Asymmetric Hydroboration Approach to the Scalable Synthesis of ((1R,3S)-1-Amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol (BMS-986104) as a Potent S1P1 Receptor Modulator

Article abstract of DOI:10.1021/acs.jmedchem.6b01433

We describe a highly efficient route for the synthesis of 4a (BMS-986104). A key step in the synthesis is the asymmetric hydroboration of trisubstituted alkene 6. Particularly given the known difficulties involved in this type of transformation (6 → 7), the current methodology provides an efficient approach to prepare this class of compounds. In addition, we disclose the efficacy of 4a in a mouse EAE model, which is comparable to 4c (FTY720). Mechanistically, 4a exhibited excellent remyelinating effects on lysophosphatidylcholine (LPC) induced demyelination in a three-dimensional brain cell culture assay.

Full text of DOI:10.1021/acs.jmedchem.6b01433

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Article
Asymmetric Hydroboration Approach to the Scalable Synthesis of
((1R, 3S)-1-Amino-3-((R)-6-hexyl-5, 6, 7, 8-tetrahydronaphthalen-2-
1
yl)cyclopentyl)methanol (BMS-986104) as a Potent S1P Receptor Modulator
Michael G. Yang, Zili Xiao, T. G. Murali Dhar, Hai-Yun Xiao, John L Gilmore, David Marcoux,
Jenny H. Xie, Kim W. McIntyre, Tracy L. Taylor, Virna Borowski, Elizabeth Heimrich, Yu-
Wen Li, Jianlin Feng, Alda Fernandes, Zheng Yang, Praveen V. Balimane, Anthony M.
Marino, Georgia Cornelius, Bethanne M. Warrack, Arvind Mathur, Dauh-Rurng Wu, Peng
Li, Anuradha Gupta, Bala Pragalathan, Ding Ren Shen, Mary Ellen Cvijic, Lois D. Lehman-
McKeeman, Luisa M. Salter-Cid, Joel C. Barrish, Percy H Carter, and Alaric J. Dyckman
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01433 • Publication Date (Web): 23 Nov 2016
Downloaded from http://pubs.acs.org on November 27, 2016
Just Accepted
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
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Development, Leads Discovery & Optimization
Lehman-McKeeman, Lois; Bristol-Myers Squibb Company, Investigative
Toxicology (MS H24-03)
Salter-Cid, Luisa; Bristol-Myers Squibb Pharmaceutical Research and
Development, Discovery Biology
Barrish, Joel; Achillion Pharmaceuticals Inc
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Carter, Percy; Bristol-Myers Squibb Company
Dyckman, Alaric; Bristol-Myers Squibb, Discovery Chemistry
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Asymmetric Hydroboration Approach to the Scalable Synthesis of ((1R, 3S)-1-
Amino-3-((R)-6-hexyl-5, 6, 7, 8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol
(BMS-986104) as a Potent S1P1 Receptor Modulator
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Michael G. Yang,* Zili Xiao, T. G. Murali Dhar, HaiꢀYun Xiao, John L. Gilmore, David Marcoux, Jenny
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H. Xie, Kim W. McIntyre, Tracy L. Taylor, Virna Borowski, Elizabeth Heimrich, YuꢀWen Li, Jianlin Feng,
Alda Fernandes, Zheng Yang, Praveen Balimane, Anthony M. Marino, Georgia Cornelius, Bethanne M.
Warrack, Arvind Mathur, DauhꢀRurng Wu, Peng Li, Anuradha Gupta, Bala Pragalathan, Ding Ren Shen,
Mary Ellen Cvijic, Lois D. LehmanꢀMcKeeman, Luisa SalterꢀCid, Joel C. Barrish, Percy H. Carter, and
Alaric J. Dyckman*
Research and Development, BristolꢀMyers Squibb Company, Princeton, New Jersey 08543ꢀ4000, United States
ABSTRACT: We describe a highly efficient route for the synthesis of 4a (BMSꢀ986104). A key step in
the synthesis is the asymmetric hydroboration of trisubstituted alkene 6. Particularly given the known
difficulties involved in this type of transformation (6 → 7), the current methodology provides an efficient
approach to prepare this class of compounds. In addition, we disclose the efficacy of 4a in a mouse EAE
model, which is comparable to 4c (FTY720). Mechanistically, 4a exhibited excellent remyelinating effects
on lysophosphatidylcholine (LPC)ꢀinduced demyelination in a threeꢀdimensional brain cell culture assay.
INTRODUCTION
Multiple sclerosis (MS) is a chronic, debilitating disease of the central nervous system (CNS), which
affects more than two million people worldwide.1 Studies have shown that B and T cells play a significant
role in MS pathogenesis and T cells are significantly elevated during disease development.2,3 Upon
entering the CNS, T cells are reactivated by activated antigenꢀpresenting cells resulting in overproduction
of cytokines and initiation of inflammatory processes.4,5 Sphingosineꢀ1ꢀphosphate (S1P) is known to
transduce extracellular signals through its interactions with five related Gꢀproteinꢀcoupled receptors (S1P1ꢀ
5).6 Studies have shown that the S1P1 receptor is essential for regulating T cell egress from secondary
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lymphoid organs.7 FTY720 (4c) is a onceꢀdaily oral drug for the treatment of MS.8 The phosphate form of
FTY720 (4cꢀP) is a full agonist of S1P1, showing maximal response in in vitro assays compared to the
endogenous ligand S1P, and it induces complete S1P1 receptor internalization.9 Unlike S1P, agonism with
4cꢀP (FTY720ꢀP) induces prolonged desensitization without recycling, thus acting as powerful functional
antagonist to block S1P/S1P1 signaling. As a result of this functional antagonism, 4c (FTY720) inhibits T
cell egress from thymus and lymph nodes, thereby reducing T cell infiltration into the CNS.10 Compound
4c is also known to promote remyelination by acting directly on the cells of the central nervous system, an
effect that is believed to contribute to clinical efficacy of 4c in MS.11 Therapeutic efficacy of 4c has also
been demonstrated in various animal models of disease, such as rheumatoid arthritis, inflammatory bowel
disease, lupus, and atherosclerosis.12ꢀ14 Clinical studies have demonstrated an adverse pulmonary and
cardiovascular safety profile of 4c that includes decrease of pulmonary function, transient bradycardia and
sustained elevation of blood pressure.15 With the objectives of eliminating or reducing the unwanted sideꢀ
effects, we recently described the synthesis of tetrahydronaphthaleneꢀbased aminoꢀcyclopentylmethanol 4a
(BMSꢀ986104) and its associated research findings as a potent, differentiated, modulator of S1P1,4,5 (Figure
1).16,17 In vitro, the phosphorylated metabolite of 4a, formed through the action of sphingosine kinase 2,18
displayed ligandꢀbiased signaling at S1P1, including partial agonism in GTPγS and receptor internalization
assays, as well as a reduced predicted liability for heart rate effects based on a humanꢀrelevant
cardiomyocyte assay. In vivo, pulmonary edema in rodent that was noted after oral administration with full
agonists of S1P1 (including 4c) was absent with 4a.
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Figure 1. Original synthetic route for the synthesis of 4a.
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Figure 2. Improved synthetic route for the synthesis of 4a.
In our previous communication, we reported the in vivo efficacy of 4a in a rodent model of colitis and the
synthetic route used for the preparation of 4a (Figure 1).17 The described synthesis of 4a was nonꢀ
stereoselective and the overall yield was low (<1% yield in 11ꢀsteps). Furthermore, we had to rely on
multiple chiral separations to obtain the isomerically pure product (4a). Since the therapeutic efficacy of
4a was only studied in a T cell transfer colitis model at that time, we were also interested in expanding our
research to include additional animal models of disease, namely the mouse experimental autoimmune
encephalomyelitis (EAE) model for MS. An improved synthesis of 4a to provide material in support of
these studies was desired. In this publication, we describe an efficient synthesis of 4a and highlight the
methodology development of an asymmetric hydroboration of triꢀsubstituted alkene 6 (Figure 2, 6 → 7).
The current investigation also describes the in vitro findings of 4a and its in vivo results associated with the
EAE model.
Scheme 1. Synthesis of βꢀtetralone 14.
Reagents and conditions: a) stepꢀ1: K2CO3, H2O; stepꢀ2: NaBH4, MeOH, 87% yield for two steps. b) CDI, Dioxane, 97% yield. c) Zn, TMSCl, THF, 80 °C, 1h,
63% yield. d) LiHMDS, THF, 9 and 11a followed by Pd2(dba)3, Qꢀphos, Dioxane, 80 °C, 10ꢀ85% yield. e) LiHMDS, THF, 9 and 11b followed by Pd2(dba)3, Qꢀ
phos, Dioxane, 80 °C, 94ꢀ100% yield. f) TFA, CH2Cl2, 96% yield. g) stepꢀ1: oxalyl chloride; stepꢀ2: ethylene, AlCl3, CH2Cl2, ꢀ78 °C to rt, 64% yield for two
steps.
CHEMISTRY
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The revised synthetic approach for the preparation of 4a started with the synthesis of intermediate 14
(Scheme 1). Compound 5 was prepared using the procedures described in the literature.19 Treatment of
amino methyl ester HCl salt 5 with aqueous K2CO3 and then sodium borohydride provided primary alcohol
8 in 87% yield. CDIꢀpromoted cyclization gave 9 in 97% yield. Reformatsky reaction with arylbromide 9
and tertꢀbutyl chlorozincacetate (11a) completed the initial synthesis of 12. The organozinc chloride
solution of 11a is commercially available, but is costly ($780 → 50 mL, 0.5 M solution). In our
experience, 11a was only stable at low temperature and for relatively short duration. As a result, the
reaction yields of the Reformatsky reaction ranged from 10–85%, depending upon the quality of 11a. In
order to reduce the cost and control the quality of the organozinc reagent, we explored other alternatives for
the preparation of a comparable organozinc reagent. Using a slightly modified procedure from the
literature,20 we were able to make more than 150 grams of tertꢀbutyl bromozincacetate (11b) from
commercially available αꢀbromo ester 10. The synthesis of 11b was typically carried out using 15 grams
of αꢀbromo ester 10 for each trial. As a solid, tertꢀbutyl bromozincacetate 11b was easy to handle,
inexpensive to make, and stable at room temperature for weeks. More importantly, the coupling reaction of
9 (10 g, 33.8 mmol) with 11b proceeded smoothly to give the desired product 12 in 94–100% yield,
consistently in multiple trials. Deprotection of 12 with TFA gave acid 13. Reaction of 13 with oxalyl
chloride provided the corresponding acid chloride, which was treated with ethylene and AlCl3 to provide βꢀ
tetralone 14 in 64% yield for the last two steps.
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Scheme 2. Asymmetric hydroboration approach to compound 4a.
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Reagents and conditions: a) LDA, DMPU, ꢀ78 °C followed by PhN(Tf)2, ꢀ78 → 0 °C, 69% yield. b) LiHMDS, Fe(acac)3, THF/NMP, C6H13MgBr, ꢀ40 °C, 94%
yield. c) SꢀIpcꢀBH2, BF3·OEt2, ꢀ40 °C, CH2Cl2 followed by H2O2, NaOH, rt, 30 min, 87% yield. d) stepꢀ1: H2/PdꢀC, rt, MeOH; Stepꢀ2: Chiral OJꢀH (5 ꢀm),
CO2/[IPA:ACN 1:1 w 0.1% TFA] (90:10), 35 °C, 100 bars, PK1. 77% for two steps. e) NaOH, Dioxane, 95% yield.
Table 1. Asymmetric Hydrogenation or Hydroboration of 6
Entry
Reducing Agents
H2, Pd(OH)2
Reaction Conditionsa
MeOH, rt, 1 h
(R) : (S) ratio
1 : 1
1
2
3
4
5
6
7
H2, catASium®
MNXyIF(R)ꢀRh
Catecholboraneb
(S)ꢀBINAP
MeOH, rt, 900 psi, 16 h
2 : 1
THF, rt → 80 °C, 16 h
→ Step 2 & Step 3
no product
no product
3 : 1
(S)ꢀAlpineꢀ
Boramine
CH2Cl2, rt → 55 °C, 16 h
→ Step 2 & Step 3
CH2Cl2, rt, 0 °C, 2 h
→ Step 2 & Step 3
(S)ꢀIpcꢀBH2
(S)ꢀIpcꢀBH2
(S)ꢀIpcꢀBH2
CH2Cl2, ꢀ30 °C, 6 h
→ Step 2 & Step 3
8 : 1
THF, ꢀ30 °C, 6 h
→ Step 2 & Step 3
no product
aStepꢀ2: H2O2, NaOH, rt, 30 min. Stepꢀ3: H2/PdꢀC, H2, rt, MeOH. bBis(1,5ꢀcyclooctadiene)rhodium(I) tetrafluoroborate.
The asymmetric synthesis of compound 4a from 14 is outlined in Scheme 2. The LDAꢀpromoted
triflation reaction of 14 with phenyl triflimide gave 15 in 69% yield. Intermediate 15 was then treated with
LiHMDS followed by Feꢀcatalyzed coupling with hexylmagnesium bromide to give 6 in 94% yield, which
was used for the next reaction without further purification. With intermediate 6 in hand we were in a
position to explore chiral reagentꢀbased asymmetric reactions. Upon close examination of structure 6, it
was clear that achieving high enantioꢀselectivity in the transformation of 6 to 7 would be quite challenging
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since all the stereocenters and potential coordinating groups in molecule 6 were remote from the reaction
5
6
site.
Therefore, it was difficult for a chiral reagent to effectively distinguish between the two
7
8
diastereotopic faces of the alkene.
9
For comparison, both asymmetric hydrogenation and hydroboration reaction conditions were studied.
Representative reactions with selected chiral reagents are summarized in Table 1. As expected, the control
reaction of 6 with hydrogen and palladium hydroxide on carbon gave no appreciable level of diastereoꢀ
selectivity, (Entry 1, R : S = 1:1). Subsequently, we surveyed a large number of chiral catalysts for their
use in the asymmetric hydrogenation reaction (data not shown). Most of these reactions proved to be nonꢀ
selective and the best ratio in selectivity was 2:1 favoring the R isomer (Entry 2). Catecholborane and Sꢀ
alpine boramineꢀpromoted hydroboration reactions did not provide the desired product. However, the
reaction of 6 with Sꢀisopinocampheyl borane (SꢀIpcꢀBH2) in CH2Cl2 at 0 °C went smoothly and exhibited a
low level of diastereoꢀselectivity in favor of the R isomer (Entry 5, ratio 3:1). It is worth noting that SꢀIpcꢀ
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BH2 was generated freshly from Sꢀalpine boramine and BF3•OEt2 in THF at rt for 1.5 h. After significant
optimization, the hydroboration reaction conditions that gave the most favorable results were determined to
be SꢀIpcꢀBH2 in CH2Cl2 at ꢀ30 °C for 6 h, affording 7 with a very good level of enantioꢀselectivity in favor
of the R isomer after hydrogenolysis (Entry 6, ratio 8:1). It is important to note that the same reaction did
not produce any desired product when THF was used as the solvent in place of CH2Cl2 (Entry 7).
Using the optimal reaction conditions (SꢀIpcꢀBH2 in CH2Cl2 at ꢀ30 °C for 6 h), the hydroborationꢀ
oxidation reaction of 6 gave the intermediate alcohol 6a in 87% yield after a column purification. The
stereochemistry of the alcohol resulting from the hydroborationꢀoxidation sequence was not determined,
since it was inconsequential for the next step. Hydrogenolysis of 6a followed by a chiral supercritical fluid
chromatography (SFC) provided 7a as a single isomer in 77% yield and with >99% purity. Saponification
of 7a gave the homochiral analog 4a in 95% yield with isomeric purity >99.8%. In 12 steps, the overall
synthetic yield of 4a from 5 is ~21%, a significant improvement compared to the original synthesis (<1%,
11 steps). Using the improved synthetic route, we prepared more than 15 grams of 4a which was used for
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supporting various in vivo studies (vide infra). Previously, we determined the absolute stereochemistry of
4a by single crystal Xꢀray structure.17 The NMR and chiral HPLC analytical data of 4a matched perfectly
with the original analytical data. Saponification of diastereoisomer 7b provided the minor isomer 4b. To
complete the structure–activity relationship (SAR) study, we were interested in profiling both
diastereoisomers 4a and 4b.
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10
S1P1 Compounds in Balb/c Mice:
Peripheral Blood Lymphocyte Reduction
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*
4hr
24hr
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*
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*
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66%
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Vehicle
4a
4a
Vehicle
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4b
(0.5 mg/kg) (30 mg/kg)
(1 mg/kg) (30 mg/kg)
* p<.05 vs. Vehicle
ANOVA w/ Dunnett's
Mean +/- SEM
n=4/group
Figure 3. Lymphocyte reduction in mouse comparing 4a and diastereoisomer 4b.
Table 2. In Vitro Characterization of 4aꢀP vs 4bꢀP
Assay
4a-P
4b-P
hS1P1 HLE GTPγS Binding
(EC50, nM), Ymax (agonist)
6.4 ± 7.6
(n = 11), 72%
60 ± 9
(n = 6), 47%
hS1P3 GTPγS (EC50, nM)
(agonist)
>1000 (n = 7)
>1000 (n = 6)
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hS1P3 GTPγS (IC50, nM),
(antagonist)
>1000 (n = 3)
>1000 (n = 4)
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RESULTS AND DISCUSSION
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As discussed above, lymphopenia induced by the functional antagonism at S1P1 is a key aspect of the
clinical efficacy of 4c.21,22 We therefore studied the effects of 4a and 4b on the reduction of circulating
lymphocytes in the mouse Blood Lymphocyte Reduction assay (BLR) (Figure 3). Mice (four per group)
were dosed orally with the compounds dissolved in polyethylene glycol and peripheral blood lymphocyte
counts were assessed at 4 and 24 hours later, respectively. A trend of greater lymphocyte reduction at 24ꢀ
hour than that measured at the 4ꢀhour time point was evident for both 4a and 4b at each dose level (Figure
3). This was consistent with the observation that the appearance of the active metabolite (4aꢀP or 4bꢀP) for
these compounds was delayed, with greater phosphate concentration in blood at 24ꢀhour as compared to 4ꢀ
hour.23 Treatment with 4a (0.5 or 30ꢁmg/kg) resulted in a dose dependent reduction of peripheral blood
lymphocytes of 68–90% at the 24ꢀhour time point. It is worth noting that 4a revealed higher levels of
lymphocyte reduction at 0.5 mg/kg than that of 4b at 1 mg/kg with respect to their own vehicle (e.g.,
lymphopenia at 24 h: 4aꢀ68% vs. 4bꢀ60%). A similar trend was also recorded for the groups dosed at 30
mg/kg (e.g., lymphopenia at 24 h: 4aꢀ90% vs. 4bꢀ84%). The data derived from the BLR assay as shown in
Figure 3 indicated that 4a, with an EC50 of 7.9 nM for the corresponding active phosphate 4aꢀP, was more
potent than its isomer 4b (phosphate 4bꢀP EC50 of 35 nM). The fact that 4a was more efficacious than 4b
in the BLR assay indicates the importance of stereochemistry of the nꢀhexyl side chain on the reduction of
blood lymphocytes. The observed in vivo results are also in agreement with our in vitro findings (Table 2).
Phosphates 4aꢀP and 4bꢀP,24 the active metabolites of 4a and 4b, respectively, were characterized in the
GTPγS assay for both hS1P1 and hS1P3. Against hS1P1 both 4aꢀP and 4bꢀP were found to be partial
agonists (Ymax 74% and 47% respectively) with 4aꢀP being more potent (Table 2). Both 4aꢀP and 4bꢀP
were inactive in the hS1P3 GTPγS assay in either agonist or antagonist mode. Compound 4a was profiled
in vitro in liability assays and advanced for in vivo evaluation in rodent models of chronic inflammation.
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S1P1 Compounds in Balb/c Mice:
Peripheral Blood Lymphocyte Reduction
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0
*
HO
NH2
OR
C6H13
4hr
4c: R = H
24hr
*
4cꢀP: R = PO3H2
52%
*
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80%
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68%
*
88%
80%
*
89%
*
91%
*
90%
*
90%
*
93%
*
*
Vehicle
4c
(1 mg/kg)
4c
4a
4a
4a
(10 mg/kg) (0.5 mg/kg) (10 mg/kg) (30 mg/kg)
* p < 0.05 vs. Vehicle
Mean +/- SEM
ANOVA w/ Dunnett's
n=4/group
Figure 4. Lymphocyte reduction in mouse comparing 4a and 4c.
Table 3. General Profiles of 4c and 4a
Assay
4c
4a
796 ± 233
(n = 3)
2065 ± 67
(n = 3)
hS1P1 GTPγS (EC50, nM)
hERG patch clamp:
28
36
% Inh @ 0.3 ꢀM
Na+ flux IC50, ꢀM
> 80
> 80
10.4 / 13.3
3.2 / 10.7 / 25
19 / > 20
5.0 / >20 / >20
CYP IC50, ꢀM: 3A4 / 2D6
2C8 / 2C9 / 2C19
120 / 120
120 / 35
107 / 120
120 / 120
LM T1/2, min: human / rat
mouse / monkey
Protein binding (h/r/m/c/d)
all < 1% free
all < 1% free
Figure 4 illustrates the comparative effects of 4a and 4c on the reduction of circulating lymphocytes in
the mouse BLR assay. A single oral dose of 4c (1 or 10ꢁmg/kg) significantly reduced peripheral
lymphocyte counts by 80–93% at 24 hours. Notably, the maximal degree of lymphopenia was comparable
between 4a and 4c, as was the EC50 of the corresponding phosphates (7.9 nM for 4aꢀP vs. 5.6 nM for 4cꢀP).
Compound 4a was profiled for S1P1 activity, CYP inhibition, channel selectivity, and halfꢀlife (T1/2)
determination in liver microsomes (Table 3). As expected, the parent compound 4a carried little activity
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against S1P1 receptor in the GTPγS assay (IC50 = 2065 nM). With regard to ion channel selectivity, 4a
showed 36% inhibition at 0.3 ꢀM in a hERG patch clamp assay and IC50 > 80 ꢀM in a Na+ channel flux
assay. The CYP450 inhibition IC50 profile of 4a was in the micromolar range. At clinically relevant
concentrations, drug interactions involving the inhibition of the major drugꢀmetabolizing CYPs are not
anticipated with 4a. The halfꢀlives of 4a upon incubation with human, rat, and mouse liver microsomes
were long (T1/2 >100 min) and 4a was also found to be highly protein bound in all tested species (<1% free
in human, rat, mouse, cyno and dog). In general, we found the in vitro profiles of 4a and 4c to be similar
(Table 3).
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(A): EAE Model
4
100%
3
Vehicle
-33%
4a
2
(0.5 mg/kg, q.a.d)
*
-55%
*
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*
*
*
(4 mg/kg, q.a.d)
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(1 mg/kg, q.d)
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* P <0.05 vs.Vehicle Group
Mann-Whitney t Test
Days
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0
(B): Spinal cord histology
Inflammation/Damage
* P <0.05 vs. Vehicle group
Oneꢀway ANOVA
*
*
*
Vehicle
4c
4a
4a
1 mg/kg
0.5 mg/kg
4 mg /kg
Figure 5. 4a in MOG peptide induced mouse EAE model, clinical score (A) and histological evaluation (B). Dosing Regimen: 4a (every other day), 4c (daily).
The in vivo efficacy of 4a was evaluated in a chronic model of MOG peptide induced mouse EAE
(Figure 5). For comparison, 4c was also included in this study and dosed at 1 mg/kg (daily, po). The
mouse in vivo T1/2 of 4a was longer than that of 4c (4a: 98 h vs. 4c: 25 h). With the long T1/2 of 4a, it was
dosed every other day (q.a.d.) to avoid excessive accumulation, while 4c was dosed once daily. Oral
dosing of 4a at 0.5 and 4 mg/kg (q.a.d.) was initiated on day 1, and cumulative clinical scores were
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determined. The data indicated that the level of clinical efficacy obtained was proportional to the dose of
4a administered, and there was a significant improvement in clinical scores of the mice in the highest
dosage group, with the lower dose giving intermediate effects (Figure 5, A). The pattern of clinical
response for 4a at 4 mpk q.a.d. was similar to that of 4c at 1 mpk q.d. and revealed 55% reduction in
clinical score at day 22 relative to the vehicle (vs. 76% for 4c, Figure 5, A).
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Histological evaluation of lumbar spinal cords were performed on day 22 to investigate the effect of 4a
on the formation of inflammatory lesions in the CNS. Inflammatory lesions were readily detectable in
control mice, whereas the spinal cords from mice administered 4a exhibited a marked reduction in central
nervous system damage (Figure 5, B). When comparing all groups using one way ANOVA and Tukey’s
post test, 4c and 4a were significantly different from the control group in the categories of tissue damage
and inflammation (p < 0.05). Therefore, inhibition of EAE development by 4a could be a result of
inhibiting encephalitogenic T cell responses and/or their migration into the CNS. These findings have
identified 4a as a possible therapeutic agent for the treatment of multiple sclerosis.
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(E): *p < 0.05 vs. LPC; ANOVA
followed by Dunnett test, n= 3-5.
140
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*
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0
veh LPC 0.01 0.1
LPC + 4c-P
10
0.01 0.1
LPC + 4a-P
10
Figure 6. Effects of 4cꢀP and 4aꢀP on LPCꢀinduced demyelination in organotypic cerebellar slices.
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To understand the mechanism involved in suppressing EAE by administering 4a, we evaluated 4aꢀP, the
active phosphorylated form of 4a, in a threeꢀdimensional brain cell culture assay to test if it attenuated
demyelination (Figure 6).25,26 During the development of MS, myelin is attacked and destroyed by an
autoimmune response, resulting in demyelination and subsequent axonal degeneration.27 Unlike EAE,
which aims to approximate the pathophysiology of MS, the organotypic cerebellar slice culture system was
used to study the remyelinating effects of 4aꢀP on lysophosphatidylcholine (LPC)ꢀinduced demyelination
without the complex systemic immune system interactions observed in intact animals. Organotypic
cerebellar slices retain the astrocyte and microglia elements that allow us to identify their contribution to
myelinationꢀassociated processes. Therefore, the interpretation and analysis of a remyelinating effect
should be more straightforward than with EAE.
Figure 6 illustrates the effects of 4cꢀP and 4aꢀP on LPCꢀinduced demyelination in the organotypic
cerebellar slice. Most of the myelin sheaths around axonal fibers lost their integrity in the slice treated with
LPC in comparison to that treated with vehicle (Figure 6, A vs. B). To validate the organotypic cerebellar
slice culture system, we included 4cꢀP as a positive control since 4cꢀP is known to exhibit remyelination
activity in this model.11,28 In a recent study, both astrocytes and microglia were shown to be important for
remyelination.11 In the same report, 4c was found to increase the number of astrocytes and microglia,
enhancing remyelination primarily through S1P1/S1P5 signaling pathways. In our remyelination studies,
the organotypic cerebellar slices were incubated with 0.5 mg/mL LPC overnight and then transferred to the
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culture medium for 24 h. Slices were then treated with 4cꢀP or 4aꢀP for 7 days before being processed for
immunohistochemistry. As shown in Figure 6, C and D, most myelinated axons remained myelinated, and
were not affected by LPCꢀinduced demyelination with treatment of 4cꢀP or 4aꢀP. Myelination index values
were then used to quantify the remyelination effects of 4cꢀP and 4aꢀP (Figure 6, E). The myelination index
value of LPCꢀtreated slices was ~50% using analysis of variance (ANOVA) followed by Dunnett test (P <
0.05). Compound 4aꢀP promoted Purkinje cell axonal remyelination at 0.01–10 nM concentration range
and myelination index values of 4cꢀP and 4aꢀP were both ~100% (Figure 6, E).
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CONCLUSIONS
Efforts aimed at improving the synthesis of 4a led to the discovery of the asymmetric hydroborationꢀ
oxidation reaction of 6 (Table 1). The observed high levels of diastereoꢀselectivity of this reaction are
remarkable (Table 1, entry 6), given what has been known in the literature that alkenes lacking
coordinating groups have long been notoriously difficult to hydrogenate or hydroborate with high stereoꢀ
selelctivity 29,30
.
The current methodology provides an efficient approach to prepare this class of
compounds. Expanding upon our previous work,17 which supported the potential development of 4a in the
area of inflammatory bowel disease, the data outlined in this paper supports the development of 4a in
multiple sclerosis. Here, we have evaluated the impact of 4a in the MOG induced mouse EAE model and
found its efficacy at 4 mg/kg q.a.d. to be similar to 4c at 1 mg/kg q.d. (Figure 5). Consistent with previous
studies in colitis models, although a partial agonist of S1P1 in several in vitro assays, 4a was able to achieve
efficacy similar to 4c in the EAE model. To understand the mechanism involved in suppressing EAE by
administering 4a, we evaluated 4aꢀP in the organotypic cerebellar slice culture system and showed
remarkable remyelinating effects on LPCꢀinduced demyelination (Figure 6). Reductions in EAE clinical
scores were paralleled by reductions in demyelination, axonal loss, and astrogliosis. Taken together, these
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findings demonstrate that administration of 4a effectively prevents the development of disease in the mouse
5
6
EAE model and therefore could find utility in the treatment of human MS.
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EXPERIMENTAL SECTION
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Chemistry. All commercially available chemicals and solvents were used without further purification.
Reactions are performed under an atmosphere of nitrogen. All new compounds gave satisfactory 1H NMR,
LC/MS and/or HRMS, and mass spectrometry results. 1H NMR spectra were obtained on a Bruker 400
MHz or a Jeol 500 MHz NMR spectrometer using residual signal of deuterated NMR solvent as internal
reference. Electrospray ionization (ESI) mass spectra were obtained on a Water Micromass ESIꢀMS single
quadrupole mass spectrometer. The purity of tested compounds determined by analytical HPLC was
>95%. Analytical HPLC conditions A: analytical HPLC was performed on a Shimadzu instrument:
column, 1ꢀWaters Sunfire C18 2.1 x 30 mm; gradient elution 0–100% B over 4 min with 1 min hold
(solvent A, 90% water/10% MeOH/0.1% TFA; solvent B, 90% MeOH/10% water/0.1% TFA; flow rate, 1
mL/min; 220 nm as the detection wavelength. Analytical HPLC conditions B: analytical HPLC was
performed on a Shimadzu instrument: column, YMC Combiscreen ODSꢀA, 4.6 mm × 50 mm; gradient
elution 0–100% B over 4 min with 1 min hold (solvent A, 90% water/10% MeOH/0.2% H3PO4; solvent B,
90% MeOH/10% water/0.2% H3PO4; flow rate, 4 mL/min; 220 nm as the detection wavelength.
((1R,3S)-1-Amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol
(4a,
Scheme 2). To a solution of 7a (2.35 g, 6.61 mmol) in dioxane (40 mL) was added water (10 mL) and
lithium hydroxide hydrate (2.77 g, 66.1 mmol). The reaction mixture was stirred at 100 °C for 20 h. LCꢀ
MS indicated that the reaction was done and the mixture was cooled to 50 °C and filtered. The solid was
washed with 1,4ꢀdioxane (3 x 5 mL) and filtered each time, the combined filtrates were added water (45
mL) dropwise with stirring. The solid was collected with filtration and washed with water (3 x 5 mL),
dried with high vacuum to give 4a as a white solid (2.10 g 6.31 mmol, 95% yield). 1H NMR (400 MHz,
METHANOLꢀd4) δ ppm: 7.06 ꢀ 6.90 (m, 3H), 3.54 ꢀ 3.40 (m, 2H), 3.01 (dd, J = 11.2, 7.1, 4.0 Hz, 1H), 2.87
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ꢀ 2.71 (m, 3H), 2.35 (dd, J = 16.3, 10.3 Hz, 1H), 2.21 (dd, J = 13.1, 7.4 Hz, 1H), 2.09 ꢀ 1.87 (m, 3H), 1.84 ꢀ
5
6
7
8
1.63 (m, 3H), 1.60 ꢀ 1.49 (m, 1H), 1.46 ꢀ 1.29 (m, 11H), 0.98 ꢀ 0.87 (m, 3H). ESIꢀMS:
H+]). HPLC:
m/z 330.35 ([M +
t
R = 3.66 min (analytical HPLC conditions A).
9
((1R,3S)-1-Amino-3-((S)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol
(4b,
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Scheme 2) A procedure similar to that described in the synthesis of 4a was used to prepare 4b from 7b
(The minor product 7b was obtained from the previous Chiral SFC preparative separation step used for the
1
synthesis of 7a). H NMR (400 MHz, CHLOROFORMꢀd) δ ppm: 7.07 ꢀ 6.89 (m, 3H), 3.56 ꢀ 3.33 (m, 2H),
3.20 ꢀ 2.90 (m, 1H), 2.87 ꢀ 2.73 (m, 3H), 2.35 (dd,
J = 16.3, 10.8 Hz, 1H), 2.26 (dd, J = 13.1, 8.0 Hz, 1H),
2.12 ꢀ 2.00 (m, 1H), 1.98 ꢀ 1.84 (m, 2H), 1.79 ꢀ 1.63 (m, 3H), 1.53 ꢀ 1.45 (m, 1H), 1.43 ꢀ 1.22 (m, 12H),
0.95 ꢀ 0.84 (m, 3H). ESIꢀMS: m/z 330.27 ([M + H+]). HPLC: tR = 3.77 min (analytical HPLC conditions
B).
(5R,7S)-7-(6-Hexyl-7,8-dihydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one (6, Scheme 2).
To a solution of 15 (3.5g, 8.39 mmol) and NMP (8.07 ml, 84 mmol) in THF (200 mL) was added LiHMDS
(8.39 ml, 8.39 mmol) at ꢀ 40 °C and stirred at the same temperature for 25 min followed by Ferric
acetylacetonate (0.296 g, 0.839 mmol) and hexylmagnesium bromide (8.39 mL, 16.77 mmol) dropwise.
The reaction mixture was stirred at ꢀ 40 to ꢀ 20 °C for 45 min then warmed to 0 °C and stirred for 15 min.
The reaction mixture was diluted with water (150 mL), extracted with EtOAc (300 mL), and then washed
with sat. NaHCO3 (3 x 100 mL). The organic layer was collected, left on the bench for overnight at rt. The
white solid was filtered off, and the mother liquor was dried over Na2SO4, concentrated on the rotavapor to
give a crude product 6 which was used for the next reaction without further purification (crude yield 94%).
1H NMR (400 MHz, CHLOROFORMꢀd), δ ppm: 7.04 ꢀ 6.91 (m, 3H), 6.21 (s, 1H), 5.06 (br s, 1H), 4.42 ꢀ
4.25 (m, 2H), 3.15 ꢀ 2.98 (m, 1H), 2.80 (t,
J = 8.0 Hz, 2H), 2.42 ꢀ 2.09 (m, 7H), 2.05 ꢀ 1.93 (m, 2H), 1.90 ꢀ
1.78 (m, 1H), 1.54 ꢀ 1.45 (m, 2H), 1.39 ꢀ 1.29 (m, 6H), 0.99 ꢀ 0.85 (m, 3H). ESIꢀMS:
H+]). HPLC:
m/z 354.30 ([M +
t
R = 4.23 min (analytical HPLC conditions A).
(5R,7S)-7-((6R)-6-Hexyl-5-hydroxy-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-
azaspiro[4.4]nonan-2-one (6a, Scheme 2). To a solution of 6 (2.1 g, 5.94 mmol) in CH2Cl2 (10 mL) was
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added dropwise to a mixture of ((1R,2S,3R,5R)ꢀ2,6,6ꢀtrimethylbicyclo[3.1.1]heptanꢀ3ꢀyl)borane (SꢀIpcꢀ
5
6
7
8
BH2, 0.892 g, 5.94 mmol) in 30 mL of CH2Cl2 (under N2) at ꢀ35 °C. The mixture was stirred at ꢀ35 °C for
4 h and then at ꢀ20 °C for 2 h. MeOH (3.61 mL, 89 mmol) was added dropwise, the mixture was then
stirred at ꢀ10 °C for 10 min and at 0 °C for 10 min. At 0 °C, 25 mL of THF was added then NaOH (9.90
mL, 59.4 mmol) was added dropwise followed by H2O2 (6.07 mL, 59.4 mmol), the mixture was stirred at rt
for 16 h. The mixture was diluted with CH2Cl2 (100 mL) and water (50 mL), filtered through a pad of
celite and the cake was washed with CH2Cl2 (3 x 100 mL), and the filtrate was separated and the aqueous
layer was extracted with CH2Cl2 (50 mL). The combined CH2Cl2 was washed with water (100 mL) and
brine (100 mL), dried over Na2SO4 and concentrated under vacuo. The residue was purified with a 40 g
isco column: EtOAc/Hexane = 0 ꢀ 50%, gradient time = 55 min, the product came out at 45% EtOAc. The
isolated fractions were concentrated and dried in vacuo to give 6a as a solid (1.9 g, 5.11 mmol, 87% yield).
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1H NMR (400 MHz, METHANOLꢀd4) 7.38 (d,
1H), 4.44 ꢀ 4.25 (m, 3H), 3.11 ꢀ 2.98 (m, 1H), 2.76 (t,
ꢀ 2.04 (m, 3H), 2.01 ꢀ 1.63 (m, 5H), 1.57 ꢀ 1.46 (m, 2H), 1.35 (d,
ꢀ 0.88 (m, 3H). Instead of 6a, the related dehydroxylation product (5R,7S)ꢀ7ꢀ(6ꢀhexylꢀ7,8ꢀ
J
= 8.1 Hz, 1H), 7.10 (dd,
= 6.4 Hz, 2H), 2.30 (dd,
= 3.5 Hz, 7H), 1.27 ꢀ 1.18 (m, 1H), 0.96
J
= 7.9, 1.5 Hz, 1H), 6.99 (s,
J
J
= 12.9, 7.2 Hz, 1H), 2.20
J
dihydronaphthalenꢀ2ꢀyl)ꢀ3ꢀoxaꢀ1ꢀazaspiro[4.4]nonanꢀ2ꢀone was observed in ESIꢀMS:
H+]). HPLC:
m/z 354.23 ([M +
t
R = 3.75 min (analytical HPLC conditions A).
(5R,7S)-7-((R)-6-Hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one
(7a, Scheme 2). To a solution of 6a (3.2 g, 8.61 mmol) in MeOH (30 mL) was added PdꢀC (1.1 g, 1.034
mmol), the mixture was placed under vacuum and back filled with H2 with a H2 balloon. The reaction
mixture was stirred at rt for 6 h. A solid was precipitated out. To the mixture was added EtOAc (20 mL)
and the reaction was hydrogenated for 16 h. The reaction mixture was filtered through a pad of celite and
the cake was washed with EtOAc, CH2Cl2/MeOH and EtOAc. The combined organic solvents were
concentrated under vacuo and purified by preparative chiral SFC
: Preparative Column: Chiralpak ASꢀH
(3×25 cm, 5µm); Flow rate: 165 mL/min; 35 °C and 100 bars; Mobile Phase: CO2/MeOH (65/35)
;
Detector Wavelength: 210 nm; Separation Program: Stack injection; Injection: 0.9 mL with cycle time 165
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sec; Sample preparation: 0.71 g / 30 mL MeOH : DCM (9 : 1), ~24 mg/mL. The chiral separation
provided 7a (2.35 g, 6.61 mmol, 77% yield). 1H NMR (400 MHz, METHANOLꢀd4) δ ppm: 7.02 ꢀ 6.91
7
8
(m, 3H), 4.43 ꢀ 4.24 (m, 2H), 3.02 (tt, J = 11.0, 7.2 Hz, 1H), 2.88 ꢀ 2.72 (m, 3H), 2.41 ꢀ 2.23 (m, 2H), 2.17
9
ꢀ 2.04 (m, 2H), 2.01 ꢀ 1.62 (m, 5H), 1.49 ꢀ 1.30 (m, 11H), 0.98 ꢀ 0.88 (m, 3H). ESIꢀMS:
H+]). HPLC:
m/z 356.30 ([M +
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t
R = 4.33 min (analytical HPLC conditions A).
((1R,3S)-1-Amino-3-(4-bromophenyl)cyclopentyl)methanol (8, Scheme 1). K2CO3 (3 g, 21.71
mmol) was dissolved in water (30 mL). 5 (4.4 g, 13.15 mmol, see reference 19 for its synthesis) and ethyl
acetate (40 mL) were then added. The mixture was stirred at rt for 20 min. The aqueous layer was
separated and extracted with ethyl acetate (4 x 20 mL). The combined ethyl acetate solutions were dried
over Na2SO4 and concentrated under reduced pressure to give 5a as a free base of 5. The liquid 5a was
dissolved in ethanol (40 mL), NaBH4 (1.244 g, 32.9 mmol) was added at 0 °C portionwise. The mixture
was stirred at rt for 18 h. The reaction mixture was cooled to 0 °C and 6 N aqueous HCl was added
dropwise to quench the reaction. The suspension was stirred at rt for 30 min before 30% aqueous NaOH
was added at 0 °C. The mixture was stirred at rt for 1 h and then concentrated to remove organic solvents.
Ethyl acetate (50 mL) was added. Water (30 mL) was added to dissolve the solid and the aqueous layer was
separated and extracted with ethyl acetate (3 x 20 mL). The combined ethyl acetate solutions were dried
over K2CO3 and Na2SO4 and concentrated under reduced pressure to give 8 (3.1 g, 11.47 mmol, 87 %
yield) as a white solid. 1H NMR (400 MHz, DMSOꢀd6), δ ppm: 7.51ꢀ7.40 (m, 2H), 7.27 (d,
J
= 8.4 Hz,
2H), 3.32ꢀ3.20 (m, 2H), 3.09ꢀ2.92 (m, 1H), 2.11 (dd,
J
= 12.9, 8.7 Hz, 1H), 1.98ꢀ1.87 (m, 1H), 1.80 (qd, J =
11.1, 7.9 Hz, 1H), 1.69ꢀ1.58 (m, 1H), 1.48 (ddd,
J
= 12.4, 7.9, 2.2 Hz, 1H), 1.32 (dd, J = 12.8, 10.1 Hz,
1H). ESIꢀMS:
m
/z
270.16 ([M + H+]). HPLC:
tR = 1.85 min (analytical HPLC conditions B).
(5R,7S)-7-(4-Bromophenyl)-3-oxa-1-azaspiro[4.4]nonan-2-one (9, Scheme 1). To a cloudy solution
of 8 (3.1 g, 11.47 mmol) in THF (50 mL) was added 1,1'ꢀCarbonyldiimidazole (2.79 g, 17.21 mmol)
portionwise. Pyridine (1.392 mL, 17.21 mmol) was then added. The reaction was stirred at rt under
nitrogen overnight. 6 N aqueous HCl (~12 mL) was added dropwise with water bath cooling to make pH
~2. The mixture was stirred at rt for 1.5 h before ethyl acetate (50 mL) was added. The aqueous layer was
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separated and extracted with ethyl acetate (2 x 25 mL). The combined organic solutions were washed with
1 N aqueous HCl (25 mL), brine (25 mL), and saturated aqueous sodium bicarbonate solution (25 mL) till
pH ~8, dried over Na2SO4, and concentrated under reduced pressure. The residue was dissolved in ethyl
acetate (~50 mL) with heating and mixed with hexanes (~50 mL). The solid was filtered, washed with
ethyl acetate, and dried to give 9 (3.3 g, 11.14 mmol, 97% yield) as a white solid. 1H NMR (400 MHz,
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CHLOROFORMꢀd), δ ppm: 7.45 (d,
J = 8.6 Hz, 2H), 7.12 (d, J = 8.4 Hz, 2H), 6.42 (br. s., 1H), 4.41ꢀ4.21
(m, 2H), 3.17ꢀ2.91 (m, 1H), 2.34 (dd,
J = 13.3, 7.4 Hz, 1H), 2.23ꢀ2.11 (m, 2H), 2.01ꢀ1.90 (m, 2H), 1.88ꢀ
1.74 (m, 1H). ESIꢀMS:
m/
z
296.14 ([M + H+]). HPLC:
tR = 2.78 min (analytical HPLC conditions B).
(2-(tert-Butoxy)-2-oxoethyl)zinc(II) bromide (11b, Scheme 1). To a mixture of Zn (1.760 g, 26.9
mmol) dust and THF (15 mL), TMSꢀCl (0.082 mL, 0.641 mmol) was added and the mixture was stirred at
rt for 1 h. tertꢀButyl 2ꢀbromoacetate (5 g, 25.6 mmol) was then added dropwise to bring the temp to 50 °C
and maintained at the temperature during the addition (25 min). The reaction mixture was stirred at 50 °C
for 1 h. The reaction mixture was then allowed to cooled down to 10 °C and kept there for 1 h. The solid
was collected with filtration and washed with THF (3 x 2 mL), dried under vacuo to give 11b (5.4 g, 16.24
1
mmol, 63% yield) as a white solid. H NMR (400 MHz, METHANOLꢀd4)
δ ppm: 3.75 (t, J = 6.3 Hz, 4H),
1
1.97 ꢀ 1.93 (m, 2H), 1.92 ꢀ 1.87 (m, 4H), 1.46 (s, 9H). The H NMR was taken as a complex of 11b
associated with one equivalent of THF (THF coordinated to the Zn in the molecule 11b).
tert-Butyl 2-(4-((5R,7S)-2-oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)phenyl)acetate (12, Scheme 1). To
a pressure flask (350 mL) containing 9 (5 g, 16.88 mmol) anhydrous THF (100 mL) was charged with N2,
then LiHMDS (18.57 mL, 18.57 mmol) was added with stirring. After stirred for 30 min, 1,2,3,4,5ꢀ
pentaphenylꢀ1'ꢀ(diꢀtꢀbutylphosphino)ferrocene (0.360 g, 0.506 mmol), Pd2(dba)3 (0.464 g, 0.506 mmol) and
11b (16.84 g, 50.6 mmol) were added respectively. The mixture was flashed with N2 for 5 min, sealed and
stirred at 80 °C for 16 h. After cooling, the reaction mixture was filtered through a pad of celite and the
cake was washed with EtOAc (200 mL), then water (50 mL) and 1 N HCl (100 mL) were added. The
organic layer was collected and washed with 1 N HCl (2 x 100 mL), brine (2 x100 mL), dried over Na2SO4
and concentrated under vacuo to give the crude product 12 which was used for the next reaction as is (5.6 g,
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16.90 mmol, 100% yield) as a white solid. H NMR (400 MHz, CHLOROFORMꢀd), δ ppm: 7.26 ꢀ 7.16
5
6
(m, 4H), 5.59 ꢀ 5.52 (m, 1H), 4.38 ꢀ 4.27 (m, 2H), 3.53 (s, 2H), 3.14 ꢀ 3.03 (m, 1H), 2.41 ꢀ 2.31 (m, 1H),
7
8
9
2.15 (s, 2H), 1.97 (d,
H+]). HPLC:
J = 1.5 Hz, 2H), 1.89 ꢀ 1.79 (m, 1H), 1.49 ꢀ 1.45 (m, 9H). ESIꢀMS: m/z 332.18 ([M +
t
R = 3.09 min (analytical HPLC conditions A).
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2-(4-((5R,7S)-2-Oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)phenyl)acetic acid (13, Scheme 1). To a
mixture of 12 (5.6 g, 16.90 mmol) in CH2Cl2 (50 mL) was added TFA (25 mL). The reaction mixture was
stirred at rt for 1.5 h. The solvent was removed under vacuo and the residue was dissolved in EtOAc (150
mL) and 1 N NaOH (100 mL). The organic layer was separated, collected, and extracted with 1 N NaOH
(100 mL). The aqueous layers were combined and acidified with conc. HCl to pH = 1ꢀ2 and then was
extracted with EtOAc (2 x 100 mL), which was washed with brine (100 mL), dried over Na2SO4 and
concentrated under vacuo to give 13 (4.45 g, 16.16 mmol, 96% yield) as a white solid. 1H NMR (400
MHz, CHLOROFORMꢀd), δ ppm: 7.31 (s, 1H), 7.25 ꢀ 7.17 (m, 3H), 4.38 ꢀ 4.29 (m, 2H), 3.69 ꢀ 3.65 (m,
2H), 3.16 ꢀ 3.04 (m, 1H), 2.36 (dd,
J
= 13.8, 8.5 Hz, 1H), 2.21 ꢀ 2.12 (m, 2H), 1.99 (dd,
J = 13.8, 10.2 Hz,
1H), 1.93 ꢀ 1.79 (m, 2H). ESIꢀMS:
m/z
276.12 ([M + H+]). HPLC: tR = 2.13 min (analytical HPLC
conditions A).
(5R,7S)-7-(6-Oxo-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one
(14,
Scheme 1). To a mixture of 13 (0.5 g, 1.816 mmol) in CH2Cl2 (13 mL) was added oxalyl chloride (0.477
mL, 5.45 mmol) followed by a few drops of DMF. After 1 h, an aliquot was quenched in MeOH and
checked by LCMS for the methyl ester. LCMS showed a complete conversion to the acid chloride.
Reaction mixture was concentrated in vacuo and dried. Residue was reꢀdissolved in CH2Cl2 (13 mL) in a
glass pressure vessel. Ethylene was bubbled through for 3 min at ꢀ78 °C before aluminum chloride (0.727
g, 5.45 mmol) was added. Ethylene was bubbled through for 7 more min then the reaction was sealed and
allowed the reaction to slowly warm to rt over 1 h. Sonication broke down the chunk. The mixture was
stirred at rt for 1.5 h. The reaction mixture was poured on to ice. The reaction mixture was diluted with
dichloromethane. The aqueous layer was extracted with CH2Cl2. The combined organic layers were
washed with 1M HCl, dried over Na2SO4, filtered and conc. The crude material was purifed on a silica gel
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cartridge (12 g) using an ethyl acetate / hexanes gradient (20ꢀ100%) to give 14 (0.33 g, 1.157 mmol, 64%
1
yield) as a yellow solid. H NMR (400 MHz, CHLOROFORMꢀd), δ ppm: 7.20ꢀ7.00 (m, 3H), 5.49 (br. s.,
7
8
1H), 4.45ꢀ4.25 (m, 2H), 3.59 (s, 2H), 3.08 (t,
J
= 6.8 Hz, 3H), 2.58 (t,
J
= 6.7 Hz, 2H), 2.38 (dd,
J = 13.2,
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7.3 Hz, 1H), 2.27ꢀ2.11 (m, 2H), 2.05ꢀ1.92 (m, 2H), 1.92ꢀ1.74 (m, 1H). ESIꢀMS:
m/z
286.14 ([M + H+]).
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HPLC:
tR = 2.18 min (analytical HPLC conditions B).
6-((5R,7S)-2-Oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)-3,4-dihydronaphthalen-2-yl
trifluoromethanesulfonate (15, Scheme 2). To a mixture of 14 (1.8 g, 6.31 mmol) and DMPU (2.282 mL,
18.92 mmol) in THF (50 mL) was added LDA (9.46 mL, 18.92 mmol) dropwise. The reaction mixture was
stirred for 30 min then 1,1,1ꢀtrifluoroꢀNꢀphenylꢀNꢀ(trifluoromethyl)sulfonyl methanesulfonamide (4.51 g,
12.62 mmol) in THF (10 mL) was added. The reaction mixture was warmed to 0 °C and stirred for 1 h.
LCMS showed conversion to be complete. The reaction mixture was diluted with water (5 mL), ethyl
acetate (40 mL) and washed with sat NaCl (20 mL). The organic layer was collected, dried over MgSO4,
and concentrated. The crude material was purified on a silica gel cartridge (80 g) using an EtOAc/Hex
gradient (0ꢀ100% EtOAc over 20 minutes). Isolated productꢀcontaining fractions, concentrated and dried
in vacuo to give 15 as an oil (1.8 g, 4.31 mmol, 68.4 % yield). 1H NMR (400 MHz, CHLOROFORMꢀd)
δ
7.05 (s, 2H), 7.02 (s, 1H), 6.49 (s, 1H), 5.66 (s, 1H), 4.42 ꢀ 4.23 (m, 2H), 3.07 (t, J = 8.4 Hz, 2H), 2.80 ꢀ
2.64 (m, 2H), 2.35 (dd,
J = 13.4, 7.3 Hz, 1H), 2.23 ꢀ 2.09 (m, 2H), 2.05 ꢀ 1.88 (m, 3H), 1.89 ꢀ 1.77 (m, 1H).
ESIꢀMS:
m
/z
418.06 ([M + H+]). HPLC:
tR = 3.57 min (analytical HPLC conditions A).
Biological Assays. Receptor HLE [35S] GTPγS Binding Assays. Compounds were loaded in a 384
Falcon Vꢀbottom plate (0.5 ꢂL/well in a 3ꢀfold dilution). Membranes prepared from S1P1/CHO cells or
EDG3ꢀGa15ꢀbla HEK293T cells were added to the compound plate (40 ꢂL/well, final protein 3 ꢂg/well)
with multidrop. [35S]GTP (1250 Ci/mmol, PerkinElmer) was diluted in assay buffer: 20 mM HEPES, pH
7.5, 10 mM MgCl2, 150 mM NaCl, 1 mM EGTA, 1 mM DTT, 10 ꢂM GDP, 0.1% fatty acid free BSA, and
10 ꢂg/mL saponin to 0.4 nM. Then 40 ꢂL of the [35S] GTP solution was added to the compound plate with
a final concentration of 0.2 nM. The reaction was kept at room temperature for 45 min. At the end of
incubation, all the mixtures in the compound plate were transferred to a 384ꢀwell FB filter plates via GPCR
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robot system. The filter plate was washed with water four times by using the modified manifold Embla
plate washer and dried at 60 °C for 45 min. Then 30 ꢂL of MicroScint 20 scintillation fluid was added to
each well for counting at Packard TopCount. EC50 is defined as the agonist concentration that corresponds
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to 50% of the Ymax (maximal response) obtained for each individual compound tested.
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Blood Lymphocyte Reduction Assay (BLR) in Rodents. Balb/c mice were dosed orally with test article
(as a solution or suspension in the vehicle) or vehicle alone (polyethylene glycol 300, PEG300). Blood was
drawn at 4 h and at 24 h by retroꢀorbital bleeding. 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 4 and
24 h measurements. The results represent the average results of all animals within each treatment group (n
= 3−4).
hERG Patch Clamp Assay. Wholeꢀcell patchꢀclamp was used to directly measure hERG currents in
HEK 293 cells stably expressing the cloned hERG potassium channel α subunit. The compound was tested
in an aqueous buffer with pH 7.4 at room temperature. Repetitive test pulses (0.05 Hz) were applied from a
holding potential of 80 mV to +20 mV for 2 seconds and tail currents were elicited following the test pulses
by stepping the voltage to 65 mV. The effects from the compound were calculated by measuring inhibition
of peak tail current
Mouse Experimental Autoimmune Encephalomyelitis Assay (EAE). Mice (C57BL/6 female, 6ꢀ8 weeks
of age, Charles River, n=10 treatment group) were immunized subcutaneously with 150 mg MOG35ꢀ55
emulsified 1:1 with Incomplete Freund’s Adjuvant (Sigma) supplemented with 150 mg mycobacterium
tuberculosis H37RA (Difco Laboratories). Then 400 ng of pertussis toxin (CalBiochem) was injected
intraperitoneally on the day of immunization and 2 days later. Clinical scoring and body weight were taken
3 times per week. Clinical scoring system: 0.5: partial tail weakness; 1: limp tail or waddling gait with tail
tonicity; 1.5: waddling gait with partial tail weakness; 2: waddling gait with limp tail (ataxia); 2.5: ataxia
with partial limb paralysis; 3: full paralysis of one limb; 3.5: full paralysis of one limbs with partial
paralysis of a second limb; 4: full paralysis of two limbs; 4.5: moribund; 5: death. Mean clinical score was
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calculated by averaging the scores of all mice in each group. Upon termination of the study, spinal
columns were excised and submersion fixed in 10% Neutral Buffered Formalin. Three transverse sections
of lumbar spinal cord were routinely processed, paraffin embedded (RPPE) and were sectioned at 3µm and
6um, stained with hematoxylin and eosin and the myelinꢀspecific stain Luxol Fast Blue, respectively.
Slides were analyzed in a blinded fashion for severity of inflammation, tissue damage, and demyelination.
Lesion activity was scored on a semiꢀquantitative 0ꢀ5 scale, and overall mean histological score of
inflammation, damage, and demyelination from each group was calculated.
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Organotypic Cerebellar Slice Culture Assay. Newborn (P1ꢀ2) wildꢀtype CD1 mouse pups were
obtained from Charles River. After decapitation, the cerebellum was isolated and cut into 350 um
parasagittal slices. Slices were then transferred to culture inserts (Millipore MillicellꢀCM organotypic
culture inserts) in a 6ꢀwell plate with 1 ml culture medium, and incubated at 36–37◦C with 5% CO2.
Culture medium was composed of 50% minimal essential media, 25% heatꢀinactivated horse serum, 25%
Earle’s balanced salt solution, 6.5 mg/ml glucose (SigmaꢀAldrich, StꢀLouis, MO), and penicillin and
glutamine supplements (All from Invitrogen, Carlsbad, CA), and was replaced every 2–3 days. Slices were
cultured for 13 days in vitro (DIV13) to allow clearance of debris and myelination to occur. For
remyelination studies, slices were incubated with 0.5 mg/ml LPC for overnight, and then transferred to the
culture medium for 24 h. Afterward, slices were treated with 4cꢀP or 4aꢀP for 7 days before being
processed for immunohistochemistry.
Cerebellar slices were fixed with 4% paraformaldehyde and incubated with rat antiꢀmyelin basic protein
(MBP, 1:1000, Abcam) and rabbit antiꢀcalbindin Dꢀ28K (CBD, 1: 4000, Swant, Switzerland) at 4 °C
overnight. MBP and CBD were used for visualizing myelin protein and Purkinje cell bodies and axonal
processes, respectively. The slices were incubated with 1:500 goat anti rat IgG Alexa Fluor 594 and goat
antiꢀrabbit IgG Alexa Fluor 488 (Life Technologies), and then mounted and cover slipped. A SP8 confocal
microscopy was used for immunofluorescence image acquisition. MBP and CBD immunostaining was imaged at
×40 magnification, and multiple images were captured from each slices. The areas covered by axonal processes
singly stained for CBD or doubly stained for CBD and MBP immunoreactivity were analyzed using a macro with
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ImageꢀJ (NIH). The Myelination Index was calculated by dividing CBD+MBP double immunoreactivity/area unit
with CBD single immunoreactivity/area unit as a measure of the amount of myelination per axon area. Quantitative
data were analyzed statistically using ANOVA, followed by the Dunnett's Multiple Comparison Test.
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AUTHOR INFORMATION
Corresponding Authors
*M.G.Y.: Phone, 609ꢀ252ꢀ3234; Eꢀmail, michael.yang@bms.com
*A.J.D.: Phone, 609ꢀ252ꢀ3593; Eꢀmail, alaric.dyckman@bms.com
ACKNOWLEDGMENTS
The authors are also grateful to the members of the Department of Discovery Synthesis and the
Biocon/BMS Research Center for their collaboration on this project.
ABBREVIATIONS USED
BLR, blood lymphocyte reduction assay; CDI, carbonyldiimidazole; CNS, central nervous system; EAE,
experimental autoimmune encephalomyelositis;
SꢀIpcꢀBH2, Sꢀisopinocampheyl borane; LiHMDS, lithium
bis(trimethylsilyl)amide; LDA, lithium diisopropylamide; LPC, lysophosphatidylcholine; MS, multiple
sclerosis; S1P, sphingosineꢀ1ꢀphosphate; SFC, supercritical fluid chromatography.
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Quackenbush, E.; Xie, J.; Milligan, J.; Thornton, R.; Shei, GꢀJ.; Card, D.; Keohane, CA.; Rosenbach,
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Fingolimod (FTY720) enhances remyelination following demyelination of organotypic cerebellar
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