), 6.96-7.00 (m, 2H), 6.69 (d, J = 8.0 Hz, 1H), 4.80 (d, J = 6.0 Hz, 1H), 4.48 (d, J = 6.4 Hz, 1H), 2.92-2.97 (m, 2H), 2.35-2.41 (m, 2H), 1.47 (s, 3H). MS (ESI) m/z 339 (M+H)+. HRMS m/z: [M + H]+ calcd for C17H18F3N2O2, 339.13149; found, 339.13124.

Compound 76 was prepared analogously to 74a by using (R)-71a instead of 71a.

cis-3-[(R)-Hydroxy(pyridin-2-yl)methyl]-1-methyl-3-[4-(trifluoromethyl)pyridin-2yl]cyclobutanol (76). 1H NMR (400 MHz, CD3CN) δ 8.59 (d, J = 5.1 Hz, 1H), 8.28 (d, J = 4.8 Hz, 1H), 7.49 (td, J = 7.7, 1.3 Hz, 1H), 7.37 (d, J = 5.1 Hz, 1H), 7.18 (s, 1H), 7.12 (dd, J = 7.4, 4.9 Hz, 1H), 6.72 (d, J = 7.9 Hz, 1H), 4.98 (d, J = 6.3 Hz, 1H), 4.60 (d, J = 6.3 Hz, 1H), 3.41 (s, 1H), 2.85 (d, J = 12.4 Hz, 1H), 2.74 (d, J = 12.6 Hz, 1H), 2.63 (dd, J = 12.4, 3.3 Hz, 1H), 2.55 (dd, J = 12.6, 3.3 Hz, 1H), 0.97 (s, 3H). MS (ESI) m/z 339 (M+H)+. [α]D20 = +43.1 º (c = 1.0, MeOH). HRMS m/z: [M + H]+ calcd for C17H18F3N2O2, 339.13149; found, 339.13169.


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TRPV3 In Vitro Activity: Ca2+ Flux Assay. Experiments were performed using a fluorometric imaging plate reader (FLIPRTETRA) high-throughput cellular screening system. One day before the experiment, recombinant HEK293 cells stably expressing human TRPV3 (NCBI accession NP_659505 with an I25V substitution) were plated in growth medium [high glucose (4.5 mg/mL)-containing Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum] in black-walled, clear-bottom, 384-well poly-D-lysine-coated assay plates (Greiner BioOne GmbH, Frickenhausen, Germany) by using a Multidrop dispenser (Thermo Fisher Scientific, Waltham, MA) and incubated in a humidified 5% CO2 incubator at 37 °C. On the day of the experiment, growth medium was removed and 30 μL of no-wash FLIPR Calcium 4 dye (Molecular Devices, Sunnyvale, CA)(λEX 470−495 nm; λEM 515−575 nm) prepared in modified D-PBS (Ca2+- and Mg2+-free, supplemented with 20 mM HEPES and pH adjusted to 7.4 with NaOH) were added to each well by using a Multidrop dispenser. Cells were incubated for 1-2 hr in the dark at room temperature. Test compounds were dissolved in DMSO, and plates were prepared by robotic dispensing. An initial 3 min baseline period was monitored following the addition of 10 μL of 5X concentrated compound serial dilutions which were suspended in D-PBS (Ca2+ and Mg2+ free, supplemented with 20 mM HEPES and adjusted to pH 7.4 with NaOH). A DMSO stock of 2-APB (Tocris Bioscience, Bristol, UK) was initially diluted at 5X concentration prepared in modified D-PBS (with 0.9 mM Ca2+ and 0.5 mM Mg2+, supplemented with 20 mM HEPES and adjusted to pH 7.4 with NaOH). TRPV3 channels were then activated by addition of 10 μL of 2-APB (60 µM final concentration) to the cells, and the resulting Ca2+ flux was measured for an additional 3 min. 2-APB concentration response curves were generated on each



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plate for Kb determinations. The intensity of the fluorescence was measured and the peak increase in fluorescence over baseline (relative fluorescence units) was calculated and expressed as the percentage of the maximal agonist response. Compound potency was calculated from curve fits using a four-parameter logistic Hill equation.

Associated Content 2-APB concentration-response curve, pharmacological selectivity assay data for 74a, cardiovascular and in vivo efficacy study protocols and results for 74a, structure elucidation data by NMR, and crystallographic analysis (CIF). Molecular formula strings (CSV)

Accession Codes Coordinates, anisotropic temperature factors, bond lengths, and bond angles have been deposited at the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, United Kingdom, http://www.ccdc.cam.ac.uk, under the following deposition numbers: 1455331 (5a), 1455332 (5a HCl), 1455333 ((R)-5h), 1455334 (15 HCl), 1455335 (37a)

Author Information *Phone: 847-935-4214. E-mail: [email protected]

Disclosures


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All authors are employees, former employees, or retirees of Abbott/AbbVie. This study was sponsored by AbbVie. AbbVie contributed to the study design, research, and interpretation of data, writing, reviewing, and approving the publication.

Acknowledgements We thank Melissa Vos and Marc Lake for preparation of the pcDNA3.1/human TRPV3 expression vector and the recombinant HEK293 cells used to characterize in vitro pharmacology of TRPV3 antagonists. Abbreviations Used 2-APB, 2-aminoethyl diphenylborinate; AUC0-8 , area under plasma concentration time curve from time 0 to 8h; Clint u, microsomal intrinsic clearance unbound; F(po), oral bioavailability; FLIPR, fluorescent imaging plate reader; fSP3, fraction of SP3-hibridized carbon atoms; HLM, human liver microsomes; KHMDS, potassium hexamethyldisilazide; LLE, ligand-lipophilicity efficiency;

MPO,

multi-parameter

optimization;

PFI,

property

forecast

index;

PK,

pharmacokinetic; RLM, rat liver microsomes; T1/2, terminal half-life; TPSA, topological polar surface area; TRPV3, transient potential vanilloid 3; References

1. Peier, A.M.; Reeve, A.J.; Andersson, D.A.; Moqrich, A.; Earley, T.J.; Hergarden, A.C.; Story, G. M.; Colley, S.; Hogenesch, J.B.; McIntyre, P.; Bevan, S.; Patapoutian, A. A heatsensitive TRP channel expressed in keratinocytes. Science 2002, 296, 2046–2049. 2. Smith, G.D.; Gunthorpe, M.J.; Kelsell, R.E.; Hayes, P.D.; Reilly P.; Facer, P.; Wright J.E.; Jerman, J.C.; Walhin, J.P.; Ooi, L; Egerton, J.; Charles, K.J.; Smart, D.; Randall, A.D.;



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Anand, P.; Davis, J.B. TRPV3 is a temperature-sensitive vanilloid receptor-like protein. Nature 2002, 418, 186-90. 3. Xu, H.; Ramsey, I.S.; Kotecha, S.A.; Moran, M.M.; Chong, J.A.; Lawson, D.; Ge, P.; Lilly, J.; Silos-Santiago, I.; Xie, Y.; DiStefano, P.S.; Curtis, R.; Clapham, D.E. TRPV3 is a calcium-permeable temperature-sensitive cation channel. Nature 2002, 418, 181-186. 4. Facer, P.; Casula, M.A.; Smith, G.D.; Benham, C.D.; Chessell, I.P.; Bountra, C.; Sinisi, M.; Birch, R.; Anand, P. Differential expression of the capsaicin receptor TRPV1 and related novel receptors TRPV3, TRPV4 and TRPM8 in normal human tissues and changes in traumatic and diabetic neuropathy. BMC Neurol. 2007, 7, 11. 5. Gopinath, P.; Wan, E.; Holdcroft, A.; Facer, P.; Davis, J.B.; Smith G.D.; Bountra, C.; Anand, P. Increased capsaicin receptor TRPV1 in skin nerve fibres and related vanilloid receptors TRPV3 and TRPV4 in keratinocytes in human breast pain. BMC Women’s Health 2005, 5, 2. 6. Bang, S.; Yoo, S.; Yang, T.J.; Cho, H.; Hwang, S.W. Farnesyl pyrophosphate is a novel painproducing molecule via specific activation of TRPV3. J. Biol. Chem. 2010, 285, 1936219371. 7. Chung, M.K.; Lee, H.; Mizuno, A.; Suzuki, M.; Caterina, M.J. 2-aminoethoxydiphenyl borate activates and sensitizes the heat-gated ion channel TRPV3. J. Neurosci. 2004, 24, 5177-5182. 8. Borbíró, I.; Lisztes, E.; Tóth, B.I.; Czifra, G.; Oláh, A.; Szöllosi, A.G.; Szentandrássy, N.; Nánási, P.P.; Péter, Z.; Paus, R.; Kovács, L.; Bíró, T. Activation of transient receptor potential vanilloid-3 inhibits human hair growth. J. Invest. Dermatol. 2011, 131, 1605-1614. 9. Cheng, X.; Jin, J.; Hu, L.; Shen, D.; Dong, X.P.; Samie, M.A.; Knoff, J.; Eisinger, B.; Liu, M.L.; Huang, S.M.; Caterina, M.J.; Dempsey, P.; Michael, L.E.; Dlugosz, A.A.; Andrews,


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Table of Contents graphic

OH N

OH

Cl

N

N

CF3

Cl HO

74a

5a TRPV3 FLIPR Kb = 0.08 µM TRPV3 FLIPR Kb = 12.0 µM (plasma adjusted) rat/human Clint u (L/h/kg) = 151 / 41

TRPV3 FLIPR Kb = 0.56 µM TRPV3 FLIPR Kb = 0.62 µM (plasma adjusted) rat/human Clint u (L/h/kg) = 6 / 2


Synthesis and Pharmacology of (Pyridin-2-yl)methanol Derivatives as

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