Identification and Preclinical Pharmacology of ((1R, 3S)-1-amino-3-((S

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Identification and Preclinical Pharmacology of ((1R,3S)-1-amino-3((S)-6-(2-methoxyphenethyl)-5,6,7,8-tetrahydronaphthalen-2yl)cyclopentyl)methanol (BMS-986166): A Differentiated Sphingosine-1Phosphate (S1P1) Receptor Modulator Advanced into Clinical Trials John Gilmore, Hai-Yun Xiao, T. G. Murali Dhar, Michael G. Yang, Zili Xiao, Jenny H. Xie, Lois D. LehmanMcKeeman, Lei L Gong, Huadong Sun, Lloyd LeCureux, Cliff Chen, Dauh-Rurng Wu, Marta Dabros, Xiaoxia Wang, Tracy Taylor, Xia D Zhou, Elizabeth Heimrich, Rochelle Thomas, Kim W. McIntyre, Virna Borowski, Bethanne M. Warrack, Yuwen Li, Hong Shi, Paul C. Levesque, Zheng Yang, Anthony M. Marino, Georgia Cornelius, Celia J. D'Arienzo, Arvind Mathur, Richard A. Rampulla, Anuradha Gupta, Bala Pragalathan, Ding Ren Shen, Mary Ellen Cvijic, Luisa M. Salter-Cid, Percy H Carter, and Alaric J. Dyckman J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01695 • Publication Date (Web): 20 Feb 2019 Downloaded from http://pubs.acs.org on February 20, 2019

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Journal of Medicinal Chemistry

Yang, Zheng; Bristol-Myers Squibb Pharmaceutical Research and Development, Metabolism and Pharmacokinetic Department, Pharmaceutical Candidate Optimization Marino, Anthony; Bristol-Myers Squibb Pharmaceutical Research and Development, PCO-MAP Cornelius, Georgia; Bristol-Myers Squibb Pharmaceutical Research and Development, PCO D'Arienzo, Celia; Bristol Myers Squibb Co, Discovery Chemistry Mathur, Arvind; Bristol-Myers Squibb Pharmaceutical Research and Development, Discovery Chemistry Rampulla, Richard; Bristol-Myers Squibb Pharmaceutical Research and Development, Discovery Chemistry Gupta, Anuradha; Syngene International Ltd, Biocon BMS Research Center Pragalathan, Bala; Syngene International Pvt Ltd, Biocon BMS Research Center Plot 2 & 3 Bommasandra IV Phase Shen, Ding; Bristol Myers Squibb Co, Immunology Biology Cvijic, Mary; Bristol-Myers Squibb Pharmaceutical Research and Development, Leads Discovery & Optimization Salter-Cid, Luisa; Bristol Myers Squibb Co, Discovery Chemistry Carter, Percy; BMS, R&D Dyckman, Alaric; Bristol Myers Squibb Co, Discovery Chemistry

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Journal of Medicinal Chemistry 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

Identification and Preclinical Pharmacology of ((1R,3S)-1-amino-3-((S)-6-(2methoxyphenethyl)-5,6,7,8-tetrahydronaphthalen2-yl)cyclopentyl)methanol (BMS-986166): A Differentiated Sphingosine-1-Phosphate (S1P1) Receptor Modulator Advanced into Clinical Trials John L. Gilmore*, Hai-Yun Xiao, T. G. Murali Dhar, Michael Yang, Zili Xiao, Jenny Xie, Lois D. Lehman-McKeeman, Lei Gong, Huadong Sun, Lloyd Lecureux, Cliff Chen, Dauh-Rurng Wu, Marta Dabros, Xiaoxia Yang, Tracy L. Taylor, Xia D. Zhou, Elizabeth M. Heimrich, Rochelle Thomas, Kim W. McIntyre, Virna Borowski, Bethanne M. Warrack, Yuwen, Li, Hong Shi, Paul C. Levesque, Zheng Yang, Anthony M. Marino, Georgia Cornelius, Celia J. D’Arienzo, Arvind Mathur, Richard Rampulla, Anuradha Gupta, Bala Pragalathan, Ding Ren Shen, Mary Ellen Cvijic, Luisa M. Salter-Cid, Percy H. Carter, and Alaric J. Dyckman

Bristol-Myers Squibb Research and Development, P.O Box 4000, Princeton, NJ 08543-4000

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ABSTRACT

Recently, our research group reported the identification of BMS-986104 (2) as a differentiated S1P1 receptor modulator. In comparison to fingolimod (1), a full agonist of S1P1 currently marketed for the treatment of relapse remitting multiple sclerosis (RRMS), 2 offers several potential advantages having demonstrated improved safety multiples in preclinical evaluations against undesired pulmonary and cardiovascular effects. In clinical trials, 2 was found to exhibit a pharmacokinetic half-life (T1/2) longer than that of 1, as well as a reduced formation of the phosphate metabolite that is required for activity against S1P1. Herein, we describe our efforts to discover highly potent, partial agonists of S1P1 with a shorter T1/2 and increased in vivo phosphate metabolite formation. These efforts culminated in the discovery of BMS-986166 (14a), which was advanced to human clinical evaluation. The pharmacokinetic/pharmacodynamic (PK/PD) relationship as well as pulmonary and cardiovascular safety assessments will be discussed. Furthermore, efficacy of 14a in multiple preclinical models of autoimmune disease are presented.

INTRODUCTION Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite formed through the phosphorylation of sphingosine by sphingosine kinases.1,2

S1P regulates a multitude of

physiological processes including lymphocyte trafficking, cardiac function, vascular development, and inflammation. These biological functions are mediated through the interaction of S1P with five G-protein coupled receptors S1P1-5.3 Agonism of the S1P1 receptor has been shown to block lymphocyte trafficking from the thymus and secondary lymph nodes resulting in immunosuppression.4-8 Because of the ability of S1P1 receptor agonists to suppress lymphocyte 2 ACS Paragon Plus Environment


egress, they have great potential as therapeutic agents in a variety of autoimmune diseases including rheumatoid arthritis, lupus, multiple sclerosis, and inflammatory bowel disease. In 2010, Fingolimod (1) was approved by the FDA for the treatment of relapsing-remitting multiple sclerosis (RRMS).9 Compound 1 is a non-selective S1P receptor agonist which is dosed as a pro-drug and subsequently metabolized in vivo via phosphorylation to form 1-P, in a manner similar to the conversion of sphingosine to S1P.10 It has been reported that 1-P induces S1P1 receptor internalization and degradation on lymphocytes and behaves as a functional antagonist to inhibit S1P induced cell migration.11 As a result, lymphocytes are sequestered in the thymus and secondary lymphoid organs, leading to a marked peripheral lymphopenia. In a variety of S1P1 functional assays, such as GTPS, ERK phosphorylation, receptor internalization, cAMP and calcium mobilization, 1-P is a full agonist, eliciting a response comparable to that of the endogenous ligand S1P. Dosing with 1 has been shown to cause a transient, dose-dependent decrease of heart rate and a decline in pulmonary function in clinical studies.12,13 While these side effects were originally attributed to the off-target activity at the S1P3 receptor, recent clinical evaluations of S1P3-sparing S1P1 full agonists have implicated agonism of S1P1 as contributing to at least the heart rate effects.14


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NH2 OH NH2

OR

OR 1, Fingolimod, (R = H) 1-P (R= -PO3H2)

2, BMS-986104, (R = H) 2-P (R= -PO3H2) selective S1P1 partial agonist

non-selective S1P1 full agonist

NH2

OH N O

R1

N O

O

n

BMS-824

Cl

Ph

OH

N

F3C N

COOH

S

8-17 R1 = H, OMe n = 0-2

OH NH2

NH2 OH

OH

KRP-203

R2 Representative S1P1 Inhibitors utilizing aryl and heteroaryl groups in place of alkyl tail

23-30 R2 = heteroaryls

Figure 1. Incorporation of aryl and heteroaryl side chains

Previous research efforts from our laboratories led to the discovery of the clinical compound BMS-986104 (2).15a

This compound is a novel S1P1 receptor modulator that

demonstrates ligand-biased signaling through S1P1 and excellent selectivity against S1P3. Despite being a partial agonist of S1P1 in certain S1P1/CHO cell assays such as receptor internalization, 2 was shown to block S1P induced T cell migration completely in vitro and to induce a comparable level of lymphopenia and efficacy in rodents comparable to 1. These results provided evidence that efficacy and lymphopenia can be fully achieved without complete desensitization of the S1P1 receptor. More importantly, 2 is differentiated from 1 in terms of pulmonary safety based on preclinical rodent studies and in cardiovascular safety based on preclinical studies using human cardiomyocytes.15a However, 2 was found to exhibit a T1/2 in human clinical studies longer than was observed for 1 (18 days vs. 7-9 days). Additionally, these clinical studies showed the 4 ACS Paragon Plus Environment


formation of the active phosphate metabolite, 2-P, to be lower than needed to drive the desired level of lymphocyte reduction at the top dose evaluated.

Herein, we describe our medicinal

chemistry efforts to find novel compounds in this series with a reduced human T1/2 and improved in vivo formation of the active phosphate metabolite while maintaining efficacy as well as an improved safety profile. Our strategy involved using the pharmacokinetic T1/2 and phosphate to parent ratios in rodents along with lymphocyte reduction for the initial selection criteria.16 Compound 1 exhibited greater formation of the active phosphate metabolite in rat when compared to 2 and was subsequently found to also show a higher ratio in additional nonclinical species and ultimately in humans (see table 7). We retained the cyclopentyl amino-alcohol head group of 2 as this moiety has been shown to provide the desired S1P1 biased agonist profile by us15a,15b and others15c and we focused on modifications to the hexyl side chain and tetralin framework. Our initial tetralin modifications were previously disclosed15b, and this work focuses on the evaluation of aryl and heteroaryl replacements for the hexyl side chain (Figure 1). Previously discovered S1P1 full agonists, such as BMS-824 discovered in our labs15d as well as KRP-203 and others 11, established the tolerance for aryl and heteroaryl groups in this region. We commenced an exploration to determine if aryl and heteroaryl modifications would lead to the desired improvements over 2. CHEMISTRY The synthesis of compounds 8-16 is depicted in Scheme 1. Starting from methyl (1R,3S)amino-3-(4-bromophenyl)cyclopentane-1-carboxylate hydrochloride (3)17, reduction of the ester followed by the reaction of the resulting amino alcohol with carbonyldiimidazole (CDI) afforded carbamate 4. The bromophenyl group of this carbamate was then reacted with 2-(tert-butoxy)-2oxoethyl)zinc(II) bromide using a catalyst system of Pd2(dba)3 and 1,2,3,4,5-pentaphenyl-1′-(di5 ACS Paragon Plus Environment

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Scheme 1a: Synthesis of 6-substituted-((1R,3S)-1-amino-3-(5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol compounds H N

NH2-HCl O Br

O

a,b Br

3

H N

O

O

h,i 6

O

7

TfO

O O

O O

4

O

a

c,d

O

Me

H N

O

H N

O

e,f,g

5

R-MgBr or R j,k,l,m

NH2

NH2

OH

OH

R

8a-16a

R

8b-16b

Reagents and conditions: a) NaBH4, MeOH, 91%; b) CDI, Dioxane, 87%; c) LiHMDS, (2-(tert-butoxy)-2-

oxoethyl)zinc(II) bromide, THF; d) Pd2(dba)3, Q-phos, 80 oC, 85%; e) TFA, DCM; f) oxalyl chloride; g) ethylene, AlCl3, DCM, 92%; h) LDA, DMPU; i) PhN(Tf)2, THF, -78 to 0 oC, 60% (2 steps); j) LiHMDS, Co(acac)3, THF/NMP, RMgBr, -40 oC or CuI, Pd(PPh3)2Cl2, TEA, alkyne; k) Pd(OH)2, MeOH, H2; l) Chiral SFC separation; m) 1 N NaOH, dioxane.

tert-butylphosphino)ferrocene (Q-phos) to give compound 5. The tert-butyl ester of 5 was deprotected with trifluoroacetic acid (TFA) and the resulting acid was converted to the acid chloride using oxalyl chloride. An intramolecular Friedels-Craft alkylation was performed on this acid chloride in the presence of ethylene and aluminum chloride resulting in the formation of tetralone 6.18 This tetralone was then converted to an enol triflate using phenyl triflimide. Reaction of enol triflate 7 with a variety of Grignard reagents using Co(acac)3 as a catalyst followed by a reduction of the alkene using 10% Pd(OH)2 yielded various aryl compounds with an alkyl linker of 0, 1, or 2 methylene groups as a mixture of diastereoisomers. Alternatively, triflate 7 could be coupled with an appropriately substituted terminal alkyne using Sonogashira conditions19 followed by reduction to yield the desired compounds. Subsequent chiral chromatographic separation of these mixtures followed by basic hydrolysis of the carbamate group afforded the individual diastereoisomers 8a-16a and 8b-16b. 6 ACS Paragon Plus Environment


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Scheme 2a: Alternate synthesis of 14a and 15a. H N

H N

O O

Br

4

HO2C

H N

O

O O

O

O

CO2H a,b,c,d

H N

O

e,f,g

O

TsO

17 O

18a

TsO

18b

h,i NH2

NH2

OH

OH OMe MeO 14a

a

15a

Reagents and conditions: a) Pd(OAc)2, P(o-Tol)3, K2CO3, H2O/MeCN, 80 oC b) 10% Pd/C, MeOH, H2; c) H2SO4,

4 h, then MeOH; d) 10% Pd/C, MeOH, H2, 55% yield from 4; e) LiBH4, THF, 60 oC, 90%; f) Chiral SFC separation g) TsCl, pyridine, 80-95%; h) CuBr-DMS, RMgCl, Et2O, 93%; i) 1 N NaOH, dioxane, 57%.

An alternate route was devised for the synthesis of compound 14a and 15a as shown in Scheme 2. In this route, Heck reaction of 4 with itaconic acid and reduction of the double bond was followed by cyclization, facilitated using sulfuric acid, and the resulting keto-acid was esterified by the addition of MeOH to the reaction mixture. Pd-catalyzed benzylic deoxygenation under hydrogenation conditions yielded 17. Compound 17 was then separated using chiral SFC conditions. Each of these isomers was reduced to the corresponding alcohols using lithium borohydride and subsequently converted to tosylates 18a and 18b. The absolute stereochemistry of compound 18b was determined to be S at the site of the tosylate attachment by X-ray crystallography.20 The R-tosylate (18a) was then coupled with (2-methoxybenzyl)magnesium chloride using copper(I) bromide dimethyl sulfide complex and subsequently hydrolyzed to afford 14a. The absolute stereochemistry of 14a was also confirmed by X-ray crystallography.21 Compound 15a was synthesized in a similar manner using (3-methoxybenzyl)magnesium chloride and 18a. 7 ACS Paragon Plus Environment

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Compounds 22-29 were synthesized using routes shown in Scheme 3. Compound 17 was first reduced using lithium borohydride then oxidized to the aldehyde (19). This aldehyde was then converted to a terminal alkyne using dimethyl (1-diazo-2-oxopropyl)phosphonate. The terminal alkyne intermediate was separated using chiral SFC conditions to yield 20a and 20b. The R-alkyne (20a) and S-alkyne (20b)22 intermediates were coupled with an appropriately substituted heteroarylbromides using Sonogashira conditions.19

Subsequent reduction and hydrolysis

afforded 22-25 and 29. An alternate route was employed to synthesize 27 and 28 using a JuliaKocienski olefination.23 Compound 18a was coupled with 1-phenyl-1H-tetrazole-5-thiol then

Scheme 3a: Synthesis of 6-substituted-heterophenethyl compounds. H N

H N

O O

O a,b

17

H N

O

O 20b

20a

O O

e,f,g H N

O O

TsO

a

O O

c,d

19

O

H N

O

18a

NH2 OH

h,i,j

R

21

e,f,g

NH2 f,g

NH2

OH

R

22a-29a

R

OH

22b-29b

Reagents and conditions: a) LiBH4, THF, 60 oC, 90%; b) Swern oxidation, 84%; c) K2CO3, dimethyl (1-diazo-2-

oxopropyl)phosphonate, 82% d) Chiral SFC separation, 30-35% of 20a and 20b; e) ArBr, Pd(amphos)Cl2, Cs2CO3, MeCN, 80 oC, 40-50%; f) 10% Pd(OH)2, MeOH, H2, 95%; g) 1 N NaOH, dioxane; h) 1-phenyl-1H-tetrazole-5-thiol, K2CO3, DMF, 80 oC, 90%; i) H2O2, ammonium molybdate tetrahydrate, 0 °C, 99%; j) pyrazole aldehydes, KHMDS, 30-40% .



oxidized to the sulfone with hydrogen peroxide and ammonium molybdate tetrahydrate. This intermediate was coupled with the appropriately substituted pyrazole aldehyde to give 21 which was then reduced and hydrolyzed as previously described to afford 27 and 28. RESULTS AND DISCUSSION Since binding and other in vitro functional assays require the synthesis of the active phosphate metabolite and are not predictive of the compounds’ ability to form the active metabolites in vivo, 8a/b, 9a/b and 10a were initially assessed using a mouse blood lymphocyte reduction (BLR) pharmacodynamic/pharmacokinetic (PD/PK) model to determine whether these side chain modifications would result in an acceptable amount of circulating lymphocyte reduction (>65%) as well as an in vivo phosphate metabolite to parent ratio which was greater than that of 2 (ratios of 0.5 at 30 mg/kg and 1.0 at 0.5 mg/kg). The first set of compounds evaluated were derivatives with an alkyl linker of 0, 1, or 2 methylenes and a terminal phenyl group in place of the hexyl side chain (8-10). The results of these studies are shown in Table 1. The compounds were evaluated at a low dose (1 mg/kg) and a high dose (15, 30, or 50 mg/kg) for each of the individual isomers and the lymphocyte count was measured at 4 and 24 h. The two isomers with a directly attached phenyl group (8a, 8b) exhibited poor lymphocyte reduction at the 1 mg/kg dose at both timepoints. At a 30 mg/kg dose, both isomers showed good reduction of lymphocytes at both the 4 and 24 h timepoints; however, concentrations at 24 h indicated poor formation of the phosphate metabolite with a phosphate to parent ratio of 0.14. Compounds 9a and 9b22, which have a benzyl moiety as the side chain, similarly had poor lymphocyte reduction at the lower dose and acceptable levels of reduction at the higher dose for both isomers. The phosphate to parent ratio of 9a and 9b were both better than that of 2 at the 30 mg/kg dose with ratios of 1.4 and 1 respectively versus a ratio 0.5 for 2. Lastly, one of the isomers of the phenethyl derivatives (10a)22 was assessed in the mouse 9 ACS Paragon Plus Environment

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BLR PK/PD assay. At the 1 mg/kg dose, 10a exhibited poor lymphocyte reduction at the 4 h timepoint, however at the 24 h timepoint, the compound showed 52% lymphocyte reduction. The high dose for 10a demonstrated good lymphocyte reduction at both timepoints. This compound also demonstrated a phosphate to parent ratio in the range of 2.3 to 2.8 which was a marked improvement over 2. Since the benzyl and phenethyl derivatives both demonstrated favorable lymphocyte reduction and phosphate metabolite formation, we chose to further assess derivatives in these series. For the next set of benzyl and phenethyl analogs, metabolic “soft spots” were incorporated into the side chain and the compounds were evaluated in a BLR PK/PD study in Lewis rats (Table 2). In this study, the percent reduction of circulating lymphocytes was measured at 4 and 24 h post dose (2 mg/kg, p.o). Additionally, the corresponding levels of parent and active phosphate metabolite were measured at these timepoints. For compounds with greater than 65% Table 1: Effects on blood lymphocyte counts in BALB-c mice NH2 OH

R

8-10

4h Compd

2

R

24h

Lymph Red. (dose)

Parent (nM) a

Phos. (nM) a

Lymph Red. (dose)

Parent (nM) a

17% (0.5 mg/kg)

BLQ

BLQ

73% (0.5 mg/kg)

19

Phos. Phos./parent (nM) a Ratio (24h) 20

1

88% (30 mg/kg) 1000±420

150±80

90% (30 mg/kg) 1200±490 620±220

0.5

8a

75

1.2

125h

5

38h

>125h

8

14h

46h

6

10h

28h

3

N

27

N

28

N

N

N

N

29 OMe

Pred. = predicted; Phos. = Phosphate

Table 4. Effects of compounds 14-16 on BALa protein levels

Compd

BALa Protein mg/dL(dose)

Fold over

Rat PD 24h ED50

Fold over


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Vehicleb

(mg/kg)c

ED50

12.5±1.0 (3 mg/kg)

3.1

0.1

30x

BMS-824 24.3±1.0 (1 mg/kg)

4.3

0.1

10x

14a

14±1.5 (10 mg/kg) 8.8 (3 mg/kg)

3.1 1.6

0.1

100x 30x

14b

29±0.1 (10 mg/kg) 10.5± (2 mg/kg)

6.4 1.8

0.05

200x 40x

15a

12.7±0.9 (10 mg/kg) 10±1 (3 mg/kg)

3.2 2.1

0.05

200x 60x

15b

17.5±0.9 (10 mg/kg) 10±1 (2 mg/kg)

4.4 1.6

0.05

200x 40x

16a

26±1.0 (25 mg/kg)

4.7

0.5

50x

16b

24.3±0.3 (25 mg/kg)

4.5

0.6

40x

1

Compounds tested in Lewis rat (n = 3-4); aBAL = Bronchoalveolar Lavage; bChange in BAL protein levels of treated group relative to vehicle (PEG300) control. c Estimated ED50 for lymphocyte reduction in rat at 24 h.

mg/kg) for each of the individual isomers and the results of the BAL protein elevation is reported as both relative to vehicle and relative to an estimated in vivo ED50. In addition, the results from a previously disclosed S1P3 sparing/S1P1 full agonist, BMS-824 (dosed at 1 mg/kg), as well as 1, are shown in the study for comparative purposes. At the higher dose, the 2-methoxy (14a/14b) compounds had similar relative increases with a 3.1 fold increase in BAL protein at 100 fold over ED50 and a 6.4 fold increase in BAL protein at 200 fold over ED50 respectively. The 3-methoxy (15a/15b) compounds were both at 200 fold over ED50 and exhibited a 3.2 and 4.4 fold increase respectively. The 4-methoxy compounds (16a/ 16b) were least favorable from the phenethyl series with a 4.5-4.7 fold increase in BAL levels at 40-50 fold over ED50. When evaluated at lower doses, the 2-methoxy and 3-methoxy analogs demonstrated favorable results, with 14a, 14b, and 15b leading to BAL protein increases of less than two-fold at 30-40 fold over ED50. All the 2-methoxy 16 ACS Paragon Plus Environment


and 3-methoxy compounds exhibited significantly less BAL protein elevation as compared to our S1P3 sparing/S1P1 full agonist, BMS-824, which elicited a BAL elevation of 4.3 fold at 10 fold over ED50. The transient heart rate lowering reported with 1 (S1P1,3,4,5 agonist) attributed to off-target activity on S1P3 was later observed in clinical trials with S1P3 sparing S1P1 full agonists.14 This was despite the fact that cardiovascular evaluations in nonclinical species did not predict for bradycardia with the S1P3 sparing compounds.14

Given this differential cardiovascular

pharmacology between human and nonclinical species, a human cellular assay was utilized to guide our selection of molecules that would have reduced potential for heart rate effects in the clinic. The phosphates of isomers 14a/b, 15a/b, and 16a were prepared and evaluated in cultured human cardiomyocytes derived from inducible pluripotent stem cells (iPSCs). Cardiomyocytes differentiated from iPSCs show a concentration-dependent response to cardioactive drugs that is consistent with observed clinical effects.27 The rate of beating in these cells have been shown to be reduced by exposure to S1P1 agonists independent of S1P3 activity, with more limited reduction in beat rate being demonstrated by biased agonists of S1P1, such as 2.15a,15b Compounds 14a-P, 14b-P, 15a-P, 15b-P, 16a-P were evaluated at 1 nM and 10 nM (Figure 2) and compared to both 1-P, an S1P1/S1P3 full agonist as well as BMS-824, an S1P1 full agonist that is S1P3 sparing. We considered the maximum desirable beating rate reduction at these concentrations to be 15% and 30%, respectively. All the methoxy phenethyl analogs were significantly improved over 1-P and BMS-824 in this assay at both concentrations. Compounds 14a-P and 15a-P were the only compounds with less than 15% rate reduction at 1 nM. At 10 nM, the compounds that exhibited an acceptable rate reduction of lower than 30% were 14a-P, 14b-P and 15b-P. While 14b-P and 15b-P both showed impressive results at 10 nM neither compound 17 ACS Paragon Plus Environment

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gave a desirable active phosphate metabolite-to-parent ratio (see Table 2). Compound 14a-P, with 11% and 27% rate reduction at 1 nM and 10 nM respectively, was the only compound to show the desired profile at both concentrations. This improvement of 14a-P over 1-P in the cardiomyocyte assay is notable given that 14a-P exhibits a 2-fold potency increase over 1-P in a human lymphocyte chemotaxis assay (see Table 5).

Figure 2. Reduction in beating rate of human iPSC derived cardiomyocytes in the presence of full S1P1 agonists with varying S1P3 agonist profiles versus partial S1P1 agonists sparing of S1P3 (Average of 2 experiments)

Based upon the assessments for lymphopenia, projected T1/2, phosphorylation potential as well as pulmonary and cardiovascular safety, 14a was chosen for further evaluation as it had the best overall profile from this series. The phosphate of 14a was then extensively characterized in a variety of in vitro assays and its profile was compared to 1-P as well as to the phosphate of our previous clinical candidate, 2-P. As is clear from Table 5, all three compounds have equivalent potency in the S1P1 binding assay and act as full agonists in the cyclic AMP functional assay; however, as we found with 2-P15a, 14a-P is differentiated from 1-P in the S1P1 GTPγS, ERK-P, 18 ACS Paragon Plus Environment


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and S1P1 internalization assays. The GTPγS assay shows 14a-P to have a maximum efficacy that reaches ~80% of the endogenous ligand while 1-P reaches close to 100%. In the ERK-P assay, while all three exhibit full agonism, the potencies of 14a-P and 2-P are 100-500 fold weaker than 1-P, and finally, in the S1P1 internalization assay, 14a-P and 2-P are partial agonists whereas 1-P Table 5. 14a-P in vitro pharmacology

Assay

1-P

2-P

14a-P

hS1P-1 binding (IC50, nM), Ymax

0.008 ± 0.0014 (n=6), 101%

0.010 ± 0.004 (n=7), 100%

0.014 ± 0.003 (n=6), 101%

hS1P-1 cAMP (EC50, nM), Ymax

0.007 ± 0.003 (n=3), 100%

0.006 ± 0.002 (n=3), 98%

0.008 ± 0.004 (n=6), 101%

hS1P-1 GTPS (EC50, nM), Ymax

0.70 ± 0.28 (n=6), 96%

0.90 ± 0.36 (n=7), 81%

0.60 ± 0.08 (n=6), 79%

hS1P-1 internalization (EC50, nM), Ymax

0.070 ± 0.02 (n=6), 102%

0.11 ± 0.02 (n=5), 68%

0.11 ± 0.06 (n=5), 72%

hS1P-1 ERK-P (EC50, nM), Ymax

0.02 ± 0.006 (n=6), 100%

8.2 ± 3.6 (n=4), 101%

3.5 ± 1.7 (n=6), 99%

hS1P-3 GTPS (EC50, nM)

3.6 ± 2.80 (n=7)

>1000 (n=7)

>1000 (n=7)


1.6 ± 0.43 (n=3)

10.5 (n=2)

3.4 ± 2.2 (n=3)


0.67 ± 0.24 (n=3)

10.7 (n=2)

3.5 ± 0.98 (n=3)

Lymphocyte chemotaxisa (IC50, nM)

1.2 (n=2)

2.5 (n=2)

0.6 (n=2)

hS1P = human sphingosine 1-phosphate; Ymax = Normalized maximal response aantagonist mode

exhibits full agonist activity in this assay. This partial agonist profile of 14a and 2 in the internalization assay may be related to the improved pulmonary safety observed in preclinical studies (lower BAL protein elevation) since maintaining some level of S1P/S1P1. signaling has been postulated to be crucial for controlling vascular tone.26d,28 It has also been postulated that 19 ACS Paragon Plus Environment

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transient activation of S1P1 by fingolimod and other S1P1/S1P3 selective compounds in atrial myocytes before functional antagonism of S1P1R underlies the first-dose bradycardia observed in some patients.29 This bradycardia effect is linked to S1PR-dependent activation of G proteincoupled inwardly-rectifying potassium (GIRK) channels on atrial myocytes. The nature of S1PR activation that leads to this heart rate effect is now defined. We hypothesize the weaker potency in ERK phosphorylation for 14a-P and 2-P may contribute to the their weaker potency in the cardiomyocyte beating rate assay. Compound 14a also maintains excellent selectivity over S1P3 while showing a full potent agonism of S1P4 and S1P5. Finally, the compounds were assessed in a lymphocyte chemotaxis assay, a functional primary cell assay that correlates closely with in vivo lymphopenia.

Compound 1-P does not induce T-cell chemotaxis on its own, but completely

blocks the S1P induced T-cell migration with an IC50 of 1.2 nM. Compound 14a-P also completely blocks this S1P induced T-cell migration with an IC50 of 0.6 nM. Table 6 shows the pharmacokinetic parameters for 14a derived from whole blood concentrations via dried blood spot (DBS) analysis. The average absolute oral bioavailability (F%) was 53% across all species (range = 23-76%). The time to maximum concentration (Tmax) occurred at 6-7 hours for 14a and for the active phosphate metabolite (14a-P) likely reflecting a slow rate of absorption. Compound 14a has large volume of distribution, with an average blood steady-state volume of distribution (Vss) of 16 L/kg (range = 10-21 L/kg) in nonclinical species. The terminal T1/2 of 14a after IV dosing ranged from 38 to 165 h, which was much shorter than that for 2 (131 to 210 h). Low clearance from blood (CLTb) was observed in all the species tested, Table 6. Pharmacokinetic parameters for compound 14a

species

IV CL Dose (mg/kg)a (mL/min/kg) Vss (L/kg)

IV T1/2 (h)


F% (PO)


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SpragueDawley rat

IV: 1 (n = 3) PO: 1 (n = 3)

9.7± 0.57

16± 0.92

38± 2.7

23%

Beagle dog

IV: 1 (n = 6) PO: 0.3 (n = 3)

1.9± 1.1

21± 7.5

165± 34

61%

Cynomolgus monkey

IV: 1 (n = 3) PO: 1 (n = 3)

3.8± 0.95

10± 1.9

46± 1.4

76%

16

83

53%

Animal Average

18.4% hydroxypropyl--cyclodextrin in 13.8 mM citric acid as vehicle. F = bioavailability; Cl = clearance, Vss = volume of distribution at steady state; AUC = area under curve a

Table 7. Phosphate/parent ratio for 1, 2 and 14a

AUC ratio of phosphate to parent

a

species

1

2

14a

SpragueDawley rat

5.6

2.5

5.2

Beagle dog

3.7

2.4

3.1

Cynomolgus monkey

5.9

0.3

1.0

Human

0.42

n/aa

--

Due to phosphate 2-P levels being 20 M, 2B6: 17 M 2C9: 14 M, 2C19 6.9 M 2D6: 18 M, 3A4: 5.5 M 2C8: 8.6 M

Protein Binding (%

All >99.9% (h, r, m, c, d)

bound) LMb T1/2 (min)

242/97/372/110 (h, r, d, c)

pKa

9.5

Log D (pH 6.5)

5.05

CYP = cytochrome P450 LM = liver microsomes; h= human; r = rat; m = mouse; c = cynomolgus monkey; d = dog

a

b

half-lives of 14a upon incubation with human, rat, cyno and dog liver microsomes were long (all T1/2 > 97 min), and 14a was found to be highly protein bound in all tested species (>99.9% bound in human, rat, mouse, cyno, and dog).



We next evaluated the suppressive effect of 14a on experimental autoimmune encephalomyelitis (EAE), a preclinical animal model for multiple sclerosis (MS) therapy. The graphs in Figure 3 demonstrate the effects of orally administered 14a as well as 1 in this EAE study.

Compound 14a was administered as once-daily doses of 0.1 and 0.5 mg/kg with p.o.

dosing initiated at the time of immunization. Compound 14a showed a dose-dependent reduction in clinical scores for inflammation (Figure 3A) and both doses were efficacious lowering the EAE clinical scores taken from Day 12 until the completion of the study when compared to the vehicle treated group. To investigate the effects of 14a on the formation of inflammatory lesions in the CNS, a histological evaluation of the spinal cord was performed on day 21 of the EAE study (A) Effect of compound treatment on EAE Clinical Score

Clinical Scores

5 4

Vehicle 14a (0.1 mg/kg) 14a (0.5 mg/kg)

3

1 (1 mg/kg)

2 1 0

9

* * *

12

14

* * 16

* *

* *

19

21

Days

(B) Effect of compound treatment on EAE Spinal Cord Histology 4

Average Score

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

Vehicle 14a (0.1mg/kg) 14a (0.5mg/kg) 1 (1.0 mg/kg)

3

*

2

*

1 0

Vehicle

0.1

0.5 14a

*

1.0 1

Figure 3a. Efficacy of 14a (BMS-986166: 0.1 and 0.5 mg/kg) and 1 (FTY: 1.0 mg/kg) vs. vehicle in a mouse experimental autoimmune encephalomyelitis model (EAE), clinical score (A), and histological evaluation (B).


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Page 25 of 81

aMice

(n=10/group) were administered 14a PO/QD starting on Day 0 at the time of immunization with

MOG-peptide (myelin oligodendrocyte glycoprotein). Vehicle: PEG300. *p value < 0.05 compared to vehicle treatment group.

(E) Myelination

*

140

Myelination Index (% veh)

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


120 100

*

*

*

*

0.1

10

80 60 40 20 0

a

veh LPC

0.01

0.1

10

LPCa then 1-P (nM)

Figure 4.

*

0.01

LPCa then 14a-P (nM)

Effect of compounds in lysophosphatidylcholine (LPC)-induced

demyelination model.

Immunohistochemical staining showing myelination of

organotypic cerebellar slices in vehicle (A), in slices incubated overnight with LPC (B) and in slices incubated overnight with LPC followed by treatment with 1-P (C) or 14a-P (D) for 7 days. Remyelination is induced by subsequent treatment. Myelination index of 1-P and 14a-P after overnight incubation with LPC followed by 7 days of treatment (E). aIncubated

with 0.5 mg/mL LPC overnight; *p value 0.05 vs. LPS; ANOVA followed

by Dunnett test, n = 3-5.

(Figure 3B). Again, a dose-dependent reduction in the histological score was noted. Based on clinical scores and spinal cord histology, a dose of 0.5 mg/kg/day of 14a demonstrates maximal efficacy and its clinical score was comparable to the 1.0 mg/kg/day dose of 1. To further understand the effect of 14a on EAE and its potential as a treatment for MS, the active metabolite, 14a-P, was assessed in an organotypic cerebellar slice culture assay to examine 24 ACS Paragon Plus Environment


Page 26 of 81

the effect on demyelinated axons (Figure 4).30,31 During the development of MS, myelin is attacked and destroyed by autoimmune responses, resulting in demyelination and subsequent axonal degeneration.32 Compound 1-P has been shown to promote remyelination by acting directly on demyelinated neuronal axons in the central nervous system, an effect that is believed to contribute to its clinical efficacy in MS.32 Figure 4 illustrates the effects of 14a-P and 1-P on lysophosphatidylcholine (LPC)-induced demyelination in the organotypic cerebellar slice. Most of axonal fibers in the slice treated with LPC lost their myelin sheaths as compared to those treated with vehicle (Figure 4, A vs B); however, treatment with 14a-P or 1-P reversed LPC-induced demyelination (Figure 4C/D).

Similar results were reported with 2-P.33

To quantify the

remyelination effect of 14a-P and 1-P, myelination index values were generated (Figure 4E). The myelination index value of LPC-treated slices was ∼50% of vehicle; treatment with either 14a-P or 1-P in the LPC-treated slices lifted the myelination index up to ∼100% (Figure 4, C and D), suggesting that both 14a-P or 1-P promoted remyelination of demyelinated neuronal axons in the brain. The in vivo efficacy of 14a was further demonstrated in a collagen-induced arthritis (CIA) model (Figure 5). DBA/1 mice received a primary collagen immunization to initiate disease development and were concurrently dosed orally with 14a at single once-daily doses of 0.1 or 0.5 mg/kg or injected intraperitoneally (i.p.) with mCTLA-4Ig twice per week at 1 mg/kg. CTLA-4Ig is a soluble receptor-IgG fusion protein that interferes with T-cell co-stimulation and has been shown to be clinically efficacious in the treatment of rheumatoid arthritis.34 Compound 14a dose dependently reduced both the clinical score as well as the paw histology score following the dosing regimens. Based on these evaluations, a dose of 0.5 mg/kg/day of 14a demonstrated maximal efficacy and its clinical score was comparable to the 1.0 mg/kg dose of mCTLA4Ig. 25 ACS Paragon Plus Environment

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(A)

15

(B)

Vehicle 14a (0.1 mg/kg) 14a (0.5 mg/kg) mCTLA4Ig (1.0 mg/kg)

5

10 Paw Histology Score

Clinical score

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


5

0 30

40

50

days post primary immunization

4 41% 3 2

80% 92%

1 0

***

*** Veh

0.1

0.5 14a

1 mCTLA4-Ig

Figure 5. Efficacy of 14a (0.1 and 0.5 mg/kg) and mCTLA4Ig (1.0 mg/kg) vs. vehicle in a Collagen Induced Arthritis model (CIA), clinical score (A) and histological evaluation (B). ***p 99% d.e.; tr = 22.6


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min. (Method: ChiralCel AD-H (25 x 0.46 cm, 5 m). Flow rate: 2 mL/min; Mobile phase: CO2/(MeOH + 0.1%DEA) in 85:15 ratio isocratic); []D20 +53.77 ( c 0.315, MeOH).

The following compounds were prepared using the procedures described for 14a/14b:

((1R,3S)-1-Amino-3-((R)-6-(3-methoxyphenethyl)-5,6,7,8-tetrahydronaphthalen-2yl)cyclopentyl)methanol (15a). 36 Obtained

((1R,3S)-1-amino-3-((R)-6-(3-methoxyphenethyl)-5,6,7,8-tetrahydronaphthalen-2-

yl)cyclopentyl)methanol, TFA as a white solid (14 mg, 67% yield). Isomer a, 15a: 1H NMR (400 MHz, MeOD)  7.18 (t, J=7.8 Hz, 1H), 7.04 - 6.97 (m, 3H), 6.85 - 6.77 (m, 2H), 6.74 (dd, J=8.3, 1.9 Hz, 1H), 3.79 (s, 3H), 3.71 - 3.57 (m, 2H), 3.20 - 3.02 (m, 1H), 2.89 (dd, J=16.5, 3.3 Hz, 1H), 2.83 - 2.75 (m, 2H), 2.75 - 2.69 (m, 2H), 2.50 - 2.36 (m, 2H), 2.19 - 2.06 (m, 1H), 2.06 - 1.88 (m, 4H), 1.81 - 1.64 (m, 4H), 1.44 (dtd, J=12.8, 10.4, 5.9 Hz, 1H); MS (M+H)+ at m/z 380; HPLC tr = 8.89 minutes (Method D); HPLC Purity = 95%.

((1R,3S)-1-Amino-3-((S)-(6-(3-methoxyphenethyl)-5,6,7,8-tetrahydronaphthalen-2yl)cyclopentyl)methanol (15b).36 Obtained

((1R,3S)-1-amino-3-((S)-6-(3-methoxyphenethyl)-5,6,7,8-tetrahydronaphthalen-2-

yl)cyclopentyl)methanol, TFA as a white solid (14 mg, 67% yield). Isomer b, 15b: 1H NMR (400 MHz, MeOD)  7.18 (t, J=7.8 Hz, 1H), 7.05 - 6.96 (m, 3H), 6.85 - 6.76 (m, 2H), 6.74 (dd, J=8.3, 42 ACS Paragon Plus Environment


1.9 Hz, 1H), 3.79 (s, 3H), 3.71 - 3.56 (m, 2H), 3.19 - 3.02 (m, 1H), 2.89 (dd, J=16.4, 3.6 Hz, 1H), 2.83 - 2.75 (m, 2H), 2.75 - 2.68 (m, 2H), 2.50 - 2.35 (m, 2H), 2.20 - 2.06 (m, 1H), 2.06 - 1.87 (m, 4H), 1.82 - 1.61 (m, 4H), 1.53 - 1.36 (m, 1H); ); MS (M+H)+ at m/z 380; HPLC tr = 8.91 minutes (Method D); HPLC Purity = 99%.

((1R,3S)-1-Amino-3-((R)-6-(4-methoxyphenethyl)-5,6,7,8-tetrahydronaphthalen-2yl)cyclopentyl)methanol (16a).36 Obtained

((1R,3S)-1-amino-3-((R)-6-(4-methoxyphenethyl)-5,6,7,8-tetrahydronaphthalen-2-

yl)cyclopentyl)methanol, TFA as a white solid (53 mg, 72% yield). Isomer a, 16a: 1H NMR (400 MHz, MeOD)  7.13 (d, J=8.6 Hz, 2H), 7.05 - 6.96 (m, 3H), 6.84 (d, J=8.6 Hz, 2H), 3.77 (s, 3H), 3.71 - 3.56 (m, 2H), 3.20 - 3.01 (m, 1H), 2.97 - 2.74 (m, 3H), 2.75 - 2.61 (m, 2H), 2.42 (dd, J=14.2, 7.8 Hz, 2H), 2.21 - 2.07 (m, 1H), 2.05 - 1.86 (m, 4H), 1.80 - 1.57 (m, 4H), 1.43 (dtd, J=12.9, 10.4, 5.9 Hz, 1H); ); MS (M+H)+ at m/z 380; HPLC tr = 8.88 minutes (Method D); HPLC Purity = 99.3%

((1R,3S)-1-Amino-3-((S)-6-(4-methoxyphenethyl)-5,6,7,8-tetrahydronaphthalen-2yl)cyclopentyl)methanol (16b).36 Obtained


yl)cyclopentyl)methanol, TFA as a white solid (55 mg, 73% yield). Isomer b, 16b: 1H NMR (400 MHz, MeOD)  7.13 (d, J=8.8 Hz, 2H), 7.03 - 6.96 (m, 3H), 6.84 (d, J=8.8 Hz, 2H), 3.77 (s, 3H), 3.70 - 3.57 (m, 2H), 3.19 - 3.03 (m, 1H), 2.96 - 2.74 (m, 3H), 2.73 - 2.63 (m, 2H), 2.42 (dd, J=14.1,


Page 44 of 81

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7.9 Hz, 2H), 2.20 - 2.07 (m, 1H), 2.06 - 1.87 (m, 4H), 1.79 - 1.60 (m, 4H), 1.43 (dtd, J=12.7, 10.6, 5.9 Hz, 1H); MS (M+H)+ at m/z 380; HPLC tr = 8.90 minutes (Method D); HPLC Purity = 98%.

Methyl 6-((5R,7S)-2-oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)-1,2,3,4-tetrahydronaphthalene2-carboxylate (17). 36 To a mixture of potassium carbonate (523 mg, 3.8 mmol), (5R,7S)-7-(4-bromophenyl)-3oxa-1-azaspiro[4.4]nonan-2-one (4, 800 mg, 2.7 mmol), itaconic acid (456 mg, 3.5 mmol), and acetonitrile (8 mL) was added water (2.4 mL). The mixture was stirred until the evolution of carbon dioxide stopped and then was sparged with nitrogen for 3 min. After palladium(II) acetate (30 mg, 0.14 mmol) and tri-o-tolylphosphine (82 mg, 0.27 mmol) were added, the mixture was sparged with nitrogen for an additional 3 min. The mixture was stirred at 80 °C for 20 h and then concentrated in vacuo. The residue was mixed with water (40 mL), basified with potassium carbonate and filtered. The filtrate was washed with diethyl ether (2 x 15), then the aqueous layer was acidified to ~pH 2 with 6 N aqueous hydrochloric acid. The resulting solid was separated and the aqueous solution was extracted with a mixture of THF/EtOAc (3:1; 4 x 10 mL). The solid and the extracts were combined and concentrated. MS (M+H-H2O)+ at m/z 328. The residue was mixed with THF (5 mL), ethyl acetate (5 mL), methanol (20 mL), and 10% Pd-C (400 mg, 0.376 mmol) then hydrogenated under a balloon of H2 overnight. The catalyst was filtered away and the filtrate was concentrated and lyophilized to give a white solid. MS (M+H-H2O)+ at m/z 330. The solid was mixed with 98% sulfuric acid (15 mL, 281 mmol), and the clear solution was stirred at room temperature for 4 h. MeOH (8 mL, 198 mmol) was added slowly with water44 ACS Paragon Plus Environment


bath cooling. The mixture was stirred at room temperature for 1 h before being poured onto ice (150 g). The mixture was extracted with ethyl acetate (4 x 40 mL). The combined ethyl acetate extracts were washed with sat. aqueous sodium bicarbonate solution (20 mL), dried over Na2SO4, and concentrated in vacuo. Flash chromatography purification (24g silica gel column, gradient elution from 10 to 100% ethyl acetate in hexanes) afforded methyl 4-oxo-6-((5R,7S)-2-oxo-3-oxa1-azaspiro[4.4]nonan-7-yl)-1,2,3,4-tetrahydronaphthalene-2-carboxylate (440 mg; MS (M+HH2O)+ at m/z 344. A mixture of methyl 4-oxo-6-((5R,7S)-2-oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)-1,2,3,4tetrahydronaphthalene-2-carboxylate (440 mg, 1.281 mmol), MeOH (15 mL), acetic acid (1.5 mL), and 10% Pd-C (200 mg, 0.188 mmol) was hydrogenated under a balloon of H2 over 3 days. The mixture was filtered through a membrane filter. The filtrate was concentrated in vacuo to give methyl

6-((5R,7S)-2-oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)-1,2,3,4-tetrahydronaphthalene-2-

carboxylate (337 mg, 55% for 4 steps) as a white solid. MS (M+H)+ at m/z 330. 1H NMR (400 MHz, CDCl3) δ 7.06 (s, 1H), 7.02 - 6.92 (m, 2H), 6.39 - 6.26 (m, 1H), 4.37 - 4.25 (m, 2H), 3.75 (s, 3H), 3.02 - 2.97 (m, 2H), 2.91 - 2.81 (m, 2H), 2.80 - 2.67 (m, 1H), 2.35 - 2.27 (m, 1H), 2.26 2.06 (m, 3H), 2.03 - 1.91 (m, 2H), 1.91 - 1.75 (m, 3H).

(5R,7S)-7-((R)-6-(Hydroxymethyl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1azaspiro[4.4]nonan-2-one

(18a)

and

(5R,7S)-7-((S)-6-(hydroxymethyl)-5,6,7,8-

tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (18b). 36 A

mixture

of

methyl

6-((5R,7S)-2-oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)-1,2,3,4-

tetrahydronaphthalene-2-carboxylate (337 mg, 1.023 mmol), anhydrous THF (3 mL), and a 2N 45 ACS Paragon Plus Environment

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solution of lithium borohydride (2.56 mL, 5.12 mmol) in THF was stirred at 70 °C for 4 h. Saturated aqueous ammonium chloride solution was added slowly at 0 °C to quench the reaction then water and ethyl acetate were added. The aqueous solution was extracted with ethyl acetate. The combined ethyl acetate solutions were dried over Na2SO4 and concentrated in vacuo to afford (5R,7S)-7-(6-(hydroxymethyl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan2-one (300 mg, 0.995 mmol, 97% yield). MS (M+H)+ at m/z 302. Chiral SFC separation (AD-H (46 x 25cm), 45% MeOH in CO2, 3mL/min, 220 nm, 35 oC) gave isomers 1 and 2 as white solids. (5R,7S)-7-((R)-6-(hydroxymethyl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1azaspiro[4.4]nonan-2-one (isomer 2, 690 mg, 2.289 mmol) was dissolved in dry pyridine (5 mL) and p-toluenesulfonyl chloride (1.3 g, 6.9 mmol) was added in one portion. The resulting mixture was stirred at room temperature for 4 h. The solvent was removed in vacuo, and the crude material was purified on a silica gel cartridge using an EtOAc/Hex gradient (20-100% EtOAc over 12 column volumes) to afford ((R)-6-((5R,7S)-2-oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)-1,2,3,4tetrahydronaphthalen-2-yl)methyl 4-methylbenzenesulfonate (18a, 860 mg, 1.89 mmol, 82% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.89 - 7.79 (m, 2H), 7.40 (s, 2H), 6.99 (br s, 3H), 6.45 - 6.32 (m, 1H), 4.42 - 4.23 (m, 2H), 4.07 - 3.96 (m, 2H), 3.08 - 2.93 (m, 1H), 2.90 2.70 (m, 3H), 2.51 - 2.48 (m, 3H), 2.46 - 2.38 (m, 1H), 2.36 - 2.25 (m, 1H), 2.21 - 2.07 (m, 3H), 2.03 - 1.89 (m, 3H), 1.87 - 1.74 (m, 1H), 1.52 - 1.37 (m, 1H); MS (M+H)+ at m/z 456; HPLC tr = 2.01 minutes (Method A). The above procedure was repeated using isomer 1 to afford ((S)-6-((5R,7S)-2-oxo-3-oxa1-azaspiro[4.4]nonan-7-yl)-1,2,3,4-tetrahydronaphthalen-2-yl)methyl 4-methylbenzenesulfonate 46 ACS Paragon Plus Environment


(18b, 800 mg, 95% yield). 1H NMR (400 MHz, CDCl3) δ 7.84 - 7.79 (m, 2H), 7.39 - 7.33 (m, 2H), 7.00 - 6.90 (m, 3H), 6.48 - 6.41 (m, 1H), 4.34 - 4.24 (m, 2H), 4.06 - 3.94 (m, 2H), 3.04 - 2.91 (m, 1H), 2.87 - 2.72 (m, 3H), 2.46 (s, 3H), 2.44 - 2.35 (m, 1H), 2.33 - 2.21 (m, 1H), 2.20 - 2.03 (m, 3H), 2.00 - 1.88 (m, 3H), 1.84 - 1.78 (m, 1H), 1.48 - 1.37 (m, 1H); MS (M+H)+ at m/z 456; HPLC tr = 2.05 minutes (Method A).

6-((5R,7S)-2-Oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)-1,2,3,4-tetrahydronaphthalene-2carbaldehyde (19).36 A solution of oxalyl chloride (260 µl, 3 mmol) in DCM (5mL) was stirred under N2 and cooled to -78 °C. DMSO (425 µL, 6 mmol) was then added dropwise and the reaction mixture was stirred for 1 h at -78 °C.

A solution of (5R,7S)-7-((R)-6-(hydroxymethyl)-5,6,7,8-

tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (600 mg, 2 mmol) in DCM/DMSO (3:1, 4 mL) was added dropwise. After 30 min, TEA (1.1 mL, 8 mmol) was added dropwise and the reaction mixture was stirred for 15 min at -78 °C then warmed to room temperature and stirred for another 15 min. The mixture was quenched with water (1 mL) at 0°C, then diluted with EtOAc (50mL) and washed with sat. NH4Cl (2 x 30mL). The organic layers were combined, dried (Na2SO4) and concentrated in vacuo. The residue was purified on a silica gel cartridge using an EtOAc/Hex gradient (0-100% EtOAc over 15 min) to afford 6-((5R,7S)-2-oxo-3-oxa-1azaspiro[4.4]nonan-7-yl)-1,2,3,4-tetrahydronaphthalene-2-carbaldehyde (500 mg, 84% yield). 1H NMR (400 MHz, CDCl3) δ 9.89 - 9.71 (m, 1H), 7.15 - 7.10 (m, 1H), 7.03 - 6.98 (m, 1H), 6.97 6.93 (m, 1H), 5.16 - 5.02 (m, 1H), 4.38 - 4.27 (m, 2H), 3.11 - 3.01 (m, 1H), 3.01 - 2.95 (m, 2H),


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2.91 - 2.84 (m, 2H), 2.78 - 2.66 (m, 1H), 2.39 - 2.30 (m, 1H), 2.29 - 2.21 (m, 1H), 2.16 - 2.09 (m, 2H), 2.05 - 1.91 (m, 2H), 1.89 - 1.76 (m, 2H); MS (M+H)+ at m/z 300.

(5R,7S)-7-6-Ethynyl-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (20a, 20b).36 To

a

mixture

of

6-((5R,7S)-2-oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)-1,2,3,4-

tetrahydronaphthalene-2-carbaldehyde (718 mg, 2.4 mmol) and potassium carbonate (995 mg, 7.20 mmol) in MeOH (3 mL) was added dimethyl (1-diazo-2-oxopropyl)phosphonate (0.540 mL, 3.60 mmol). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with ethyl acetate and washed with sat. NaCl. The organic layer was dried with MgSO4, filtered and concentrated. The crude material was purified on a silica gel cartridge using an EtOAc/Hex gradient (20-100% EtOAc over 12 column volumes) to afford 580 mg of (5R,7S)-76-ethynyl-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one

(82%

yield).

This diastereomeric mixture was separated by SFC using a Chiralpak OJ-H, 25 X 3 cm ID, 5m column and eluting with 80/20 CO2/MeOH at 280 mL/min and each isomer was obtained with > 98% d.e. Peak 1 was isolated to afford (5R,7S)-7-((S)-6-ethynyl-5,6,7,8-tetrahydronaphthalen-2yl)-3-oxa-azaspiro[4.4]nonan-2-one (isomer a, 20a, 225 mg, 32% yield).

1H

NMR (400 MHz,

CDCl3) δ 7.09 - 7.02 (m, 1H), 7.01 - 6.93 (m, 2H), 5.90 - 5.72 (m, 1H), 4.32 (d, J=12.3 Hz, 2H), 3.11 - 2.74 (m, 6H), 2.37 - 2.27 (m, 1H), 2.10 (d, J=2.4 Hz, 4H), 2.04 - 1.76 (m, 4H); MS (M+H)+ at m/z 296; HPLC tr = 0.94 minutes (Method A).



Peak 2 was isolated and recovered to afford (5R,7S)-7-((R)-6-ethynyl-5,6,7,8tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (isomer b, 20b, 245 mg, 35% yield). 1H NMR (400 MHz, CDCl3) δ 7.09 - 6.90 (m, 3H), 6.77 - 6.59 (m, 1H), 4.40 - 4.20 (m, 2H), 3.20 - 2.70 (m, 6H), 2.36 - 2.24 (m, 1H), 2.11 (br d, J=1.8 Hz, 4H), 2.04 - 1.76 (m, 4H); MS (M+H)+ at m/z 296; HPLC tr = 0.94 minutes (Method A). Stereochemistry was assigned by converting one of the isomers to the precursor of 15a and performing a chiral analysis to match the isomers.

((1R,3S)-1-Amino-3-((S)-6-(2-(pyridin-2-yl)ethyl)-5,6,7,8-tetrahydronaphthalen-2yl)cyclopentyl)methanol (22a). 36 An oven dried flask was charged with cesium carbonate (66.2 mg, 0.203 mmol), bis(ditert-butyl(4- dimethylaminophenyl)phosphine)dichloropalladium(II) (3 mg, 4.24 µmol) and MeCN (1 mL) under nitrogen. It was then degassed 3x under vacuum, followed by step wise addition of 2-bromopyridine (10 µl, 0.095 mmol) and (5R,7S)-7-((R)-6-ethynyl-5,6,7,8tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (20a, 20 mg, 0.068 mmol). The reaction mixture was heated at 80 °C overnight. The solvents were removed in vacuo and the residue was dissolved in MeOH (2 mL). Pearlman's catalyst (5 mg, 0.036 mmol) was added and the reaction mixture was hydrogenated under a balloon of H2 for 1 h. The catalyst was filtered away and 1N NaOH (2 mL) was added to the filtrate. The reaction mixture was heated at 95 °C for 6 h then neutralized with TFA and filtered. The filtrate was purified by HPLC (Method B) to afford

((1R,3S)-1-amino-3-((S)-6-(2-(pyridin-2-yl)ethyl)-5,6,7,8-tetrahydronaphthalen-2-

yl)cyclopentyl)methanol, TFA (13 mg, 0.033 mmol, 49% yield) as an off white solid. 1H NMR 49 ACS Paragon Plus Environment

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(400MHz, MeOD)  8.44 (dd, J=5.1, 0.9 Hz, 1H), 7.77 (td, J=7.7, 1.8 Hz, 1H), 7.37 (d, J=7.9 Hz, 1H), 7.26 (ddd, J=7.5, 5.1, 1.1 Hz, 1H), 6.99 (s, 3H), 3.62 - 3.46 (m, 2H), 3.14 - 2.98 (m, 1H), 2.93 (t, J=7.8 Hz, 2H), 2.89 - 2.69 (m, 2H), 2.45 (dd, J=16.3, 9.7 Hz, 1H), 2.31 (dd, J=13.2, 6.4 Hz, 1H), 2.12 - 1.98 (m, 2H), 1.97 - 1.87 (m, 3H), 1.87 - 1.72 (m, 4H), 1.63 (t, J=12.5 Hz, 1H), 1.46 (dtd, J=12.8, 10.4, 5.9 Hz, 1H) ); MS (M+H)+ at m/z 351; HPLC tr = 3.66 minutes (Method D); HPLC Purity = 95%.

((1R,3S)-1-Amino-3-((R)-6-(2-(pyridin-2-yl)ethyl)-5,6,7,8-tetrahydronaphthalen-2yl)cyclopentyl)methanol (22b).36 An oven dried flask was charged with cesium carbonate (66 mg, 0.20 mmol), bis(di-tertbutyl(4- dimethylaminophenyl)phosphine)dichloropalladium(II) (3 mg, 4.24 µmol) and MeCN (1 mL) under nitrogen. It was degassed 3x under vacuum, followed by stepwise addition of (5R,7S)7-((S)-6-ethynyl-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (20b, 20 mg, 0.068 mmol) and 2-bromopyridine (10 µL, 0.095 mmol). The reaction mixture was heated at 80 °C for 1 h, then cooled and quneched with water. The reaction mixture was diluted with ethyl acetate and washed with sat. NaCl. The organic layer was dried with MgSO4, filtered and concentrated. The crude material was dissolved in MeOH (2 mL) and Pearlman's catalyst (5 mg, 0.036 mmol) was added. The reaction mixture was hydrogenated under a balloon of H2 for 1 h. The catalyst was filtered away and 1N NaOH (2 mL) was added to the filtrate. The reaction mixture was heated at 95 °C for 6 h then neutralized with TFA and filtered. The filtrate was purified by HPLC (Method B) to afford ((1R,3S)-1-amino-3-((R)-6-(2-(pyridin-2-yl)ethyl)-5,6,7,8tetrahydronaphthalen-2-yl)cyclopentyl)methanol, TFA (14 mg, 0.036 mmol, 54% yield) as an off 50 ACS Paragon Plus Environment


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white solid. 1H NMR (400 MHz, MeOD)  8.44 (d, J=4.2 Hz, 1H), 7.78 (td, J=7.7, 1.8 Hz, 1H), 7.37 (d, J=7.9 Hz, 1H), 7.26 (ddd, J=6.9, 5.7, 0.9 Hz, 1H), 7.08 - 6.90 (m, 3H), 3.70 - 3.53 (m, 2H), 3.21 - 3.01 (m, 1H), 2.98 - 2.84 (m, 3H), 2.84 - 2.72 (m, 2H), 2.53 - 2.33 (m, 2H), 2.05 (s, 4H), 1.83 - 1.62 (m, 5H), 1.55 - 1.39 (m, 1H); MS (M+H)+ at m/z 351; HPLC tr = 3.62 minutes (Method D); HPLC Purity = 95.1%.

The following compounds were prepared using the procedures described for 22a/22b: ((1R,3S)-1-Amino-3-((S)-6-(2-(pyridin-3-yl)ethyl)-5,6,7,8-tetrahydronaphthalen-2yl)cyclopentyl)methanol (23a). 36 Obtained

((1R,3S)-1-amino-3-((S)-6-(2-(pyridin-3-yl)ethyl)-5,6,7,8-

tetrahydronaphthalen-2-yl)cyclopentyl)methanol, TFA (35 mg, 0.096 mmol, 71% yield) as an off white solid. 1H NMR (400 MHz, MeOD)  8.43 (d, J=1.8 Hz, 1H), 8.37 (dd, J=4.8, 1.3 Hz, 1H), 7.81 - 7.71 (m, 1H), 7.38 (dd, J=7.5, 5.3 Hz, 1H), 7.06 - 6.94 (m, 3H), 3.69 - 3.54 (m, 2H), 3.15 3.02 (m, 1H), 2.99 - 2.74 (m, 5H), 2.55 - 2.32 (m, 2H), 1.95 - 1.85 (m, 5H), 1.79 - 1.63 (m, 4H), 1.57 - 1.39 (m, 1H); MS (M+H)+ at m/z 351; HPLC tr = 3.86 minutes (Method D); HPLC Purity = 95.5%.

((1R,3S)-1-Amino-3-((R)-6-(2-(pyridin-3-yl)ethyl)-5,6,7,8-tetrahydronaphthalen-2yl)cyclopentyl)methanol (23b). 36 Obtained

((1R,3S)-1-amino-3-((R)-6-(2-(pyridin-3-yl)ethyl)-5,6,7,8-

tetrahydronaphthalen-2-yl)cyclopentyl)methanol, TFA (4.5 mg, 0.012 mmol, 36% yield). 51 ACS Paragon Plus Environment

1H

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NMR (400 MHz, MeOD) δ 8.47 - 8.42 (m, 1H), 8.39 - 8.33 (m, 1H), 7.78 - 7.73 (m, 1H), 7.40 7.35 (m, 1H), 7.02 - 7.00 (m, 2H), 7.00 - 6.98 (m, 1H), 3.19 - 3.04 (m, 1H), 2.88 - 2.75 (m, 4H), 2.55 - 2.34 (m, 2H), 2.20 - 1.99 (m, 3H), 1.83 - 1.67 (m, 4H), 1.54 - 1.40 (m, 2H), 1.33 - 1.25 (m, 4H); MS (M+H)+ at m/z 351; HPLC tr = 3.82 minutes (Method D); HPLC Purity = 96.5%.

((1R,3S)-1-Amino-3-((R)-6-(2-(pyridin-4-yl)ethyl)-5,6,7,8-tetrahydronaphthalen-2yl)cyclopentyl)methanol (24). 36 Obtained

((1R,3S)-1-amino-3-((R)-6-(2-(pyridin-4-yl)ethyl)-5,6,7,8-

tetrahydronaphthalen-2-yl)cyclopentyl)methanol, TFA (4 mg, 10.7 µmol, 32% yield) as a clear oil. 1H NMR (400 MHz, MeOD) δ 8.42 (d, J=5.1 Hz, 2H), 7.34 (d, J=5.9 Hz, 2H), 7.06 - 6.95 (m, 3H), 3.77 - 3.47 (m, 2H), 3.20 - 3.04 (m, 1H), 2.99 - 2.73 (m, 4H), 2.57 - 2.33 (m, 2H), 1.82 - 1.62 (m, 4H), 1.39 - 1.20 (m, 7H); MS (M+H)+ at m/z 351; HPLC tr = 3.77 minutes (Method D); HPLC Purity = 94.2%.

((1R,3S)-1-Amino-3-((S)-6-(2-(pyrimidin-2-yl)ethyl)-5,6,7,8-tetrahydronaphthalen-2yl)cyclopentyl)methanol (25a). 36 Obtained

((1R,3S)-1-amino-3-((S)-6-(2-(pyrimidin-2-yl)ethyl)-5,6,7,8-

tetrahydronaphthalen-2-yl)cyclopentyl)methanol, TFA (8 mg, 0.022 mmol, 16% yield) as an off white solid. 1H NMR (400MHz, MeOD) δ 8.74 (d, J=4.8 Hz, 2H), 7.35 (t, J=5.0 Hz, 1H), 7.07 6.94 (m, 3H), 3.75 - 3.53 (m, 2H), 3.13 - 3.01 (m, 2H), 2.95 - 2.63 (m, 3H), 2.52 - 2.34 (m, 2H),



2.16 - 1.99 (m, 2H), 1.94 - 1.84 (m, 6H), 1.79 - 1.62 (m, 2H), 1.55 - 1.39 (m, 1H); MS (M+H)+ at m/z 352; HPLC tr = 5.48 minutes (Method E); HPLC Purity = 95.4%.

((1R,3S)-1-Amino-3-((R)-6-(2-(pyrimidin-2-yl)ethyl)-5,6,7,8-tetrahydronaphthalen-2yl)cyclopentyl)methanol (25b). 36

Obtained

((1R,3S)-1-amino-3-((R)-6-(2-(pyrimidin-2-yl)ethyl)-5,6,7,8-

tetrahydronaphthalen-2-yl)cyclopentyl)methanol, TFA (3 mg, 5.80 µmol, 27% yield) as an off white solid. 1H NMR (400 MHz, MeOD) δ 8.72 (d, J=5.1 Hz, 2H), 7.34 (t, J=5.1 Hz, 1H), 7.03 6.93 (m, 3H), 3.71 - 3.51 (m, 2H), 3.15 - 3.00 (m, 3H), 2.96 - 2.70 (m, 3H), 2.52 - 2.34 (m, 2H), 2.16 - 1.81 (m, 7H), 1.77 - 1.62 (m, 2H), 1.44 (dtd, J=12.8, 10.6, 5.9 Hz, 1H); MS (M+H)+ at m/z 352; HPLC tr = 4.94 minutes (Method D); HPLC Purity = 96.8%.

The following compounds were prepared using the procedures described for 14a/14b:

((1R,3S)-1-Amino-3-(6-(2-(1-methyl-1H-imidazol-5-yl)ethyl)-5,6,7,8-tetrahydronaphthalen2-yl)cyclopentyl)methanol (26).36 Obtained

((1R,3S)-1-amino-3-(6-(2-(1-methyl-1H-imidazol-5-yl)ethyl)-5,6,7,8-

tetrahydronaphthalen-2-yl)cyclopentyl)methanol, TFA (isomer b, 12 mg, 0.023 mmol, 49% yield) as a clear oil. 1H NMR (400 MHz, MeOD)  7.53 (s, 1H), 7.10 - 6.92 (m, 3H), 6.73 (d, J=0.9 Hz, 53 ACS Paragon Plus Environment

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1H), 3.71 - 3.55 (m, 5H), 3.18 - 3.03 (m, 1H), 2.98 - 2.77 (m, 3H), 2.76 - 2.66 (m, 2H), 2.55 - 2.32 (m, 2H), 2.20 - 1.98 (m, 2H), 1.99 - 1.85 (m, 4H), 1.85 - 1.64 (m, 3H), 1.47 (dtd, J=12.8, 10.4, 6.4 Hz, 1H); MS (M+H)+ at m/z 354; HPLC tr = 4.46 minutes (Method E); HPLC Purity = 95.3%.

((1R,3S)-1-Amino-3-((S)-6-(2-(1-methyl-1H-pyrazol-3-yl)ethyl)-5,6,7,8tetrahydronaphthalen-2-yl)cyclopentyl)methanol (27).36 To

a

mixture

of

((R)-6-((5R,7S)-2-oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)-1,2,3,4-

tetrahydronaphthalen-2-yl)methyl 4-methylbenzenesulfonate (1 g, 2.195 mmol) and K2CO3 (0.910 g, 6.59 mmol) in DMF (10) was added 1-phenyl-1H-tetrazole-5-thiol (0.782 g, 4.39 mmol). The reaction mixture was heated at 80 °C overnight. The reaction mixture was diluted with ethyl acetate and washed with sat. NaCl. The organic layer was dried with MgSO4, filtered and concentrated. The crude material was purified on a silica gel cartridge using an EtOAc/Hex gradient (0-100% EtOAc over 13 column volumes) to afford (5R,7S)-7-((R)-6-(((1-phenyl-1Htetrazol-5-yl)thio)methyl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (0.94 g, 2.04 mmol, 93% yield). HPLC retention time = 1.02 min (Method A); MS (M+H)+ at m/z 462. To H2O2 (8.3 mL, 81 mmol) at 0 °C was added ammonium molybdate tetrahydrate (0.50 g, 0.40 mmol). The resulting solution was then added to a mixture of (5R,7S)-7-((R)-6-(((1phenyl-1H-tetrazol-5-yl)thio)methyl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1azaspiro[4.4]nonan-2-one (0.94 g, 2.04 mmol) in THF (15 mL) at 0 °C. The reaction mixture was stirred overnight. The reaction mixture was diluted with ethyl acetate and washed with sat. NaCl. The organic layer was dried with MgSO4, filtered and concentrated to afford (5R,7S)-7-((R)-654 ACS Paragon Plus Environment


(((1-phenyl-1H-tetrazol-5-yl)sulfonyl)methyl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1azaspiro[4.4]nonan-2-one (1 g, 2.03 mmol, 99% yield) which was used without further purification. HPLC retention time = 0.96 min (Method A); MS (M+H)+ at m/z 494. 1M KHMDS in THF (425 µl, 0.425 mmol) was added dropwise to a solution of (5R,7S)7-((R)-6-(((1-phenyl-1H-tetrazol-5-yl)sulfonyl)methyl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3oxa-1-azaspiro[4.4]nonan-2-one (35 mg, 0.071 mmol) and 1-methyl-1H-pyrazole-3-carbaldehyde (39 mg, 0.36 mmol) in DMF (1 mL) and THF (1 mL) at -78 °C. The resultant solution was stirred at that temperature for 2 h then allowed to warm to room temperature and stirred for 16 h,. The reaction mixture was diluted with ethyl acetate and washed with sat. NaHCO3 (2 x 15mL) and sat. NaCl (20mL). The organic layer was dried with Na2SO4, filtered and concentrated. The crude material was purified on a silica gel cartridge using an EtOAc/Hex gradient (0-65% EtOAc over 13 column volumes) to afford (5R,7S)-7-((R)-6-((E)-2-(1-methyl-1H-pyrazol-3-yl)vinyl)-5,6,7,8tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (21, 10 mg, 0.026 mmol, 37% yield). HPLC retention time = 1.14 min (Method A); MS (M+H)+ at m/z 378.3. To a mixture of (5R,7S)-7-((R)-6-((E)-2-(1-methyl-1H-pyrazol-3-yl)vinyl)-5,6,7,8tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (10 mg, 0.026 mmol) in MeOH (5 mL) was added Pd-C (14 mg, 0.013 mmol). The reaction mixture was then hydrogenated under a balloon of H2. The mixture was stirred for 2 h. The catalyst was then filtered away and the filtrate was concentrated in vacuo. The crude product was treated with 1,4-dioxane (2 mL) and 0.5 mL of water with lithium hydroxide hydrate (17 mg, 0.4 mmol). The mixture was stirred at 100 °C oovernight, and then the solvent was removed under a stream of N2. The residue was purified by HPLC (Method B) to afford ((1R,3S)-1-amino-3-((S)-6-(2-(1-methyl-1H-pyrazol-3yl)ethyl)-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol, TFA as an off white solid (2 55 ACS Paragon Plus Environment

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mg, 15% yield). 1H NMR (400 MHz, MeOD) δ 7.48 (d, J=2.2 Hz, 1H), 7.02 - 7.00 (m, 2H), 7.00 - 6.97 (m, 1H), 6.12 (d, J=2.2 Hz, 1H), 3.84 (s, 3H), 3.75 - 3.52 (m, 2H), 3.17 - 3.01 (m, 1H), 2.96 - 2.64 (m, 5H), 2.50 - 2.32 (m, 2H), 2.20 - 1.86 (m, 5H), 1.82 - 1.62 (m, 4H), 1.53 - 1.35 (m, 1H); MS (M+H)+ at m/z 354; HPLC tr = 5.26 minutes (Method D); HPLC Purity = 97.6%.

The following compound was prepared using the procedures described for 27:

((1R,3S)-1-Amino-3-((S)-6-(2-(1-methyl-1H-pyrazol-4-yl)ethyl)-5,6,7,8tetrahydronaphthalen-2-yl)cyclopentyl)methanol (28).36 Obtained

((1R,3S)-1-amino-3-((S)-6-(2-(1-methyl-1H-pyrazol-4-yl)ethyl)-5,6,7,8-

tetrahydronaphthalen-2-yl)cyclopentyl)methanol, TFA as an off-white solid (3 mg, 23% yield). 1H

NMR (400 MHz, MeOD) δ 7.42 (s, 1H), 7.34 (s, 1H), 7.01 (s, 2H), 6.99 (d, J=1.1 Hz, 1H), 3.85

(s, 3H), 3.71 - 3.57 (m, 2H), 3.21 - 3.03 (m, 1H), 3.00 - 2.71 (m, 3H), 2.69 - 2.56 (m, 2H), 2.50 2.34 (m, 2H), 2.19 - 2.07 (m, 1H), 2.05 - 1.85 (m, 4H), 1.80 - 1.57 (m, 4H), 1.43 (dtd, J=12.8, 10.5, 5.9 Hz, 1H); MS (M+H)+ at m/z 354; HPLC tr = 5.56 minutes (Method D); HPLC Purity = 96.4%.

The following compound was prepared using the procedures described for 22a/22b:



((1R,3S)-1-Amino-3-((R)-6-(2-(4-methoxypyridin-2-yl)ethyl)-5,6,7,8-tetrahydronaphthalen2-yl)cyclopentyl)methanol (29).36

Obtained

((1R,3S)-1-amino-3-((R)-6-(2-(4-methoxypyridin-2-yl)ethyl)-5,6,7,8-

tetrahydronaphthalen-2-yl)cyclopentyl)methanol, TFA (6 mg, 0.012 mmol, 34% yield) as a clear oil. 1H NMR (400MHz, MeOD) δ 8.51 (d, J=7.0 Hz, 1H), 7.47 (d, J=2.9 Hz, 1H), 7.39 (dd, J=7.0, 2.6 Hz, 1H), 7.08 - 6.96 (m, 3H), 4.15 (s, 3H), 3.74 - 3.55 (m, 2H), 3.21 - 3.04 (m, 3H), 2.99 - 2.76 (m, 3H), 2.61 - 2.36 (m, 2H), 2.17 - 2.02 (m, 2H), 2.00 - 1.89 (m, 3H), 1.89 - 1.80 (m, 3H), 1.74 (t, J=12.8 Hz, 1H), 1.58 - 1.42 (m, 1H); MS (M+H)+ at m/z 352; HPLC tr = 4.94 minutes (Method D); HPLC Purity = 97.1%.

Preparation of phosphates:

((1R,3S)-1-Amino-3-((S)-6-(2-methoxyphenethyl)-5,6,7,8-tetrahydronaphthalen-2yl)cyclopentyl)methyl dihydrogen phosphate (14a-P). 36 To

a

mixture

of

((1R,3S)-1-amino-3-(6-(2-methoxyphenethyl)-5,6,7,8-

tetrahydronaphthalen-2-yl)cyclopentyl)methanol, TFA (2 mg, 4.05 µmol) in acetonitrile (0.5 mL) was added pyrophosphoryl chloride (30 µl, 0.217 mmol). Stirred for 1 h then quenched with water and purified by HPLC (Method B) to afford ((1R,3S)-1-amino-3-((S)-6-(2-methoxyphenethyl)5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methyl dihydrogen phosphate (1.5 mg, 82% yield). 1H

NMR (500 MHz, CDCl3 + CF3OD (2 drops)) δ 7.22 - 7.15 (m, 2H), 7.06 (d, J=7.9 Hz, 1H),


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6.99 -6.87 (m, 4H), 4.18 (br d, J=7.3 Hz, 2H), 3.86 (s, 3H), 3.21 - 3.08 (m, 1H), 2.93 (br dd, J=16.6, 4.4 Hz, 1H), 2.85 - 2.69 (m, 4H), 2.56 - 2.39 (m, 2H), 2.27 - 2.10 (m, 2H), 2.10 - 1.88 (m, 4H), 1.84 - 1.72 (m, 1H), 1.66 (qd, J=7.5, 2.6 Hz, 2H), 1.44 (dtd, J=12.8, 10.7, 6.1 Hz, 1H); MS (M+H)+ at m/z 460; HPLC tr = 6.94 minutes (Method D); HPLC Purity = 98.4%.

The following phosphates were prepared using the procedure for 14a-P:

((1R,3S)-1-Amino-3-((R)-6-(2-methoxyphenethyl)-5,6,7,8-tetrahydronaphthalen-2yl)cyclopentyl)methyl dihydrogen phosphate (14b-P). 36 Recovered

((1R,3S)-1-amino-3-((R)-6-(2-methoxyphenethyl)-5,6,7,8-

tetrahydronaphthalen-2-yl)cyclopentyl)methyl dihydrogen phosphate (1.4 mg, 77% yield). MS (M+H)+ at m/z 460; HPLC tr = 0.88 minutes (Method A); HPLC Purity = 95%.

((1R,3S)-1-Amino-3-((R)-6-(3-methoxyphenethyl)-5,6,7,8-tetrahydronaphthalen-2yl)cyclopentyl)methanol dihydrogen phosphate (15a-P).36 Recovered

((1R,3S)-1-amino--((R)-6-(3-methoxyphenethyl)-5,6,7,8-

tetrahydronaphthalen-2-yl)cyclopentyl)methyl dihydrogen phosphate(1 mg, 55% yield). MS (M+H)+ at m/z 460; HPLC tr = 0.86 minutes (Method A); HPLC Purity = 95%.



((1R,3S)-1-Amino-3-((S)-6-(3-methoxyphenethyl)-5,6,7,8-tetrahydronaphthalen-2yl)cyclopentyl)methanol dihydrogen phosphate (15b-P).36 Recovered

((1R,3S)-1-amino-3-((S)-6-(3-methoxyphenethyl)-5,6,7,8-

tetrahydronaphthalen-2-yl)cyclopentyl)methyl dihydrogen phosphate (1.4 mg, 77% yield). MS (M+H)+ at m/z 460; HPLC tr = 0.86 minutes (Method A); HPLC Purity = 95%.

((1R,3S)-1-Amino-3-((R)-6-(4-methoxyphenethyl)-5,6,7,8-tetrahydronaphthalen-2yl)cyclopentyl)methanol dihydrogen phosphate (16a-P). 36 Recovered

((1R,3S)-1-amino-3-((R)-6-(4-methoxyphenethyl)-5,6,7,8-

tetrahydronaphthalen-2-yl)cyclopentyl)methanol dihydrogen phosphate (1.5 mg, 83% yield). MS (M+H)+ at m/z 460; HPLC tr = 0.86 minutes (Method A); HPLC Purity = 95%.

((1R,3S)-1-Amino-3-((S)-6-(4-methoxyphenethyl)-5,6,7,8-tetrahydronaphthalen-2yl)cyclopentyl)methanol dihydrogen phosphate (16b-P).36 Recovered


yl)cyclopentyl)methyl dihydrogen phosphate (1.5 mg, 83% yield). MS (M+H)+ at m/z 460; HPLC tr = 0.86 minutes (Method A); HPLC Purity = 95%.

Biological Methods. All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee and conformed to the “Guide for the Care and Use 59 ACS Paragon Plus Environment

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of Laboratory Animals” published by the National Institutes of Health (NIH Publication No. 8523, revised 2011). Phosphates were dissolved in 0.3M NaOH as a 3 mM stock prior to being assayed.

SIP1 Binding Assay: Membranes were prepared from CHO cells expressing human S1P1 . Cell pellets (l x 109 cells/pellet) were suspended in buffer containing 20 mM HEPES (4-(2hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.5, 50 mM NaCl, 2 mM EDTA (ethylenediaminetetraacetic acid) and Protease Inhibitor cocktail (Roche), and disrupted on ice using the Polytron homogenizer. The homogenate was centrifuged at 20,000 rpm (48,000 g) and the supernatant was discarded. The membrane pellets were resuspended in buffer containing 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl2, 2 mM EDTA and stored in aliquots at -80 °C after protein concentration determination. Membranes (2 µg/well) and 0.03 nM final concentration of 33P-S1P ligand (1 mCi/mL, Perkin elmer or American Radiolabeled Chemicals) diluted in assay buffer (50 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% fatty acid free BSA (bovine serum albumin), 1 mM NaF) were added to the compound plates (384 Falcon v-bottom plate (0.5 µL/well in a 11 point, 3-fold dilution). Binding was performed for 45 minutes at room temperature, terminated by collecting the membranes onto 384- well Millipore FB filter plates, and radioactivity was measured by TOPCOUNT®. The competition data of the test compounds over a range of concentrations was plotted as percentage inhibition of radioligand specific binding.

Receptor [35S] GTPγS Binding Assays (S1P1 GTPγS / S1P3 GTPγS): Compounds were loaded in a 384 Falcon v-bottom plate (0.5 µL/well in a 11 point, 3- fold dilution). Membranes prepared from 60 ACS Paragon Plus Environment


S1P1/CHO cells or EDG3-Ga15-bla HEK293T cells (EDG3 equivalent S1P3) were added to the compound plate (40 µL/well, final protein 3 µg/well) with MULTIDROP®. [35S]GTP (1250 Ci/mmol, Perkin - 3 - Elmer) was diluted in assay buffer: 20 mM HEPES, pH7.5, 10 mM MgCl2, 150 mM NaCl, 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM dithiothreitol, 10 µM GDP, 0.1% fatty acid free BSA, and 10 µg/mL Saponin to 0.4 nM. 40 µL of the [35S]GTP solution was added to the compound plate with a final concentration of 0.2 nM. The reaction was kept at room temperature for 45 min. At the end of incubation, all the mixtures in the compound plate were transferred to Millipore 384-well FB filter plates via the VELOCITY l ® Vprep liquid handler. The filter plate was washed with water 4 times by using the manifold Embla plate washer and dried at 60 °C for 45 min. MicroScint 20 scintillation fluid (30 µL) was added to each well for counting on the Packard TOPCOUNT®. EC50 is defined as the agonist concentration that corresponds to 50% of the Ymax (maximal response) obtained for each individual compound tested.

hS1P1 cAMP assay: Test compounds were prediluted in 1-µM forskolin (Sigma Aldrich, MO, USA) and 100-µM isobutyl methyl xanthine (IBMX; Sigma Aldrich, MO, USA) in PBS buffer on 384-well Proxi-plates (Perkin Elmer, MA, USA). hS1P-1/CHO cells were plated in F12 medium (Gibco, NY, USA) and incubated with test compound for 30 minutes at room temperature. cAMP was measured using cAMP homogeneous time-resolved fluorescence (HTRF) reagents (CISBIO, MA, USA) per manufacturer’s instructions. Briefly, cells were lysed using HTRF lysis buffer, incubated with anti-cAMP cryptate for 1 h at room temperature, and then read on Perkin Elmer Envision. A standard curve was run on each plate to obtain the absolute concentration of cAMP. The IC50 was defined as the concentration of test compound which produces 50% of the maximal


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response (defined by 1 M S1P) and was quantified using the 4-parameter logistic equation to fit the data.

hS1P1 ERK phosphorylation: hS1P1/CHO cells were plated in BD Amine 384-well plates the day before the assay. On the day of the assay, growth medium was removed and replaced with serumfree medium (Ham’s F-12; Invitrogen) and incubated for 2 h. Test compounds prediluted in HBSS/20 mM HEPES (Gibco, NY, USA) were transferred to the cell plates and incubated for 7 minutes at 37 °C. Cells were lysed in a lysis buffer (Perkin Elmer, MA, USA), and phospho-ERK was measured using the SureFire pERK kit (Perkin Elmer, MA, USA) per manufacturer’s instructions. Data were plotted as percentage activation of the test compound relative to the efficacy of 10 µM S1P. The EC50 was defined as the concentration of test compound which produces 50% of the maximal response and was quantified using the 4-parameter logistic equation to fit the data.

S1P1 Internalization Assay: For quantification of S1P1 expression using a high content system, GFP was fused to hS1P1 at its C-terminus and a stable CHO cell line expressing hS1P1/GFP was established. Cells were suspended in assay medium (F12 medium with 5 % Charcoal-Dextran treated FBS, 20 mM HEPES, and 1X Pen/Strep) at 7.5 x 104 cells/mL and plated into 384 well plates (in 20 L, 1,500 cells/well final) with a Multidrop liquid handler (Thermo Scientific, Waltham, MA). Cell plates were incubated at 37 ºC for 48 h. Titrated S1P and test compounds were dispensed into the cell plates (concentration range from 100 M to 0.005 μM) and the plates were further incubated at 37 ºC for 50 min. Cells were fixed by adding 10 μL of fixation buffer 62 ACS Paragon Plus Environment


(7.4% v/v formaldehyde containing 15 μM Draq5 in DPBS) directly onto the assay medium and incubated for 15 min at room temperature followed by two washes with DPBS. This was followed by the addition of 50 μl DPBS to the cell layer and the plates were sealed with a transparent plate seal (Perkin Elmer #6005185). Imaging was carried out using the Evotec Opera High Content Confocal System (Perkin Elmer, Boston, MA) equipped with 3 diode lasers, peltier cooled CCD camera detector and a Nipkow spinning disk. Cell images were quantified using a membrane fluorescent scoring algorithm. Data was analyzed using a customized HTS data analysis software package. EC50 values were determined using XL-Fit software program.

Chemotaxis assay: Human T cells were isolated from peripheral blood mononuclear cells by rosetting with sheep red blood cells for 1 h at 4 oC. The sheep red blood cells were removed by lysis using ACK lysing buffer (Gibco). Human T cells from two donors were cultured for 72 h in RPMI-1640 containing 0.5% fatty-acid free BSA (Calbiochem) and then labeled with the fluorescent dye Calcein-AM (10 g/mL; Molecular Probes) in chemotaxis buffer (phenol red-free RPMI-1640, containing 0.5% fatty-acid free BSA) at room temperature for 45 min. The labeled cells were then washed and resuspended in chemotaxis buffer at 1.2x107/mL. Chemotaxis was measured using a chemotaxis plate with 5 m pores (Neuroprobe). Labeled cells (3 x 105 in 25 L of chemotaxis buffer) were pre-incubated with test compound (final assay concentrations ranged from 0.01 to 50 nM) for 10 min at room temperature before loaded to each of the upper wells of a plate. The bottom wells of the plate were loaded with S1P at concentration that is known to give 90% maximal chemotaxis without presence of test compounds. The membrane was placed over the lower wells and the plate was covered and incubated at 37 C for 120 min. Following 63 ACS Paragon Plus Environment

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incubation the upper wells were washed twice with PBS to remove un-migrated cells and fluorescence in the lower wells was read at 485 nm/530 nm excitation/emission on a Cytofluor 4000 (Perceptive Biosystems). The inhibition of chemotaxis achieved by titrated concentrations of compounds was calculated as a percentage of the compound-free S1P control signal after plate background and buffer background signal subtraction. The IC50 is defined as the concentration of compound required to reach 50% inhibition of chemotaxis and was calculated by the XLFit software program.

Blood Lymphocyte Reduction (BLR) assay in rodent: Balb-C mice or Lewis rats were dosed orally with vehicle alone (polyethylene glycol 300) or with test compounds. Compounds were dosed as a solution or suspension in the vehicle, adjusted to reflect the free amount of test article in the event that salt forms are utilized. Blood was drawn at different time points and blood lymphocyte counts were determined on an ADVIA 120 Hematology Analyzer (Siemens Healthcare Diagnostics). The results were measured as a reduction in the percentage of circulating lymphocytes as compared to the vehicle treated group at the time of measurement. The results represent the average results of all animals within each treatment group (n = 2-4).

In Vivo Phosphate Ester Formation: BALB/c mice or Lewis rats were dosed orally with compounds. Blood was drawn at 24 h, spotted onto Dried Blood Spot (DBS) Cards. DBS Cards were stored at ambient temperature in sealed plastic bags with desiccant added. When ready for analysis, a 6 mm punch (equivalent to 12.5 L of wet blood) was taken at n = 1 and placed in a shallow 96-well filter plate. Next, 105 L of a mixture of 75% acetonitrile and 25% water 64 ACS Paragon Plus Environment


containing internal standard was added and gently vortexed for 30 min then centrifuged. Supernatant was separated from the protein pellet and 5 l was injected. The parent (alcohol) and active phosphate ester compounds were quantitatively analyzed, using a DBS calibration curve, by LC/MS/MS on a Triple Quadrapole Instrument. The area ratios of the phosphorylated compound to the parent (alcohol) compound were determined. A larger value for the ratio of the phosphorylated compound to the parent (alcohol) compound indicated greater phosphate ester compound formation from the parent (alcohol) compound. Reference material (parent alcohol and phosphate ester) was analyzed to optimize the LC-MS/MS assay and enable data reporting in concentrations. DBS standard curves containing both parent alcohol and phosphate ester compounds were prepared and analyzed in the same manner as the study samples and analyzed by the optimized LC-MS/MS to quantify the amount of phosphate ester compound formed. The results represent the average results of all animals within each treatment group (n = 2-4).

T1/2 microsome assay: S1P1 compounds were added to 0.1 M Tris-HCl Buffer (pH 7.4, 37 °C) containing 1 mg/mL liver microsomal protein and 1 mM MgCl2 in a shaking water bath, to yield a final compound concentration of 0.5 µM. The reaction was initiated by adding an NADPH-regenerating system. The samples were taken at 0, 60, and 120 minutes and the reaction was stopped by the addition of acetonitrile to precipitate proteins. The samples were then centrifuged and prepared for analysis via ultra-performance liquid chromatography (UPLC) with tandem mass spectrometry (LC/MS/MS). %remaining (%Rem) was quantified by peak ratio of analyte to internal standard at time t to time 0. In vitro T1/2 was estimated by

𝐼𝑛 𝑣𝑖𝑡𝑟𝑜 𝑇1/2 = 0.693/𝑘 %𝑅𝑒𝑚 = 100 𝑒 ―𝑘𝑡 65 ACS Paragon Plus Environment

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where k denotes the rate constant of compound elimination from in vitro microsomal incubation. In vivo T1/2 was estimated by 𝐼𝑛 𝑣𝑖𝑣𝑜 𝑇1/2 = 0.693 𝑉/𝐶𝐿 V denotes the volume of distribution and was rendered a value of 13 L/kg based on the similar volume of distribution of the compounds in this series. CL (clearance) is scaled up from in vitro T1/2 based on an established method - Ref: Obach, R.S.; Baxter, J.G.; Liston, T. E.; Silber, B. M.; Jones, B. C.; MacIntyre, F.; Rance, D. J.; Wastall, P. The prediction of human pharmacokinetic parameters from preclinical and in vitro metabolism data. J Pharmacol Exp Ther 1997, 283, 4658.

In Vitro Phosphate Ester Formation:

The rates of ATP-dependent phosphorylation were

determined using recombinant sphingosine kinase 1 and 2 (SK1 and SK2). Test compounds (30 mM stock in DMSO) were diluted to 0.3 mM in 0.2% fatty acid-free BSA in 1 mM Tris-HCl (pH 7.4). The phosphorylation reaction consisted of 80 L assay buffer (100 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM MgCl2, 1 mM sodium vanadate, 10 mM sodium fluoride, and 0.5 mM 4deoxypyridoxine-HCl), 10 L 0.3 mM substrate/BSA, 5 L kinase enzyme (SK1, human recombinant, #10348, 0.44 mg/mL (0.22 g/assay) Cayman Chemical Company, Ann Arbor, MI; SK2, human recombinant, #E-K069, 0.5 mg/mL (0.25 g/assay) Echelon Biosciences, Inc, Salt Lake City, UT), and 5 L 20 mM Mg-ATP (containing 1 Ci [33P]ATP, pH 7.4). The reaction was incubated for 30 minutes in a 37 oC shaking water bath, and stopped by addition of cold, 350 L methanol-HCl (150:1), 250 l 2M KCl, and 350 L chloroform. The tubes were vigorously 66 ACS Paragon Plus Environment


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mixed, and placed on ice for 15 minutes. The chloroform phase was separated from the aqueous phase by centrifugation at 10,000 g, for 10 minutes. The upper aqueous phase was discarded and the remaining lipid/chloroform phase was evaporated over a nitrogen stream. The remaining pellet was resuspended in 40 L 1-butanol, and completely spotted at the origin of a thin-layer chromatography (TLC) plate. The product was resolved in 1-butanol:glacial acetic acid:water (3:1:1) for 2.5 h in a chromatography tank. Standards were prepared from the [33P]ATP assay stock (200 nCi/L), and diluted 1:10 (20 nCi/L), 1:100 (2.0 nCi/L), 1:1000 (0.2 nCi/L), and 1:10,000 (0.02 nCi/L) in PBS. Each dilution standard was spotted at the top of the TLC plate at 0.2, 0.4, 0.6, 0.8, and 1.0 L, and produced a linear standard curve on each plate. The TLC plate, including the resolved lipid products and spotted standards, were enclosed in plastic wrap, and exposed for 18 h to a phosphor-imaging storage screen. The captured area-volume of radiation was determined with an imaging scanner (Storm®, GE Healthcare, Sunnyvale, CA). Quantitation of the standards and resolved product were determined by ImageQuant® software

Pulmonary Toxicity Assay: The analysis of protein levels in bronchoalveolar lavage (BAL) fluid obtained from an animal were used to gauge pulmonary side effects.

Post study rats were

euthanized with intraperitoneal barbiturate overdose. The animals were placed in a supine position, a skin incision was made and blunt dissection followed to expose the trachea. The trachea was incised and a catheter was inserted 4-6 mm into the trachea. Phosphate-buffered saline (PBS; 1 mL/mouse) was infused into the lungs and then aspirated. The concentration of the protein in the recovered BAL fluid was determined on an Advia 1800 Chemistry Analyzer (Siemens


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Healthcare Diagnostics). The results represent the average results of all animals within each treatment group (n = 2-4).

Multi-Electrode Array (MEA) Electrophysiology Studies in Human-Inducible Pluripotent Stem Cell-Derived Cardiomyocytes: Human-inducible pluripotent stem cell-derived cardiomyocytes (hiPSC CMs) were purchased from Cellular Dynamics International (Madison, WI). qPCR analysis showed similar RNA expression levels of S1P1, S1P2 and S1P3 in hiPSC CMs as in adult human heart tissue (results not shown). hiPSC CMs were cultured with 7% CO2 on 0.1% gelatin treated 6-well culture plates for 7 days, then trypsinized and diluted with cardiac fibroblasts (10%). Suspensions of hiPSC CMs and fibroblasts were then co-cultured on laminin-coated 9-well multielectrode arrays (MEA) plates (256-9 well MEA300/30iRITO-mq; Multichannel Systems; Atlanta, GA). After 7 days culture on MEA plates, cells formed a spontaneously beating monolayer over recording electrodes imbedded in each well. Spontaneous extracellular field potentials (FPs) were recorded from 28 electrodes/well at a sampling frequency of 10 kHz using an USB-MEA256-System and MC Rack acquisition software (Multi Channel Systems). Following a 20-60 minute equilibration period in a humidified environment at 37 oC with constant 5% CO2 and 95% O2 supply, compounds were added to each well in 300 μL maintenance medium with final DMSO or NaOH vehicle concentration less than 0.1% or 30 μM. Dilute NaOH (30 μM was used as control in these studies and had no significant effect on beating rate of hiPSC CMs. Effects of test agents on field potentials were evaluated for at least 2 h. Data were analyzed with MC_DataTool and custom software written in MatLab (Mathworks; Natick, MA).



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Mouse Experimental Autoimmune Encephalomyelitis Assay (EAE): Mice (C57BL/6 female, 6-8 weeks of age, Charles River, n=10-12 treatment group) were immunized subcutaneously with 150 g MOG35-55 emulsified 1:1 with incomplete Freund’s adjuvant (sigma) supplemented with 150 g Mycobacterium tuberculosis H37RA (Difco Laboratories).

400 ng of pertussis toxin

(CalBiochem) was injected intraperitoneally on the day of immunization and two days later. Animals were dosed once daily with the test article (as a solution or suspension in the vehicle) or vehicle alone (polyethylene glycol 300, “PEG300”) starting from 1 day after immunization. Clinical scoring and body weight were taken 3 times per week until the end of the study on day 24. Clinical scoring system: 0.5: partial tail weakness; 1: limp tail or waddling gait with tail tonicity; 1.5: waddling gait with partial tail weakness; 2: waddling gait with limp tail (ataxia); 2.5: ataxia with partial limb paralysis; 3: full paralysis of one limb; 3.5: full paralysis of one limbs with partial paralysis of a second limb; 4: full paralysis of two limbs; 4.5: moribund; 5: death. Upon termination of the study, spinal columns were excised and submersion fixed in 10% neutral buffered formalin. Three transverse sections of lumbar spinal cord were routinely processed, paraffin embedded (RPPE) and were sectioned at 3 μm and 6um, stained with hematoxylin and eosin and the myelin-specific stain Luxol Fast Blue, respectively. Slides were analyzed in a blinded fashion for severity of inflammation, tissue damage, and demyelination. Lesion activity was scored on a semiquantitative 0−5 scale, and overall mean histological score of inflammation, damage, and demyelination from each group was calculated.

Organotypic Cerebellar Slice Culture Assay: Newborn (P1-2) wild-type CD1 mouse pups were obtained from Charles River. After decapitation, the cerebellum was isolated and cut into 350 μm parasagittal slices. Slices were then transferred to culture inserts (Millipore Millicell-CM 69 ACS Paragon Plus Environment

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organotypic culture inserts) in a 6-well plate with 1 mL of culture medium and incubated at 36−37 °C with 5% CO2. Culture medium was composed of 50% minimal essential media, 25% heat-inactivated horse serum, 25% Earle’s balanced salt solution, 6.5 mg/mL glucose (SigmaAldrich, St. Louis, MO), and penicillin and glutamine supplements (all from Invitrogen, Carlsbad, CA) and was replaced every 2−3 days. Slices were cultured for 13 days in vitro (DIV13) to allow clearance of debris and myelination to occur. For remyelination studies, slices were incubated with 0.5 mg/mL LPC overnight and then transferred to the culture medium for 24 h. Afterward, slices were treated with 1-P or 14a-P for 7 days before being processed for immunohistochemistry.

Cerebellar slices were fixed with 4% paraformaldehyde and incubated with rat anti-myelin basic protein (MBP, 1:1000, Abcam) and rabbit anti-calbindin D-28K (CBD, 1:4000, Swant, Switzerland) at 4 °C overnight. MBP and CBD were used for visualizing myelin protein and Purkinje cell bodies and axonal processes, respectively. The slices were incubated with 1:500 goat anti-rat IgG Alexa Fluor 594 and goat anti-rabbit IgG Alexa Fluor 488 (Life Technologies) and then mounted and cover-slipped. A SP8 confocal microscopy was used for immunofluorescence image acquisition. MBP and CBD immunostaining was imaged at ×40 magnification, and multiple images were captured from each slice. The areas covered by axonal processes singly stained for CBD or doubly stained for CBD and MBP immunoreactivity were analyzed using a macro with Image-J (NIH). The myelination index was calculated by dividing CBD + MBP double immunoreactivity/area unit with CBD single immunoreactivity/area unit as a measure of the amount of myelination per axon area. Quantitative data were analyzed statistically using ANOVA, followed by the Dunnett’s multiple comparison test. 70 ACS Paragon Plus Environment


Collagen-Induced Arthritis in Mice: DBA/1 male mice (8-10 wk of age; Harlan) were immunized subcutaneously at the base of the tail on Day 0 and again on Day 21 with 200 ug bovine type II collagen admixed with reconstituted Sigma Adjuvant System (SAS; Sigma-Aldrich). For “preventative” dosing, mice were dosed daily (beginning on Day 0) by oral gavage with vehicle (EtOH:TPGS:PEG300; 5:5:90) or with 14a. mCTLA4Ig was injected intraperitoneally twice per week at 1 mg/kg. Following the booster immunization, disease activity was monitored and scored twice per week using standard criteria (0: normal; 1: mild, but definite redness and swelling of the ankle or wrist, or apparent redness and swelling limited to individual digits regardless of number of affected digits; 2: moderate redness and swelling of ankle or wrist; 3: severe redness and swelling of the entire paw including digits; 4: maximally inflamed limb with involvement of multiple joints). Clinical paw scores for all four paws were summed for each mouse, and mean ± SEM was calculated for each treatment group. For histological evaluation, right hind paws were fixed, decalcified and embedded in paraffin. Sections were cut in the sagittal plane, stained in H &E and evaluated microscopically without knowledge of treatment group. Lesions were scored on a severity scale of 0 (normal) to 4 in two separate categories, inflammation (cellular infiltration, pannus formation and edema) and bone resorption. For inflammation/edema, the scoring was as follows: Grade 0, unremarkable; grade 1, minimal change; grade 2, mild change; grade 3, moderate change; grade 4, marked change. For bone resorption, the grading was as follows: Grade 0, unremarkable; grade 1, minimal focal to multifocal cortical or trabecular bone resorption; grade 2, mild focal to multifocal cortical or trabecular bone resorption; grade 3, moderate resorption of cortical or trabecular bone with rare full-thickness resorption; grade 4, full-thickness resorption with distortion of bone architecture. 71 ACS Paragon Plus Environment

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Statistical analysis for all the parameters were compared by one-way analysis of variance and Dunnett’s multiple comparison test.

ASSOCIATED CONTENT Supporting Information Available. The Supporting Information is available free of charge on the ACS Publications website. X-ray crystallographic structure of 18b X-ray crystallographic structure of 14a Molecular formula strings (CSV) AUTHOR INFORMATION * To whom correspondence should be addressed. Phone: 609-252-3153. E-mail: [email protected].

ABBREVIATIONS USED S1P, Sphingosine-1-phosphate; S1P1-5, Sphingosine-1-phosphate receptors 1-5; RRMS, relapsing-remitting multiple sclerosis; BLR, blood lymphocyte reduction; BAL, bronchoalveolar lavage; CIA, collagen induced arthritis; EAE, experimental autoimmune encephalomyelitis.



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R.; Cyster, J. G. Finding a way out: lymphocyte egress from lymphoid organs. Nat. Immunol. 2007, 8, 1295-1301. (c) Rosen, H.; Goetzl, E. J. Sphingosine 1-phosphate and its receptors: an autocrine and paracrine network. Nat. Rev. Immunol. 2005, 5, 560-570. (8)

Pappu, R.; Schwab, S. R.; Cornelissen, I.; Pereira, J. P.; Regard, J. B.; Xu, Y.; Camerer, E.; Zheng, Y.-W.; Huang, Y.; Cyster, J. G.; Coughlin, S. R. Promotion of lymphocyte egress into blood and lymph by distinct sources of sphingosine-1-phosphate. Science 2007, 316, 295–298.

(9)

Singer, B.; Ross A. P.; Tobias, K. Oral fingolomid for the treatment of patients with relapsing forms of multiple sclerosis. Int. J. Clin. Pract. 2011, 65, 887-895.

(10) Sanchez, T.; Estrada-Hernandez, T.; Paik, J.-H.; Wu, M.-T.; Venkataraman, K.; Brinkmann, V.; Claffey, K.; Hla, T. Phosphorylation and action of the immunomodulatory FTY720 inhibits vascular endothelial cell growth factor-induced vascular permeability. J Biol. Chem. 2003, 47, 47281-47290. (11)

(a) Dyckman, A. Modulators of sphingosine-1-phosphate pathway biology: recent advances in the discovery and development of sphingosine-1-phosphate-1 receptor agonists. J. Med. Chem., 2017, 60, 5267–5289 and references cited therein. (b) Roberts, E.; Guerrero, M.; Urbano, M.; Rosen, H. Sphingosine 1-phosphate receptor agonists: a patent review (2010-2012). Expert Opin. Ther. Pat. 2013, 23, 817-841 and references cited therein.

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Kappos, L.; Antel, J.; Comi, G.; Montalban, X.; O’Connor, P.; Polman, C. H.; Haas, T.; Korn, A. A.; Karlsson, G.; Radue, E. W. Oral fingolimod (FTY720) for relapsing multiple sclerosis. N. Engl. J. Med. 2006, 355, 1124-1140.

(13)

David, O. J.; Kovarik J. M,; Schmouder R. L. Clinical pharmacokinetics of fingolimod.



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Clin. Pharmacokinet. 2012, 51, 15-28. (14)

Gergely, P.; Nuesslein-Hildesheim, B.; Guerini, D.; Brinkmann, V.; Traebert, M.; Bruns, C.; Pan, S.; Gray, N. S.; Hinterding, K.; Cooke, N. G.; Groenewegen, A.; Vitaliti, A.; Sing, T.; Luttringer, O.; Yang, J.; Gardin, A.; Wang, N.; Crumb, W. J. Jr.; Saltzman, M.; Rosenberg, M.; Wallstrom, E. The selective sphingosine 1-phosphate receptor modulator BAF312 redirects lymphocyte distribution and has species-specific effects on heart rate. Br. J. Pharm. 2012, 167, 1035-1047.

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(a) Dhar, T. G. M.; Xiao, H.-Y.; Xie, J.; Lehman-McKeeman, L. D.; Wu, D.-R.; Dabros, M.; Yang, X.; Taylor, T. L.; Zhou, X. D.; Heimrich, E. M.; Thomas, R.; McIntyre, K. W.; Warrick, B.; Shi, H.; Levesque, P. C.; Zhu, J. L.; Hennan, J.; Balimane, P.; Yang, Z.; Marino, A.; Cornelius, G.; Darienzo, C.; Mathur, A.; Shen, D. R.; Cvijic, M. E.; Salter-cid, L.; Barrish, J. C.; Carter, P. C.; Dyckman, A. J. pharmacology

of

clinical

candidate

Identification and preclinical

((1R,3S)-1-amino-3-((R)-6-hexyl-5,6,7,8-

tetrahydronaphthalen-2-yl)cyclopentyl)methanol (BMS-986104) - a differentiated S1P1 receptor modulator. ACS Med. Chem. Lett. 2016, 7, 283–288. (b) Marcoux, D.; Xiao, H.Y.; Dhar, T. G. M.; Xie, J.; Lehman-McKeeman, L. D.; Wu, D.-R.; Dabros, M.; Yang, X.; Taylor, T. L.; Zhou, X. D.; Heimrich, E. M.; Thomas, R.; McIntyre, K. W.; Shi, H.; Levesque, P. C.; Sun, H.; Yang, Z.; Marino, A.; Cornelius, G.; Darienzo, C.; Gupta, A.; Pragalathan, B.; Rampulla, R.; Mathur, A.; Shen, D. R.; Cvijic, M. E.; Salter-cid, L.; Lombardo, L. J.; Carter, P. C.; Dyckman, A. J. Identification of potent tricyclic prodrug S1P1 receptor modulators Med. Chem. Commun. 2017, 8, 725. (c) Zhu, R.; Snyder, A. H.; Kharel, Y.; Schaffter, L.; Sun, Q.; Kennedy, P. C.; Lynch, K. R.; Macdonald, T. L. Asymmetric synthesis of conformationally constrained fingolimod analogues--discovery 75 ACS Paragon Plus Environment

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of an orally active sphingosine 1-phosphate receptor type-1 agonist and receptor type-3 antagonist. J. Med. Chem. 2007, 50, 6428-6435. (d) Gilmore, J. L.; Sheppeck, J. E.; Watterson, S. H.; Haque, L.; Mukhopadhyay, P.; Tebben, A. J.; Galella, M. A.; Shen, D. R.; Yarde, M.; Cvijic, M. E.; Borowski, V.; Gillooly, K. M.; Taylor, T.; McIntyre, K. W.; Warrack, B.; Levesque, P. C.; Li, J. P.; Cornelius, G.; D’Arienzo, C. J.; Marino, A. M.; Balimane, P. V.; Salter-Cid, L.; Barrish, J. C.; Pitts, W. J.; Carter, P. H.; Xie, J.; Dyckman, A. J. Discovery and structure activity relationship (SAR) of a series of ethanolamine-based direct acting agonists of sphingosine-1-phosphate (S1P1). J. Med. Chem. 2016, 59, 6248−6264. (16)

Protein sequence alignment shows sphinosine kinase (SK) and sphingosine-1-phosphate phosphatase (SGPP) share high homology between rat and human, with 77-79% for SK1, 79-80% for SK2, 81% for SGPP1, and 82-85% for SGPP2.

(17)

Wallace, G. A.; Gordon, T. D.; Hayes, M. E.; Konopacki, D. B.; Fix-Stenzel, S. F.; Zhang, X.; Grongsaard, P.; Cusack, K. P.; Schaffter, L. M.; Henry, R, F.; Stoffel, R. H. Scalable synthesis and isolation of the four stereoisomers of methyl 1-amino-3-(4bromophenyl)cyclopentanecarboxylate, useful intermediates for the synthesis of S1P1 receptor agonists. J. Org. Chem., 2009, 74, 4886-4889.

(18)

Silveira, C. C.; Braga, A. L.; Kaufmanb, T. S.; Lenardao, E. J. Synthetic approaches to 2tetralones. Tetrahedron, 2004, 60, 8295-8328.

(19)

Sonogashira, K.; Tohda, Y.; Hagihara, N. A convenient synthesis of acetylenes: catalytic substitutions

of

acetylenic

hydrogen

with

bromoalkenes,

bromopyridines. Tet. Lett. 1975, 16, 4467-4470. 76 ACS Paragon Plus Environment

iodoarenes

and


(20)

Page 78 of 81

The absolute and relative stereochemistry of compound 18b was unambiguously assigned ((S)-6-((5R,7S)-2-oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)-1,2,3,4-tetrahydronaphthalen-2yl)methyl 4-methylbenzenesulfonate by X-ray crystal structure. Crystallographic data for the structures 18b reported in this paper has been deposited with the Cambridge Crystallographic Data Centre as supplementary publication nos. CCDC 1876258. Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: (‡44) 1223-336-033; e-mail: [email protected]). The absolute and relative stereochemistry of 18a was assigned ((R)-6-((5R,7S)-2-oxo-3oxa-1-azaspiro[4.4]nonan-7-yl)-1,2,3,4-tetrahydronaphthalen-2-yl)methyl

4-

methylbenzenesulfonate based on the results for the crystal structure of 18b. (21)

The absolute and relative stereochemistry of compound 14a was unambiguously assigned ((1R,3S)-1-amino-3-((S)-6-(2-methoxyphenethyl)-5,6,7,8-tetrahydronaphthalen-2yl)cyclopentyl)methanol by X-ray crystal structure. Crystallographic data for the structures 14a reported in this paper has been deposited with the Cambridge Crystallographic Data Centre as supplementary publication nos. CCDC 1876257. Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: (‡44) 1223-336-033; e-mail: [email protected]).

(22)

The absolute and relative stereochemistry of compounds 9a/9b, 10a/10b and 20a/20b were assigned by chiral HPLC analysis using material with known stereochemistry synthesized using scheme 2. This analysis indicated that 9a has the S configuration and 9b has the R configuration at the benzyl attachment point. This analysis indicated that 10a has the R configuration and 10b has the S configuration at the phenethyl attachment point. This


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analysis indicated that 20a has the R configuration and 20b has the S configuration at the alkyne attachment point. (23)

Blakemore, P. R.; Cole, W. J.; Kocieński, P. J.; Morley, A. A stereoselective synthesis of trans-1,2-disubstituted alkenes based on the condensation of aldehydes with metallated 1-phenyl-1H-tetrazol-5-yl sulfones. Synlett, 1998, 1, 26-28.

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Siow D. L. and Wattenberg B. W. An assay system for measuring the acute production of sphingosine 1-phosphate in intact monolayers, Anal. Biochem. 2007, 371, 184-193.

(25) Barnett, N. and Ware, L. B. Biomarkers in acute lung injury – marking forward progress. Crit. Care Clin. 2011, 27, 661-683. (26) (a) Shea, B. S.; Brooks, S. F.; Fontaine, B. A.; Chun, J.; Luster, A. D.; Tager, A. Prolonged exposure to sphingosine 1−phosphate receptor-1 agonists exacerbates vascular leak, fibrosis, and mortality after lung lnjury. Am. J. Respir. Cell Mol. Biol. 2010, 43, 662−673. (b) Sammani, S.; Moreno-Vinassco, L.; Mirzapoiazova, T.; Singleton, P. A.; Chiang, E. T.; Evenoski, C. L.; Wang, T.; Mathew, B.; Husain, A.; Moitra, J.; Sun, X.; Nunez, L.; Jacobson, J. R.; Dudek, S. M.; Natarajan, V.; Garcia, J. G. N. Differential effects of sphingosine 1-phosphate on airway and vascular barrier function in the murine lung. Am. J. Respir. Cell Mol. Biol. 2010, 43, 394−402. (c) Muller H.C.; Hocke A.C.; Hellwig, K,; Gutbier, B.; Peters H.; Schonrock, S. M.; Tschernig, T.; Schmiedl, A.; Hippenstiel, S.; N’Guessan, P. D.; Rosseau, S.; Suttorp, N.; Witzenrath, M. The sphingosine-1–phosphate receptor agonist FTY720 dose dependently affected endothelial integrity in vitro and aggravated ventilator-induced lung injury in mice. Pulm. Pharmacol. Ther. 2011, 24, 377– 385. (d) Oo, M. L.; Chang, S-H, Thangada, S.; Wu, M.-T.; Rezaul, K.; Blaho, V.; Hwang,



S.-I.; Han, D. K.; Hla, T. Engagement of S1P(1)-degradative mechanisms leads to vascular leak in mice. J. Clin. Invest. 2011, 121, 2290-2300. (27)

Khan, J. M.; Lyon, A. R.; Harding, S. E. The case for induced pluripotent stem cell-derived cardiomyocytes in pharmacological screening. Br. J. Pharamacol. 2013, 169, 304-317.

(28)

Gong, G. L.; Bhaskaran, V.; Lecureux, L.; Lehman-McKeeman, L. D. Sphingosine-1Phosphate Receptor 1 (S1PR1) Agonists Cause Proliferative and Pro-fibrotic Changes in Rat Lung. Presented at Society of Toxicology Meeting, Baltimore, Maryland, March 12– 16, 2017, paper 1668.

(29)

Camm, J.; Hla, T.; Bakshi, R. Brinkmann, V. Cardiac and vascular effects of fingolimod: mechanistic basis and clinical implications American Heart Journal, 2014, 168, 632-644.

(30)

Merrill, J. E. In vitro and in vivo pharmacological models to assess demyelination and remyelination. Neuropsychopharmacology 2009, 34, 55-73.

(31)

Sheridan, G. K.; Dev, K. K. S1P1 receptor subtype inhibits demyelination and regulates chemokine release in cerebellar slice cultures. Glia 2012, 60, 382-392.

(32)

(a) Miller, R. H.; Mi, S. Dissecting demyelination. Nat. Neurosci. 2007, 10, 1351−1354. (b) Slowik, A.; Schmidt, T.; Beyer, C.; Amor, S.; Clarner, T.; Kipp, M. The sphingosine 1-phosphate receptor agonist FTY720 is neuroprotective after cuprizone-induced CNS demyelination. Br. J. Pharmacol. 2015, 172, 80−92.

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Yang, M. G.; Xiao, Z.; Dhar, T. G. M.; Xiao, H.-Y.; Gilmore, J. L.; Marcoux, D.; Xie, J. H.; McIntyre, K. W.; Taylor, T. L.; Borowski, V.; Heimrich, E. M.; Li, Y-W.; Feng, J.; Fernandez, A.; Yang, Z.; Balimane, P.; Marino, A.; Cornelius, G.; Warrick, B.; Mathur, A.; Wu, D.-R.; Li, P.; Gupta, A.; Pragalathan, B.; Shen, D. R.; Cvijic, M. E.; LehmanMcKeeman, L. D.; Salter-cid, L.; Barrish, J. C.; Carter, P. C.; Dyckman, A. J. Assymetric


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hydroboration approach to the scalable synthesis of ((1R,3S)-1-amino-3-((R)-6-hexyl5,6,7,8-tetrahydronaphalen-2-yl)cyclopentyl)methanol (BMS-986104) as a potent S1P receptor modulator J. Med. Chem. 2016, 59, 11138−11147. (34)

Rozelle, A. L.; Genovese, M. C. Efficacy results from pivotal clinical trials with abatacept. Clin. Exp. Rheumatol. 2007 (5 Suppl 46): S30-S34.

(35)

Bihorel, S.; Singhal, S.; Shevell, D.; Sun, H.; Xie, J.; Basdeo, S.; Liu, A.; Dutta, S.; Huang, H.; Lin, K.; Throup, J.; Girgis, I. Population pharmacokinetic analysis of BMS-986166, a novel selective sphingosine-1-phosphate 1 receptor modulator, and exposure-response assessment of its effects on lymphocyte counts and heart rate in healthy participants Clinical Pharmacology in Drug Development unpublished results.

(36) Xiao, H.-Y.; Dyckman, A. J.; Xiao, Z.; Yang, M. G.; Dhar, T. G. M.; Gilmore, J. L.; Marcoux, D.; Substituted Bicyclic Compounds. U.S. Pat. Appl. 0052888, 2016. WO201628959.



TOC Graphic NH2 OR EAE Model:

OMe 4

14a, BMS-986166, (R = H) 14a-P (R= -PO3H2) -selective S1P1 partial agonist -improved CV and pulmonary safety profile -improved in vivo phosphate formation -Highly efficacious in EAE model of MS

Clinical Scores

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Vehicle 14a (0.1 mg/kg) 14a (0.5 mg/kg)

3 2

Fingolimod (1.0 mg/kg)

1 0

9

12

* * *

* **

14

16

Days


* *

*

19

21

Identification and Preclinical Pharmacology of ((1R, 3S)-1-amino-3-((S

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