Bicyclic Ligand-Biased Agonists of S1P1: Exploring Side Chain Modifications to Modulate the PK, PD, and Safety Profiles

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Bicyclic Ligand-Biased Agonists of S1P : Exploring Side Chain

1

Modi ﬁ cations to Modulate the PK, PD, and Safety Pro ﬁ les

John L. Gilmore,* Hai-Yun Xiao, T. G. Murali Dhar, Michael Yang, Zili Xiao, Xiaoxia Yang,

Tracy L. Taylor, Kim W. McIntyre, Bethanne M. Warrack, Hong Shi, Paul C. Levesque,

Anthony M. Marino, Georgia Cornelius, Arvind Mathur, Ding Ren Shen, Jian Pang, Mary Ellen Cvijic,

Lois D. Lehman-McKeeman, Huadong Sun, Jenny Xie, Luisa Salter-Cid, Percy H. Carter,

and Alaric J. Dyckman

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sı Supporting Information

ABSTRACT: Sphingosine-1-phosphate (S1P) binds to a family of

sphingosine-1-phosphate G-protein-coupled receptors (S1P₁₋₅).

The interaction of S1P with these S1P receptors has a fundamental

role in many physiological processes in the vascular and immune

systems. Agonist-induced functional antagonism of S1P has been

1

shown to result in lymphopenia. As a result, agonists of this type

hold promise as therapeutics for autoimmune disorders. The

previously disclosed diﬀerentiated S1P modulator BMS-986104

1

(

1) exhibited improved preclinical cardiovascular and pulmonary safety proﬁles as compared to earlier full agonists of S1P ; however,

1

it demonstrated a long pharmacokinetic half-life (T₁_/₂18 days) in the clinic and limited formation of the desired active phosphate

metabolite. Optimization of this series through incorporation of oleﬁns, ethers, thioethers, and glycols into the alkyl side chain

aﬀorded an opportunity to reduce the projected human T₁_/₂and improve the formation of the active phosphate metabolite while

maintaining eﬃcacy as well as the improved safety proﬁle. These eﬀorts led to the discovery of 12 and 24, each of which are highly

potent, biased agonists of S1P . These compounds not only exhibited shorter in vivo T in multiple species but are also projected

1

1/2

to have signiﬁcantly shorter T₁_/₂values in humans when compared to our ﬁrst clinical candidate. In models of arthritis, treatment

with 12 and 24 demonstrated robust eﬃcacy.

INTRODUCTION

prodrug which is converted to an active phosphate species via

in vivo phosphorylation, akin to the bioconversion of

■

1

,2

Sphingosine-1-phosphate (S1P), a key regulator of numer-

ous vascular and immune functions, elicits its eﬀects in large

10

sphingosine to S1P. Action on S1P by ﬁngolimod-phosphate

1

has been linked to its beneﬁcial immunomodulatory properties,

part via signaling through ﬁve G-protein-coupled receptors

3

whereas concurrent agonism of S1P was initially associated

3

(

S1P₁₋₅). Interaction of S1P with the S1P₁receptor in

with several safety issues, such as cardiovascular and

particular has been shown to regulate traﬃcking of T and B

cells, with agonist-induced functional antagonism of S1P₁

blocking lymphocyte egress from the thymus and secondary

11,12

pulmonary eﬀects.

Subsequently, there was a signiﬁcant

amount of research aimed at the identiﬁcation of S1P agonists

1

13

4

−8

that were sparing of S1P activity. Recently, the S1P -sparing

3 3

lymphoid organs. Agonist-induced functional antagonism of

S1P full agonist, siponimod, was approved for the treatment of

1

S1P is the consequence of prolonged receptor internalization,

1

secondary progressive MS. However, clinical evaluations of this

as well as other S1P -sparing S1P full agonists have indicated

followed by degradation rather than recycling back to the

surface, resulting in the deletion of the S1P receptor from the

3

1

6

a

that agonism of S1P contributes to the cardiovascular side

1

cell surface. Many lymphocyte subsets rely on cell surface

1

4

eﬀects in humans. Our research eﬀorts shifted to the

S1P to detect the gradient of S1P that is maintained from the

1

discovery of third-generation S1P receptor modulators that

1

lymphatic system to the blood in order to migrate into

6

b

circulation. In response to this functional antagonism, these

cells lose the ability to migrate and are entrapped in the

thymus and secondary lymphoid organs resulting in

Received: June 29, 2020

Published: January 25, 2021

8

immunosuppresion.

The S1P modulators ﬁrst gained validation as therapeutics

with the approval of ﬁngolimod for the treatment of multiple

9

sclerosis. Fingolimod is a non-selective S1P receptor agonist

©

2021 American Chemical Society

https://dx.doi.org/10.1021/acs.jmedchem.0c01109

1

454

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Figure 1. Incorporation of alkene and heteroalkyl side chains. ^aCompounds with an a/b designation were obtained by separation of the

diastereoisomers using SFC conditions with a chiral column. The stereochemistry at the side chain connection was not unambiguously determined

18

for these analogues.

demonstrate ligand-biased signaling through S1P , in addition

humans, the initial triage of compounds involved using a rat

blood lymphocyte [blood lymphocyte reduction (BLR)] PK/

PD study to determine if our compounds met the desired

1

to maintaining excellent selectivity against S1P , which was the

3

hallmark of second-generation compounds. As detailed for our

15a,17

16,17

earlier compounds,

the phosphorylated forms demon-

parameters.

strated comparable potency and eﬃcacy in some GPCR

signaling assays, while in others, they showed either reduced

Y_m_a_xvalues (partial agonism) or reduced potency. This ligand-

biased proﬁle is believed to be relevant to the improved proﬁle

observed in the pulmonary and cardiovascular safety

evaluations. These eﬀorts led to the discovery of the clinical

As the cyclopentyl amino alcohol has been shown to exhibit

15,17

the desired S1P

-biased agonist proﬁle,

1

this fragment was

left unchanged in our new analogues, and herein, we describe

modiﬁcations of the hexyl side chain of the tetralin moiety

(Figure 1). Our initial strategy involved using the same relative

stereochemistry and side chain length as 1 and incorporating

oleﬁns (2−9), ethers (10−13, 20), thioethers/sulfoxides/

sulfones (14−18), and glycols (19) into the side chain in an

eﬀort to produce compounds with the desired PK, PD, and

safety proﬁles (see Table 1). We anticipated that adding these

potential sites of metabolism in the side chain of our

^c^o^m^p^o^uⁿ^d^s^w^o^u^l^d^l^e^a^d^t^o^a^r^e^d^u^c^tⁱ^oⁿ^o^f^t^h^eⁱ^r^T1/2 values

due to a reduction in intrinsic clearance. Next, within the

terminal methoxy series, the length of the side chain was

modiﬁed from the initial hexyl side chain to include side chains

with lengths ranging from four to eight atoms (21−25) and

both side chain isomers in this series were studied (see Table

15a

compound BMS-986104 (1). Despite being a partial agonist

of S1P in several in vitro assays, the bioactive phosphate

1

metabolite of 1 (1-P) was shown to fully block S1P-induced T

cell migration in vitro and to induce a comparable level of

lymphopenia and eﬃcacy as ﬁngolimod in rodent studies.

Furthermore, 1 is diﬀerentiated from ﬁngolimod in preclinical

15a

studies of pulmonary and cardiovascular safety. However, in

clinical studies, the pharmacokinetic half-life of 1 was found to

be long (18 days) and the conversion of 1 to 1-P was lower

than expected, leading to a suboptimal level of lymphocyte

reduction. Eﬀorts in our labs then shifted to discovering novel

compounds with an improved bioconversion to the active

phosphate metabolite as well as a reduced human pharmaco-

kinetic T₁_/₂. The compounds must also maintain the improved

safety proﬁle of 1 and have comparable or better eﬃcacy.

Because ﬁngolimod exhibited a superior conversion to the

active phosphate metabolite in rat studies when compared to 1

and was subsequently found to also have a higher ratio in

2

). Last, a variety of ethers, thioethers, and glycols were

prepared with varying lengths and side chain stereochemistry

26−36).

(

CHEMISTRY

■

The synthesis of the alkene compounds 2−9 is shown in

Scheme 1. Starting from (5R,7S)-7-((R)-6-(hydroxymethyl)-

1

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Scheme 1. Synthesis of 6-Alkene-Substituted-((1R,3S)-1-amino-3-(5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol

a

Compounds

a

Reagents and conditions: (a) oxalyl chloride, DMSO, TEA, DCM, −78 °C, 84%; (b) Julia−Kocienski oleﬁnation: step 1; R−OH, DEAD,1-

phenyl-1H-tetrazole-5-thiol, Ph P, THF, 0 °C, 77%. Step 2; ammonium molybdate tetrahydrate, 30% H O , 0 °C, 5-(R-thio)-1-phenyl-1H-

3

2

1

tetrazole, EtOH, 0 °C, 95%. Step 3; KHMDS, 5-(R -sulfonyl)-1-phenyl-1H-tetrazole, THF, 20−30%; (c) Wittig oleﬁnation: R-(PPh )Br, n-BuLi,

3

THF, −78 °C, 50−70%; (d) LiOH, water, dioxane or 1 N NaOH, DMSO, MeOH, 100 °C, 50−90%; (e) triethyl phosphonoacetate, THF, 0 °C;

(

f) Pd(OH) , MeOH, H , 80% (two steps); (g) LiBH , THF, 80 °C; (h) chiral SFC separation; (i) Dess−Martin periodinane, DCM, 95%; (j) p-

2

4

t

TsCl, pyridine, 82%; (k) allyl-MgBr, copper(I) bromide, THF, −78 °C, 76−96%; (l) OsO , 50% NMO in BuOH, THF, NaIO in water, 57%; (m)

4

trans-3-hexene, second-generation Grubbs catalyst, −78 °C, 74%; (n) LiBr, THF, 50 °C, 100%; (o) pent-4-en-1-yl-MgBr, copper(I) bromide, THF,

78 °C, 77%.

−

5

,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]-

of this alcohol provided the aldehyde, which was then

converted to the trans- (4) and cis- (5) alkenes using the

same methods as described above. Compound 8 was also

synthesized from 40 by ﬁrst converting the alcohol to a tosylate

and then reacting this tosylate with allylmagnesium bromide in

the presence of copper(I) bromide. The resulting terminal

alkene was oxidized using osmium tetroxide to provide

aldehyde 41, which was converted to 8 using the methods

described above. To synthesize additional alkene compounds,

37 was ﬁrst converted to tosylate 42. To prepare the two

hex-3-ene derivatives, this tosylate was reacted with allylmag-

nesium bromide, as previously described to form 43. An oleﬁn

metathesis reaction was performed between trans-3-hexene and

43 using the second-generation Grubbs catalyst to aﬀord the

17a

nonan-2-one (37), a Swern oxidation of the alcohol aﬀorded

aldehyde 38. The hex-1-ene compounds were then synthesized

from this aldehyde using two methods. A Julia−Kocienski

1

9

oleﬁnation, followed by base-catalyzed hydrolysis furnished

20

trans-alkene isomer 2, or a Wittig oleﬁnation, followed by

base-catalyzed hydrolysis provided the cis-isomer 3. The hex-2-

ene compounds (4, 5) and the hex-4-ene derivative (8) were

synthesized starting from tetralone 39. The tetralone was

reacted with triethyl phosphonoacetate using a Horner−

Emmons reaction to yield the homologated ethyl ester, which

was then reduced to the alcohol using lithium borohydride.

Chiral SFC separation aﬀorded compound 40 after determi-

17a

21

nation of the desired diastereomer. A Dess−Martin oxidation

1

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Scheme 2. Synthesis of 6-Ether/Thioether-Substituted-((1R,3S)-1-amino-3-(5,6,7,8-tetrahydronaphthalen-2-

a

yl)cyclopentyl)methanol Compounds

a

Reagents and conditions: (a) 1-pentanol, p-TsOH, trimethoxymethane, 100 °C, 70%; (b) 10% Pd/C, MeOH, H ; (c) chiral SFC separation; (d)

2

t

LiOH, water, dioxane or 1 N NaOH, DMSO, MeOH, 100 °C, 50−72%; (e) n-butanol or butyl mercaptan, KO Bu, THF, 70 °C and then step d,

t

7

0−90%; (f) L-10-(−)-camphor sulfonic acid, m-CPBA, DCM and then step d, 62−64%; (g) 2-methoxyethanol or but-3-en-1-ol, KO Bu, DMF/

THF, 70 °C and then step d, 70−90%; (h) copper(I) bromide, THF, −78 to 0 °C and then step d, 40−60%; (i) allyl-MgBr, copper(I) bromide-

t

DMS complex, THF, −78 °C, 65%; (j) OsO , 50% NMO in BuOH, THF, NaIO in water, 78%; (k) ethoxytrimethylsilane, triethylsilane, FeCl ,

4

3

CH NO , 0 °C, 73% and then step d, 65%; (l) p-TsCl, pyridine, 60−82%; (m) propane-1-thiol, 2 N NaOH, dioxane, 90 °C, 98%; (n) NaBH ,

3

2

4

EtOH, DCM; (o) ethanethiol, 2 N NaOH, dioxane, 90 °C, 85%; (p) CuI, bis(triphenylphosphine)palladium(II) chloride, TEA, various alkynes, 60

o

°C, 37−80%; (q) Na , MeOH, 30%; (r) Mg , 1,2- dibromoethane, 1-bromo-4-methoxybutane, 60 °C.

cis- and trans-hex-3-ene derivatives as a mixture. Chiral SFC

hydrogenated with Pd/C to aﬀord the penultimate compound

as a mixture of diastereomers. Chiral SFC separation of the two

diastereomers followed by base-catalyzed hydrolysis aﬀorded

compounds 10a and 10b. Tosylate 42 was used to prepare

several of the ether and thioether analogues. Compounds 11

and 14 were synthesized by heating this tosylate with n-butanol

or n-butyl mercaptan in the presence of base, followed by the

addition of water and additional base to hydrolyze the

carbamate. Compound 14 was then oxidized using m-CPBA

to form sulfoxide 15 and sulfone 16. Compound 17 was

synthesized in a manner similar to 11 and 14 starting from

separation of these two isomers followed by base-promoted

22

hydrolysis aﬀorded compounds 6 and 7. Compound 9 was

also synthesized from 42 using a copper(I) bromide mediated

coupling with pent-4-en-1-ylmagnesium bromide and base

hydrolysis.

Scheme 2 shows the methods used to synthesize the various

ether, thioether, and glycol compounds (10−25). Compounds

10a and 10b were prepared by heating tetralone 39 with

pentanol in the presence of trimethoxymethane and p-

toluenesulfonic acid. The resulting intermediate was then

1

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Table 1. PK/PD/Tox Screening of Compounds with the Same Relative Stereochemistry and Side Chain Length.

a

b

c

Compounds tested in Lewis rats (n = 2−4), *p < 0.05 versus vehicle ANOVA w/Dunnett’s test. Blood concentration at the time indicated. The

d

in vivo T₁is estimated from PK proﬁles from oral dosing observed at four time points: 4, 24, 48, and 72 h (see Table S3 ). For the cardiomyocyte

/2

evaluations, the phosphate of each compound was prepared and assayed. Beat rate reductions are an average of three or more replicates at each

concentration unless otherwise noted; *n = 2. Lymph Red. = lymphocyte reduction; Phos. = phosphate; BAL = bronchoalveolar lavage, CM =

cardiomyocyte; conc = concentration; blq = below limit of quantitation; nt = not tested; and nd = not able to determine.

alcohol 40. To move the ether and thioether to the 4-position

of the alkyl side chain, 42 was converted to 44 by ﬁrst reacting

with allylmagnesium bromide and copper(I) bromide, followed

by an oxidation of the resulting terminal alkene to aﬀord the

aldehyde. A reductive etheriﬁcation of 44 was then employed

using ethoxytrimethylsilane in the presence of triethylsilane

and iron chloride to form 12 after hydrolysis. The thioether

(18) was synthesized by converting 44 to tosylate 45 and then

1

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reacting with ethanethiol. Finally, glycol 19 and ether 20 were

prepared from tosylate 42 by heating this tosylate with 2-

methoxyethanol or but-3-en-1-ol in the presence of base,

followed by a hydrolysis. The initial syntheses of the terminal

methoxy analogues involved their preparation as a mixture of

diastereomers, which were then separated via chiral HPLC. To

synthesize most of the terminal methoxy compounds, a

Sonogashira coupling between 46 and a variety of terminal

alkynes was utilized. The resulting coupled products were then

hydrogenated, and the diastereomers were separated using

chiral SFC conditions. Subsequent hydrolysis aﬀorded 13a,

for heart rate eﬀects in the clinic, the phosphate metabolites of

our compounds were prepared and evaluated in cultured

cardiomyocytes derived from human-inducible pluripotent

stem cells. The concentration-dependent responses of these

cells to cardioactive drugs are consistent with observed clinical

25

eﬀects. Exposure of these cardiomyocytes to S1P agonists

1

has been shown to reduce the rate of beating regardless of their

17a

activity on S1P . Compounds with a biased agonist proﬁle

3

demonstrated a more limited reduction in this cardiomyocyte

1

5a,b

assay.

Analogues with a beat rate change in the

cardiomyocyte assay of less than 15% at 1 nM and less than

30% at 10 nM were deemed to be progressible. Many of the

compounds tested in the cardiomyocyte assay were also

1

3b, 22a, 22b, 23, 24, 25a, and 25b. Compound 24 was

17a

subsequently resynthesized using a chiral intermediate, 47.

Compound 47 was converted to tosylate 48, which was then

reacted with 4-methoxybutylmagnesium bromide in the

presence of copper(I) bromide. A base hydrolysis of the

resulting compound aﬀorded 24 as a homochiral product. The

ﬁnal set of terminal methoxy derivatives (21a and 21b) were

synthesized by converting diastereomeric-40 to the corre-

sponding tosylate and reacting this material with sodium

methoxide. The coupled product was then separated using

chiral SFC conditions and each diastereomer was hydrolyzed

to aﬀord 21a and 21b. The compounds in Table 3 (26−36)

were prepared using the various methods described above (see

Scheme S1). All phosphate metabolites were prepared by

reacting the ﬁnal compounds with pyrophosphoryl chloride

and then quenching with water.

screened in a functional S1P GTPγS assay, and, as is shown in

1

Table S2 , increased activity on S1P is not by itself suﬃcient to

1

elicit an increase in the cardiomyocyte response.

Table 1 shows the results for compounds which have the

same side chain length and relative stereochemistry as 1 since

previously published work indicated this to be the optimal

stereoisomer based on in vitro evaluation of phosphorylation

15a

potential, followed by an in vivo PK/PD/Tox screening. In

our eﬀort to shorten the T₁_/₂and maintain the eﬃcacy and

safety proﬁle of 1, a series of compounds were prepared in

which the hexyl side chain was replaced with a variety of

hexene derivatives (1−9). With the exception of 3 and 8, the

ability of the compounds to reduce circulating lymphocytes

was similar to 1. In general, the change to the various hexene

side chains did result in compounds with shorter estimated

^T1/2 values. Compounds 3, 6, and 7 were signiﬁcantly shorter

^wⁱ^t^h^T1/2 values of 4 to 5 h. Many of these compounds also

provided signiﬁcant improvement in their ability to form the

active phosphate metabolite with the most notable being 5, 6,

and 9, which had phosphate-to-parent ratios of 8.6, 5.8, and 19,

respectively. Compound 6 was the only compound that met

the criteria for lymphocyte reduction, estimated T1/2, and

phosphate metabolite formation, so the level of BAL protein

elevation was measured, and it was found to be negligible,

similar to what had been found with 1. Of the compounds

from this series tested in the cardiomyocyte assay, 4 and 5

were the only ones that had a beat rate reduction we deemed

progressible. Compound 6 did meet the criteria at the lower

concentration; however, it was slightly above the 30% rate

reduction at the higher concentration, again similar to 1. None

of the hexene derivatives met all the speciﬁed criteria.

RESULTS AND DISCUSSION

■

Because our compounds require a bioconversion to form the

active phosphate metabolites, new analogues were initially

assessed directly in vivo using a pharmacodynamic/pharma-

cokinetic/tox screening approach. A rat BLR PD/PK model

determined whether the modiﬁcations to the side chain

resulted in an acceptable degree of lymphocyte reduction.

Based on the lymphocyte reduction of 1 at various doses (see

Table S1), we considered compounds tested at 1 mg/kg or

greater with at least 70% reduction 24 h post-dose and

compounds tested at 0.3 mg/kg or 0.5 mg/kg with at least 45

or 60% reduction 24 h post-dose, respectively, to be

acceptable. This model also provided the level of phosphate

metabolite formation and an estimate of the pharmacokinetic

T₁_/₂from oral dosing. Compound 1 exhibited a pharmacoki-

netic T₁_/₂over 100 h in rat and was subsequently found to

have a long T₁_/₂in a human clinical trial; therefore, we looked

for compounds with an estimated T₁_/₂of 30 h or less in our

study. In addition to the long T₁_/₂, 1 was also found to have

poorer than expected phosphate metabolite formation in

humans, so we sought compounds with a phosphate-to-parent

ratio in the rat PK/PD study based on the 24 h concentration

levels better than that of 1 (ratio of 1.2). Maximizing the

phosphate-to-parent ratio in rodents was hoped to lead to an

increase in the phosphate metabolite formation in humans,

which would drive the desired pharmacology while minimizing

exposure to the parent and any oﬀ-target eﬀects that it might

carry. To assess the potential for pulmonary toxicity, the level

of protein in bronchoalveolar lavage (BAL) ﬂuid, a marker for

An ether linkage was incorporated along the hexyl side chain

to aﬀord various derivatives (10−13). The pentoxy com-

pounds (10a and 10b) which have the ether linkage at the

position adjacent to the tetralin moiety were prepared, and

while both diastereomers were isolated, the stereochemistry of

18

the side chain was not determined. Both compounds were

eﬀective at reducing lymphocyte count at 24 h with 78 and

67% reductions, respectively; however, the phosphate-to-

parent ratios were poor for each. While neither isomer

increased levels of BAL protein in vivo, the one isomer tested

(10b) performed poorly in the cardiomyocyte assay at the

higher concentration. The butoxymethyl compound 11 was

similar to the pentoxy derivatives in that it had desirable results

for lymphocyte reduction and BAL protein elevation but also

exhibited a low phosphate-to-parent ratio. Next, the ethox-

ypropyl analogue 12 was analyzed. This compound met the

speciﬁed criteria for all the assays with an 83% reduction of

lymphocytes at 24 h, an estimated T₁_/₂of 26 h, a phosphate-to-

parent ratio of 1.8, little to no BAL protein elevation, and an

23,24

vascular leakage and pulmonary edema,

was measured at

the conclusion of the study. Compound 1 exhibited very little

BAL protein elevation and we strove to identify compounds

that gave similar results. In order to reﬂect the intrinsic activity

of the compounds, the correlation to BAL eﬀects was made

based on the apparent in vivo PD ED . To assess the potential

50

1

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Table 2. PK/PD/Tox Screening of Terminal Methoxy Series

a

b

Compounds tested in Lewis rats (n = 2−4) at 2 mg/kg, *p < 0.05 versus vehicle ANOVA w/Dunnett’s test. Blood concentration at the time

c

d

indicated. The in vivo T is estimated from oral dosing over 72 h (see Table S3). For the cardiomyocyte evaluations, the phosphate of each

1

/2

compound was prepared and assayed. Beat rate reductions are an average of three or more replicates at each concentration unless otherwise noted;

n = 2. Lymph Red. = lymphocyte reduction; Phos. = phosphate; BAL = bronchoalveolar lavage, CM = cardiomyocyte; conc = concentration; blq

below limit of quantitation; and nt = not tested.

*

=

acceptable beat rate change in the cardiomyocyte assay (−14 ±

.4% at 1 nM and −29 ± 0.6% at 10 nM). Last, in the ether

acceptable 24 h lymphocyte reduction, the phosphate-to-

parent ratio was low and no further studies were warranted.

The compounds in Table 2 represent analogues in the

terminal methoxy series in which the side chain length was

varied from four to eight atoms. Because the side chain was

modiﬁed in both composition and length, each stereoisomer at

3

series, the two methoxybutyl analogues (13a and 13b) were

assayed. These two diastereomers performed quite well in the

BLR assay exhibiting 87 and 78% reduction in the lymphocyte

count at 24 h. Compound 13b also had a desirable T₁_/₂of 16 h

and performed well in the cardiomyocyte assay; however, it

had a low parent-to-phosphate ratio and a marginal result in

BAL protein elevation assay, with a 2-fold elevation of protein

relative to vehicle control.

18

the side chain connection for this series was studied. The

methoxyethyl compounds (21a and 21b) represent the

shortest chain length investigated, and these compounds

showed little to no lymphocyte reduction at either time

point. The compounds with one additional atom in the side

chain (22a and 22b), however, displayed fairly robust

lymphocyte reduction at both the 4 and 24 h time points.

Additionally, both compounds had desirable T₁_/₂values (13−

14 h). The phosphate-to-parent ratio was signiﬁcantly better

for 22a in comparison to 22b. The phosphate metabolite of

22b was also tested in the cardiomyocyte assay and showed

favorable percent beat rate change at the higher concentration

(−25 ± 4.3%) but higher than desired at the lower

concentration (−20 ± 2.4%). The compounds with a side

chain length of six atoms, 13a and 13b, were included in this

table for comparison purposes as they were discussed in the

previous section. The next set of compounds studied had a side

chain length of seven atoms (23 and 24). Both diastereomers

again displayed robust lymphocyte reductions and short T₁_/₂

values in comparison to 1. Furthermore, these compounds

both had good to excellent phosphate metabolite formation

Compounds 14, 17, and 18 represent analogues in which a

sulfur is incorporated into the hexyl side chain instead of the

oxygen linkage. In general, these compounds performed rather

poorly in the cardiomyocyte assay and had low phosphate-to-

parent ratios. For the butylthiomethyl derivative 14, the

corresponding sulfoxide 15 and sulfone 16 were also prepared.

These two compounds had very poor lymphocyte reduction at

the 4 h time point and 16 was also poor at the 24 h time point.

While the sulfoxide compound (15) exhibited an improved

phosphate-to-parent ratio (2.3) and modest BAL protein

elevation (1.5 fold over vehicle), the compound was not

studied further due to the low 24 h phosphate metabolite

concentrations at a dose of 5 mg/kg. The glycol variant, 19,

was found to have poor lymphocyte reduction and was not

tested in the BAL protein or cardiomyocyte assays. Finally, the

compound with an ether linkage combined with a terminal

alkene was studied (20), and while the compound had

1

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Table 3. PK/PD/Tox Screening of Miscellaneous Compounds

a

b

Compounds tested in Lewis rats (n = 2−4) at 0.5, 1, 2, or 5 mg/kg, *p < 0.05 versus vehicle ANOVA w/Dunnett’s test. Blood concentration at

c

d

the time indicated. The in vivo T is estimated from oral dosing over 72 h (see Table S3). For the cardiomyocyte evaluations, the phosphate of

1

/2

each compound was prepared and assayed. Beat rate reductions are an average of three or more replicates at each concentration unless otherwise

noted; *n = 2. Lymph Red. = lymphocyte reduction; Phos. = phosphate; BAL = bronchoalveolar lavage, CM = cardiomyocyte; conc =

concentration; and nt = not tested.

(

ratios of 8.2 and 2.6, respectively), showing a signiﬁcant

aspect. Additionally, 24 demonstrated more favorable results

versus 23 in the pulmonary toxicity assay (1.5- vs 1.9-fold BAL

protein increase) as well as in the cardiovascular safety assay

(−7 ± 2.8% at 1 nM and −23 ± 0.01% at 10 nM vs −20 ±

3.9% at 1 nM and −40 ± 5.3% at 10 nM).

improvement over 1 (ratio of 1.2). At a dose of 2 mg/kg, the

amount of BAL protein elevation was low to moderate. In the

cardiomyocyte assay, the phosphate metabolite of 24 exhibited

a favorable result at both concentrations tested (−7 ± 2.8% at

1

nM and −23 ± 0.01% at 10 nM), while 23 performed rather

poorly at both concentrations (−20 ± 3.9% at 1 nM and −40

5.3% at 10 nM). Last, the compounds with eight atoms in

Because of the diﬀering eﬃcacy and toxicity proﬁles

observed in the diastereomers of the terminal methoxy series,

where both the stereochemistry and chain length were

modiﬁed, the diastereomer of some compounds from Table

1 as well as other sets of diastereomers with longer side chain

length were studied (Table 3). The ﬁrst four compounds are

thioether analogues. Compounds 26 and 27 represent the

diastereomers of 14 and 17, respectively. For 26, much like the

diﬀerence in the two diastereomers 23 and 24 from the

terminal methoxy series, the cardiomyocyte results were

signiﬁcantly improved versus 14 (−14 ± 0.01% at 1 nM and

−25 ± 1% at 10 nM vs −28 ± 7.1 at 1 nM and −46 ± 8.3 at

10 nM); however, 26 had modest 24 h lymphocyte reduction

±

the side chain were studied (25a and 25b). These compounds

both performed well in the lymphocyte assay at 24 h, had good

phosphate-to-parent ratios, and exhibited low BAL protein

elevation; however, the T₁_/₂values were slightly longer than

the other compounds in this series (26−31 h), and the

cardiomyocyte results did not meet the speciﬁed criteria for

either compound. From this series, 24 had the best overall

proﬁle. While 23, the diastereomer with the same relative

stereochemistry as 1, had a superior phosphate-to-parent ratio,

2

4 still exhibited a 2-fold improvement relative to 1 in this

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a

Table 4. In Vitro Pharmacology

assay

FTY720-P

1-P

12-P

24-P

hS1P binding (IC , nM), Y

max

0.008 ± 0.0014 101%

0.70 ± 0.28 96%

0.070 ± 0.02 102%

0.02 ± 0.006 100%

3.6 ± 2.8

0.010 ± 0.004 100%

0.90 ± 0.36 81%

0.11 ± 0.02 68%

8.2 ± 3.6 101%

>1000

0.04 ± 0.017 98%

2.1 ± 0.53 83%

0.4 53%

5.2 ± 2.4 99%

>1000

10 ± 8.8

0.016 ± 0.004 98%

2.1 ± 0.92 77%

4.9 77%

2.7 ± 3.8 100%

>1000

1

50

hS1P GTPγS (EC , nM), Y

max

1

50

c

hS1P internalization (EC , nM), Y

max

1

50

hS1P ERK-P (EC , nM), Y

max

1

50

hS1P GTPγS(EC , nM)

3

50

d

hS1P GTPγS (EC , nM)

1.6 ± 0.43

10.5

10.7

d

5.6 ± 2.9

3.6 ± 4.7

4

50

d

hS1P GTPγS (EC , nM)

0.67 ± 0.24

4.9 ± 2.6

5

50

d

human lymphocyte chemotaxis (IC , nM)

1.2

2.5

4.1

0.7

5

0

a

b

c

d

n ≥ 3 unless otherwise noted. Antagonist mode. n = 1. n = 2; hS1P = human sphingosine 1-phosphate; Y = Normalized maximal response.

max

Table 5. Pharmacokinetic Parameters for Compounds 12 and 24

1

2

a

species

dose (mg/kg)

IV CL (mL/min/kg)

V_s_s(L/kg)

T₁_/₂(h)

F % (PO)

rat

IV:1 PO:1

6.0 ± 0.3

7.4 ± 0.7

2.1

12 ± 1.1

8.4 ± 0.5

10

29 ± 5.5

21 ± 1.9

79

45%

76%

cynomolgus monkey

projected human

2

4

a

b

species

dose (mg/kg)

IV CL (mL/min/kg)

V_s_s(L/kg)

T₁_/₂(h)

F % (PO)

69%

rat

IV:2

IV:1 PO:1

PO:1

10 ± 2.7

9.7 ± 3.8

13 ± 3.8

9.9 ± 3.1

19 ± 0.9

17 ± 0.7

81 ± 13

<30

cynomolgus monkey

dog

projected human

a

b

n = 3 on males; Rat: Sprague-Dawley unless otherwise noted. 18.4% hydroxypropyl-β-cyclodextrin in 13.8 mM citric acid as vehicle. F =

bioavailability, Cl = clearance, and V = volume of distribution at steady state.

ss

and rather poor phosphate metabolite formation, similar to 14.

Based on the assessments for reduction of circulating

This improvement in the cardiomyocyte assay was not

observed for 27 as it had equally poor results as 17. The last

two compounds in the thioether series have a chain length of

seven atoms (28 and 29). The 24 h lymphocyte reductions for

these compounds were 63 and 56%, respectively, despite both

compounds having rather low phosphate metabolite concen-

trations. Of this pair, only compound 29 was tested in the

cardiomyocyte assay and it performed poorly (−29 ± 0.01 at 1

nM and −41 ± 1.7 at 10 nM).

The next set of compounds 30−36 are ether analogues in

which the ether position and chain length have been varied.

The ﬁrst four compounds in this series have seven atoms in the

side chain and the ether in the ﬁrst or second position relative

to the tetralin core. With the exception of 32, these

compounds showed good lymphocyte reductions, but

relatively poor formation of the phosphate metabolite

lymphocytes, projected in vivo T₁_/₂, potential for phosphor-

ylation, as well as pulmonary and cardiovascular safety, 12 and

24 were chosen for further evaluation. Previous work showed

our third-generation partial S1P agonists to have a pulmonary

1

and cardiovascular safety proﬁle superior to that of

15a,b,17

ﬁngolimod.

These clinical compounds were found to

be partial agonists in two of the in vitro assays. Thus, the

phosphate metabolites of compounds 12 (12-P) and 24 (24-

P) were characterized in these in vitro assays and compared to

the phosphate metabolite of our ﬁrst clinical candidate (1-P)

to ascertain if they had a similar partial agonist in vitro proﬁle

(Table 4). The compounds all have equivalent potency in the

S1P binding assay; however, as we found with 1-P, 12-P and

1

24-P are diﬀerentiated from ﬁngolimod phosphate (FTY720-

P) in the S1P GTPγS and S1P internalization assays where

1

they exhibit a partial agonist proﬁle. Our improved preclinical

pulmonary safety (lower BAL protein elevation) is postulated

to be associated with the partial agonist proﬁle of the

compounds, as studies have indicated that maintaining some

(

phosphate/parent <1.0). Additionally, the one compound of

this group that was assayed for cardiovascular safety (30a) did

not meet the desired criteria. The last two compounds in this

mono-ether series possess side chains of eight atoms (33a and

degree of S1P/S1P signaling is critical for controlling vascular

1

23,25,26

33b). These diastereomers both showed impressive 24 h

tone.

Additionally, 12-P and 24-P are diﬀerentiated

lymphocyte reduction (both 80%) and reasonable phosphate-

to-parent ratios (1.6 and 1.7); furthermore, each compound

elicited little to no BAL protein elevation. The estimated T₁_/₂

for 33a (29 h) was longer than desired. The ﬁnal four

compounds in this table are sets of glycol side chains

containing seven or eight atoms. For these compounds, only

one of the diastereomers from each set had reasonable 24 h

lymphocyte reduction (34a and 35; both 70%). These

compounds also showed signiﬁcant phosphate metabolite

formation and modest BAL protein elevation; however, neither

progressed to cardiovascular safety evaluation or estimated T₁_/₂

determination.

from FTY720-P in the ERK-P assay. While 12-P and 24-P are

both full agonists in this assay like FTY20-P, they exhibit 130-

to 260-fold weaker potency than FTY720-P. The bradycardia

eﬀect in some patients receiving ﬁngolimod is likely a function

of their full agonist activity of S1P in atrial myocytes prior to

1

the subsequent functional antagonism (receptor down-

27

regulation) in those cells. Additionally, the S1P-dependent

activation of G protein-coupled inwardly rectifying potassium

(GIRK) channels on atrial myocytes has also been described as

a contributing factor. The weaker potency in ERK phosphor-

ylation for the third-generation agonists may contribute to the

reduced activity in the cardiomyocyte beating rate assay.

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1

7

Compounds 12-P and 24-P both maintain excellent selectivity

these strains of rats. In monkeys, however, 12 was improved

over 1 (0.73 vs 0.3). A similar improvement in the monkey

phosphate-to-parent ratio (∼3-fold) was noted for our second

clinical candidate, and this ultimately led to better phosphor-

over S1P . The compounds were also tested for activity against

3

S1P and S1P where they both exhibited full agonism with

4

5

IC values of 10 nM (S1P ) and 4.9 nM (S1P ) for 12-P and

5

0

4

5

1

7

5

2

.6 nM (S1P ) and 3.6 nM (S1P ) for 24-P. Finally, 12-P and

4-P were tested in a human chemotaxis assay, a functional

ylation in humans. The phosphate-to-parent ratio of 24 was

found to be lower than 1 in both monkeys (<0.1) and dogs

(0.31).

4

5

primary cell assay that correlates well with in vivo

lymphopenia. Much like 1-P, these compounds both

completely block the S1P-induced T-cell migration with an

IC₅₀of 4.1 and 0.7 nM, respectively.

The in vivo pharmacokinetic parameters for 12 and 24 were

investigated in rats, monkeys, and dogs, following intravenous

Next, we evaluated 12 and 24 in an in vivo eﬃcacy model.

Figure 2 shows the eﬀect of treatment with compounds 12 or

24 in a rat adjuvant-induced arthritis model (rat AA) in Lewis

rats. In rats dosed with vehicle only, there was an increase in

paw volume beginning at day 11, and this swelling reached a

near-maximum level by day 15 to 19 (1.5−3.0 mL increase).

Rats given 12 at once daily doses of 0.1, 0.5, and 2.5 mg/kg

exhibited a dose-dependent reduction in paw edema. The rats

dosed at 0.5 mg/kg exhibited slight paw edema beginning at

day 18 and at day 22 had roughly 75% reduction in swelling as

compared to the vehicle group. In the groups treated with 2.5

mg/kg of 12, there was no appreciable paw volume increase

observed throughout the duration of the study. From this

study, 12 was determined to have an ED of 0.3 mg/kg in the

rat AA model. Rats given 24 at once daily doses of 0.1, 0.3, and

1.0 mg/kg also exhibited a dose-dependent reduction in paw

edema. The rats dosed at 0.3 mg/kg exhibited paw edema

beginning at day 13 and at day 20 had roughly 50% reduction

in swelling as compared to the vehicle group. In the groups

treated with 1.0 mg/kg of 24, there was no appreciable paw

volume increase observed throughout the duration of the

study. From this study, 24 also had an ED₅₀of 0.3 mg/kg in

the rat AA model.

(

i.v.) and oral (p.o.) administration (Table 5). For 12, the

results demonstrated favorable drug characteristics with

acceptable bioavailability in both rats (F = 45%) and monkeys

(F = 76%) using 1 mg/kg oral administration. Compound 12

has a large volume of distribution, with a steady-state volume

of distribution (V ) of 12 ± 1.1 L/kg in rats and 8.4 ± 0.5 L/

ss

kg in dogs. The terminal T₁_/₂of 12 after IV dosing was 29 ±

5

.5 h in rats and 21 h in monkeys, and these were signiﬁcantly

shorter than that for 1 (131 to 210 h). Low clearance from

blood was observed in all the species tested. Using the

estimated clearance that was generated from human liver

microsomal data and adjusted with IVIVC ratio, the projected

human T₁_/₂of 12 is 79 h with a volume of distribution of 10

L/kg and clearance of 2.1 mL/min/kg. For 24, the results

again demonstrated favorable drug characteristics with accept-

able bioavailability in monkeys (F = 69%) following oral

administration. Compound 24 also had a large volume of

5

0

28

distribution, with V of 13 ± 3.8 L/kg in rats and 9.9 ± 3.1 L/

ss

kg in dogs. The terminal T₁_/₂of 24 was shorter than that of 12

(

19 ± 0.9 in rats and 17 ± 0.7 in monkeys) and again low

CONCLUSIONS

■

clearance from blood was observed. The projected human T

of 24 is less than 30 h.

The phosphate-to-parent blood AUC ratios in rats, dogs,

and monkeys after oral dosing for ﬁngolimod, 1, 12, and 24 are

shown in Table 6. Because the phosphate metabolite is the

1

/2

In summary, after an extensive exploration of alkenes, ethers,

thioethers, and glycols as replacements for the hexyl side chain

of our previous clinical candidate (1), our eﬀorts led to the

discovery of 12 and 24, each of which are highly potent, biased

28

agonists of S1P . Furthermore, in comparison to our ﬁrst

1

clinical candidate, both compounds not only exhibited shorter

ⁱⁿ^vⁱ^v^o^T1/2 in multiple species but are also projected to have

signiﬁcantly shorter T₁_/₂values in humans. These compounds

both eﬃciently reduced blood lymphocyte counts in rats, had

acceptable liability proﬁles, and showed excellent pharmaco-

kinetic properties across multiple species. Studies in rodent

models of arthritis demonstrated robust eﬃcacy with 12 and

Table 6. Phosphate/Parent Ratios for Fingolimod, 1, 12,

and 24

a

species

Fingolimod

1

12

24

Sprague-Dawley rat

cynomolgus monkey

beagle dog

5.6

5.9

3.7

0.42

2.5

0.3

2.4

0.9

0.73

nt

0.3

<0.1

0.31

nt

2

4 treatment. In preclinical evaluations, both compounds

human

∼0.1

nt

demonstrated good projected safety proﬁles of undesired

cardiovascular and pulmonary eﬀects. Despite the positive

attributes of 24, poor formation of the desired phosphate

metabolite was observed in all species tested, leading to

deprioritization of this compound. Compound 12, however,

exhibited an improved phosphate ratio in cynomolgus

monkeys in comparison to 1. This improvement in the

phosphate metabolite formation in monkeys, along with a

reduced projected human T₁_/₂, a robust eﬃcacy in a model of

arthritis, and an improved safety proﬁle led to further

evaluation of 12.

a

Phosphate-to-parent ratios calculated using the AUC ratio from PO

dosing, nt = not tested.

active species, a compound with greater eﬃciency of

bioconversion to the active phosphate species could aﬀord a

lower overall dose, reducing the risk of oﬀ-target eﬀects in

addition to being more easily formulated. Based on the

available data of ﬁngolimod, it appears that although the extent

of phosphorylation in humans was reduced in comparison to

non-clinical species, an improved phosphorylation in non-

clinical species translated to improved phosphorylation in

humans. In contrast to the phosphate-to-parent ratios from the

PK/PD studies in Lewis rats (see Tables 1 and 2), the ratios

for 12 (0.9) and 24 (0.3) in Sprague-Dawley rats were

signiﬁcantly lower than 1 (2.5). These lower ratios in Sprague-

Dawley rats were not observed in our previous clinical

candidates, both of which had consistent results between

EXPERIMENTAL SECTION

■

Chemical Methods. All commercially available chemicals and

solvents were used without further puriﬁcation. Reactions were

performed under an atmosphere of nitrogen. All ﬂash column

chromatography was performed on EM Science silica gel 60 (particle

1

size of 40−60 μm). All new compounds gave satisfactory H NMR,

1

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Figure 2. Eﬃcacy of 12 (0.1, 0.5, and 2.5 mg/kg) and 24 (0.1, 0.3, and 1.0 mg/kg) vs vehicle in an adjuvant arthritis model (AA) in Lewis rats. n =

in each group.

8

LC/MS, and mass spectrometry results. ¹H NMR spectra were

obtained on a Bruker 400 MHz or a JEOL 500 MHz NMR

spectrometer using the residual signal of deuterated NMR solvent as

internal reference. Electrospray ionization mass spectra were obtained

on a Waters ZQ single quadrupole mass spectrometer. High-

resolution mass spectral analysis was performed on an LTQ-FT

mass spectrometer interfaced to a Waters Acquity ultraperformance

liquid chromatography.

HPLC analyses were performed using the following conditions. All

ﬁnal compounds had an HPLC purity of ≥95% unless otherwise

stated.

Method A (analytical): Waters Acquity UPLC, BEH C18 2.1 × 50

mm, 1.7 μm particles; mobile phase A: 98:2 water/ACN 0.05%TFA;

mobile phase B: 2:98 water/ACN 0.05%TFA; column temp 50 °C;

gradient 2−98%B over 1 min then 0.5 min hold at 100% B. Flow 0.8

mL/min; detection: UV 200 nm.

Ammonium molybdate tetrahydrate (680 mg, 0.550 mmol) was

added to 30% H O (407 mL, 40 mmol) at 0 °C and the resultant

2

solution was added dropwise to a solution of 5-(pentylthio)-1-phenyl-

1H-tetrazole (650 mg, 2.6 mmol) in EtOH (20 mL) at 0 °C. The

mixture was allowed to warm to rt and stirred at rt for 16 h. Next, 30

mL of brine was added and the mixture was extracted with EtOAc (80

mL), washed with brine, dried (Na

SO ), and concentrated in vacuo

2

4

to aﬀord 5-(pentylsulfonyl)-1-phenyl-1H-tetrazole (700 mg, 95%

+

1

yield). LC/MS M = 328. HPLC t = 3.36 (method C).

r

KHMDS (420 μL, 0.21 mmol) was added dropwise to a solution of

5

(

-(pentylsulfonyl)-1-phenyl-1H-tetrazole (26 mg, 0.092 mmol) and

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

hydronaphthalene-2-carbaldehyde (38, 25 mg, 0.084 mmol) in THF

(

3 mL) at −78 °C. The reaction mixture was stirred at −78 °C for 1

h. Water (1 mL) was then added and the mixture was warmed to rt.

The reaction mixture was diluted with EtOAc (80 mL), washed with

brine, dried (Na SO ), and concentrated in vacuo to aﬀord (5R,7S)-7-

Method B (analytical): Waters Acquity UPLC BEH C18 2.1 × 50

mm, 1.7 μm particles; mobile phase A: 90:10 water/ACN 0.1%TFA;

mobile phase B: 10:90 water/ACN 0.1%TFA; temperature: 50 °C;

gradient: 0−100% B over 2 min, then a 0.5 min hold at 100% B; ﬂow:

2

4

(

1

(R)-6-((E)-hex-1-en-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-

1

-azaspiro[4.4]nonan-2-one (5 mg, 17% yield). H NMR (400 MHz,

MeOD): δ 7.02−6.90 (m, 3H), 5.55−5.46 (m, 1H), 4.44−4.25 (m,

H), 3.14−2.94 (m, 1H), 2.88−2.67 (m, 3H), 2.53 (dd, J = 16.3, 10.1

Hz, 1H), 2.42−2.32 (m, 1H), 2.29 (dd, J = 13.1, 7.2 Hz, 1H), 2.19−

2

1.0 mL/min; detection: UV at 220 nm.

Method C (analytical): Waters Sunﬁre C18 2.1 × 50 mm 5 μ, 1.7

μm particles; mobile phase A: 90:10 water/ACN 0.1% TFA; mobile

phase B: 10:90 water/ACN 0.1% TFA; temperature: 40 °C; gradient:

2

(

3

.01 (m, 4H), 1.97−1.87 (m, 3H), 1.84−1.71 (m, 2H), 1.62−1.46

+1

m, 1H), 1.43−1.32 (m, 4H), 1.01−0.86 (m, 3H); LC/MS M

54.

=

0

−100% B over 4 min, then a 0.5 min hold at 100% B; ﬂow: 1.0 mL/

(

(1R,3S)-1-Amino-3-((R)-6-((Z)-hex-1-en-1-yl)-5,6,7,8-tetrahydro-

min; detection: UV at 220 nm.

29

naphthalen-2-yl)cyclopentyl)methanol (3). To a solution of

pentyltriphenylphosphonium, bromide salt (182 mg, 0.44 mmol) in

THF (3 mL) at −78 °C was added n-butyllithium (168 μL, 0.421

mmol) dropwise. The reaction was allowed to warm to 0 °C and

maintained at 0 °C for 1 h and then cooled back to −78 °C. A

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

Method D (analytical purity long run): Shimadzu HPLC, Sunﬁre

C18 (3.0 × 150 mm, 3.5 μm particles; mobile phase A: 5:95

acetonitrile/water with 0.05% TFA; mobile phase B: 95:5

acetonitrile/water with 0.05 TFA; temperature: 25 °C; gradient: 0−

100% B over 12 min, then a 3 min hold at 100% B; ﬂow: 1.0 mL/min;

detection: UV at 254 nm.

Method E (analytical): Column Waters Acquity BEH C18 2.1 × 50

1

,2,3,4-tetrahydronaphthalene-2-carbaldehyde (38, 60 mg, 0.200

mmol) in THF (2 mL) was then added and the reaction was allowed

to warm at rt and stirred for 1.5 h. The reaction was quenched with

mm 1.7 μm; linear gradient of 0−100% solvent B over 3 min, then

0.75 min hold at 100% B; ﬂow rate: 1.11 mL/min; solvent A: 5:95

saturated NH Cl (5 mL) and extracted with EtOAc (50 mL). The

4

acetonitrile/water with 10 mM ammonium acetate; solvent B: 95:5

acetonitrile/water with 10 mM ammonium acetate; temperature = 50

organic extract was washed with saturated NH Cl (3 × 20 mL), dried

4

over sodium sulfate, ﬁltered, and concentrated in vacuo. The crude

material was puriﬁed on a silica gel cartridge using an EtOAc/Hex

gradient (0−45% EtOAc over 25 min) to aﬀord (5R,7S)-7-((R)-6-

°

C; products detected at 220 wavelength w/positive ionization mode.

(

(1R,3S)-1-Amino-3-((R)-6-((E)-hex-1-en-1-yl)-5,6,7,8-tetrahydro-

29

naphthalen-2-yl)cyclopentyl)methanol (2). DEAD (730 μL, 4.6

(

(Z)-hex-1-en-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-

mmol) was added dropwise to a solution of pentan-1-ol (300 mg, 3.40

^m^m^o^l⁾^,¹^-^p^h^eⁿ^y^l^-¹^H^-^t^e^t^r^a^z^o^l^e^-⁵^-^t^hⁱ^o^l⁽⁷⁴⁰^m^g^,⁴^.²^m^m^o^l⁾^,^aⁿ^d^P^h3^P

1

azaspiro[4.4]nonan-2-one (40 mg, 57% yield) as a white solid. H

NMR (400 MHz, MeOD): δ 6.98 (s, 3H), 5.52−5.26 (m, 2H), 4.37

s, 1H), 4.31 (s, 1H), 3.03 (tt, J = 11.0, 7.3 Hz, 1H), 2.83 (dd, J = 7.9,

(1.09 g, 4.2 mmol) in THF (20 mL) at 0 °C. The mixture was stirred

(

at a temperature range from 0 °C to rt for 16 h. The mixture was

diluted with EtOAc (30 mL) and washed with brine (2 × 20 mL) and

water (20 mL), then dried (Na SO ), and concentrated in vacuo. The

4.6 Hz, 2H), 2.72 (s, 2H), 2.58−2.38 (m, 1H), 2.29 (dd, J = 12.9, 7.2

Hz, 1H), 2.19−2.05 (m, 4H), 2.01−1.70 (m, 4H), 1.61−1.49 (m,

+

1

2

4

1H), 1.39 (s, 4H), 1.08−0.72 (m, 3H); LC/MS M = 354.3; HPLC

crude material was puriﬁed on a silica gel cartridge using an EtOAc/

Hex gradient (0−50% EtOAc over 12 column volumes) to aﬀord 5-

t = 1.7 (method B).

r

To a solution of (5R,7S)-7-((R)-6-((Z)-hex-1-en-1-yl)-5,6,7,8-

tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (45

mg, 0.127 mmol) in dioxane (2 mL) was added water (0.5 mL)

+

1

(pentylthio)-1-phenyl-1H-tetrazole (650 mg, 77% yield). LC/MS M

=

249.2. HPLC t = 3.36 (method C).

r

1

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Article

and lithium hydroxide hydrate (53 mg, 1.3 mmol). The mixture was

concentrated. The crude material was puriﬁed on a silica gel cartridge

(12 g) using an 20% MeOH/DCM/DCM gradient (0−25% 20%

MeOH/DCM over 17 CV) to aﬀord (5R,7S)-7-((S)-6-((Z)-hex-2-en-

stirred at 100 °C for 16 h under N . After cooling, the mixture was

2

ﬁltered and washed with MeOH, the combined solvents were

evaporated, and the residue was puriﬁed by HPLC: column

Phenomenex Luna C18 5μ 21.2 × 100 mm. Solvent A: 10%

MeOH−90% H O−0.1% TFA; solvent B: 90% MeOH−10% H O−

1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-

1

2-one (14 mg, 0.040 mmol, 28% yield). H NMR (400 MHz, CDCl ):

3

δ 7.07−7.01 (m, 1H), 6.99−6.91 (m, 2H), 5.58 (br s, 1H), 5.54−5.42

(m, 2H), 4.40−4.23 (m, 2H), 3.11−2.95 (m, 1H), 2.91−2.73 (m,

3H), 2.43 (br dd, J = 16.4, 10.7 Hz, 1H), 2.32 (dd, J = 13.2, 7.3 Hz,

1H), 2.22−2.09 (m, 4H), 2.09−2.01 (m, 2H), 2.01−1.91 (m, 3H),

1.91−1.71 (m, 2H), 1.46−1.38 (m, 3H), 0.94 (t, J = 7.3 Hz, 3H);

2

0

.1% TFA. Gradient time = 15 min. Start B = 0%, ﬁnal B 100%. Stop

time 20 min. The collected fraction was basiﬁed with saturated

NaHCO , concentrated under vacuo and the aqueous layer was

3

extracted with DCM (3 × 20 mL), which was dried (Na SO ) and

2

4

+

1

concentrated under vacuo. The residue was freeze-dried to aﬀord

LC/MS M = 354.2; HPLC t = 1.23 (method A).

r

(

(1R,3S)-1-amino-3-((R)-6-((Z)-hex-1-en-1-yl)-5,6,7,8-tetrahydro-

To a mixture of (5R,7S)-7-((S)-6-((Z)-hex-2-en-1-yl)-5,6,7,8-

tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (14

mg, 0.040 mmol) in MeOH (1 mL) and DMSO (1 mL) was

added 1 N NaOH (1 mL). The reaction mixture was heated at 95 °C

for 4 h, then cooled, and acidiﬁed with TFA. The crude product was

ﬁltered and puriﬁed by HPLC. HPLC conditions: Phenomenex Luna

5μ C18 column (30 × 100 mm); MeCN (0.1% TFA)/water (0.1%

TFA); 20−100% gradient over 15 min; and 30 mL/min. Isolated

fractions with the correct mass were freeze-dried overnight to aﬀord

((1R,3S)-1-amino-3-((S)-6-((Z)-hex-2-en-1-yl)-5,6,7,8-tetrahydro-

naphthalen-2-yl)cyclopentyl)methanol, TFA (11 mg, 0.022 mmol,

1

naphthalen-2-yl)cyclopentyl)methanol (32 mg, 69% yield). H NMR

(400 MHz, MeOD): δ 7.12−6.89 (m, 3H), 5.49−5.27 (m, 2H), 3.63−

3

4

1

.47 (m, 2H), 3.07 (ddd, J = 10.9, 7.3, 4.1 Hz, 1H), 2.83 (dd, J = 8.0,

.7 Hz, 2H), 2.79−2.64 (m, 2H), 2.57−2.39 (m, 1H), 2.40−2.24 (m,

H), 2.21−2.00 (m, 3H), 1.98−1.77 (m, 4H), 1.70−1.46 (m, 2H),

+

1

.45−1.32 (m, 4H), 1.00−0.84 (m, 3H); LC/MS M = 328.3;

HPLC t = 8.75 (method D).

r

(

(1R,3S)-1-Amino-3-((S)-6-((E)-hex-2-en-1-yl)-5,6,7,8-tetrahydro-

29

naphthalen-2-yl)cyclopentyl)methanol (4). To a mixture of

5R,7S)-7-((R)-6-(2-hydroxyethyl)-5,6,7,8-tetrahydronaphthalen-2-

(

1

yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (40, 80 mg, 0.254 mmol) in

DCM (5 mL) was added Dess−Martin periodinane (140 mg, 0.330

mmol). The reaction mixture was stirred 1 h, diluted with DCM, and

washed with 1 N NaOH. The organic layer was dried with MgSO₄,

ﬁltered, and concentrated. The compound was used without further

puriﬁcation.

57% yield). H NMR (400 MHz, MeOD): δ 6.99 (s, 3H), 5.49 (t, J =

4.7 Hz, 2H), 3.72−3.56 (m, 2H), 3.20−3.03 (m, 1H), 2.90−2.71 (m,

3H), 2.49−2.36 (m, 2H), 2.17−2.10 (m, 3H), 2.10−2.02 (m, 2H),

2.02−1.88 (m, 4H), 1.83−1.66 (m, 2H), 1.51−1.33 (m, 3H), 0.94 (t,

+

1

J = 7.4 Hz, 3H); LC/MS M = 328; HPLC t = 8.28 (method D).

r

Purity = 90%.

1

M KHMDS in THF (0.36 mL, 0.36 mmol) was added dropwise

((1R,3S)-1-Amino-3-((R)-6-((E)-hex-3-en-1-yl)-5,6,7,8-tetrahydro-

naphthalen-2-yl)cyclopentyl)methanol (6) and ((1R,3S)-1-Amino-

3-((R)-6-((Z)-hex-3-en-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)-

to a solution of 5-(butylsulfonyl)-1-phenyl-1H-tetrazole (57 mg, 0.22

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

yl)-1,2,3,4-tetrahydronaphthalen-2-yl)acetaldehyde (45 mg, 0.144

mmol) in THF (3 mL) at −78 °C and the resultant solution was

stirred at the same temperature for 2 h. Water (1 mL) was added and

the mixture was warmed to rt, diluted with water (10 mL), and

extracted with EtOAc (30 mL). The organic layer was washed with

saturated NH Cl (2 × 15 mL), brine (20 mL), dried (MgSO ), and

2

9

cyclopentyl)methanol (7). Nitrogen was bubbled through a

mixture of trans-3-hexene (5.8 mL, 47 mmol), (5R,7S)-7-((R)-6-

(

but-3-en-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro-

[4.4]nonan-2-one (43, 1.5 g, 4.6 mmol), and DCM (50 mL) for 3

min at −78 °C before Grubbs second-generation catalyst (0.25 g,

0.294 mmol) was added. The bubbling was continued for 2 min and

4

concentrated under vacuo to aﬀord the desired product, which was

puriﬁed by HPLC. HPLC conditions: Phenomenex Luna 5μ C18

column (30 × 100 mm); MeCN (0.1% TFA)/water (0.1% TFA);

then the mixture was stirred under N at 40 °C for 3.5 h. The mixture

2

was concentrated and puriﬁed by ﬂash chromatography (24 g silica gel

column, gradient elution from 0 to 40% of EtOAc in DCM) to aﬀord

(5R,7S)-7-((R)-6-(hex-3-en-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)-

3-oxa-1-azaspiro[4.4]nonan-2-one (1.2 g, 3.4 mmol, 74% yield) as a

3

0−100% gradient over 15 min; and 30 mL/min. Isolated fractions

with the correct mass were freeze-dried overnight to provide (5R,7S)-

-((S)-6-((E)-hex-2-en-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-

7

solid. SFC separation (20% MeOH in CO , ADH column; 40 °C; 140

2

oxa-1-azaspiro[4.4]nonan-2-one (11 mg, 0.031 mmol, 22% yield).

To a mixture of (5R,7S)-7-((S)-6-((E)-hex-2-en-1-yl)-5,6,7,8-

tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (11

mg, 0.031 mmol) in MeOH (1 mL) and DMSO (1 mL) was

added 1 N NaOH (1 mL). The reaction mixture was heated at 95 °C

for 4 h, then cooled, and acidiﬁed with TFA. The crude product was

ﬁltered and puriﬁed by HPLC. HPLC conditions: Phenomenex Luna

bar BPR) gave PK1: (5R,7S)-7-((R)-6-((E)-hex-3-en-1-yl)-5,6,7,8-

tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (0.8 g,

1

2.263 mmol, 49.1% yield). H NMR (500 MHz, CDCl

): δ 7.08−7.01

3

(m, 1H), 7.00−6.91 (m, 2H), 6.36 (s, 1H), 5.62−5.34 (m, 2H),

4.37−4.25 (m, 2H), 3.09−2.96 (m, 1H), 2.91−2.76 (m, 3H), 2.40

(dd, J = 16.4, 10.5 Hz, 1H), 2.31 (dd, J = 13.3, 7.2 Hz, 1H), 2.21−

2.08 (m, 4H), 2.08−1.91 (m, 5H), 1.90−1.79 (m, 1H), 1.79−1.69

5

μ C18 column (30 × 100 mm); MeCN (0.1% TFA)/water (0.1%

(m, 1H), 1.53−1.32 (m, 3H), 1.00 (t, J = 7.5 Hz, 3H); HPLC t =

r

+

1

TFA); 20−100% gradient over 15 min; and 30 mL/min. Isolated

fractions with the correct mass were freeze-dried overnight to provide

4.11 min (condition C); LC/MS M = 354); and PK2: (5R,7S)-7-

((R)-6-((Z)-hex-3-en-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-

1

(

(1R,3S)-1-amino-3-((S)-6-((E)-hex-2-en-1-yl)-5,6,7,8-tetrahydro-

1-azaspiro[4.4]nonan-2-one (0.1 g, 0.283 mmol, 6.14% yield). H

naphthalen-2-yl)cyclopentyl)methanol, TFA (13 mg, 0.028 mmol,

NMR (500 MHz, CDCl ): δ 7.04 (d, J = 7.8 Hz, 1H), 6.98−6.91 (m,

3

1

9

0% yield). H NMR (400 MHz, MeOD): δ 6.99 (s, 3H), 5.53−5.46

2H), 5.60 (br s, 1H), 5.44−5.30 (m, 2H), 4.39−4.23 (m, 2H), 3.11−

2.96 (m, 1H), 2.91−2.77 (m, 3H), 2.41 (dd, J = 16.2, 10.7 Hz, 1H),

2.32 (dd, J = 13.3, 7.2 Hz, 1H), 2.22−2.04 (m, 6H), 2.01−1.89 (m,

3H), 1.89−1.80 (m, 1H), 1.79−1.70 (m, 1H), 1.50−1.37 (m, 3H),

(m, 2H), 3.71−3.55 (m, 2H), 3.19−3.03 (m, 1H), 2.89−2.73 (m,

3

1

H), 2.48−2.33 (m, 2H), 2.19−1.87 (m, 9H), 1.81−1.68 (m, 2H),

+1

.51−1.31 (m, 3H), 0.94 (t, J = 7.4 Hz, 3H); LC/MS M = 328;

HPLC t = 8.42 (method D).

1.00 (t, J = 7.5 Hz, 3H); HPLC t

M

r

= 4.08 min (condition C); LC/MS

= 354). See the Supporting Information for additional NMR

analysis.

Each isomer was dissolved independently in dioxane (0.5 mL) and

r

+

1

(

(1R,3S)-1-Amino-3-((S)-6-((Z)-hex-2-en-1-yl)-5,6,7,8-tetrahydro-

2

9

naphthalen-2-yl)cyclopentyl)methanol (5). To a mixture of

butyltriphenylphosphonium, bromide salt (172 mg, 0.43 mmol) in

THF (1 mL) was added LiHMDS (0.43 mL, 0.43 mmol). The

reaction mixture was stirred for 30 min, then 2-((R)-6-((5R,7S)-2-

oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)-1,2,3,4-tetrahydronaphthalen-2-

yl)acetaldehyde (45 mg, 0.144 mmol) in THF (1 mL) was added, and

the reaction mixture was stirred for 1 h. The reaction mixture was

quenched with water, then diluted with EtOAc, and washed with

treated with NaOH (0.93 g, 23 mmol) in water (7 mL) at 90 °C

under N for 1.5 days. Each mixture was independently extracted with

2

EtOAc (4 × 4 mL). The combined EtOAc extracts were dried

(Na SO ) and concentrated. The resulting residue was puriﬁed using

2

4

reverse-phase HPLC [Phenomenex Luna 5μ 30 × 100 mm (Axia);

gradient over 6 min from 40 to 100% of solvent B and holding @

saturated NaCl. The organic layer was dried with MgSO , ﬁltered, and

100% of solvent B for 6 min; solvent A: 10% MeOH: 90% H O: 0.1%

4

2

1

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J. Med. Chem. 2021, 64, 1454−1480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

+

1

TFA; solvent B: 90% MeOH, 10% H O, 0.1% TFA]. The appropriate

(m, 3H), 1.42−1.35 (m, 3H); LC/MS M = 328.4; HPLC t = 9.89

2

r

fractions were concentrated, neutralized with 2 N aqueous NaOH,

and extracted with EtOAc. The organic fraction was then

concentrated to aﬀord each isomer:

min (method D).

((1R,3S)-1-Amino-3-((R)-6-(hex-5-en-1-yl)-5,6,7,8-tetrahydro-

2

9

naphthalen-2-yl)cyclopentyl)methanol (9). To a mixture of ((R)-

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

dronaphthalen-2-yl)methyl 4-methylbenzenesulfonate (42, 150 mg,

0.33 mmol) in dry THF (2 mL) was added lithium bromide (143 mg,

1.646 mmol). The resulting mixture was stirred at 50 °C for 16 h and

quenched with water (3 mL). After cooling, the mixture was taken up

Obtained ((1R,3S)-1-amino-3-((R)-6-((E)-hex-3-en-1-yl)-5,6,7,8-

tetrahydronaphthalen-2-yl)cyclopentyl)methanol (6, 11 mg, 52%

1

yield) as a solid. H NMR (400 MHz, CDCl ): δ 7.05−6.93 (m,

3

2

1

H), 5.56−5.36 (m, 2H), 3.53−3.40 (m, 2H), 3.09−2.96 (m, 1H),

.88−2.74 (m, 3H), 2.38 (dd, J = 16.4, 10.5 Hz, 1H), 2.28 (dd, J =

3.3, 8.0 Hz, 1H), 2.15−1.85 (m, 8H), 1.83−1.62 (m, 2H), 1.52 (dd,

J = 13.2, 11.0 Hz, 1H), 1.46−1.32 (m, 3H), 0.97 (t, J = 7.5 Hz, 3H);

HPLC t = 3.50 min (method C); LC/MS M = 328 (see the

in EtOAc (20 mL), washed with saturated NaHCO

(Na SO ), and concentrated under vacuo to aﬀord (5R,7S)-7-((R)-6-

(bromomethyl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro-

[4.4]nonan-2-one (120 mg, 0.330 mmol, 100% yield). H NMR (400

(10 mL), dried

3

2

4

+1

r

1

Supporting Information for NMR conﬁrmation of the E isomer).

Obtained ((1R,3S)-1-amino-3-((R)-6-((Z)-hex-3-en-1-yl)-5,6,7,8-

MHz, MeOD): δ 7.01 (s, 2H), 7.00−6.99 (m, 1H), 4.42−4.27 (m,

2H), 3.51 (d, J = 6.2 Hz, 2H), 3.10−3.00 (m, 1H), 3.00−2.90 (m,

1H), 2.87−2.79 (m, 2H), 2.54 (dd, J = 16.2, 10.0 Hz, 1H), 2.29 (dd, J

= 13.0, 7.0 Hz, 1H), 2.18−2.02 (m, 5H), 2.01−1.87 (m, 2H), 1.87−

1.74 (m, 1H).

A stirred suspension of copper(I) bromide (31.5 mg, 0.220 mmol)

and (5R,7S)-7-((R)-6-(bromomethyl)-5,6,7,8-tetrahydronaphthalen-

2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (40 mg, 0.110 mmol) in

THF (5 mL) was cooled with a dry ice bath in acetone and treated

with pent-4-en-1-ylmagnesium bromide (2.2 mL, 1.1 mmol). The

resulting mixture was warmed slowly to rt and stirred for 3 h. The

mixture was quenched with water (3 mL) at 0 °C, and the mixture

was taken up in EtOAc (20 mL). The layers were separated and the

tetrahydronaphthalen-2-yl)cyclopentyl)methanol (7, 5 mg, 48%

1

yield) as a solid. H NMR (400 MHz, CDCl ): δ 7.03 (s, 2H), 7.00

3

(

2

1

s, 1H), 5.44−5.33 (m, 2H), 3.58−3.43 (m, 2H), 3.13−2.98 (m, 1H),

.92−2.77 (m, 3H), 2.41 (br dd, J = 16.2, 10.7 Hz, 1H), 2.30 (dd, J =

3.4, 7.7 Hz, 1H), 2.23−2.03 (m, 6H), 1.85−1.63 (m, 4H), 1.56 (dd,

J = 13.0, 11.4 Hz, 1H), 1.50−1.34 (m, 3H), 1.00 (t, J = 7.6 Hz, 3H);

LC/MS M⁺¹= 328; HPLC t = 3.50 min (method C). Purity 90%

r

(

see the Supporting Information for NMR conﬁrmation of Z isomer).

(

(1R,3S)-1-Amino-3-((R)-6-((Z)-hex-4-en-1-yl)-5,6,7,8-tetrahydro-

2

9

naphthalen-2-yl)cyclopentyl)methanol (8). To a stirred mixture of

ethyltriphenylphosphonium bromide (650 mg, 1.76 mmol) and

anhydrous THF (5 mL) was added a 1 M THF solution of KOtBu

(1.8 mL, 1.8 mmol) dropwise at 0 °C. The mixture was stirred at the

organic layer was washed with saturated NaHCO (10 mL), dried

3

same temperature for 1 h before a solution of 4-((R)-6-((5R,7S)-2-

oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)-1,2,3,4-tetrahydronaphthalen-2-

yl)butanal (41, 120 mg, 0.35 mmol) in anhydrous THF (5 mL) was

added dropwise at 0 °C. The mixture was stirred at 0 °C for 40 min.

(Na SO ), and concentrated under vacuo to aﬀord (5R,7S)-7-((R)-6-

(hex-5-en-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro-

2

4

[4.4]nonan-2-one (30 mg, 0.085 mmol, 77% yield). HPLC t = 1.64

r

+

1

min (method B); LC/MS M = 354.

Saturated aqueous NH Cl solution (2 mL) was added dropwise to

(5R,7S)-7-((R)-6-(hex-5-en-1-yl)-5,6,7,8-tetrahydronaphthalen-2-

yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (30 mg, 0.085 mmol) was

dissolved in dioxane (1.5 mL) and water (0.5 mL) and lithium

hydroxide hydrate (36 mg, 0.85 mmol) were added. The reaction

4

quench the reaction. Water (1 mL) and hexanes (8 mL) were added.

The aqueous layer was separated and extracted with EtOAc (3 × 2

mL). The combined organic fractions were dried over Na SO and

2

4

concentrated under reduced pressure. Flash chromatography

puriﬁcation using ISCO (12 g of silica gel column, gradient elution

from 15 to 80% of EtOAc in hexanes) aﬀorded (5R,7S)-7-((R)-6-

mixture was stirred under N at 100 °C for 16 h. The mixture was

2

concentrated and puriﬁed by preparative HPLC: Column Phenom-

enex Luna C18 5μ 21.2 × 100 mm. Solvent A: 10% MeOH−90%

H O−0.1% TFA; solvent B: 90% MeOH−10% H O−0.1% TFA.

(

hex-4-en-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro-

2

[

4.4]nonan-2-one (115 mg, 0.33 mmol, 89% yield) as a white solid.

Gradient time = 15 min. Start B = 0%, ﬁnal B 100%. Stop time 20

min. The collected fraction was basiﬁed with saturated NaHCO₃,

concentrated under vacuo, and the aqueous layer was extracted with

DCM (3 × 20 mL). The combined organic layers were dried

(Na SO ) and concentrated under vacuo to aﬀord ((1R,3S)-1-amino-

This product was further puriﬁed using SFC conditions (ChiralPak

AD-H 25 × 5 cm ID, 5 μm; Flow rate: 150.0 mL/min; mobile phase:

7

0/30 CO /MeOH) to aﬀord (5R,7S)-7-((R)-6-((Z)-hex-4-en-1-yl)-

2

5

,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one

2

4

1

(

1

86 mg, 69% yield). H NMR (400 MHz, CDCl ): δ 7.11−7.01 (m,

3-((R)-6-(hex-5-en-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)-

3

H), 7.00−6.90 (m, 2H), 5.78 (br s, 1H), 5.57−5.36 (m, 2H), 4.43−

.23 (m, 2H), 3.13−2.96 (m, 1H), 2.93−2.73 (m, 3H), 2.49−2.25

cyclopentyl)methanol (20 mg, 0.055 mmol, 65% yield) as a white

1

4

solid. H NMR (400 MHz, MeOD): δ 6.97 (d, J = 1.5 Hz, 3H), 5.83

(

1

3

m, 2H), 2.21−2.04 (m, 4H), 2.03−1.91 (m, 3H), 1.89−1.80 (m,

(ddt, J = 17.1, 10.3, 6.8 Hz, 1H), 5.00 (dq, J = 17.2, 1.8 Hz, 1H), 4.93

(ddd, J = 10.2, 2.2, 1.2 Hz, 1H), 3.63−3.48 (m, 2H), 3.12−2.95 (m,

1H), 2.89−2.72 (m, 3H), 2.42−2.23 (m, 2H), 2.16−1.99 (m, 3H),

1.98−1.75 (m, 4H), 1.75−1.53 (m, 2H), 1.47−1.25 (m, 9H); HPLC

H), 1.64 (br d, J = 5.9 Hz, 3H), 1.54−1.43 (m, 3H), 1.44−1.33 (m,

H); LC/MS M⁺¹= 354.4; HPLC t = 4.17 min (method C).

r

A mixture of (5R,7S)-7-((R)-6-((Z)-hex-4-en-1-yl)-5,6,7,8-tetrahy-

dronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (40 mg, 0.11

mmol), 2 N aqueous NaOH (1.1 mL, 2.3 mmol), and dioxane (0.5

+

1

t = 8.33 min (method D); LC/MS M = 328.3.

r

((1R,3S)-1-Amino-3-(6-(pentyloxy)-5,6,7,8-tetrahydronaphtha-

2

9

mL) was stirred under N in a sealed vial at 100 °C for 4 h. The

len-2-yl)cyclopentyl)methanol (10a, 10b). A mixture of 1-

pentanol (10 mL, 92 mmol), p-toluenesulfonic acid monohydrate

(8 mg, 0.042 mmol), and trimethoxymethane (0.61 mL, 5.6 mmol)

2

mixture was cooled and extracted with EtOAc (4 × 1 mL), and the

combined EtOAc extracts were dried (Na SO ) and concentrated.

2

4

The residue was puriﬁed using reverse-phase HPLC (Phen Luna 5μ

0 × 100 mm (Axia); gradient over 7 min from 35 to 100% of solvent

was stirred at 100 °C for 2 h with a slow N stream to remove the

2

3

methanol byproduct. The residual liquid was mixed with (5R,7S)-7-

(6-oxo-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]-

nonan-2-one (39, 400 mg, 1.4 mmol) and stirred at 100 °C under N₂

for 2.5 h. Next, 10% Pd−C (400 mg) was added at rt, followed by

EtOAc (5 mL). The mixture was vigorously stirred under a hydrogen

balloon for 4 h. The mixture was ﬁltered through a membrane ﬁlter

and the ﬁltrate was concentrated. Flash chromatography puriﬁcation

(12 g silica gel column, gradient elution from 0 to 100% EtOAc in

hexanes) aﬀorded (5R,7S)-7-(6-(pentyloxy)-5,6,7,8-tetrahydronaph-

thalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (350 mg, 0.98 mmol,

B; solvent A: 10% MeOH: 90% H O: 0.1% TFA; solvent B: 90%

2

MeOH, 10% H O, 0.1% TFA). The appropriate fractions were

2

concentrated, neutralized with 2 N aqueous NaOH and extracted with

EtOAc. The EtOAc layer was concentrated to aﬀord ((1R,3S)-1-

amino-3-((R)-6-((Z)-hex-4-en-1-yl)-5,6,7,8-tetrahydronaphthalen-2-

yl)cyclopentyl)methanol (35 mg, 0.104 mmol, 92% yield) as a white

1

solid. H NMR (400 MHz, CDCl ): δ 7.02 (s, 2H), 6.99 (s, 1H),

3

5

.57−5.37 (m, 2H), 3.58−3.40 (m, 2H), 3.04 (tt, J = 10.7, 7.5 Hz,

1

H), 2.93−2.71 (m, 3H), 2.39 (br dd, J = 16.4, 10.7 Hz, 1H), 2.29

+

1

(dd, J = 13.4, 7.9 Hz, 1H), 2.09 (br d, J = 6.5 Hz, 3H), 1.97−1.89 (m,

70% yield) as a sticky solid. LC/MS M = 358. Chiral separation

4H), 1.81−1.67 (m, 4H), 1.63 (dd, J = 6.1, 0.8 Hz, 3H), 1.55−1.44

(Lux-Amy-2 (3 × 25 cm), 25% MeOH, 120 mL/min, 220 nm, 45 °C)

1

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of the solid aﬀorded two isomers. Each isomer was hydrolyzed in the

following fashion.

Isomer 1: A mixture of (5R,7S)-7-(6-(pentyloxy)-5,6,7,8-tetrahy-

dronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (110 mg, 0.31

mmol), lithium hydroxide monohydrate (155 mg, 3.7 mmol), dioxane

mL, 6.7 mmol) in nitromethane (10 mL) at 0 °C was added ferric

chloride (22 mg, 0.134 mmol). The mixture was stirred at 0 °C for 15

min and then at rt for 12 h. The reaction mixture was diluted with

EtOAc and washed with saturated NaCl. The organic layer was dried

with MgSO , ﬁltered, and concentrated and then puriﬁed by HPLC.

4

(

1 mL), and water (1 mL) was stirred at 90 °C under N for 15 h.

HPLC conditions: Phenomenex Luna 5μ C18 column (30 × 100

mm); MeCN (0.1% TFA)/water (0.1% TFA); 20−100% gradient

over 15 min; and 30 mL/min. Isolated fractions with the correct mass

were freeze-dried overnight to aﬀord (5R,7S)-7-((S)-6-(3-ethoxy-

propyl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]-

2

The mixture was cooled and extracted with EtOAc (4 × 1 mL). The

combined EtOAc extracts were dried over anhydrous Na SO ,

concentrated under reduced pressure, and the resulting reside was

puriﬁed using reverse-phase HPLC (Phenomenex Luna 5μ 30 × 100

mm (Axia); gradient over 8 min from 30 to 100% of solvent B;

2

4

1

nonan-2-one (350 mg, 0.98 mmol, 73% yield). H NMR (400 MHz,

solvent A: 10% MeOH: 90% H O: 0.1% TFA; solvent B: 90% MeOH,

MeOD): δ 6.97 (s, 2H), 6.96 (s, 1H), 4.42−4.26 (m, 2H), 3.58−3.46

(m, 4H), 3.02 (tt, J = 11.1, 7.2 Hz, 1H), 2.90−2.72 (m, 3H), 2.37

(dd, J = 16.3, 10.3 Hz, 1H), 2.28 (dd, J = 13.0, 6.8 Hz, 1H), 2.19−

2.03 (m, 2H), 2.01−1.88 (m, 3H), 1.86−1.76 (m, 1H), 1.75−1.64

2

1

0% H O, 0.1% TFA). Fractions with product were pooled and

2

basiﬁed with K CO , then extracted with EtOAc, dried (Na SO ), and

2

3

2

4

concentrated to aﬀord ((1R,3S)-1-amino-3-(6-(pentyloxy)-5,6,7,8-

tetrahydronaphthalen-2-yl)cyclopentyl) methanol (10a, 60 mg, 0.17

(m, 3H), 1.52−1.33 (m, 3H), 1.20 (t, J = 7.0 Hz, 3H); HPLC t =

r

1

+1

mmol, 50% yield) as a white solid. H NMR (400 MHz, CDCl ): δ

13.6 min (method D); LC/MS M = 358.1.

3

7

2

1

.03−6.95 (m, 3H), 3.73−3.63 (m, 1H), 3.57−3.42 (m, 4H), 3.09−

.97 (m, 2H), 2.95−2.85 (m, 1H), 2.82−2.68 (m, 2H), 2.27 (dd, J =

3.3, 7.8 Hz, 1H), 2.13−2.01 (m, 2H), 1.97−1.46 (m, 7H), 1.37−

To a solution of (5R,7S)-7-((S)-6-(3-ethoxypropyl)-5,6,7,8-tetra-

hydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (400 mg,

1.12 mmol) in dioxane (40 mL) was added 1 N NaOH (40 mL).

The reaction mixture was heated at 100 °C overnight and then

cooled, poured into water, and extracted with EtOAc. The organic

.29 (m, 4H), 0.94−0.87 (m, 3H); HPLC t = 7.19 min (method D);

r

+1

LC/MS M = 332.3.

Isomer 2: A solution of (5R,7S)-7-(6-(pentyloxy)-5,6,7,8-tetrahy-

dronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (68 mg, 0.19

mmol) and lithium hydroxide monohydrate (96 mg, 2.3 mmol) in

layer was dried with MgSO , ﬁltered, and concentrated to aﬀord

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

4

thalen-2-yl)cyclopentyl)methanol (250 mg, 0.73 mmol, 65% yield).

1

dioxane (1 mL) and water (1 mL) was stirred at 90 °C under N for

H NMR (400 MHz, MeOD): δ 7.03−6.92 (m, 3H), 3.57−3.42 (m,

2

1

5 h. The mixture was cooled and extracted with EtOAc (4 × 1 mL).

6H), 3.01 (tt, J = 11.1, 7.2 Hz, 1H), 2.89−2.75 (m, 3H), 2.37 (dd, J =

16.2, 10.5 Hz, 1H), 2.20 (dd, J = 13.2, 7.0 Hz, 1H), 2.05−1.85 (m,

3H), 1.83−1.75 (m, 1H), 1.75−1.64 (m, 4H), 1.60−1.50 (m, 1H),

The combined EtOAc extracts were dried (Na SO ) and concen-

2

4

trated under reduced pressure to aﬀord ((1R,3S)-1-amino-3-(6-

(

pentyloxy)-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol

47 mg, 0.14 mmol, 72% yield) as a yellowish solid. H NMR (400

1.47−1.33 (m, 3H), 1.20 (t, J = 7.0 Hz, 3H); HPLC t = 6.63 min

r

1

+1

(method D); LC/MS M = 358.1.

MHz, CDCl ): δ 7.03−6.95 (m, 3H), 3.73−3.64 (m, 1H), 3.57−3.49

(

((1R,3S)-1-Amino-3-((S)-6-(3-ethoxypropyl)-5,6,7,8-tetrahydro-

3

m, 2H), 3.48−3.40 (m, 2H), 3.09−2.96 (m, 2H), 2.95−2.86 (m,

H), 2.81−2.69 (m, 2H), 2.25 (dd, J = 13.2, 7.9 Hz, 1H), 2.13−2.00

naphthalen-2-yl)cyclopentyl)methyl Dihydrogen phosphate (12-

2

9

1

P). To a mixture of ((1R,3S)-1-amino-3-((S)-6-(3-ethoxypropyl)-

5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol (40 mg,

0.121 mmol) in acetonitrile (4 mL) was added pyrophosphoryl

chloride (0.1 mL, 0.723 mmol). After stirring at rt for 1 h, the reaction

mixture was quenched with water (0.5) mL) and then puriﬁed by

HPLC to aﬀord ((1R,3S)-1-amino-3-((S)-6-(3-ethoxypropyl)-5,6,7,8-

tetrahydronaphthalen-2-yl)cyclopentyl)methyl dihydrogen phosphate

(20 mg, 0.46 mmol, 37% yield). HPLC conditions: Phenomenex

Luna 5 μm C18 column (30 × 100 mm); MeCN (0.1% TFA)/water

(0.1% TFA); 20%−100% gradient over 15 min; and 30 mL/min. LC/

(m, 2H), 1.95−1.83 (m, 1H), 1.82−1.55 (m, 5H), 1.48 (dd, J = 13.2,

1

1.0 Hz, 1H), 1.37−1.29 (m, 4H), 0.93−0.87 (m, 3H); HPLC t =

r

1

7

.27 min (method D); LC/MS M⁺= 332.3.

(

(1R,3S)-1-Amino-3-((R)-(6-(butoxymethyl)-5,6,7,8-tetrahydro-

29

naphthalen-2-yl)cyclopentyl)methanol (11). To a mixture of n-

butanol (500 μL, 5.5 mmol) and a 1 N potassium tert-butoxide in

THF (33 μL, 0.33 mmol) was added ((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 (42, 15 mg, 0.033 mmol). The mixture was

stirred at 70 °C for 6 h and then quenched with a saturated aqueous

+

1

MS M = 412.4; HPLC t = 1.07 (method A).

r

NH Cl solution (1 mL) and water (1 mL). The reaction mixture was

((1R,3S)-1-Amino-3-(6-(4-methoxybutyl)-5,6,7,8-tetrahydro-

4

2

9

extracted with EtOAc (3 × 2 mL). The combined EtOAc extracts

naphthalen-2-yl)cyclopentyl)methanol (13a, 13b). To a mixture

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

naphthalen-2-yl triﬂuoromethanesulfonate (46, 400 mg, 0.96

mmol), copper(I) iodide (18 mg, 0.096 mmol), and bis-

(triphenylphosphine)palladium(II) chloride (67 mg, 0.096 mmol)

in TEA (3 mL) was added 4-methoxybut-1-yne (161 mg, 1.92 mmol).

The reaction mixture was heated at 60 °C for 1 h, diluted with EtOAc,

were dried (Na SO ) and concentrated under reduced pressure to

2

4

aﬀord a solid. The solid was mixed with water (0.5 mL), lithium

hydroxide monohydrate (27.6 mg, 0.659 mmol), and dioxane (1 mL).

The resulting mixture was stirred at 100 °C under N for 7 h. The

2

mixture was extracted with EtOAc (4 × 1 mL) and the combined

EtOAc extracts were concentrated and then puriﬁed using reverse-

phase HPLC (Waters Xbridge C18 19 × 100 mm; gradient over 8

and washed with 1 M HCl. The organic layer was dried with MgSO ,

4

min from 30 to 100% of solvent B; solvent A: 10% MeOH: 90% H O:

ﬁltered, and concentrated. The crude material was puriﬁed on a silica

gel cartridge (24 g) using an EtOAc/Hex gradient (0−100% EtOAc

over 13 CV) to aﬀord (5R,7S)-7-(6-(4-methoxybut-1-yn-1-yl)-7,8-

2

0

.1% TFA; solvent B: 90% MeOH, 10% H O, 0.1% TFA). Fractions

2

with a desired product were basiﬁed with K CO and then extracted

2

3

with EtOAc to aﬀord ((1R,3S)-1-amino-3-((R)-(6-(butoxymethyl)-

dihydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (126 mg,

1

5

0

,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol (9.8 mg,

.029 mmol, 88% yield) as a white solid. H NMR (500 MHz,

38% yield). H NMR (400 MHz, CDCl

): δ 7.06−6.92 (m, 3H),

3

1

6.73−6.67 (m, 1H), 6.38−6.32 (m, 1H), 4.38−4.24 (m, 2H), 3.58 (t,

J = 6.9 Hz, 2H), 3.07−2.93 (m, 1H), 2.81 (t, J = 8.1 Hz, 2H), 2.68 (t,

J = 6.9 Hz, 2H), 2.47−2.36 (m, 2H), 2.31 (dd, J = 13.3, 7.2 Hz, 1H),

2.22−2.08 (m, 3H), 2.03−1.91 (m, 2H), 1.92−1.76 (m, 2H); HPLC

CDCl ): δ 7.08−6.97 (m, 3H), 3.47 (br t, J = 6.7 Hz, 4H), 3.40 (dd, J

=

dd, J = 16.4, 10.5 Hz, 1H), 2.29 (br dd, J = 13.3, 7.8 Hz, 1H), 2.20 (br

s, 2H), 2.07 (s, 3H), 1.99−1.85 (m, 1H), 1.82−1.66 (m, 2H), 1.65−

1

3

8.9, 6.7 Hz, 2H), 3.14−2.98 (m, 1H), 2.92−2.71 (m, 3H), 2.47 (br

+

1

t

= 0.94 min (method A); LC/MS M = 352.2.

r

.56 (m, 2H), 1.56−1.50 (m, 1H), 1.48−1.37 (m, 3H), 0.96 (t, J =

(5R,7S)-7-(6-(4-Methoxybut-1-yn-1-yl)-7,8-dihydronaphthalen-2-

+

1

7

.5 Hz, 3H); HPLC t = 7.19 min (method D); LC/MS M = 332.1.

yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (126 mg) was dissolved in

MeOH (10 mL) and then Pearlman’s catalyst (40 mg, 0.29 mmol)

was added. The reaction mixture was stirred under a blanket of

hydrogen (balloon pressure) for 1 h. The catalyst was then ﬁltered oﬀ

and the mixture was concentrated in vacuo, and the resulting residue

was puriﬁed by HPLC. HPLC conditions: Phenomenex Luna 5μ C18

r

(

(1R,3S)-1-Amino-3-((S)-6-(3-ethoxypropyl)-5,6,7,8-tetrahydro-

29

naphthalen-2-yl)cyclopentyl)methanol (12). To a stirred solution

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

tetrahydronaphthalen-2-yl)propanal (44, 440 mg, 1.34 mmol),

ethoxytrimethylsilane (1 mL, 6.7 mmol), and triethylsilane (1.07

1

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column (30 × 100 mm); MeCN (0.1% TFA)/water (0.1% TFA);

4H), 1.67−1.57 (m, 3H), 1.56−1.38 (m, 4H); HPLC t = 8.91 min

+1

r

2

0−100% gradient over 15 min; and 30 mL/min. Isolated fractions

with the correct mass were freeze-dried overnight to aﬀord (5R,7S)-7-

6-(4-methoxybutyl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-

azaspiro[4.4]nonan-2-one (130 mg, 100% yield). HPLC t = 1.04 min

(method D); LC/MS M = 348.1.

((1R,3S)-1-Amino-3-((6R)-6-((butylsulﬁnyl)methyl)-5,6,7,8-tetra-

2

9

(

hydronaphthalen-2-yl)cyclopentyl)methanol (15). To a stirred

clear solution of ((1R,3S)-1-amino-3-((R)-6-((butylthio)methyl)-

r

M); LC/MS M⁺¹= 358.3. This product was then separated into

diastereomers using SFC chiral separation: chiral AS-H 25 × 3 cm ID,

mm; ﬂow rate: 85.0 mL/min; mobile phase: 75/25 CO /MeOH,

5

0

,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol (10 mg,

.029 mmol) in DMSO (0.061 mL, 0.86 mmol) was added L-10-

(

−)-camphor sulfonic acid (33 mg, 0.14 mmol) in DCM (0.5 mL)

5

2

and methanol (0.2 mL) at −78 °C, followed by 77% m-CPBA (6.5

mg, 0.030 mmol). The temperature was raised to 0 °C over 30 min.

The mixture was stirred at 0 °C for 30 min and rt for 30 min. The

mixture was concentrated and the residue was puriﬁed using reverse-

phase HPLC (Waters Xbridge C18 19 × 100 mm; gradient over 10

and the two fractions were concentrated and dried. Isomer 1:

obtained 32 mg and isomer 2: obtained 33 mg.

To a mixture of (5R,7S)-7-(6-(4-methoxybutyl)-5,6,7,8-tetrahy-

dronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (isomer 2, 33

mg, 0.097 mmol) in MeOH (1 mL) and DMSO (0.5 mL) was added

min from 10 to 100% of solvent B; solvent A: 10% MeOH: 90% H O:

1

N NaOH (0.5 mL). The reaction mixture was heated at 95 °C for 4

2

0

.1% TFA; solvent B: 90% MeOH, 10% H O, 0.1% TFA). Fractions

h, then cooled, and acidiﬁed with TFA. The crude product was

ﬁltered and puriﬁed by HPLC. HPLC conditions: Phenomenex Luna

2

with product were pooled, basiﬁed with aqueous 1 N NaOH to pH =

12, and then extracted with EtOAc. The organic layer was dried

Na SO ) and concentrated to aﬀord ((1R,3S)-1-amino-3-((6R)-6-

∼

5μ C18 column (30 × 100 mm); MeCN (0.1% TFA)/water (0.1%

(

TFA); 20−100% gradient over 15 min; and 30 mL/min. Isolated

fractions with the correct mass were freeze-dried overnight to aﬀord

2

4

(butylsulﬁnyl)methyl)-5,6,7,8-tetrahydronaphthalen-2-yl)-

1

cyclopentyl)methanol (7 mg, 0.018 mmol, 64% yield) as a solid. H

NMR (400 MHz, MeOD): δ 7.16−6.88 (m, 3H), 3.56−3.35 (m, 2H),

3.16−2.94 (m, 2H), 2.93−2.73 (m, 6H), 2.60 (br dd, J = 16.2, 10.0

Hz, 1H), 2.44−2.26 (m, 1H), 2.25−2.09 (m, 2H), 2.09−1.82 (m,

3

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

len-2-yl)cyclopentyl)methanol, TFA (13a, 33 mg, 0.073 mmol, 74%

1

yield). H NMR (400 MHz, MeOD): δ 7.18−6.72 (m, 3H), 3.73−

3

1

2

1

.56 (m, 2H), 3.44 (t, J = 6.4 Hz, 2H), 3.35 (s, 3H), 3.21−3.03 (m,

H), 2.92−2.75 (m, 3H), 2.49−2.30 (m, 2H), 2.20−2.07 (m, 1H),

.05−1.87 (m, 4H), 1.73 (t, J = 12.7 Hz, 2H), 1.66−1.56 (m, 2H),

H), 1.80−1.64 (m, 4H), 1.61−1.45 (m, 3H), 0.99 (t, J = 7.3 Hz,

H); HPLC t = 6.36 min (method D); LC/MS M = 364.1.

+1

r

(

(1R,3S)-1-Amino-3-((R)-6-((butylsulfonyl)methyl)-5,6,7,8-tetra-

.55−1.45 (m, 2H), 1.45−1.27 (m, 3H); HPLC t = 7.57 min

29

r

hydronaphthalen-2-yl)cyclopentyl)methanol (16). To a stirring

solution of ((1R,3S)-1-amino-3-((R)-6-((butylthio)methyl)-5,6,7,8-

tetrahydronaphthalen-2-yl)cyclopentyl)methanol (10 mg, 0.029

mmol) and L-10-(−)-camphor sulfonic acid (33 mg, 0.14 mmol) in

DCM (0.5 mL) and methanol (0.2 mL) at −78 °C was added 77%

mCPBA (13 mg, 0.058 mmol). The temperature was raised to 0 °C

over 30 min. The mixture was stirred at 0 °C for 30 min and at rt for

1

(

method D); LC/MS M⁺= 332.

To a mixture of (5R,7S)-7-(6-(4-methoxybutyl)-5,6,7,8-tetrahy-

dronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (isomer 1, 32

mg, 0.090 mmol) in MeOH (1 mL) and DMSO (0.5 mL) was added

1

N NaOH (0.5 mL). The reaction mixture was heated at 95 °C for 4

h, then cooled, and acidiﬁed with TFA. The crude product was

ﬁltered and puriﬁed by HPLC. HPLC conditions: Phenomenex Luna

30 min. The mixture was concentrated and the residue was puriﬁed

5μ C18 column (30 × 100 mm); MeCN (0.1% TFA)/water (0.1%

using reverse-phase HPLC (Waters Xbridge C18 19 × 100 mm;

gradient over 10 min from 10 to 100% of solvent B; solvent A: 10%

MeOH: 90% H O: 0.1% TFA; solvent B: 90% MeOH, 10% H O,

TFA); 20−100% gradient over 15 min; and 30 mL/min. Isolated

fractions with the correct mass were freeze-dried overnight to aﬀord

2

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

0

.1% TFA). Fractions with product were pooled and basiﬁed to pH =

12 with 1 N NaOH and then extracted with EtOAc. The organic

layer was dried (Na SO ) and concentrated to aﬀord ((1R,3S)-1-

len-2-yl)cyclopentyl)methanol, TFA (13b, 29 mg, 0.064 mmol, 71%

∼

1

yield). H NMR (400 MHz, MeOD): δ 7.17−6.78 (m, 3H), 3.72−

2

4

3

1

2

1

3

.55 (m, 2H), 3.44 (t, J = 6.5 Hz, 2H), 3.35 (s, 3H), 3.21−3.02 (m,

H), 2.95−2.70 (m, 3H), 2.52−2.29 (m, 2H), 2.18−2.06 (m, 1H),

.02−1.86 (m, 4H), 1.70 (d, J = 12.5 Hz, 2H), 1.66−1.56 (m, 2H),

amino-3-((R)-6-((butylsulfonyl)methyl)-5,6,7,8-tetrahydronaphtha-

len-2-yl)cyclopentyl)methanol (7 mg, 0.018 mmol, 62% yield) as a

1

solid. H NMR (400 MHz, MeOD): δ 7.09−6.92 (m, 3H), 3.55−3.44

+

1

.55−1.29 (m, 5H); HPLC t = 7.64 min (method D); LC/MS M

=

r

(m, 2H), 3.21−3.11 (m, 4H), 3.11−2.99 (m, 2H), 2.90−2.83 (m,

32.

2

1

H), 2.74−2.60 (m, 1H), 2.59−2.43 (m, 1H), 2.31−2.13 (m, 2H),

(

(1R,3S)-1-Amino-3-((R)-6-((butylthio)methyl)-5,6,7,8-tetrahy-

.07−1.88 (m, 2H), 1.87−1.76 (m, 3H), 1.76−1.63 (m, 2H), 1.60−

29

dronaphthalen-2-yl)cyclopentyl)methanol (14). To a stirred

.46 (m, 3H), 1.01 (t, J = 7.4 Hz, 3H); HPLC t = 7.00 min (method

r

mixture of butyl mercaptan (0.7 mL, 1.6 mmol), anhydrous THF

+1

D); LC/MS M = 380.3.

(

1

(

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

,2,3,4-tetrahydronaphthalen-2-yl)methyl 4-methylbenzenesulfonate

42, 240 mg, 0.53 mmol) was added a 1 N THF solution of

(

(1R,3S)-1-Amino-3-((R)-6-(2-(propylthio)ethyl)-5,6,7,8-tetrahy-

29

dronaphthalen-2-yl)cyclopentyl)methanol (17). (5R,7S)-7-((R)-

-(2-Hydroxyethyl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-

6

potassium tert-butoxide (1.6 mL, 1.6 mmol) dropwise. The resulting

mixture was stirred at rt for 1.5 h and then at 50 °C for 1 h before 2 N

aqueous NaOH (5.3 mL, 10.6 mmol) was added. The mixture was

concentrated to remove THF and dioxane (3 mL) was added. The

resulting mixture was stirred at 90 °C overnight, cooled to rt, and

extracted with EtOAc (5 mL, 2 × 2 mL). The combined EtOAc

extracts were dried (Na SO ), concentrated, and the resulting residue

azaspiro[4.4]nonan-2-one (40, 180 mg, 0.57 mmol) was dissolved in

dry pyridine (1 mL) and p-toluenesulfonyl chloride (326 mg, 1.7

mmol) was added in one portion. The resulting mixture was stirred at

rt for 2 h and the solvent was removed in vacuo. The residue was

dissolved in DCM and loaded onto the column. Flash chromatog-

raphy puriﬁcation (4 g of silica gel column, 0−100% EtOAc in DCM)

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

1,2,3,4-tetrahydronaphthalen-2-yl)ethyl 4-methylbenzenesulfonate

2

4

was puriﬁed using reverse-phase HPLC (Phen Luna 5μ 30 × 100 mm

Axia); gradient over 8 min from 30 to 100% of solvent B; solvent A:

0% MeOH: 90% H O: 0.1% TFA; solvent B: 90% MeOH, 10%

(

(220 mg, 0.47 mmol, 82% yield) as a solid. HPLC t = 1.04 min

r

+

1

(method A); LC/MS M = 470.4.

2

H O, 0.1% TFA). Fractions with product were pooled, basiﬁed to pH

To a stirred mixture of propane-1-thiol (0.017 mL, 0.19 mmol), 2-

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

rahydronaphthalen-2-yl)ethyl 4-methylbenzenesulfonate (30 mg,

0.064 mmol), and dioxane (0.5 mL) was added 2 N aqueous

NaOH (0.64 mL, 1.3 mmol), and the resulting mixture was stirred at

90 °C overnight. The mixture was extracted with EtOAc (4 × 1 mL)

and the combined EtOAc extracts were dried (Na SO ) and

2

=

∼12 with aqueous 1 N NaOH, and then extracted with EtOAc. The

organic layer was dried (Na SO ) and concentrated to aﬀord

2

4

(

(1R,3S)-1-amino-3-((R)-6-((butylthio)methyl)-5,6,7,8-tetrahydro-

naphthalen-2-yl)cyclopentyl)methanol (153 mg, 0.42 mmol, 79%

1

yield) as a white solid. H NMR (400 MHz, CDCl ): δ 7.11−6.98 (m,

3

H), 3.95 (br d, J = 5.5 Hz, 1H), 3.56−3.42 (m, 2H), 3.14−2.95 (m,

H), 2.90−2.80 (m, 2H), 2.69−2.46 (m, 5H), 2.30 (dd, J = 13.5, 7.6

Hz, 1H), 2.17−2.05 (m, 2H), 2.02−1.87 (m, 2H), 1.83−1.76 (m,

2

4

2

concentrated and then puriﬁed using reverse-phase HPLC [Phen

Luna 5μ 30 × 100 mm (Axia); gradient over 8 min from 30 to 100%

1

468

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J. Med. Chem. 2021, 64, 1454−1480

Journal of Medicinal Chemistry

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Article

mmol). After stirring at rt for 30 min, ((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 (42, 40 mg, 0.088 mmol) in DMF (2 mL)

was added and the reaction mixture was stirred at rt for 2 h. The

reaction mixture was quenched with water (5 mL) at 0 °C, then taken

2

up in EtOAc (30 mL), and washed with saturated NaHCO (3 × 20

3

mL). The organic layer was dried (Na SO ) and concentrated under

2

4

.02 (s, 2H), 6.99 (s, 1H), 3.63−3.36 (m, 2H), 3.15−2.98 (m, 1H),

vacuo. The resulting residue was dissolved in dioxane (1.5 mL) and

water (0.5 mL), and then lithium hydroxide hydrate (37 mg, 0.88

mmol) was added. The reaction mixture was stirred at 100 °C for 16

h. The mixture was concentrated and puriﬁed with preparative HPLC.

HPLC conditions: Phenomenex Luna C18 5μ 21.2 × 100 mm.

.93−2.76 (m, 3H), 2.69−2.60 (m, 2H), 2.57−2.48 (m, 2H), 2.42

r

+1

= 348.4.

Solvent A: 10% MeOH−90% H O−0.1% TFA; solvent B: 90%

2

(

(1R,3S)-1-Amino-3-((S)-6-(3-(ethylthio)propyl)-5,6,7,8-tetrahy-

MeOH−10% H O−0.1% TFA. Gradient time = 15 min. Start B = 0%,

2

9

ﬁnal B 100%. Stop time 20 min. The collected fraction was basiﬁed

with saturated NaHCO , concentrated under vacuo, and the aqueous

3

layer was extracted with DCM (3 × 20 mL), dried (Na SO ), and

2

4

concentrated in vacuo to aﬀord ((1R,3S)-1-amino-3-((R)-6-((but-3-

dioxane (1 mL) was added 2 N aqueous NaOH (0.217 mL, 0.434

mmol) at 0 °C. The resulting mixture was stirred at 90 °C overnight.

The mixture was cooled to rt, extracted with EtOAc (4 × 1 mL), and

the combined EtOAc extracts were dried (Na SO ) and concentrated.

en-1-yloxy)methyl)-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)-

methanol (15 mg, 47% yield). H NMR (400 MHz, MeOD): δ 7.05−

1

6.93 (m, 3H), 5.88 (ddt, J = 17.2, 10.3, 6.8 Hz, 1H), 5.11 (dq, J =

17.2, 1.7 Hz, 1H), 5.04 (ddt, J = 10.3, 2.1, 1.2 Hz, 1H), 3.58−3.44 (m,

4H), 3.43 (d, J = 6.4 Hz, 2H), 3.03 (tt, J = 11.1, 7.2 Hz, 1H), 2.88−

2.73 (m, 3H), 2.44 (dd, J = 16.5, 10.3 Hz, 1H), 2.36 (qt, J = 6.7, 1.3

Hz, 2H), 2.23 (br dd, J = 13.1, 7.4 Hz, 1H), 2.11−1.97 (m, 3H),

1.97−1.85 (m, 1H), 1.85−1.68 (m, 2H), 1.56 (br t, J = 12.3 Hz, 1H),

2

4

The resulting residue was puriﬁed using reverse-phase HPLC [Phen

Luna 5μ 30 × 100 mm (Axia); gradient over 8 min from 30 to 100%

of solvent B; solvent A: 10% MeOH: 90% H O: 0.1% TFA; solvent B:

2

9

0% MeOH, 10% H O, 0.1% TFA]. Fractions with product were

2

+

1

pooled and basiﬁed to pH = ∼12 with 1 N NaOH and then extracted

1.44 (dtd, J = 12.8, 10.7, 6.7 Hz, 1H); LC/MS M = 330.3; HPLC t_r

= 6.62 (method D).

with EtOAc. The organic layer was dried (Na SO ) and concentrated

2

4

to aﬀord ((1R,3S)-1-amino-3-((S)-6-(3-(ethylthio)propyl)-5,6,7,8-

((1R,3S)-1-Amino-3-(6-(2-methoxyethyl)-5,6,7,8-tetrahydro-

2

9

tetrahydronaphthalen-2-yl)cyclopentyl)methanol (45 mg, 0.123

naphthalen-2-yl)cyclopentyl)methanol (21a, 21b). Sodium metal

(120 mg, 5.3 mmol) was added to MeOH (2 mL). The reaction

mixture was stirred until the metal was dissolved and then 2-(6-

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

naphthalen-2-yl)ethyl 4-methylbenzenesulfonate (diastereomeric-40,

125 mg, 0.27 mmol) was added and the reaction was stirred

overnight. The reaction mixture was diluted with EtOAc and washed

1

mmol, 85% yield) as a white solid. H NMR (400 MHz, MeOD): δ

7

.06−6.90 (m, 3H), 3.57−3.44 (m, 2H), 3.02 (tt, J = 11.2, 7.2 Hz,

1

H), 2.87−2.74 (m, 3H), 2.63−2.51 (m, 4H), 2.37 (dd, J = 16.2, 10.5

Hz, 1H), 2.24 (br dd, J = 12.8, 7.3 Hz, 1H), 2.09−1.78 (m, 4H),

1

.78−1.65 (m, 4H), 1.57 (br t, J = 12.4 Hz, 1H), 1.49 (dd, J = 9.4, 7.0

Hz, 2H), 1.44−1.33 (m, 1H), 1.26 (t, J = 7.4 Hz, 3H); HPLC t =

r

1

9

.14 min (method D); LC/MS M⁺= 348.3.

with water. The organic layer was dried with MgSO , ﬁltered, and

4

(

(1R,3S)-1-Amino-3-((R)-6-((2-methoxyethoxy)methyl)-5,6,7,8-

concentrated and then puriﬁed using HPLC. HPLC conditions:

Phenomenex Luna 5μ C18 column (30 × 100 mm); MeCN (0.1%

TFA)/water (0.1% TFA); 20−100% gradient over 15 min; and 30

mL/min. Isolated fractions with the correct mass were freeze-dried

overnight to aﬀord (5R,7S)-7-(6-(2-methoxyethyl)-5,6,7,8-tetrahy-

dronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (30 mg, 0.09

mmol, 30% yield). This product was then separated into individual

isomers using the following conditions: Berger SFC MGII; column:

Chiral OD-H 25 × 3 cm ID, 5 μm; ﬂow rate: 85.0 mL/min; mobile

2

9

tetrahydronaphthalen-2-yl)cyclopentyl)methanol (19). To a

stirring mixture of 2-methoxyethanol (0.043 mL, 0.549 mmol) and

a 1 N THF solution of potassium tert-butoxide (0.44 mL, 0.44 mmol)

was added ((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

42, 25 mg, 0.055 mmol). The resulting mixture was stirred at 70

C for 1.5 h and at rt for 3 days. The mixture was concentrated in

vacuo. The residue was mixed with 2 N aqueous sodium hydroxide

(

0.5 mL, 1.000 mmol) and dioxane (0.5 mL) and then heated at 90

phase: 65/35 CO /MeOH.

2

°

C overnight. The mixture was extracted with EtOAc (4 × 1 mL).

Peak 2: isomer a: recovered (5R,7S)-7-(6-(2-methoxyethyl)-

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

(13 mg, 0.039 mmol, 15% yield).

Peak 1: isomer b: recovered (5R,7S)-7-(6-(2-methoxyethyl)-

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

(12 mg, 0.036 mmol, 14% yield). Each isomer was taken individually

into the next step.

The combined EtOAc extracts were dried (Na SO ) and concen-

2

4

trated under reduced pressure. The crude material was puriﬁed via

preparative LC/MS with the following conditions: Column: Waters

XBridge C18, 19 × 150 mm, 5 μm particles; guard column: Waters

XBridge C18, 19 × 10 mm, 5 μm particles; mobile phase A: 5:95

acetonitrile/water with 10 mM ammonium acetate; mobile phase B:

9

5:5 acetonitrile/water with 10 mM ammonium acetate; gradient: 0−

To a mixture of (5R,7S)-7-(6-(2-methoxyethyl)-5,6,7,8-tetrahydro-

naphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (isomer a, 12 mg,

0.036 mmol) in DMSO (0.5 mL) and MeOH (1 mL) was added 1 N

NaOH. The reaction mixture was heated at 95 °C for 2 h, and then it

was cooled and acidiﬁed with TFA. Puriﬁed by HPLC. HPLC

conditions: Phenomenex Luna 5μ C18 column (30 × 100 mm);

MeCN (0.1% TFA)/water (0.1% TFA); 20−100% gradient over 15

min; and 30 mL/min. Isolated fractions with the correct mass were

freeze-dried overnight to aﬀord ((1R,3S)-1-amino-3-(6-(2-methox-

yethyl)-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol,

100% B over 15 min, then a 5 min hold at 100% B; ﬂow: 20 mL/min.

Fractions containing the desired product were combined and dried via

centrifugal evaporation to aﬀord ((1R,3S)-1-amino-3-((R)-6-((2-

methoxyethoxy)methyl)-5,6,7,8-tetrahydronaphthalen-2-yl)-

1

cyclopentyl)methanol (13.3 mg, 71% yield). H NMR (500 MHz,

DMSO-d ): δ 6.98 (s, 2H), 6.96 (s, 1H), 3.56−3.44 (m, 6H), 3.30 (br

6

s, 2H), 3.26 (s, 3H), 2.99−2.88 (m, 1H), 2.81−2.67 (m, 3H), 2.36

(

1

dd, J = 16.3, 10.4 Hz, 1H), 2.12 (dd, J = 12.9, 7.9 Hz, 1H), 1.99−

.83 (m, 3H), 1.86−1.73 (m, 1H), 1.71−1.63 (m, 1H), 1.62−1.53

1

(

6

m, 1H), 1.42 (dd, J = 12.9, 11.4 Hz, 1H), 1.33 (dtd, J = 12.3, 10.7,

TFA (21a, 10 mg, 0.023 mmol, 62% yield). H NMR (400 MHz,

.9 Hz, 1H); LC/MS M⁺= 333.6; HPLC t = 1.19 (method E).

1

MeOD): δ 7.03−6.97 (m, 3H), 3.70−3.58 (m, 2H), 3.55 (t, J = 6.6

Hz, 2H), 3.37 (s, 3H), 3.18−3.04 (m, 1H), 2.90−2.75 (m, 3H),

2.49−2.33 (m, 2H), 2.20−2.05 (m, 1H), 2.03−1.79 (m, 5H), 1.73 (t,

J = 12.8 Hz, 1H), 1.65 (qd, J = 6.7, 2.8 Hz, 2H), 1.49−1.35 (m, 1H);

r

(

(1R,3S)-1-Amino-3-((R)-6-((but-3-en-1-yloxy)methyl)-5,6,7,8-tet-

29

rahydronaphthalen-2-yl)cyclopentyl)methanol (20). To a mixture

of but-3-en-1-ol (32 mg, 0.44 mmol) in dry DMF (2 mL) at 0 °C was

added 1 N THF solution of potassium tert-butoxide (440 μL, 0.44

+

1

LC/MS M = 304; HPLC t = 5.57 (method D).

r

1

469

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J. Med. Chem. 2021, 64, 1454−1480

Journal of Medicinal Chemistry

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Article

To a mixture of (5R,7S)-7-(6-(2-methoxyethyl)-5,6,7,8-tetrahydro-

naphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (isomer b, 13 mg,

2.18−2.07 (m, 1H), 2.03−1.86 (m, 4H), 1.81−1.64 (m, 4H), 1.51−

+

1

1.31 (m, 3H)); LC/MS M = 318; HPLC t = 6.09 (method D).

r

0

.039 mmol) in DMSO (0.5 mL) and MeOH (1 mL) was added 1 N

To a mixture of (5R,7S)-7-(6-(3-methoxypropyl)-5,6,7,8-tetrahy-

dronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (isomer b, 13

mg, 0.038 mmol) in MeOH (1 mL) and DMSO (0.5 mL) was added

1 N NaOH (0.5 mL). The reaction mixture was heated at 95 °C for 2

h, cooled to rt, acidiﬁed with TFA, and puriﬁed by HPLC. HPLC

conditions: Phenomenex Luna 5μ C18 column (30 × 100 mm);

MeCN (0.1% TFA)/water (0.1% TFA); 20−100% gradient over 15

min; and 30 mL/min. Isolated fractions with the correct mass were

freeze-dried overnight to aﬀord ((1R,3S)-1-amino-3-(6-(3-methox-

ypropyl)-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol,

NaOH. The reaction mixture was heated at 95 °C, then cooled, and

acidiﬁed with TFA, and the reaction mixture was puriﬁed by HPLC.

HPLC conditions: Phenomenex Luna 5μ C18 column (30 × 100

mm); MeCN (0.1% TFA)/water (0.1% TFA); 20%−100% gradient

over 15 min; and 30 mL/min. Isolated fractions with the correct mass

were freeze-dried overnight to aﬀord ((1R,3S)-1-amino-3-(6-(2-

methoxyethyl)-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)-

1

methanol, TFA (21b, 10 mg, 0.022 mmol, 56.5% yield). H NMR

(

3

400 MHz, MeOD): δ 7.02−6.97 (m, 3H), 3.71−3.58 (m, 2H), 3.55

1

t, J = 6.6 Hz, 2H), 3.37 (s, 3H), 3.21−3.01 (m, 1H), 2.96−2.73 (m,

TFA (22b, 9 mg, 52% yield). H NMR (400 MHz, MeOD): δ 7.05−

H), 2.53−2.32 (m, 2H), 2.23−2.05 (m, 1H), 2.03−1.78 (m, 5H),

6

3

.92 (m, 3H), 3.73−3.55 (m, 2H), 3.44 (t, J = 6.5 Hz, 2H), 3.35 (s,

H), 3.12 (d, J = 9.2 Hz, 1H), 2.94−2.73 (m, 3H), 2.52−2.28 (m,

1

M

.78−1.56 (m, 3H), 1.42 (dtd, J = 12.8, 10.4, 6.6 Hz, 1H)); LC/MS

+1

= 304; HPLC t = 5.58 (method D).

r

2H), 2.19−2.04 (m, 1H), 2.05−1.87 (m, 4H), 1.80−1.62 (m, 4H),

(

(1R,3S)-1-Amino-3-(6-(3-methoxypropyl)-5,6,7,8-tetrahydro-

+1

1

D).

.53−1.29 (m, 3H)); LC/MS M = 318; HPLC t = 6.13 (method

2

9

r

naphthalen-2-yl)cyclopentyl)methanol (22a, 22b). To a mixture

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

naphthalen-2-yl triﬂuoromethanesulfonate (46, 100 mg, 0.240

mmol), copper(I) iodide (4.6 mg, 0.024 mmol), and bis-

(

(1R,3S)-1-Amino-3-((R)-6-(5-methoxypentyl)-5,6,7,8-tetrahydro-

29

naphthalen-2-yl)cyclopentyl)methanol (23). To a mixture of 6-

(

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

(

triphenylphosphine)palladium(II) chloride (17 mg, 0.024 mmol)

thalen-2-yl triﬂuoromethanesulfonate (46, 150 mg, 0.36 mmol),

copper(I) iodide (6.8 mg, 0.036 mmol), and bis(triphenylphosphine)-

palladium(II) chloride (25 mg, 0.036 mmol) in TEA (3 mL) was

added 5-methoxypent-1-yne (176 mg, 1.8 mmol). The reaction

mixture was heated at 60 °C for 1 h, cooled to rt, diluted with EtOAc,

and washed with 1 M HCl. The organic layer was dried with MgSO₄,

ﬁltered, and concentrated. The crude material was puriﬁed on a silica

gel cartridge (24 g) using an EtOAc/Hex gradient (0−100% EtOAc

over 13 CV) to aﬀord (5R,7S)-7-(6-(5-methoxypent-1-yn-1-yl)-7,8-

in TEA (3 mL) was added 3-methoxyprop-1-yne (0.10 mL, 1.2

mmol). The reaction mixture was heated at 60 °C for 1 h, cooled to

rt, diluted with EtOAc, and washed with 1 M HCl. The organic layer

was dried with MgSO , ﬁltered, and concentrated. The crude material

4

was puriﬁed on a silica gel cartridge (12 g) using an EtOAc/Hex

gradient (0−100% EtOAc over 20 CV) to aﬀord (5R,7S)-7-(6-(3-

methoxyprop-1-yn-1-yl)-7,8-dihydronaphthalen-2-yl)-3-oxa-1-

1

azaspiro[4.4]nonan-2-one (30 mg, 0.089 mmol, 37% yield). H NMR

(

400 MHz, MeOD): δ 7.13−7.04 (m, 2H), 7.04−6.94 (m, 1H), 6.75

s, 1H), 4.41−4.27 (m, 3H), 3.41 (s, 3H), 3.15−2.94 (m, 1H), 2.82

t, J = 8.1 Hz, 2H), 2.41 (td, J = 8.1, 1.3 Hz, 2H), 2.31 (dd, J = 13.1,

dihydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (105 mg,

1

0

(

6

.29 mmol, 80% yield). H NMR (400 MHz, CDCl ): δ 7.04−6.95

3

m, 3H), 6.69 (s, 1H), 5.62 (s, 1H), 4.38−4.27 (m, 2H), 3.52 (t, J =

7

1

.2 Hz, 1H), 2.18−2.04 (m, 3H), 2.02−1.89 (m, 2H), 1.88−1.74 (m,

.2 Hz, 2H), 3.40−3.37 (m, 3H), 3.11−2.98 (m, 1H), 2.83 (t, J = 8.1

H).

Hz, 2H), 2.50 (t, J = 7.2 Hz, 2H), 2.46−2.39 (m, 2H), 2.33 (dd, J =

To a mixture of (5R,7S)-7-(6-(3-methoxyprop-1-yn-1-yl)-7,8-

1

3.2, 7.3 Hz, 1H), 2.23−2.10 (m, 2H), 2.03−1.91 (m, 2H), 1.90−

dihydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (30 mg,

.089 mmol) in MeOH (5 mL) was added Pearlman’s catalyst (6.2

1.79 (m, 3H).

0

To a mixture of (5R,7S)-7-(6-(5-methoxypent-1-yn-1-yl)-7,8-

dihydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (105 mg,

mg, 0.044 mmol). The reaction mixture was evacuated and backﬁlled

with hydrogen and then hydrogenated under balloon pressure for 2 h.

The reaction mixture was then evacuated and backﬁlled with N and

the catalyst was ﬁltered away. The residue was puriﬁed by HPLC to

aﬀord (5R,7S)-7-(6-(3-methoxypropyl)-5,6,7,8-tetrahydronaphthalen-

2

0.29 mmol) in MeOH (10 mL) was added Pearlman’s catalyst (20

2

mg, 0.14 mmol) The reaction mixture was evacuated and backﬁlled

with hydrogen and then hydrogenated under balloon pressure for 2 h.

The reaction mixture was then evacuated and backﬁlled with N and

2

-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (25 mg). HPLC conditions:

the catalyst was ﬁltered away. The residue was puriﬁed by HPLC to

aﬀord (5R,7S)-7-(6-(5-methoxypentyl)-5,6,7,8-tetrahydronaphthalen-

Phenomenex Luna 5μ C18 column (30 × 100 mm); MeCN (0.1%

TFA)/water (0.1% TFA); 20−100% gradient over 15 min; and 30

mL/min. This product was then separated into individual isomers

using the following conditions: Berger SFC MGII; column: Chiral

AD-H 25 × 3 cm ID, 5 mm; ﬂow rate: 85.0 mL/min; mobile phase:

2

-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one. This product was then

separated into individual isomers using the following conditions:

Berger SFC MGII; column: chiral AD-H 25 × 3 cm ID, 5 mm; ﬂow

rate: 85.0 mL/min; mobile phase: 70/30 CO /MeOH.

6

0/40 CO /MeOH.

2

Isomer a: peak 2: recovered (5R,7S)-7-(6-(5-methoxypentyl)-

Peak 2: isomer a; recovered (5R,7S)-7-(6-(3-methoxypropyl)-

,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one

5

,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one

5

(

38 mg, 0.102 mmol, 36% yield).

Isomer b: peak 1: recovered (5R,7S)-7-(6-(5-methoxypentyl)-

(10 mg, 0.029 mmol, 33% yield).

Peak 1: isomer b; recovered (5R,7S)-7-(6-(3-methoxypropyl)-

,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one

5

,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one

5

(

40 mg, 0.108 mmol, 38% yield).

To a mixture of (5R,7S)-7-(-6-(5-methoxypentyl)-5,6,7,8-tetrahy-

(13 mg, 0.038 mmol, 43% yield). Each isomer was taken individually

into the next step.

dronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (isomer a, 38

mg, 0.11 mmol) in DMSO (0.5 mL) and MeOH (1 mL) was added 1

N NaOH (0.5 mL). The reaction mixture was heated at 95 °C for 2 h,

cooled to rt, acidiﬁed with TFA, and puriﬁed by HPLC. HPLC

conditions: Phenomenex Luna 5μ C18 column (30 × 100 mm);

MeCN (0.1% TFA)/water (0.1% TFA); 20−100% gradient over 15

min; and 30 mL/min. Isolated fractions with the correct mass were

freeze-dried overnight to aﬀord ((1R,3S)-1-amino-3-(6-(5-methox-

ypentyl)-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol,

To a mixture of (5R,7S)-7-(6-(3-methoxypropyl)-5,6,7,8-tetrahy-

dronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (isomer a, 10

mg, 0.038 mmol) in MeOH (1 mL) and DMSO (0.5 mL) was added

1

N NaOH (0.5 mL). The reaction mixture was heated at 95 °C for 2

h, cooled to rt, acidiﬁed with TFA, and puriﬁed by HPLC. HPLC

conditions: Phenomenex Luna 5μ C18 column (30 × 100 mm);

MeCN (0.1% TFA)/water (0.1% TFA); 20%−100% gradient over 15

min; and 30 mL/min. Isolated fractions with the correct mass were

freeze-dried overnight to aﬀord ((1R,3S)-1-amino-3-(6-(3-methox-

ypropyl)-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol,

1

TFA (26 mg, 0.055 mmol, 54% yield). H NMR (400 MHz, MeOD):

1

TFA (22a, 9 mg, 52% yield). H NMR (400 MHz, MeOD): δ 7.02−

δ 7.02−6.96 (m, 3H), 3.71−3.56 (m, 2H), 3.42 (t, J = 6.6 Hz, 2H),

3.34 (s, 3H), 3.18−3.02 (m, 1H), 2.90−2.71 (m, 3H), 2.49−2.29 (m,

2H), 2.12 (d, J = 2.9 Hz, 1H), 2.02−1.87 (m, 4H), 1.79−1.66 (m,

6

3

.96 (m, 3H), 3.71−3.57 (m, 2H), 3.44 (t, J = 6.5 Hz, 2H), 3.35 (s,

H), 3.22−3.03 (m, 1H), 2.93−2.72 (m, 3H), 2.49−2.30 (m, 2H),

1

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+

1

2

H), 1.66−1.55 (m, 2H), 1.52−1.32 (m, 7H); LC/MS M = 346;

The reaction mixture was heated at 60 °C for 1 h, cooled to rt, diluted

with EtOAc, and washed with 1 M HCl. The organic layer was dried

HPLC t = 8.16 (method D).

r

(

(1R,3S)-1-Amino-3-((S)-6-(5-methoxypentyl)-5,6,7,8-tetrahydro-

with MgSO , ﬁltered, and concentrated. The crude material was

4

2

9

naphthalen-2-yl)cyclopentyl)methanol (24). To a stirred mixture

of magnesium (0.53 g, 22 mmol) and anhydrous THF (30 mL) was

added a few drops of 1,2-dibromoethane at rt. The mixture was stirred

for 15 min before a solution of 1-bromo-4-methoxybutane (2.9 mL,

puriﬁed on a silica gel cartridge (24 g) using an EtOAc/Hex gradient

(0−100% EtOAc over 13 CV). Isolated fractions with product were

concentrated and dried in vacuo to aﬀord (5R,7S)-7-(6-(6-

methoxyhex-1-yn-1-yl)-7,8-dihydronaphthalen-2-yl)-3-oxa-1-azaspiro-

1

2

2 mmol) in anhydrous THF (10 mL) was added dropwise to keep

[4.4]nonan-2-one (35 mg, 0.092 mmol, 39% yield). H NMR (400

the reaction mixture warm and not boiling. After the addition, the

mixture was stirred at 60 °C for 3 h and cooled to rt. The solution was

separated and added to a stirred mixture of copper(I) bromide (0.63

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

yl)-1,2,3,4-tetrahydronaphthalen-2-yl)methyl 4-methylbenzenesulfo-

nate (48, 1 g, 2.2 mmol), and THF (10 mL) at −78 °C. The

mixture was stirred at −78 °C for 20 min before the temperature was

slowly raised to 0 °C. The mixture was stirred at 0 °C for 3 h and then

MHz, CDCl ): δ 7.08−6.91 (m, 3H), 6.68 (s, 1H), 5.87 (s, 1H),

3

4.48−4.20 (m, 2H), 3.44 (t, J = 6.3 Hz, 2H), 3.37 (s, 3H), 3.03 (tt, J

= 10.5, 7.2 Hz, 1H), 2.82 (t, J = 8.1 Hz, 2H), 2.47−2.38 (m, 4H),

2.32 (dd, J = 13.4, 7.3 Hz, 1H), 2.21−2.08 (m, 2H), 2.03−1.91 (m,

2H), 1.77−1.60 (m, 5H).

To a mixture of (5R,7S)-7-(6-(6-methoxyhex-1-yn-1-yl)-7,8-dihy-

dronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (34 mg, 0.090

mmol) in MeOH (5 mL) was added Pearlman’s catalyst (6.29 mg,

0.045 mmol). The reaction mixture was evacuated and backﬁlled with

hydrogen and then hydrogenated under balloon pressure for 2 h. The

at rt for 15 h. Saturated aqueous NH Cl solution (20 mL) was added

4

dropwise to quench the reaction. Water (10 mL) and hexanes (20

mL) were added to the reaction. The aqueous layer was separated and

extracted with EtOAc (2 × 15 mL). The combined organic solutions

were dried over Na SO and concentrated under reduced pressure.

reaction mixture was then evacuated and backﬁlled with N and the

2

catalyst was ﬁltered away. The residue was puriﬁed by HPLC to aﬀord

(5R,7S)-7-(6-(6-methoxyhexyl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-

oxa-1-azaspiro[4.4]nonan-2-one. This product was then separated

into individual isomers using the following conditions: Berger SFC

MGII; column: chiral AD-H 25 × 3 cm ID, 5 mm; ﬂow rate: 85.0

2

4

Flash chromatography puriﬁcation (40 g silica gel column, gradient

elution from 15 to 100% of EtOAc in hexanes) aﬀorded (5R,7S)-7-

(

1

8

(S)-6-(5-methoxypentyl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-

-azaspiro[4.4]nonan-2-one as a white solid (680 mg, 1.83 mmol,

mL/min; mobile phase: 60/40 CO /MeOH.

2

3% yield). LC/MS M⁺= 372.3; HPLC t = 1.12 (method A).

1

Peak 2: isomer a; recovered (5R,7S)-7-(6-(6-methoxyhexyl)-

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

(9 mg, 0.023 mmol, 26% yield).

Peak 1: isomer b; recovered (5R,7S)-7-(6-(6-methoxyhexyl)-

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

(8 mg, 0.021 mmol, 23% yield. Each isomer was taken individually

into the next step.

r

To a mixture of (5R,7S)-7-((S)-6-(5-methoxypentyl)-5,6,7,8-

tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (680

mg, 1.83 mmol) in MeOH (20 mL) and DMSO (5 mL) was

added 1 N NaOH (10 mL). The reaction mixture was heated at 95 °C

for 12 h, cooled to rt, diluted with EtOAc, and washed with sat NaCl.

The organic layer was dried with MgSO , ﬁltered, and concentrated.

4

The resulting white solid was suspended in EtOAc and enough

MeOH was added to dissolve using a heat gun to warm solution. HCl

gas was bubbled through for 10 s, then ether was added, and the

solution was allowed to stand in a freezer for 1 h. The solid was

collected and dried to aﬀord ((1R,3S)-1-amino-3-((S)-6-(5-methox-

ypentyl)-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol as

To a mixture of (5R,7S)-7-(6-(6-methoxyhexyl)-5,6,7,8-tetrahy-

dronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (isomer a, 9

mg, 0.023 mmol) in DMSO (0.5 mL) and MeOH (0.5 mL) was

added 1 N NaOH. The reaction mixture was heated at 95 °C for 2 h,

cooled to rt, acidiﬁed with TFA, and puriﬁed by HPLC to aﬀord

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

len-2-yl)cyclopentyl)methanol, TFA (25a, 8 mg, 0.017 mmol, 72%

yield). HPLC conditions: Phenomenex Luna 5μ C18 column (30 ×

100 mm); MeCN (0.1% TFA)/water (0.1% TFA); 20−100%

1

the HCl salt (450 mg, 1.3 mmol, 70% yield. H NMR (400 MHz,

MeOD): δ 7.04−6.90 (m, 3H), 3.55−3.38 (m, 4H), 3.34 (s, 3H), 3.00

(

tt, J = 11.1, 7.3 Hz, 1H), 2.88−2.70 (m, 3H), 2.35 (dd, J = 16.2, 10.5

1

Hz, 1H), 2.20 (dd, J = 12.8, 7.3 Hz, 1H), 2.08−1.85 (m, 3H), 1.83−

1

8

gradient over 15 min; and 30 mL/min. H NMR (400 MHz,

+1

.49 (m, 6H), 1.49−1.26 (m, 7H); LC/MS M = 346.6; HPLC t =

MeOD): δ 7.31−6.80 (m, 3H), 3.73−3.57 (m, 2H), 3.42 (t, J = 6.5

Hz, 2H), 3.34 (s, 3H), 3.19−3.02 (m, 1H), 2.93−2.71 (m, 3H),

2.50−2.28 (m, 2H), 2.21−2.05 (m, 1H), 2.03−1.86 (m, 4H), 1.82−

r

.19 (method D).

(

(1R,3S)-1-Amino-3-((S)-6-(5-methoxypentyl)-5,6,7,8-tetrahydro-

naphthalen-2-yl)cyclopentyl)methyl Dihydrogen Phosphate (24-

1.64 (m, 2H), 1.59 (quin, J = 6.8 Hz, 2H), 1.50−1.27 (m, 9H); LC/

2

9

+1

P). To a mixture of ((1R,3S)-1-amino-3-((S)-6-(5-methoxypentyl)-

MS M = 360; HPLC t = 7.59 (method D).

r

5

0

,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol (8.4 mg,

.024 mmol) in acetonitrile (1 mL) was added pyrophosphoryl

To a mixture of (5R,7S)-7-(6-(6-methoxyhexyl)-5,6,7,8-tetrahy-

dronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (8 mg, 0.021

mmol) in DMSO (0.5 mL) and MeOH (0.5 mL) was added 1 N

NaOH. The reaction mixture was heated at 95 °C for 2 h, cooled to

rt, acidiﬁed with TFA, and puriﬁed by HPLC to aﬀord ((1R,3S)-1-

amino-3-(6-(6-methoxyhexyl)-5,6,7,8-tetrahydronaphthalen-2-yl)-

cyclopentyl)methanol, TFA (25b, 7 mg, 0.015 mmol, 71% yield).

HPLC conditions: Phenomenex Luna 5μ C18 column (30 × 100

mm); MeCN (0.1% TFA)/water (0.1% TFA); 20−100% gradient

over 15 min; and 30 mL/min. Isolated fractions with the correct mass

chloride (0.135 mL, 0.972 mmol). The reaction was stirred for 1 h

and then quenched with water (0.5 mL). The reaction mixture was

puriﬁed by HPLC to aﬀord ((1R,3S)-1-amino-3-((S)-6-(5-methox-

ypentyl)-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methyl dihy-

drogen phosphate (4 mg, 8.93 μmol, 37% yield). HPLC conditions:

Phenomenex Luna 5 μm C18 column (30 × 100 mm); MeCN (0.1%

TFA)/water (0.1% TFA); 20%−100% gradient over 15 min; and 30

1

mL/min. H NMR (400 MHz, MeOD): δ 7.06−6.97 (m, 3H), 4.04−

1

3

1

.90 (m, 2H), 3.45 (t, J = 6.7 Hz, 2H), 3.35 (s, 3H), 3.22−3.09 (m,

H), 2.88−2.72 (m, 3H), 2.51 (dd, J = 13.5, 6.5 Hz, 1H), 2.34 (dd, J

were freeze-dried overnight. H NMR (400 MHz, MeOD): δ 7.03−

6.95 (m, 3H), 3.72−3.56 (m, 2H), 3.42 (t, J = 6.5 Hz, 2H), 3.34 (s,

3H), 3.21−3.00 (m, 1H), 2.92−2.70 (m, 3H), 2.51−2.27 (m, 2H),

2.22−2.04 (m, 1H), 2.03−1.86 (m, 4H), 1.81−1.64 (m, 2H), 1.59

=

16.7, 9.9 Hz, 1H), 2.19−2.09 (m, 1H), 2.08−2.01 (m, 2H), 2.01−

1

4

.89 (m, 2H), 1.79 (t, J = 12.5 Hz, 1H), 1.66 (br dd, J = 3.6, 1.7 Hz,

+

1

+1

H), 1.61 (dt, J = 14.1, 6.8 Hz, 2H), 1.49−1.32 (m); LC/MS M

=

(quin, J = 6.8 Hz, 2H), 1.50−1.24 (m, 9H); LC/MS M = 360;

26.4; HPLC t = 0.82 (method A).

HPLC t = 7.58 (method D).

r

(

(1R,3S)-1-Amino-3-(6-(6-methoxyhexyl)-5,6,7,8-tetrahydro-

((1R,3S)-1-Amino-3-((S)-6-((butylthio)methyl)-5,6,7,8-tetrahydro-

2

9

29

naphthalen-2-yl)cyclopentyl)methanol (25a, 25b). To a mixture

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

naphthalen-2-yl triﬂuoromethanesulfonate (46, 100 mg, 0.240

mmol), copper(I) iodide (4.5 mg, 0.024 mmol), and bis-

naphthalen-2-yl)cyclopentyl)methanol (26). To a stirred mixture

of butyl mercaptan (0.029 mL, 0.27 mmol) in a 1 N THF solution of

potassium tert-butoxide (0.27 mL, 0.27 mmol) was added ((S)-6-

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

naphthalen-2-yl)methyl 4-methylbenzenesulfonate (48, 25 mg, 0.055

mmol). The resulting mixture was stirred at 70 °C overnight and

(

triphenylphosphine)palladium(II) chloride (17 mg, 0.024 mmol)

in TEA (3 mL) was added 6-methoxyhex-1-yne (81 mg, 0.72 mmol).

1

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J. Med. Chem. 2021, 64, 1454−1480

Journal of Medicinal Chemistry

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concentrated in vacuo. The residue was mixed with water (0.5 mL),

lithium hydroxide monohydrate (46 mg, 1.1 mmol), and dioxane (0.5

mL) and then heated at 90 °C overnight. The mixture was extracted

with EtOAc (4 × 1 mL) and the combined EtOAc extracts were dried

mg, 0.050 mmol, 92% yield) as a white solid. H NMR (400 MHz,

CDCl ): δ 7.02 (d, J = 1.8 Hz, 2H), 6.98 (s, 1H), 3.54−3.40 (m, 2H),

3

3.11−2.92 (m, 2H), 2.88−2.77 (m, 2H), 2.61−2.43 (m, 5H), 2.28

(dd, J = 13.6, 7.9 Hz, 1H), 2.13−2.02 (m, 2H), 2.00−1.83 (m, 2H),

1.81−1.69 (m, 3H), 1.55−1.44 (m, 3H), 1.43−1.27 (m, 4H), 0.95−

(

Na SO ), concentrated, and puriﬁed by HPLC to aﬀord ((1R,3S)-1-

2 4

+

1

amino-3-((S)-6-((butylthio)methyl)-5,6,7,8-tetrahydronaphthalen-2-

yl)cyclopentyl)methanol, TFA (21 mg, 0.044 mmol, 78% yield).

HPLC conditions: Phenomenex Luna 5μ C18 column (30 × 100

0.86 (m, 3H); LC/MS M = 362.1; HPLC t = 9.66 (method D).

r

((1R,3S)-1-Amino-3-((S)-6-((pentylthio)methyl)-5,6,7,8-tetrahy-

2

9

dronaphthalen-2-yl)cyclopentyl)methanol (29). To a stirred

mixture of pentane-1-thiol (0.020 mL, 0.165 mmol), ((S)-6-

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

naphthalen-2-yl)methyl 4-methylbenzenesulfonate (48, 25 mg, 0.055

mmol), and dioxane (0.4 mL) was added 2 N NaOH (0.082 mL,

0.165 mmol) at 0 °C. The resulting mixture was stirred at 70 °C for 4

h, cooled to rt, and 2 N aqueous NaOH (0.549 mL, 1.098 mmol) was

added and the resulting mixture was stirred at 90 °C overnight. The

mixture was extracted with EtOAc (4 × 1 mL) and the combined

mm); MeCN (0.1% TFA)/water (0.1% TFA); 20−100% gradient

1

over 15 min; and 30 mL/min. H NMR (500 MHz, DMSO-d ): δ

6

7

3

2

1

.90 (br s, 2H), 7.07−6.90 (m, 3H), 5.63 (t, J = 5.0 Hz, 1H), 3.54−

.42 (m, 2H), 3.39 (s, 1H), 3.35−3.29 (m, 1H), 3.07−2.94 (m, 1H),

.88 (br dd, J = 16.3, 4.5 Hz, 1H), 2.78−2.69 (m, 2H), 2.57−2.53 (m,

H), 2.41 (dd, J = 16.3, 10.4 Hz, 1H), 2.25 (dd, J = 13.1, 7.2 Hz, 1H),

.03−1.91 (m, 2H), 1.91−1.72 (m, 4H), 1.61 (t, J = 12.6 Hz, 1H),

.57−1.44 (m, 2H), 1.43−1.31 (m, 3H), 0.88 (t, J = 7.4 Hz, 3H);

+1

LC/MS M = 348.1; HPLC t = 1.53 (method E).

EtOAc extracts were dried (Na SO ) and concentrated. The resulting

residue was puriﬁed using reverse-phase HPLC (Phen Luna 5μ 30 ×

100 mm (Axia); gradient over 8 min from 30 to 100% of solvent B;

2 4

r

(

(1R,3S)-1-Amino-3-((S)-6-(2-(propylthio)ethyl)-5,6,7,8-tetrahy-

2

9

dronaphthalen-2-yl)cyclopentyl)methanol (27). (5R,7S)-7-((S)-

-(2-Hydroxyethyl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-

6

solvent A: 10% MeOH: 90% H O: 0.1% TFA; solvent B: 90% MeOH,

2

azaspiro[4.4]nonan-2-one (40b, 45 mg, 0.14 mmol) was dissolved in

dry pyridine (0.5 mL) and p-toluenesulfonyl chloride (82 mg, 0.43

mmol) was added in one portion. The resulting mixture was stirred at

rt for 2 h and the solvent was removed in vacuo. The residue was

dissolved in DCM and loaded onto the column. Flash chromatog-

raphy puriﬁcation using ISCO (4 g of silica gel column, 0−100%

EtOAc in DCM) aﬀorded 2-((S)-6-((5R,7S)-2-oxo-3-oxa-1-azaspiro-

10% H O, 0.1% TFA). The desired fractions were basiﬁed with 2 N

2

NaOH and extracted with EtOAc. The organic layer was dried and

concentrated to aﬀord ((1R,3S)-1-amino-3-((S)-6-((pentylthio)-

methyl)-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol (16

1

mg, 0.042 mmol, 77% yield) as a white solid. H NMR (400 MHz,

MeOD): δ 7.09−6.84 (m, 3H), 3.56−3.38 (m, 2H), 3.08−2.97 (m,

1H), 2.92 (br dd, J = 16.3, 4.8 Hz, 1H), 2.86−2.71 (m, 2H), 2.59−

2.50 (m, 4H), 2.45 (br dd, J = 16.1, 10.1 Hz, 1H), 2.28−2.17 (m,

1H), 2.12−1.96 (m, 2H), 1.95−1.83 (m, 2H), 1.84−1.66 (m, 2H),

1.66−1.50 (m, 3H), 1.50−1.29 (m, 5H), 0.92 (t, J = 7.0 Hz, 3H);

[

4.4]nonan-7-yl)-1,2,3,4-tetrahydronaphthalen-2-yl)ethyl 4-methyl-

benzenesulfonate (57 mg, 0.121 mmol, 85% yield) as a solid. LC/

MS M⁺¹= 470.4; HPLC t = 1.04 (method A).

r

+

1

To a stirred mixture of propane-1-thiol (0.017 mL, 0.19 mmol), 2-

LC/MS M = 362.2; HPLC t = 9.78 (method D).

r

(

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

((1R,3S)-1-Amino-3-(6-(hexyloxy)-5,6,7,8-tetrahydronaphthalen-

2

9

hydronaphthalen-2-yl)ethyl 4-methylbenzenesulfonate (30 mg, 0.064

mmol), and dioxane (1 mL) was added 2 N NaOH (0.096 mL, 0.19

mmol) at 0 °C. The resulting mixture was stirred at 60 °C for 6 h. 2 N

aqueous NaOH (0.64 mL, 1.28 mmol) was added and the resulting

mixture was stirred at 90 °C overnight. The mixture was extracted

with EtOAc (4 × 1 mL) and the combined EtOAc extracts were dried

2-yl)cyclopentyl)methanol (30a, 30b). A mixture of n-hexanol (5

mL, 40.1 mmol), p-toluenesulfonic acid monohydrate (4 mg, 0.021

mmol), and trimethoxymethane (0.31 mL, 2.8 mmol) was stirred at

100 °C for 2 h with a slow N stream ﬂowing over the mixture to

2

remove methanol. The residual liquid was mixed with (5R,7S)-7-(6-

oxo-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-

one (39, 200 mg, 0.701 mmol) and stirred at 100 °C for 2 h. The

mixture was cooled and then 10% Pd−C (100 mg, 0.094 mmol) was

(

Na SO ) and concentrated. The resulting residue was puriﬁed using

2 4

reverse-phase HPLC (Phen Luna 5μ 30 × 100 mm (Axia); gradient

over 8 min from 30 to 100% of solvent B; solvent A: 10% MeOH:

added under N

hydrogenated under a balloon of H

2

, followed by EtOAc (3 mL). The mixture was

2

9

0% H O: 0.1% TFA; solvent B: 90% MeOH, 10% H O, 0.1% TFA).

for 12 h. The mixture was ﬁltered

2

The desired fractions were basiﬁed with 2 N NaOH and extracted

with EtOAc. The organic layer was dried and concentrated to aﬀord

through a membrane ﬁlter and the ﬁltrate was concentrated. Flash

chromatography puriﬁcation (12 g of silica gel column, 5->100%

EtOAc in hexanes) aﬀorded (5R,7S)-7-(6-(hexyloxy)-5,6,7,8-tetrahy-

dronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (220 mg, 0.59

mmol, 84% yield) as a sticky solid. This product was then separated

into individual isomers using the following conditions: Berger SFC

MGIII; column: Lux Amylose-2 3 × 250 cm, 5μ; ﬂow rate: 180.0 mL/

((1R,3S)-1-amino-3-((S)-6-(2-(propylthio)ethyl)-5,6,7,8-tetrahydro-

naphthalen-2-yl)cyclopentyl)methanol (21 mg, 0.057 mmol, 90%

1

yield) as a white solid. H NMR (400 MHz, CDCl ): δ 7.02 (s, 2H),

3

6

.99 (s, 1H), 3.55−3.41 (m, 2H), 3.16−2.98 (m, 1H), 2.91−2.76 (m,

3

H), 2.69−2.60 (m, 2H), 2.57−2.50 (m, 2H), 2.42 (dd, J = 16.2, 10.6

Hz, 1H), 2.29 (br dd, J = 13.3, 7.8 Hz, 1H), 2.17−2.04 (m, 1H),

min; mobile phase: CO /(MEOH + 0.2%DEA) = 80/20.

2

2.02−1.80 (m, 4H), 1.71−1.65 (m, 5H), 1.47−1.35 (m, 2H), 1.02 (t,

Peak 1: isomer a; recovered (5R,7S)-7-(6-(hexyloxy)-5,6,7,8-

tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (50

mg, 0.029 mmol, 23% yield).

Peak 2: isomer b; recovered (5R,7S)-7-(6-(hexyloxy)-5,6,7,8-

tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (47

mg, 0.038 mmol, 21% yield). Each isomer was taken individually

into the next step.

+1

J = 7.3 Hz, 3H); LC/MS M = 348.4; HPLC t = 9.25 (method D).

r

(

(1R,3S)-1-Amino-3-((R)-6-((pentylthio)methyl)-5,6,7,8-tetrahy-

29

dronaphthalen-2-yl)cyclopentyl)methanol (28). To a stirred

mixture of pentane-1-thiol (0.020 mL, 0.165 mmol), ((R)-6-

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

naphthalen-2-yl)methyl 4-methylbenzenesulfonate (42, 25 mg, 0.055

mmol), and dioxane (0.4 mL) was added 2 N aqueous NaOH (0.082

mL, 0.165 mmol) at 0 °C. The resulting mixture was stirred at 70 °C

for 4 h, cooled to rt, and 2 N NaOH (0.549 mL, 1.098 mmol) was

added, and the resulting mixture was stirred at 90 °C overnight. The

mixture was extracted with EtOAc (4 × 1 mL) and the combined

EtOAc extracts were dried (Na SO ) and concentrated. The resulting

A mixture of (5R,7S)-7-(6-(hexyloxy)-5,6,7,8-tetrahydronaphtha-

len-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (isomer a, 50 mg, 0.136

mmol), lithium hydroxide monohydrate (51 mg, 1.22 mmol), dioxane

(1 mL), and water (1 mL) was stirred at 90 °C for 15 h. The mixture

was cooled and extracted with EtOAc (4 × 1 mL). The combined

EtOAc extracts were dried (Na SO ) and concentrated under reduced

2

4

2

4

residue was puriﬁed using reverse-phase HPLC (Phen Luna 5μ 30 ×

00 mm (Axia); gradient over 8 min from 30 to 100% of solvent B;

solvent A: 10% MeOH: 90% H O: 0.1% TFA; solvent B: 90% MeOH,

pressure. The resulting residue was puriﬁed using reverse-phase

HPLC (Phen Luna 5μ 30 × 100 mm (Axia); gradient over 8 min

1

from 30 to 100% of solvent B; solvent A: 10% MeOH: 90% H O:

2

1

0% H O, 0.1% TFA). The desired fractions were basiﬁed with 2 N

0.1% TFA; solvent B: 90% MeOH, 10% H O, 0.1% TFA). The

2

NaOH and extracted with EtOAc. The organic layer was dried and

concentrated to aﬀord ((1R,3S)-1-amino-3-((R)-6-((pentylthio)-

methyl)-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol (19

desired fractions were basiﬁed with K CO and extracted with EtOAc.

2

3

The organic layer was then dried (Na SO ), ﬁltered, and concentrated

2

4

to aﬀord ((1R,3S)-1-amino-3-(6-(hexyloxy)-5,6,7,8-tetrahydronaph-

1

472

https://dx.doi.org/10.1021/acs.jmedchem.0c01109

J. Med. Chem. 2021, 64, 1454−1480

Journal of Medicinal Chemistry

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Article

+

1

thalen-2-yl)cyclopentyl)methanol (30a, 47 mg, 0.122 mmol, 90%

yield). H NMR (400 MHz, MeOD): δ 7.34−6.76 (m, 3H), 3.83−

1H), 1.38−1.33 (m, 3H), 0.97−0.87 (m, 3H); LC/MS M = 346.2;

1

HPLC t = 1.86 (method E).

r

3

.68 (m, 1H), 3.61−3.51 (m, 2H), 3.51−3.40 (m, 2H), 3.09−2.95

((1R,3S)-1-Amino-3-(6-(heptyloxy)-5,6,7,8-tetrahydronaphtha-

len-2-yl)cyclopentyl)methanol (33a, 33b). To a mixture of

5R,7S)-7-(6-oxo-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-

azaspiro[4.4]nonan-2-one (39, 100 mg, 0.350 mmol) and 1-heptanol

(500 μL, 3.54 mmol) in toluene (2 mL) was added p-toluenesulfonic

acid monohydrate (5 mg, 0.026 mmol). Oven dried 3A molecular

sieves were added and the mixture was heated at reﬂux overnight. The

reaction mixture was cooled to rt, diluted with EtOAc, and washed

2

9

(

7

1

m, 2H), 2.94−2.83 (m, 1H), 2.80−2.65 (m, 2H), 2.22 (dd, J = 13.1,

(

.6 Hz, 1H), 2.04 (br d, J = 2.6 Hz, 1H), 2.00−1.95 (m, 1H), 1.94−

.63 (m, 4H), 1.63−1.49 (m, 3H), 1.45−1.28 (m, 6H), 0.97−0.82

_m_,₃_H₎_;_L_C_/_M_S_M+ = 346.3; HPLC t = 8.86 (method D).

1

(

r

A mixture of (5R,7S)-7-(6-(hexyloxy)-5,6,7,8-tetrahydronaphtha-

len-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (isomer b, 47 mg, 0.13

mmol), lithium hydroxide monohydrate (51 mg, 1.2 mmol), dioxane

with saturated NaCl. The organic layer was dried MgSO , ﬁltered, and

(1 mL), and water (1 mL) was stirred at 90 °C for 15 h. The mixture

4

concentrated in vacuo. The crude material was puriﬁed on a silica gel

cartridge (40 g) using an EtOAc/Hex gradient (0−100% EtOAc over

0 min) to aﬀord (5R,7S)-7-(6-(heptyloxy)-7,8-dihydronaphthalen-2-

was cooled and extracted with EtOAc (4 × 1 mL). The combined

EtOAc extracts were dried (Na SO ) and concentrated under reduced

2

4

2

pressure. The resulting residue was puriﬁed using reverse-phase

HPLC (Phen Luna 5μ 30 × 100 mm (Axia); gradient over 8 min

yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (55 mg, 0.14 mmol, 41% yield).

+

1

LC/MS M = 384.4; HPLC t = 1.26 (method A).

r

from 30 to 100% of solvent B; solvent A: 10% MeOH: 90% H O:

2

To a mixture of (5R,7S)-7-(6-(heptyloxy)-7,8-dihydronaphthalen-

-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (53 mg, 0.138 mmol) in

0

.1% TFA; solvent B: 90% MeOH, 10% H O, 0.1% TFA). The

2

desired fractions were basiﬁed with K CO and extracted with EtOAc.

2

3

MeOH (10 mL) was added Pearlman’s catalyst (19 mg, 0.14

mmol). The reaction mixture was hydrogenated under a balloon of H₂

for 2 h. The catalyst was ﬁltered away and the mixture was

concentrated in vacuo. This product was then separated into

individual isomers using the following conditions: Berger SFC

MGII; column: chiral As−H 25 × 3 cm ID, 5μ; ﬂow rate: 85.0

The organic layer was then dried (Na SO ), ﬁltered, and concentrated

2

4

to aﬀord ((1R,3S)-1-amino-3-(6-(hexyloxy)-5,6,7,8-tetrahydronaph-

thalen-2-yl)cyclopentyl)methanol (30b, 28 mg, 0.122 mmol, 56%

1

yield). H NMR (400 MHz, MeOD): δ 7.08−6.95 (m, 3H), 3.83−

3

.73 (m, 1H), 3.63−3.55 (m, 2H), 3.54−3.42 (m, 2H), 3.09−2.97

(

7

3

m, 2H), 2.96−2.86 (m, 1H), 2.83−2.65 (m, 2H), 2.22 (dd, J = 13.2,

mL/min; mobile phase: 70/30 CO /MeOH.

2

.5 Hz, 1H), 2.14−1.89 (m, 3H), 1.88−1.66 (m, 3H), 1.65−1.50 (m,

Peak 2: isomer a; recovered (5R,7S)-7-(6-(heptyloxy)-5,6,7,8-

tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (9 mg,

+1

H), 1.46−1.26 (m, 6H), 0.97−0.88 (m, 3H); LC/MS M = 346.3;

HPLC t = 8.85 (method D).

1

r

1

7% yield); H NMR (400 MHz, CDCl ): δ 7.09−7.03 (m, 1H),

3

(

(1R,3S)-1-Amino-3-((R)-6-((pentyloxy)methyl)-5,6,7,8-tetrahy-

29

7.01−6.92 (m, 2H), 5.29 (br s, 1H), 4.40−4.26 (m, 2H), 3.78−3.67

dronaphthalen-2-yl)cyclopentyl)methanol (31). To a stirred

mixture of 1-pentanol (0.119 mL, 1.098 mmol) and a 1 N THF

solution of potassium tert-butoxide (0.549 mL, 0.549 mmol) was

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

(

1

(

m, 1H), 3.62−3.48 (m, 2H), 3.15−2.99 (m, 2H), 2.99−2.86 (m,

H), 2.85−2.68 (m, 2H), 2.33 (dd, J = 13.2, 7.3 Hz, 1H), 2.24−2.04

m, 3H), 1.96 (dd, J = 13.1, 10.9 Hz, 2H), 1.89−1.74 (m, 2H), 1.61

quin, J = 6.9 Hz, 4H), 1.35−1.29 (m, 6H), 0.93−0.87 (m, 3H).

Peak 3: isomer b; recovered (5R,7S)-7-(6-(heptyloxy)-5,6,7,8-

1,2,3,4-tetrahydronaphthalen-2-yl)methyl 4-methylbenzenesulfonate

(42, 25 mg, 0.055 mmol). The resulting mixture was stirred at 60

tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (9 mg,

7% yield); H NMR (400 MHz, CDCl ): δ 7.09−7.03 (m, 1H),

°C for 18 h and concentrated in vacuo. The residue was mixed with

1

7

1

2

3

4

3

water (0.5 mL), lithium hydroxide monohydrate (18 μL, 0.66 mmol),

and dioxane (1 mL). The resulting mixture was stirred at 100 °C for 7

h, cooled to rt, and extracted with EtOAc (4 × 1 mL). The combined

EtOAc extracts were dried (Na SO ) and concentrated and the

.01−6.92 (m, 2H), 5.25 (s, 1H), 4.40−4.25 (m, 2H), 3.80−3.66 (m,

H), 3.59−3.50 (m, 2H), 3.15−2.99 (m, 2H), 2.98−2.88 (m, 1H),

.84−2.71 (m, 2H), 2.33 (dd, J = 13.2, 7.3 Hz, 1H), 2.21−2.03 (m,

H), 2.03−1.91 (m, 2H), 1.90−1.73 (m, 2H), 1.60 (q, J = 7.0 Hz,

H), 1.36−1.29 (m, 6H), 0.95−0.87 (m, 3H).

2

4

resulting residue was puriﬁed by HPLC to aﬀord ((1R,3S)-1-amino-3-

(

(R)-6-((pentyloxy)methyl)-5,6,7,8-tetrahydronaphthalen-2-yl)-

To a mixture of (5R,7S)-7-(6-(heptyloxy)-5,6,7,8-tetrahydronaph-

1

cyclopentyl)methanol (5.5 mg, 0.016 mmol, 29% yield). H NMR

thalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (isomer a, 9 mg, 0.023

mmol) in dioxane (4 mL) was added 1 N NaOH. The reaction

mixture was heated at 100 °C overnight, cooled to rt, acidiﬁed with

TFA, and concentrated in vacuo. The residue was then ﬁltered and

puriﬁed by HPLC. HPLC conditions: Phenomenex Luna 5μ C18

column (30 × 100 mm); MeCN (0.1% TFA)/water (0.1% TFA);

20%−100% gradient over 15 min; and 30 mL/min. Isolated fractions

with the correct mass were freeze-dried overnight to aﬀord ((1R,3S)-

(

1

500 MHz, MeOD): δ 7.05−6.99 (m, 2H), 6.98 (s, 1H), 3.65−3.53

m, 2H), 3.47 (t, J = 6.7 Hz, 2H), 3.40 (d, J = 6.9 Hz, 2H), 3.10−2.98

m, 1H), 2.91−2.73 (m, 3H), 2.44 (dd, J = 16.3, 10.4 Hz, 1H), 2.36

dd, J = 13.1, 6.7 Hz, 1H), 2.08 (br s, 3H), 1.94−1.79 (m, 3H), 1.69

t, J = 12.6 Hz, 1H), 1.65−1.54 (m, 2H), 1.46−1.38 (m, 1H), 1.37−

+1

.30 (m, 4H), 0.98−0.84 (m, 3H); LC/MS M = 346.2; HPLC t =

r

.87 (method E).

(

(1R,3S)-1-Amino-3-((S)-6-((pentyloxy)methyl)-5,6,7,8-tetrahy-

1

-amino-3-(6-(heptyloxy)-5,6,7,8-tetrahydronaphthalen-2-yl)-

29

dronaphthalen-2-yl)cyclopentyl)methanol (32). To a stirred

mixture of 1-pentanol (0.12 mL, 1.1 mmol) and a 1 N THF solution

of potassium tert-butoxide (0.55 mL, 0.55 mmol) was added ((S)-6-

1

cyclopentyl)methanol, TFA (5 mg, 10 μmol, 45% yield). H NMR

400 MHz, MeOD): δ 7.03 (s, 2H), 7.00 (s, 1H), 3.83−3.73 (m, 1H),

.70−3.61 (m, 2H), 3.61−3.50 (m, 2H), 3.18−3.08 (m, 1H), 3.04

dd, J = 16.4, 4.7 Hz, 1H), 2.97−2.84 (m, 1H), 2.81−2.66 (m, 2H),

.42 (ddd, J = 13.4, 7.1, 1.1 Hz, 1H), 2.19−2.01 (m, 2H), 2.00−1.89

m, 3H), 1.88−1.77 (m, 1H), 1.73 (t, J = 12.8 Hz, 1H), 1.59 (quin, J

6.9 Hz, 2H), 1.44−1.23 (m, 8H), 0.97−0.87 (m, 3H); LC/MS M

(

3

(

2

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

naphthalen-2-yl)methyl 4-methylbenzenesulfonate (48, 25 mg, 0.055

mmol). The resulting mixture was stirred at 60 °C for 18 h,

concentrated in vacuo, and the resulting residue was mixed with water

(

=

+1

(0.5 mL), lithium hydroxide monohydrate (18 μL, 0.66 mmol), and

360.2; HPLC t = 8.34 (method D).

r

dioxane (1 mL). The resulting mixture was stirred at 100 °C for 7 h,

cooled to rt, and extracted with EtOAc (4 × 1 mL). The combined

EtOAc extracts were dried (Na SO ) and concentrated and the

To a mixture of (5R,7S)-7-(6-(heptyloxy)-5,6,7,8-tetrahydronaph-

thalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (isomer b, 8 mg, 0.021

mmol) in dioxane (4 mL) was added 1 N NaOH. The reaction

mixture was heated at 100 °C overnight, cooled to rt, acidiﬁed with

TFA, and concentrated in vacuo. The residue was ﬁltered and puriﬁed

by HPLC. HPLC conditions: Phenomenex Luna 5μ C18 column (30

× 100 mm); MeCN (0.1% TFA)/water (0.1% TFA); 20−100%

gradient over 15 min; and 30 mL/min. Isolated fractions with the

correct mass were freeze-dried overnight to aﬀord ((1R,3S)-1-amino-

3-(6-(heptyloxy)-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)-

2

4

resulting residue was puriﬁed by HPLC to aﬀord ((1R,3S)-1-amino-3-

(S)-6-((pentyloxy)methyl)-5,6,7,8-tetrahydronaphthalen-2-yl)-

(

1

cyclopentyl)methanol (10.6 mg, 0.031 mmol, 56% yield). H NMR

(

500 MHz, MeOD): δ 7.04−6.99 (m, 2H), 6.99−6.95 (m, 1H), 3.62−

3

1

.51 (m, 2H), 3.47 (t, J = 6.7 Hz, 2H), 3.40 (d, J = 6.4 Hz, 2H),

.11−2.97 (m, 1H), 2.92−2.76 (m, 3H), 2.44 (dd, J = 16.1, 10.7 Hz,

H), 2.34 (dd, J = 13.4, 6.4 Hz, 1H), 2.15−1.96 (m, 3H), 1.96−1.91

1

(m, 2H), 1.91−1.78 (m, 2H), 1.71−1.55 (m, 3H), 1.50−1.39 (m,

methanol (5 mg, 0.014 mmol, 60% yield). H NMR (400 MHz,

1

473

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J. Med. Chem. 2021, 64, 1454−1480

Journal of Medicinal Chemistry

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Article

MeOD): δ 7.07−6.98 (m, 3H), 3.83−3.73 (m, 1H), 3.71−3.51 (m,

vacuo. The residue was mixed with 2 N aqueous sodium hydroxide

(0.5 mL, 1.000 mmol) and dioxane (0.5 mL), and the resulting

mixture was stirred at 90 °C overnight. The mixture was extracted

with EtOAc (4 × 1 mL). The combined EtOAc extracts were dried

(Na SO ) and concentrated under reduced pressure. The residue was

4

H), 3.18−3.08 (m, 1H), 3.04 (dd, J = 16.6, 5.0 Hz, 1H), 2.96−2.85

(

m, 1H), 2.82−2.68 (m, 2H), 2.42 (ddd, J = 13.3, 7.1, 1.2 Hz, 1H),

.17−2.01 (m, 2H), 2.00−1.89 (m, 3H), 1.88−1.77 (m, 1H), 1.73 (t,

J = 12.8 Hz, 1H), 1.59 (quin, J = 6.9 Hz, 2H), 1.45−1.22 (m, 8H),

2

4

+1

0

D).

.96−0.86 (m, 3H); LC/MS M = 360.2; HPLC t = 8.34 (method

puriﬁed by HPLC to aﬀord ((1R,3S)-1-amino-3-((R)-6-((2-

r

propoxyethoxy)methyl)-5,6,7,8-tetrahydronaphthalen-2-yl)-

1

(

(1R,3S)-1-Amino-3-(6-(2-butoxyethoxy)-5,6,7,8-tetrahydro-

cyclopentyl)methanol (9 mg, 44% yield). H NMR (500 MHz,

2

9

naphthalen-2-yl)cyclopentyl)methanol (34a, 34b). To a solution

of 2-butoxyethanol (920 μL, 7.01 mmol) was added trimethyl

orthoformate (310 μL, 2.8 mmol) and the mixture was stirred at 100

DMSO-d ): δ 7.03−6.92 (m, 3H), 3.36−3.28 (m, 11H), 2.97−2.88

6

(m, 1H), 2.81−2.68 (m, 3H), 2.36 (br dd, J = 15.6, 11.1 Hz, 1H),

2.11 (br dd, J = 12.6, 8.2 Hz, 1H), 1.97−1.76 (m, 6H), 1.70−1.55 (m,

2H), 1.55−1.46 (m, 2H), 1.46−1.39 (m, 1H), 1.33 (dt, J = 10.3, 5.0

°

C for 2 h with the cap open. The reaction mixture was added to

+

1

(

5R,7S)-7-(6-oxo-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-

Hz, 1H), 0.93−0.77 (m, 3H); LC/MS M = 362.3; HPLC t = 1.48

r

azaspiro[4.4]nonan-2-one (39, 200 mg, 0.701 mmol) in THF (2 mL),

followed by 2-butoxyethanol (920 μl, 7.0 mmol), p-toluenesulfonic

acid monohydrate (10.00 mg, 0.053 mmol), and molecular sieves.

The reaction mixture was stirred at 110 °C for 16 h. After cooling,

(method E).

((1R,3S)-1-Amino-3-((S)-6-((2-propoxyethoxy)methyl)-5,6,7,8-tet-

29

rahydronaphthalen-2-yl)cyclopentyl)methanol (36). To a stirred

mixture of 2-propoxyethanol (0.063 mL, 0.55 mmol) and 1 N THF

solution of potassium tert-butoxide (0.55 mL, 0.55 mmol) was added

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

1

0% Pd−C (200 mg) and EtOAc (5 mL) were added. The reaction

mixture was stirred under H for 16 h and then poured onto ice,

2

17a

diluted with DCM, and washed with 1 N HCl. The organic layer was

hydronaphthalen-2-yl)methyl 4-methylbenzenesulfonate

(48, 25

collected, dried over Na SO , and concentrated to aﬀord (5R,7S)-7-

mg, 0.055 mmol). The resulting mixture was stirred at 70 °C for 1.5 h.

The mixture was concentrated in vacuo. The residue was mixed with 2

N aqueous sodium hydroxide (0.5 mL, 1 mmol) and dioxane (0.5

mL), and the resulting mixture was stirred at 90 °C overnight. The

mixture was extracted with EtOAc (4 × 1 mL). The combined EtOAc

extracts were dried (Na SO ) and concentrated under reduced

2

4

(

6-(2-butoxyethoxy)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-

azaspiro[4.4]nonan-2-one (140 mg, 0.36 mmol, 52% yield), LC/MS

M

+1

= 388.3; HPLC t = 1.05 (method A). This product was then

r

separated into individual isomers using the following conditions:

Berger SFC MGII; column: Lux-Cellulose-4 25 × 3 cm, 5 μm; ﬂow

2

4

rate: 120 mL/min; mobile phase: 65/35 CO /MeOH.

pressure. The residue was puriﬁed by HPLC to aﬀord ((1R,3S)-1-

amino-3-((R)-6-((2-propoxyethoxy)methyl)-5,6,7,8-tetrahydronaph-

2

Peak 2: isomer a; (5R,7S)-7-(6-(2-butoxyethoxy)-5,6,7,8-tetrahy-

dronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (60 mg).

Peak 1: isomer b; (5R,7S)-7-(6-(2-butoxyethoxy)-5,6,7,8-tetrahy-

dronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (60 mg).

Each isomer was taken individually into the next step.

1

thalen-2-yl)cyclopentyl)methanol (9 mg, 44% yield). H NMR (500

MHz, DMSO-d ): δ 7.02−6.94 (m, 3H), 5.52 (br s, 1H), 3.51 (br dd,

6

J = 6.9, 2.5 Hz, 4H), 3.06−2.94 (m, 1H), 2.80−2.70 (m, 7H), 2.64

(br s, 3H), 2.41−2.32 (m, 2H), 2.23 (dd, J = 13.1, 6.7 Hz, 1H), 2.03−

1.71 (m, 6H), 1.58 (br t, J = 12.9 Hz, 1H), 1.51 (sxt, J = 7.1 Hz, 2H),

To a solution of (5R,7S)-7-(6-(2-butoxyethoxy)-5,6,7,8-tetrahy-

dronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (isomer a, 60

mg, 0.155 mmol) in dioxane (3 mL) and water (1 mL) was added

lithium hydroxide (37 mg, 1.55 mmol) and the reaction mixture was

stirred at 100 °C for 16 h. The reaction mixture was diluted with

water and extracted with EtOAc (2x). The organic layer was collected,

dried over Na SO , and concentrated to aﬀord ((1R,3S)-1-amino-3-

+1

1.40−1.28 (m, 1H), 0.87 (t, J = 7.4 Hz, 3H); LC/MS M = 362.3;

HPLC t = 1.46 (method E).

r

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

tetrahydronaphthalene-2-carbaldehyde (38). A solution of oxalyl

chloride (260 μL, 3 mmol) in DCM (5 mL) was stirred for 30 min at

rt then cooled to −78 °C. DMSO (420 μL, 6 mmol) was added

dropwise and stirred for 1 h at that temperature. Next, 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 (37, 600 mg, 2 mmol) in DCM

(3 mL)/DMSO(1 mL) was added dropwise. The mixture was stirred

for 30 min at the same temperature. TEA (1.1 mL, 8 mmol) was

added dropwise and the mixture was stirred for 15 min, then warmed

to rt, and stirred for another 15 min. The reaction mixture was

quenched with water (1 mL) at 0 °C, diluted with EtOAc (50 mL),

washed with saturated NH Cl (2 × 30 mL), dried (Na SO ), ﬁltered,

2

4

(

6-(2-butoxyethoxy)-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)-

1

methanol (40 mg, 0.105 mmol, 67.9% yield). H NMR (400 MHz,

MeOD): δ 7.06−6.95 (m, 3H), 3.88−3.79 (m, 1H), 3.77−3.66 (m,

2

1

H), 3.64−3.56 (m, 2H), 3.56−3.45 (m, 4H), 3.10−2.98 (m, 2H),

.98−2.87 (m, 1H), 2.83−2.69 (m, 2H), 2.26 (dd, J = 13.2, 7.5 Hz,

H), 2.13−1.99 (m, 2H), 1.99−1.89 (m, 1H), 1.89−1.70 (m, 3H),

.65−1.51 (m, 3H), 1.47−1.33 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H);

+1

LC/MS M = 362.2; HPLC t = 7.46 (method D).

r

To a solution of (5R,7S)-7-(6-(2-butoxyethoxy)-5,6,7,8-tetrahy-

dronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (isomer b, 60

mg, 0.155 mmol) in dioxane (3 mL) and water (1 mL) was added

lithium hydroxide (37 mg, 1.55 mmol), and the reaction mixture was

stirred at 100 °C for 16 h. The reaction mixture was diluted with

water and extracted with EtOAc (2×). The organic layer was

collected, dried over Na SO , and concentrated to aﬀord ((1R,3S)-1-

4

2

4

and concentrated in vacuo. The crude material was puriﬁed on a silica

gel cartridge using an EtOAc/Hex gradient (0−100% EtOAc over 12

column volumes) to aﬀord (R)-6-((5R,7S)-2-oxo-3-oxa-1-azaspiro-

[4.4]nonan-7-yl)-1,2,3,4-tetrahydronaphthalene-2-carbaldehyde (500

1

mg, 84% yield). H NMR (400 MHz, CDCl ): δ 9.80 (d, J = 0.9 Hz,

3

1H), 7.10 (d, J = 7.9 Hz, 1H), 7.04−6.96 (m, 1H), 6.94 (s, 1H), 5.48

(br s, 1H), 4.38−4.24 (m, 2H), 3.10−2.94 (m, 3H), 2.92−2.81 (m,

2H), 2.77−2.62 (m, 1H), 2.32 (dd, J = 13.2, 7.3 Hz, 1H), 2.27−2.19

(m, 1H), 2.17−2.05 (m, 2H), 2.04−1.91 (m, 2H), 1.88−1.72 (m,

2

4

amino-3-(6-(2-butoxyethoxy)-5,6,7,8-tetrahydronaphthalen-2-yl)-

1

cyclopentyl)methanol, TFA. H NMR (400 MHz, MeOD): δ 7.04−

6

.96 (m, 3H), 3.89−3.79 (m, 1H), 3.77−3.67 (m, 2H), 3.63−3.57

+

1

(

m, 2H), 3.55−3.42 (m, 4H), 3.11−2.98 (m, 2H), 2.97−2.86 (m,

2H); LC/MS M = 300.1; HPLC t = 0.81 (method A).

r

1

2

1

7

H), 2.81−2.69 (m, 2H), 2.26−2.16 (m, 1H), 2.13−2.03 (m, 1H),

.03−1.95 (m, 1H), 1.95−1.88 (m, 1H), 1.88−1.74 (m, 2H), 1.73−

.64 (m, 1H), 1.60−1.50 (m, 3H), 1.46−1.33 (m, 2H), 0.94 (t, J =

(5R,7S)-7-((R)-6-(2-Hydroxyethyl)-5,6,7,8-tetrahydronaphthalen-

2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (40). To a mixture of

triethyl phosphonoacetate (1.68 mL, 8.4 mmol) in THF (3 mL) at

0 °C was added sodium hydride (0.34 g, 8.4 mmol) portionwise. The

reaction mixture was stirred for 30 min and then (5R,7S)-7-(6-oxo-

5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-

.4 Hz, 3H); LC/MS M⁺= 362.2; HPLC t = 7.47 (method D).

1

r

(

(1R,3S)-1-Amino-3-((R)-6-((2-propoxyethoxy)methyl)-5,6,7,8-

29

tetrahydronaphthalen-2-yl)cyclopentyl)methanol (35). To a

stirred mixture of 2-propoxyethanol (0.063 mL, 0.55 mmol) and 1

N THF solution of potassium tert-butoxide (0.44 mL, 0.44 mmol)

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

17a

one (39, 1 g, 3.5 mmol) was added. The reaction was allowed to

warm to rt and stirred for 3 h. The reaction mixture was diluted with

EtOAc and washed with saturated NaCl. The organic layer was dried

1

,2,3,4-tetrahydronaphthalen-2-yl)methyl 4-methylbenzenesulfona-

with MgSO , ﬁltered, and concentrated. The crude material was

puriﬁed on a silica gel cartridge (40 g) using an EtOAc/Hex gradient

(0−100% EtOAc over 12 CV). Isolated fractions with the desired

4

17a

te (42, 25 mg, 0.055 mmol). The resulting mixture was stirred at

0 °C for 1.5 h and at rt for 3 days before being concentrated in

7

1

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product were concentrated in vacuo. This product was dissolved in

MeOH (10 mL) and Pearlman’s catalyst (0.098 g, 0.70 mmol) was

added. The mixture was hydrogenated under a balloon of hydrogen

for 12 h. The catalyst was ﬁltered and the mixture was concentrated in

vacuo to aﬀord ethyl 2-(6-((5R,7S)-2-oxo-3-oxa-1-azaspiro[4.4]-

nonan-7-yl)-1,2,3,4-tetrahydronaphthalen-2-yl)acetate (1 g, 2.8

(1 mL) was added and the mixture was stirred vigorously at rt for 30

min. The mixture was extracted with EtOAc (3 × 2 mL). The

combined EtOAc extracts were dried (Na SO ), concentrated, and

2

4

puriﬁed using ﬂash chromatography to aﬀord 4-((R)-6-((5R,7S)-2-

oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)-1,2,3,4-tetrahydronaphthalen-2-

1

yl)butanal (27 mg, 0.079 mmol, 57% yield). H NMR (400 MHz,

mmol, 80% yield). LC/MS M⁺= 358.4; HPLC t = 0.96 (method A).

1

CDCl ): δ 9.82 (t, J = 1.7 Hz, 1H), 7.07−7.00 (m, 1H), 6.99−6.89

r

3

To a mixture of ethyl 2-(6-((5R,7S)-2-oxo-3-oxa-1-azaspiro[4.4]-

nonan-7-yl)-1,2,3,4-tetrahydronaphthalen-2-yl)acetate (650 mg, 1.82

mmol) in THF (10 mL) was added lithium borohydride in THF (5.5

mL, 5.5 mmol). The reaction mixture was heated at 80 °C overnight,

and then quenched with water and 1 N HCl. The reaction mixture

(m, 2H), 5.55 (s, 1H), 4.39−4.23 (m, 2H), 3.08−2.98 (m, 1H),

2.90−2.74 (m, 3H), 2.49 (td, J = 7.3, 1.7 Hz, 2H), 2.46−2.29 (m,

2H), 2.22−2.07 (m, 2H), 2.01−1.90 (m, 3H), 1.89−1.69 (m, 4H),

+

1

1.49−1.35 (m, 3H); LC/MS M = 342.3; HPLC t = 0.92 (method

r

A).

was extracted with EtOAc (2×), dried with MgSO , ﬁltered, and

(5R,7S)-7-((R)-6-(Hydroxymethyl)-5,6,7,8-tetrahydronaphthalen-

2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (42). (5R,7S)-7-((R)-6-

(hydroxymethyl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro-

4

concentrated. The crude material was puriﬁed on a silica gel cartridge

(40 g) using an MeOH/DCM gradient (0−10% MeOH over 15 CV)

17a

to aﬀord (5R,7S)-7-(-6-(2-hydroxyethyl)-5,6,7,8-tetrahydronaphtha-

len-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (diastereomeric-40),

which was then separated into the individual isomers under SFC

conditions: Column: chiral OD-H 25 × 3 cm ID, 5 mm; ﬂow rate:

[4.4]nonan-2-one

(37, 690 mg, 2.289 mmol) was dissolved in

pyridine (5 mL) and p-toluenesulfonyl chloride (1.3 g, 6.9 mmol) was

added in one portion. The resulting mixture was stirred at rt 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)-1,2,3,4-tetrahydronaphthalen-2-

8

5.0 mL/min, mobile phase: 65/35 CO /MeOH.

Peak 1: recovered (5R,7S)-7-((R)-6-(2-hydroxyethyl)-5,6,7,8-tetra-

2

hydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (40, 180

1

mg, 0.57 mmol, 31% yield). H NMR (400 MHz, CDCl ): δ 7.06−

yl)methyl 4-methylbenzenesulfonate (42, 860 mg, 1.9 mmol, 82%

3

1

7

3

2

.00 (m, 1H), 7.00−6.91 (m, 2H), 5.91 (s, 1H), 4.38−4.25 (m, 2H),

.81 (t, J = 6.8 Hz, 2H), 3.12−2.95 (m, 1H), 2.93−2.75 (m, 3H),

.45 (dd, J = 16.2, 10.5 Hz, 1H), 2.31 (dd, J = 13.3, 7.2 Hz, 1H),

.21−2.07 (m, 2H), 2.05−1.77 (m, 5H), 1.67 (qd, J = 6.7, 2.0 Hz,

H), 1.53−1.35 (m, 2H).

yield) as a white solid. H NMR (400 MHz, CDCl

3

): 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−

+

Peak 2: recovered (5R,7S)-7-((S)-6-(2-hydroxyethyl)-5,6,7,8-tetra-

1.37 (m, 1H); MS (M + H) at m/z 456; HPLC t

r

= 2.01 min

hydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (181 mg,

(method A).

1

0

(

3

(

.57 mmol, 32% yield). H NMR (400 MHz, CDCl ): δ 7.07−7.01

(5R,7S)-7-((R)-6-(But-3-en-1-yl)-5,6,7,8-tetrahydronaphthalen-2-

yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (43). ((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 (42, 5 g, 11 mmol) was dissolved in

THF (80 mL) and copper(I) bromide (3.15 g, 22 mmol) was added.

The mixture was stirred at rt for 30 min and then cooled to −78 °C.

Allylmagnesium bromide (55 mL, 55 mmol) was added dropwise over

a period of 30 min. The reaction mixture was gradually warmed to rt

and stirred overnight. The reaction was quenched by adding aqueous

3

m, 1H), 6.99−6.91 (m, 2H), 5.90 (br s, 1H), 4.39−4.24 (m, 2H),

.87−3.76 (m, 2H), 3.10−2.95 (m, 1H), 2.91−2.76 (m, 3H), 2.45

dd, J = 16.2, 10.5 Hz, 1H), 2.31 (dd, J = 13.2, 7.3 Hz, 1H), 2.21−

2

.06 (m, 2H), 1.99−1.81 (m, 4H), 1.78−1.61 (m, 3H), 1.54−1.36

(m, 2H).

Stereochemistry was assigned by converting the peak 2 material to

a compound with known stereochemistry (see the Supporting

Information for chiral HPLC traces).

4

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

,2,3,4-tetrahydronaphthalen-2-yl)butanal (41). To a mixture of

5R,7S)-7-((R)-6-(2-hydroxyethyl)-5,6,7,8-tetrahydronaphthalen-2-

yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (56 mg, 0.18 mmol) in pyridine

3 mL) was added p-toluenesulfonyl chloride (135 mg, 0.71 mmol),

NH Cl and then extracted with EtOAc. The organic layers were

4

1

combined and concentrated. The residue was puriﬁed on a silica gel

cartridge using an MeOH/DCM gradient to aﬀord (5R,7S)-7-((R)-6-

(but-3-en-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro-

(

[4.4]nonan-2-one (2.7 g, 8.3 mmol, 76% yield) as an oﬀ-white solid.

1

and the reaction mixture was stirred for 2 h. The analysis indicated

that the reaction was incomplete, so more p-toluenesulfonyl chloride

H NMR (400 MHz, CDCl ): δ 7.07−7.02 (m, 1H), 6.98−6.91 (m,

3

2H), 5.87 (ddt, J = 17.0, 10.3, 6.6 Hz, 1H), 5.12 (br s, 1H), 5.09−

5.03 (m, 1H), 4.98 (ddt, J = 10.2, 2.1, 1.2 Hz, 1H), 4.40−4.26 (m,

2H), 3.14−2.98 (m, 1H), 2.92−2.74 (m, 3H), 2.52−2.28 (m, 2H),

2.25−2.07 (m, 4H), 2.02−1.90 (m, 3H), 1.89−1.68 (m, 2H), 1.54−

(

135 mg, 0.71 mmol) was added, and the mixture was stirred for 1

more hour. The reaction mixture was concentrated. The crude

material was puriﬁed on a silica gel cartridge (40 g) using an EtOAc/

Hex gradient (0−100% EtOAc over 20 CV) to aﬀord 2-((R)-6-

)

+1

1.34 (m, 3H ; LC/MS M = 326.0; HPLC t = 1.25 (method A).

r

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

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

1,2,3,4-tetrahydronaphthalen-2-yl)propanal (44). To a solution of

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

rahydronaphthalen-2-yl)methyl 4-methylbenzenesulfonate (42, 1.2 g,

2.6 mmol) and copper(I) bromide-dimethyl sulﬁde complex (1.63 g,

7.90 mmol) in ether (50 mL) was added 1 M allylmagnesium

bromide in ether (40 mL, 40 mmol). The reaction mixture was stirred

naphthalen-2-yl)ethyl 4-methylbenzenesulfonate (68 mg, 0.145 mmol,

1

8

2% yield). LC/MS M⁺= 470.4; HPLC t = 1.04 (method A).

r

To a mixture of 2-((R)-6-((5R,7S)-2-oxo-3-oxa-1-azaspiro[4.4]-

nonan-7-yl)-1,2,3,4-tetrahydronaphthalen-2-yl)ethyl 4-methylbenze-

nesulfonate (65 mg, 0.138 mmol) and copper(I) bromide (40 mg,

0

.28 mmol) in THF (3 mL) at −78 °C was added allylmagnesium

bromide (2.8 mL, 2.8 mmol). The reaction was slowly allowed to

warm to rt and stirred overnight. The reaction mixture was diluted

with EtOAc and washed with saturated NaCl. The organic layer was

at rt for 16 h. The reaction was diluted with saturated NH Cl and

4

water and then extracted with EtOAc. The organic layer was collected,

dried over Na SO , and concentrated in vacuo. The crude material

2

4

dried with MgSO , ﬁltered, and concentrated to aﬀord (5R,7S)-7-

was puriﬁed on a silica gel cartridge (40 g) using an EtOAc/Hex

gradient (0−100% EtOAc over 13 CV) to aﬀord (5R,7S)-7-((R)-6-

4

(

(R)-6-(pent-4-en-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-

azaspiro[4.4]nonan-2-one (45 mg, 0.133 mmol, 96% yield). LC/MS

M

(but-3-en-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro-

+1

= 340.4; HPLC t = 1.18 (method A).

[4.4]nonan-2-one (560 mg, 1.71 mmol, 65% yield), LC/MS M

326.

=

r

To a clear solution of (5R,7S)-7-((R)-6-(pent-4-en-1-yl)-5,6,7,8-

tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (47

mg, 0.138 mmol) in THF (1.5 mL) were sequentially added 50%

NMO (0.065 mL, 0.28 mmol) and osmium tetroxide in BuOH

To a clear solution of (5R,7S)-7-((R)-6-(but-3-en-1-yl)-5,6,7,8-

tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (560

mg, 1.72 mmol) in THF (30 mL) was sequentially added 50%

t

(0.052 mL, 4.2 μmol) at rt. The solution was vigorously stirred at rt

NMO (403 mg, 3.44 mmol) and osmium tetroxide in BuOH (0.65

overnight, and then sodium periodate (118 mg, 0.55 mmol) in water

mL, 0.052 mmol). The solution was vigorously stirred overnight.

1

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Then, sodium periodate (1.47 g, 6.9 mmol) in water (15 mL) was

homogenizer. The homogenate was centrifuged at 20,000 rpm

(48,000g) and the supernatant was discarded. The membrane pellets

were resuspended in buﬀer containing 50 mM HEPES, pH 7.5, 100

added and the mixture was stirred vigorously at rt under N for 30

2

min. The mixture was extracted with EtOAc (3 × 2 mL). The

combined EtOAc extracts were dried (Na SO ), concentrated, and

mM NaCl, 1 mM MgCl , 2 mM EDTA, and stored in aliquots at −80

2

4

2

puriﬁed using silica gel chromatography puriﬁcation (20−100% of

EtOAc in hexanes) to aﬀord 3-((S)-6-((5R,7S)-2-oxo-3-oxa-1-

°C after protein concentration determination. Membranes (2 μg/

3

well) and a 0.03 nM ﬁnal concentration of P−S1P ligand (1 mCi/

azaspiro[4.4]nonan-7-yl)-1,2,3,4-tetrahydronaphthalen-2-yl)propanal

mL, PerkinElmer or American Radiolabeled Chemicals) diluted in

1

(

440 mg, 1.34 mmol, 78% yield). H NMR (400 MHz, CDCl ): δ

assay buﬀer [50 mM HEPES, pH 7.4, 5 mM MgCl , 1 mM CaCl ,

2 2

3

9

6

1

.84 (t, J = 1.7 Hz, 1H), 7.07−7.01 (m, 1H), 6.99−6.95 (m, 1H),

.94 (s, 1H), 5.30 (br s, 1H), 4.39−4.25 (m, 2H), 3.12−2.96 (m,

H), 2.91−2.78 (m, 3H), 2.57 (td, J = 7.4, 1.8 Hz, 2H), 2.43 (br dd, J

0.5% fatty acid free bovine serum albumin (BSA), and 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

min at room temperature (rt), 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.

=

16.3, 9.5 Hz, 1H), 2.33 (dd, J = 13.4, 7.3 Hz, 1H), 2.21−2.09 (m,

2

H), 2.03−1.92 (m, 3H), 1.89−1.79 (m, 1H), 1.79−1.68 (m, 3H),

+1

1

A).

.51−1.38 (m, 1H); LC/MS M = 328.2; HPLC t = 0.91 (method

r

3

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

35

1

,2,3,4-tetrahydronaphthalen-2-yl)propyl 4-Methylbenzenesulfo-

Receptor [ S] GTPγS Binding Assays (S1P GTPγS/S1P GTPγS).

1 3

nate (45). To a stirred solution of 3-((S)-6-((5R,7S)-2-oxo-3-oxa-1-

Compounds were loaded in a 384 Falcon v-bottom plate (0.5 μL/well

azaspiro[4.4]nonan-7-yl)-1,2,3,4-tetrahydronaphthalen-2-yl)propanal

in a 11 point, 3- fold dilution). Membranes prepared from S1P /CHO

1

(

42 mg, 0.128 mmol) in 100% ethanol (2 mL) and DCM (0.5 mL)

cells or EDG3-Ga15-bla HEK293T cells (EDG3 equivalent S1P₃)

was added NaBH (5 mg, 0.13 mmol). The mixture was stirred at rt

were added to the compound plate (40 μL/well, ﬁnal protein 3 μg/

4

3

5

for 1 h. The mixture was concentrated. The residue was quenched

with saturated aqueous NH Cl solution (1 mL) and water (1 mL)

well) with MULTIDROP. [ S]GTP (1250 Ci/mmol, Perkin-3-

4

Elmer) was diluted in assay buﬀer: 20 mM HEPES, pH7.5, 10 mM

and then extracted with EtOAc (4 mL, 2 × 1 mL). The combined

organic solutions were dried over sodium sulfate and concentrated

under reduced pressure to aﬀord (5R,7S)-7-((S)-6-(3-hydroxyprop-

yl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-

MgCl , 150 mM NaCl, 1 mM ethylene glycol tetraacetic acid, 1 mM

2

dithiothreitol, 10 μM GDP, 0.1% fatty acid free BSA, and 10 μg/mL

3

5

saponin to 0.4 nM. The [ S]GTP solution (40 μL) was added to the

compound plate with a ﬁnal concentration of 0.2 nM. The reaction

was kept at rt 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 four 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. EC₅₀is deﬁned as the agonist concentration that

corresponds to 50% of the Y_m_a_x(maximal response) obtained for each

individual compound tested.

+

1

one (42 mg, 0.127 mmol, 99% yield) as a white solid. LC/MS M

=

3

30.3; HPLC t = 0.87 (method A).

r

(5R,7S)-7-((S)-6-(3-hydroxypropyl)-5,6,7,8-tetrahydronaphthalen-

2

-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (42 mg, 0.13 mmol) was

dissolved in dry pyridine (1 mL) and p-toluenesulfonyl chloride (73

mg, 0.38 mmol) was added in one portion. The resulting mixture was

reacted at rt for 4 h and the solvent was removed in vacuo. The

residue was dissolved in DCM and puriﬁed using ﬂash chromatog-

raphy (4 g of silica gel column, 25−100% EtOAc in hexanes) to aﬀord

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

hS1P ERK Phosphorylation. hS1P /CHO cells were plated in BD

1

rahydronaphthalen-2-yl)propyl 4-methylbenzenesulfonate (40 mg,

Amine 384-well plates the day before the assay. On the day of the

assay, the growth medium was removed and replaced with serum-free

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 min at 37 °C.

Cells were lysed in a lysis buﬀer (PerkinElmer, MA, USA), and

phospho-ERK was measured using the SureFire pERK kit

(PerkinElmer, 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 EC₅₀was deﬁned as the

concentration of test compound, which produces 50% of the maximal

response and was quantiﬁed using the four-parameter logistic

equation to ﬁt the data.

+

1

0

.083 mmol, 65% yield) as a white solid. LC/MS M = 484.4;

HPLC t = 1.08 (method A).

r

(

5R,7S)-7-((S)-6-(Hydroxymethyl)-5,6,7,8-tetrahydronaphthalen-

-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (48). (5R,7S)-7-((R)-6-

hydroxymethyl)-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro-

2

(

[

17a

4.4]nonan-2-one

(47, 690 mg, 2.289 mmol) was dissolved in

pyridine (5 mL) and p-toluenesulfonyl chloride (1.3 g, 6.9 mmol) was

added in one portion. The resulting mixture was stirred at rt 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 ((S)-6-((5R,7S)-2-

oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)-1,2,3,4-tetrahydronaphthalen-2-

1

yl)methyl 4-methylbenzenesulfonate (48, 800 mg, 95% yield). H

S1P Internalization Assay. For quantiﬁcation of S1P expression

1

NMR (400 MHz, CDCl ): δ 7.84−7.79 (m, 2H), 7.39−7.33 (m, 2H),

using a high content system, GFP was fused to hS1P at its C-

3

1

7

3

2

1

.00−6.90 (m, 3H), 6.48−6.41 (m, 1H), 4.34−4.24 (m, 2H), 4.06−

.94 (m, 2H), 3.04−2.91 (m, 1H), 2.87−2.72 (m, 3H), 2.46 (s, 3H),

.44−2.35 (m, 1H), 2.33−2.21 (m, 1H), 2.20−2.03 (m, 3H), 2.00−

terminus and a stable CHO cell line expressing hS1P /GFP was

1

established. Cells were suspended in assay medium (F12 medium

with 5% charcoal−dextran-treated FBS, 20 mM HEPES, and 1× Pen/

Strep) at 7.5 × 104 cells/mL and plated into 384-well plates (in 20

μL, 1,500 cells/well ﬁnal) with a Multidrop liquid handler (Thermo

Scientiﬁc, Waltham, MA). Cell plates were incubated at 37 °C for 48

h. Titrated S1P and test compounds were dispensed into the cell

plates (concentration range from 100 to 0.005 μM) and the plates

were further incubated at 37 °C for 50 min. Cells were ﬁxed by adding

10 μL of ﬁxation buﬀer (7.4% v/v formaldehyde containing 15 μM

Draq5 in DPBS) directly onto the assay medium and incubated for 15

min at rt, 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 (PerkinElmer #6005185). Imaging

was carried out using the Evotec Opera High Content Confocal

System (PerkinElmer, Boston, MA) equipped with three diode lasers,

Peltier-cooled CCD camera detector, and a Nipkow spinning disk.

Cell images were quantiﬁed using a membrane ﬂuorescent scoring

+

.88 (m, 3H), 1.84−1.78 (m, 1H), 1.48−1.37 (m, 1H); MS (M + H)

at m/z 456; HPLC t = 2.05 min (method A).

r

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 of

Laboratory Animals” published by the National Institutes of Health

(

NIH Publication no. 85-23, revised 2011). Phosphate metabolites

were dissolved in 0.3 M NaOH as a 3 mM stock prior to being

assayed.

SIP Binding Assay. Membranes were prepared from CHO cells

1

expressing human S1P . Cell pellets (l × 109 cells/pellet) were

1

suspended in buﬀer containing 20 mM HEPES (4-(2- hydroxyethyl)-

1

-piperazineethanesulfonic acid), pH 7.5, 50 mM NaCl, 2 mM

ethylenediaminetetraacetic acid (EDTA) and protease inhibitor

cocktail (Roche), and disrupted on ice using the Polytron

1

476

https://dx.doi.org/10.1021/acs.jmedchem.0c01109

J. Med. Chem. 2021, 64, 1454−1480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

algorithm. Data was analyzed using a customized HTS data analysis

software package. EC₅₀values 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 °C. 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

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

Healthcare Diagnostics). The results represent the average results of

all animals within each treatment group (n = 2−4).

Multi-electrode Array 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).

(

Calbiochem) and then labeled with the ﬂuorescent dye Calcein-

AM (10 μg/mL; Molecular Probes) in chemotaxis buﬀer (phenol red-

free RPMI-1640, containing 0.5% fatty-acid free BSA) at rt for 45 min.

The labeled cells were then washed and resuspended in chemotaxis

qPCR analysis showed similar RNA expression levels of S1P

, S1P ,

1

2

7

and S1P in hiPSC CMs as in adult human heart tissue (results not

buﬀer at 1.2 × 10 /mL. Chemotaxis was measured using a chemotaxis

3

5

shown). hiPSC CMs were cultured with 7% CO on 0.1% gelatin

plate with 5 μm pores (Neuroprobe). Labeled cells (3 × 10 in 25 μL

2

treated six-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 multi-

electrode array (MEA) plates (256−9 well MEA300/30iRITO-mq;

Multichannel Systems; Atlanta, GA). After 7 days of 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

min equilibration period in a humidiﬁed environment at 37 °C with

of chemotaxis buﬀer) were preincubated with test compound (ﬁnal

assay concentrations ranged from 0.01 to 50 nM) for 10 min at rt

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

aﬀord 90% maximal chemotaxis without the 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

incubation, the upper wells were washed twice with PBS to remove

unmigrated 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 IC₅₀is deﬁned as the

concentration of compound required to reach 50% inhibition of

chemotaxis and was calculated by the XLFit software program.

BLR Assay. Lewis rats were dosed orally with vehicle alone

constant 5% CO and 95% O supply, compounds were added to each

2

well in 300 μL of 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 the beating rate of hiPSC CMs. Eﬀects of test agents on FPs were

evaluated for at least 2 h. Data were analyzed with MC_DataTool and

custom software written in MatLab (Mathworks; Natick, MA).

Rat Adjuvant Arthritis (AA) Studies. AA was induced in male

Lewis rats (∼200 g) by subcutaneous injection of 100 μL of complete

Freund’s adjuvant (Sigma-Aldrich, MO) at the base of the tail.

Volumes of both hind paws were measured with a water-based

plethysmometer (Ugo Basile, Italy). Compounds were dissolved in

PEG300 and administered daily by oral gavage (5 mL/kg) from the

time of adjuvant injection. Paw volumes were periodically measured

during the course of the study, and change in paw volumes was

calculated relative to predisease baseline measurements.

(

polyethylene glycol 300) or with test compounds. Compounds were

dosed as a solution or suspension in the vehicle and 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 Metabolite Formation. Lewis rats were dosed

orally with compounds. Blood was drawn at 24 h and spotted onto

Dried Blood Spot (DBS) Cards. The 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 μL of wet

blood) was taken at n = 1 and placed in a shallow 96-well ﬁlter plate.

Next, 105 μL of a mixture of 75% acetonitrile and 25% water

containing internal standard was added and gently vortexed for 30

min and then centrifuged. The supernatant was separated from the

protein pellet and 5 μL was injected. The parent (alcohol) and active

phosphate metabolite compounds were quantitatively analyzed, using

a DBS calibration curve, by LC/MS/MS on a Triple Quadrupole

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 metabolite formation from the

parent (alcohol) compound. Reference material (parent alcohol and

phosphate metabolite) was analyzed to optimize the LC−MS/MS

assay and enable data reporting in concentrations. DBS standard

curves containing both parent alcohol and phosphate metabolites

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 metabolite formed. The results represent the average

results of all animals within each treatment group (n = 2−4).

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

ASSOCIATED CONTENT

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01109.

■

*

Lymphocyte reduction for 1 at 4 and 24 h at various

doses; synthesis of 26−36; S1P GTPγS activity of

phosphates for select compounds; exposures at 4, 24, 48,

1

and 72 h for T₁_/₂estimation; structure analysis of 6 and

7

; analyses of 12 and 24; chiral HPLC analysis of 40b;

^a ⁿ ^d ^m ^e ^t ^h ^o ^d^f^o^r^p^r^e^dⁱ^c^tⁱⁿ^g^h^u^m^aⁿ^T1/2 and clearance

PDF )

(

Molecular formula strings (CSV )

AUTHOR INFORMATION

Corresponding Author

■

John L. Gilmore − Bristol Myers Squibb Research & Early

Development, Princeton, New Jersey 08543-4000, United

States; orcid.org/0000-0002-0520-8746 ; Phone: 609-

252-3153; Email: john.gilmore@bms.com

Authors

Hai-Yun Xiao − Bristol Myers Squibb Research & Early

Development, Princeton, New Jersey 08543-4000, United

States

1

477

https://dx.doi.org/10.1021/acs.jmedchem.0c01109

J. Med. Chem. 2021, 64, 1454−1480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

T. G. Murali Dhar − Bristol Myers Squibb Research & Early

Development, Princeton, New Jersey 08543-4000, United

States; orcid.org/0000-0003-0738-1021

Michael Yang − Bristol Myers Squibb Research & Early

Development, Princeton, New Jersey 08543-4000, United

States; orcid.org/0000-0003-2908-4060

Zili Xiao − Bristol Myers Squibb Research & Early

Development, Princeton, New Jersey 08543-4000, United

States

Xiaoxia Yang − Bristol Myers Squibb Research & Early

Development, Princeton, New Jersey 08543-4000, United

States

Tracy L. Taylor − Bristol Myers Squibb Research & Early

Development, Princeton, New Jersey 08543-4000, United

States

Kim W. McIntyre − Bristol Myers Squibb Research & Early

Development, Princeton, New Jersey 08543-4000, United

States

Bethanne M. Warrack − Bristol Myers Squibb Research &

Early Development, Princeton, New Jersey 08543-4000,

United States

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01109

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

The authors would like to thank Dauh-Rurng Wu and Peng Li

for chiral separations of intermediates, members of the

Department of Discovery Synthesis of Biocon Bristol Myers

Squibb Research Center (BBRC) for scaling up intermediates,

Sarah Traeger for NMR analyses, and Cliﬀ Chen and Zheng

Yang for in vitro liver microsome analysis.

ABBREVIATIONS USED

■

S1P, sphingosine-1-phosphate; S1P1‑5, sphingosine-1-phos-

phate receptors 1−5; RRMS, relapsing-remitting multiple

sclerosis; BLR, blood lymphocyte reduction; BAL, bronchoal-

veolar lavage

Hong Shi − Bristol Myers Squibb Research & Early

Development, Princeton, New Jersey 08543-4000, United

States

Paul C. Levesque − Bristol Myers Squibb Research & Early

Development, Princeton, New Jersey 08543-4000, United

States

Anthony M. Marino − Bristol Myers Squibb Research & Early

Development, Princeton, New Jersey 08543-4000, United

States

Georgia Cornelius − Bristol Myers Squibb Research & Early

Development, Princeton, New Jersey 08543-4000, United

States

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J. Med. Chem. 2021, 64, 1454−1480

Article Doi

Bicyclic Ligand-Biased Agonists of S1P1: Exploring Side Chain Modifications to Modulate the PK, PD, and Safety Profiles

DOI: 10.1021/acs.jmedchem.0c01109

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