Regioselective Epoxide Ring Opening for the Stereospecific Scale-Up Synthesis of BMS-960, A Potent and Selective Isoxazole-Containing S1P1 Receptor Agonist

Article abstract of DOI:10.1021/acs.oprd.6b00366

This article presents a stereospecific scale-up synthesis of (S)-1-((S)-2-hydroxy-2-(4-(5-(3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)-1,2,4-oxadiazol-3-yl)phenyl)ethyl)piperidine-3-carboxylic acid (BMS-960), a potent and selective isoxazole-containing S1P

Full text of DOI:10.1021/acs.oprd.6b00366

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Full Paper
Regioselective Epoxide Ring Opening for the Stereospecific
Scale-Up Synthesis of BMS-960, a Potent and
Selective Isoxazole-Containing S1P1 Receptor Agonist
Xiaoping Hou, Huiping Zhang, Bang-Chi Chen, Zhiwei Guo, Amarjit Singh, Animesh Goswami,
John L Gilmore, James E Sheppeck, Alaric J. Dyckman, Percy H Carter, and Arvind Mathur
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.6b00366 • Publication Date (Web): 12 Jan 2017
Downloaded from http://pubs.acs.org on January 15, 2017
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Organic Process Research & Development
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Regioselective Epoxide Ring Opening for the
Stereospecific ScaleꢀUp Synthesis of BMSꢀ960, a Potent
and Selective IsoxazoleꢀContaining S1P1 Receptor
Agonist
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Xiaoping Hou*,1, Huiping Zhang1, Bang-Chi Chen1, Zhiwei Guo2, Amarjit Singh2, Animesh Goswami2, John L.
Gilmore1, James E. Sheppeck1, Alaric J. Dyckman1, Percy H. Carter1, Arvind Mathur1
Discovery Chemistry1, BristolꢀMyers Squibb, Princeton, New Jersey, 08540, United States
Chemical & Synthetic Development2, BristolꢀMyers Squibb, New Brunswick, New Jersey, 08903,
United States
*Corresponding author: xiaoping.hou@bms.com
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ABSTRACT: This article presents a stereospecific scaleꢀup synthesis of (S)ꢀ1ꢀ((S)ꢀ2ꢀhydroxyꢀ2ꢀ(4ꢀ(5ꢀ
(3ꢀphenylꢀ4ꢀ(trifluoromethyl)isoxazolꢀ5ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)phenyl)ethyl)piperidineꢀ3ꢀcarboxylic
acid (BMSꢀ960), a potent and selective isoxazoleꢀcontaining S1P1 receptor agonist. The process
highlights an enzymatic reduction of αꢀbromo ketone toward the preparation of (S)ꢀbromo alcohol, a key
precursor of (S)ꢀ4ꢀ(oxiranꢀ2ꢀyl)benzonitrile. A regioꢀselective and stereoꢀspecific epoxide ring opening
reaction was also optimized along with improvements to 1,2,4ꢀoxadiazole formation, hydrolysis and
crystallization. The improved process was utilized to synthesize batches of BMSꢀ960 for Ames testing
and other toxicological studies.
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KEYWORDS: Enzymatic reduction, Epoxide ring opening, 1,2,4ꢀoxadiazole, Stereospecific synthesis,
S1P1 receptor agonist, Process optimization.
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INTRODUCTION
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Sphingosineꢀ1ꢀphosphate (S1P) is the endogenous ligand for the sphingosineꢀ1ꢀphophate
receptors (S1P1ꢀ5) and triggers a number of cellular responses through their stimulation. S1P and its
interaction with the S1P receptors play a significant role in a variety of biological processes including
vascular stabilization, heart development, lymphocyte homing, and cancer angiogenesis. Agonism of
S1P1, especially, has been shown to play an important role in lymphocyte trafficking from the thymus
and secondary lymphoid organs, inducing immunosuppression, which has been established as a novel
mechanism of treatment for immune diseases and vascular diseases.1ꢀ7
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During the course of our S1P1 receptor agonist program, BMSꢀ960 (Figure 1) was identified as
a lead backup candidate for further biological and toxicological studies.8 Herein, we report an efficient
stereoselective scale up synthesis of BMSꢀ960 to supply the sufficient amount of API for such studies.
Figure 1. (S)ꢀ1ꢀ((S)ꢀ2ꢀhydroxyꢀ2ꢀ(4ꢀ(5ꢀ(3ꢀphenylꢀ4ꢀ(trifluoromethyl)isoxazolꢀ5ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀ
yl)phenyl)ethyl)piperidineꢀ3ꢀcarboxylic acid, HCl (BMSꢀ960)
RESULTS AND DISCUSSIONS
Original Synthesis of (S)-1-((S)-2-hydroxy-2-(4-(5-(3-phenyl-4-(trifluoromethyl)isoxazol-5-
yl)-1,2,4-oxadiazol-3-yl)phenyl)ethyl)piperidine-3-carboxylic acid 1 (BMS-960). To prepare (S)ꢀ1ꢀ
((S)ꢀ2ꢀhydroxyꢀ2ꢀ(4ꢀ(5ꢀ(3ꢀphenylꢀ4ꢀ(trifluoromethyl)isoxazolꢀ5ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)phenyl)ethyl)
piperidineꢀ3ꢀcarboxylic acid (BMSꢀ960), a convergent synthesis (Scheme 1)8 was developed, which
required
two
advanced
intermediates,
((S)ꢀethyl
1ꢀ((S)ꢀ2ꢀhydroxyꢀ2ꢀ(4ꢀ((Z)ꢀN'ꢀ
hydroxycarbamimidoyl)phenyl)ethyl)piperidineꢀ3ꢀcarboxylate
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and
3ꢀphenylꢀ4ꢀ
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(trifluoromethyl)isoxazoleꢀ5ꢀcarboxylic acid 3. During the course of our synthesis of the first generation
candidate, BMSꢀ520,10a we developed a robust, scalable and highly regioselective [3+2]ꢀcycloaddition
and a chemoꢀselective hydrolysis, which enabled us to efficiently prepare the carboxylic acid 3 and the
corresponding acyl chloride 7 in large scale with high purity (Scheme 2).
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Scheme 1: Convergent Synthesis of BMSꢀ960
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Scheme 2: 3ꢀphenylꢀ4ꢀ(trifluoromethyl)isoxazoleꢀ5ꢀcarboxylic acid 3.10
Originally, a small amount of BMSꢀ960 was prepared for preliminary biological evaluation
through a nonꢀselective reduction and chiral separation (Scheme 3, step 2). Although the original route
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was amenable for a small scale preparation of analogues, there were a few issues to be addressed before
it could be efficiently scaled up to meet requirements of toxicological studies in terms of API quality
and quantity: a) step 1 Nꢀalkylation yield was only moderate. b) step 3 used isopropyl alcohol which
generates the ester exchange byproduct. c) step 4 oxadiazole formation yield was low, probably due to
the presence of an unprotected OH group. d) acyl fluoride was prepared from an unfavorable reagent,
cyanuric fluoride, which is very caustic and could even corrode the glassware during scaleꢀup.
Additionally, a limitation of the original route was that it could not offer a common intermediate for the
synthesis of other substituted piperidine candidates or diversification of analogues.
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Scheme 3: Original Synthesis of BMSꢀ960.8
To address the above issues, an alternative synthesis was proposed to efficiently scale up BMSꢀ960
(Scheme 7). Strategically, (S)ꢀ4ꢀ(oxiranꢀ2ꢀyl)benzonitrile (15) served as a key common intermediate,
which would allow for later stage modifications. This common intermediate was likely to expedite both
the medicinal chemistry parallel synthesis (Scheme 4, a) and later scaleꢀup campaigns (Scheme 4, b)
within the same βꢀamino alcohol chemotype.
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Scheme 4: the common intermediate approach.
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Our initial literature searches indicated that epoxides could be quickly prepared in a single step from the
corresponding aldehydes under a condition of trimethylsulfonium iodide/KOH.9 However, the yield was
only 37% for our substrate, 4ꢀformylbenzonitrile. Alternatively, we proposed a twoꢀstep method for
epoxide formation. Starting with the commercial prochiral αꢀbromoketone 8, we initially piloted a
racemic reduction with NaBH4 to quickly access the racemic secondary alcohol 14 for later stage
chemistry exploration. We noticed that the secondary alcohol 14 was unstable and was directly
converted to the racemic epoxide 15 under basic conditions with 90% yield for the two steps. However,
chiral separation was necessary to obtain the chirally pure desired (S)ꢀepoxide 15, which was not
11ꢀ13
favorable for a large scale. We therefore shifted our focus to an asymmetric reduction
to prepare
optically pure alcohol 14.
Considering the scale, cost and availability, we selected Bꢀ
14,15
chlorodiisopinocampheylborane (Aldrich: DIPꢀChlorideTM) as a chiral reducing agent
to convert
prochiral ketone 8 to the chiral alcohol 14 (Scheme 5). The preliminary results were very encouraging.
(+) DIPꢀChlorideTM gave the desired enantiomer 14 with 95% chiral purity and >85% chemical
conversion under mild conditions (0oC to RT). Despite these encouraging results, chiral separation was
still needed to remove the residual undesired enantiomer and so we decided to pursue an enzymatic
reduction with the goal of achieving the same reduction with even higher enantiomeric excess.
Scheme 5: Asymmetic chemical reduction of bromoketone 8
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To this end, the enzymatic reduction of αꢀbromoketone 8 to αꢀbromohydrin 14 followed by
facile formation of epoxide 15 now became our focus.16,17 At the beginning, we explored the enzyme
reduction of (S)ꢀethyl 1ꢀ(2ꢀ(4ꢀcyanophenyl)ꢀ2ꢀoxoethyl)piperidineꢀ3ꢀcarboxylate hydrobromide 9 to the
chiral alcohol 10 in the original synthesis of BMSꢀ960 (Scheme 3). Initial screening of a number of both
NADHꢀ and NADPHꢀdependent ketoreductases proved to be very encouraging. Ketoreductases with
(S)ꢀ and (R)ꢀdiastereoselectivity were identified producing either the desired chiral (S)ꢀalcohol 10 or the
undesired chiral (R)ꢀalcohol respectively. Eight ketoreductases (KREDꢀ106, KREDꢀ111, KREDꢀ114,
KREDꢀ115, KREDꢀ127, KREDꢀ145, ESꢀKREDꢀ109 and ESꢀKREDꢀ136) showed high conversion and
diastereomeric excess (de) for 10. In order to accommodate the needs of the common intermediate
approach (Scheme 6), we carried out enzyme screening for the reduction of αꢀbromoketone 8 with
appropriate cofactor. The reaction medium for NADPH dependent ketoreductases contained 250 mM
potassium phosphate (pH 7.0), 0.5 mM dithiothreitol, 2 mM magnesium sulfate, 1.1 mM NADP+, 80
mM Dꢀglucose, 10 U/mL glucose dehydrogenase. Whereas, screening of NADHꢀdependent enzymes
was performed in a medium which was composed of the same above components except that 1.3 mM
NAD+ rather than 1.1 mM NADP+ was used. In general, screening experiments were set up with 0.5 mL
of reaction medium, 1 mg enzyme and a stock solution of αꢀbromoketone 8 5.0 mg in 10 µL DMSO.
The reaction mixtures were stirred at 30°C and 300 RPM for 24 hours. The crude reaction mixtures
were filtered and analyzed by HPLC to determine the chemical conversion and ee. Among the
approximate 250 ketoreductase enzymes screened, seventeen ketoreductase enzymes showed >99%
conversion with >99% ee for the desired (S)ꢀalcohol 14 (Table 1). On the other hand, twenty one
ketoreductase enzymes showed >95% conversion with >95% ee for the undesired (R)ꢀalcohol 18 (Table
2). Many other ketoreductases showed lower conversions of the αꢀbromoketone 8 producing either
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alcohol 14 or 18 with varying degree of selectivities. Ultimately, ketoreductase enzyme KREDꢀNADHꢀ
110 was selected for the reduction of αꢀbromoketone 8 to (S)ꢀalcohol 14 based on its activity,
selectivity, and substrate to enzyme ratio. Though both NADH and NADPH cofactors have to be
regenerated for cost reduction, KREDꢀNADHꢀ110 also has the additional advantage of requiring the
lower cost NADH coꢀfactor.
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Table 1: Screening of Ketoreductases ꢀ Enzymes producing chiral (S)ꢀalcohol 14 preferentially
Conversion
Ketoreductase
Supplier
Cofactor
ee (%) of 14
(%)
CREDꢀA601
CREDꢀA311
CREDꢀA291
CREDꢀA401
CREDꢀA161
ESꢀKREDꢀ119
ESꢀKREDꢀ135
ESꢀKREDꢀ126
KREDꢀ136
KREDꢀ149
KREDꢀ150
KREDꢀ155
KREDꢀ162
ALMAC
ALMAC
ALMAC
ALMAC
ALMAC
EnzySource
EnzySource
EnzySource
Codexis
Codexis
Codexis
Codexis
Codexis
NADPH
NADPH
NADPH
NADPH
NADH
NADPH
NADPH
NADH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADH
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
99.7
99.7
100
99.4
99.2
99.2
100
100
100
100
100
100
100
99.1
99.2
KREDꢀ177
Codexis
Codexis
Codexis
Codexis
KREDꢀNADHꢀ110
KREDꢀNADHꢀ111
KREDꢀNADHꢀ124
NADH
NADH
Table 2: Screening of Ketoreductases ꢀ Enzymes producing chiral (R)ꢀalcohol 18 preferentially
Conversion
Ketoreductase
Supplier
Cofactor
ee (%) of 18
(%)
CREDꢀA281
CREDꢀA181
CREDꢀA371
CREDꢀA131
ESꢀKREDꢀ101
ESꢀKREDꢀ127
KREDꢀ104
KREDꢀ130
KREDꢀ131
KREDꢀ132
ALMAC
ALMAC
ALMAC
ALMAC
EnzySource
EnzySource
Codexis
Codexis
Codexis
Codexis
NADPH
NADPH
NADPH
NADH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
100
100
99
98
100
96
99
100
99
100
98
100
100
96
97
100
100
98
100
100
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KREDꢀ142
KREDꢀ148
KREDꢀ161
Codexis
Codexis
Codexis
Codexis
Codexis
Codexis
Codexis
Codexis
Codexis
Codexis
Codexis
NADPH
NADPH
NADPH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
100
100
100
98
100
100
100
100
96
96
98
100
100
100
100
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8
KREDꢀNADHꢀ102
KREDꢀNADHꢀ101
KREDꢀNADHꢀ105
KREDꢀNADHꢀ108
KREDꢀNADHꢀ112
KREDꢀNADHꢀ117
KREDꢀNADHꢀ123
KREDꢀNADHꢀ126
96
99
100
100
98
100
100
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Under basic conditions, the chiral alcohol 14 was readily transformed into (S)ꢀepoxide 15
(Scheme 6). The conditions for the reduction of αꢀbromoketone 8 to the chiral alcohol and the
conversion to the (S)ꢀepoxide 15 were subsequently optimized in terms of chemical conversion, ee,
substrate to enzyme ratio, temperature, and reaction time. A very low loading of ketoreductase KREDꢀ
NADHꢀ110 was sufficient to effect the complete reduction of 100g of αꢀbromoketone 8 in 5 hours at
40oC at a substrate concentration of 40g/L with a substrate to enzyme ratio of 200:1 and substrate to
NAD cofactor ratio of 25:1. The cofactor was regenerated by a small amount (8 kU) of the glucose
dehydrogenase enzyme and glucose (96g). The chiral alcohol 14 was extracted in MTBE, then readily
converted to (S)ꢀepoxide 15 by sodium tertꢀbutoxide. After work up, the pure chiral (S)ꢀepoxide 15 was
isolated as a stable solid in 93% yield, 99.9% AP, 100% ee.
Scheme 6. Synthesis of the common intermediate (15)
With a synthesis of the chiral epoxide inꢀhand, our attention then turned to the subsequent ringꢀ
opening reaction needed to install the aminoꢀester portion of the molecule (Scheme 7). Under a
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catalytic DMAP condition at 50oC, this proceeded as a highly regioselective and stereosepecfic process
to afford 10 which was successfully recrystallized to give the amino alcohol with high ee and chemical
purity. The secondary alcohol was now protected with a TBDMS group and as anticipated, the
oxadiazole formation yield was improved from 50% to 88% with this protecting group change. During
amidooxime formation, hydroxyl amine HCl salt was replaced with hydroxyl amine aqueous free base
to avoid the previously needed neutralization and IPA was replaced with EtOH at RT rather than reflux
to avoid ester exchange. Under the above mild conditions, the amidooxime formation proceeded with
90% yield. During the oxadiazole formation, acyl chloride rather than acyl fluoride was prepared to
avoid the corrosive reagent, cyanuric fluoride.
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Scheme 7: Alternative Synthesis of BMSꢀ960
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In conclusion, a practical enzymatic reduction to asymmetrically reduce the bromoketone 8 was
developed. Furthermore, a novel regioselective and stereospecific epoxide ring opening procedure was
established. The improved process enabled us to efficiently prepare multiple batches of BMSꢀ960 for
toxicological studies including Ames testing.
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EXPERIMENTAL SECTION
All reagents were obtained from commercial sources and used without further purification unless
otherwise stated. All reactions were carried out under a nitrogen atmosphere. All glassware was dried
and purged with nitrogen before use. Unless otherwise stated, all reactions were monitored by Shimadzu
LCMS system using the following method: Phenomenex C18 column 10 ꢁm 4.6 X 50 mm. Solvent: A =
10% methanol/90% water with 0.1% TFA; B = 90% methanol/10% water with 0.1% TFA. Gradient: 0ꢀ
100% B over 4 min. Flow: 4 mL/min, Wavelength: 220 nm. HPLC methodꢀ1 was used for achiral
analysis of enzymatic reactions and performed on a Phenomenex Synergi MAXꢀRP column (4 ꢀm, 50 ×
4.6 mm) at ambient temperature with a flow rate of 2 mL/min and a gradient of solvent A (0.01 M
NH4OAc in CH3CNꢀWater 5:95) and solvent B (0.01 M NH4OAc in CH3CN ꢀWater 95:5) from 30% to
60% solvent B over 3 min and holding at 60% B for an additional 2 min. The detection was by UV at
220 nm. The retention times were 1.7 min for 14 or 18, 1.9 min for 15 or its (R)ꢀisomer, and 2.7 min for
8. HPLC methodꢀ2 was used for chiral separation of 14 and 18 and was performed on a Chiralpak ASꢀ
RH column (5 ꢀm, 150 × 4.6 mm) with a flow rate of 0.5 mL/min at ambient temperature and a gradient
of Solvent A (0.01 M NH4OAc in CH3CNꢀWater 10:90) and Solvent B (0.01 M NH4OAc in CH3CNꢀ
Water 90:10) holding at 35% solvent B for the first 12 min, then from 35% to 60% solvent B over 3
min. The detection was by UV at 220nm. The retention times were 9.1 min for 14, 11.4 min for 18.
HPLC methodꢀ3 was used for chiral separation of 15 and its (R)ꢀisomer and was performed on a
Chiralpak ADꢀRH column (5 ꢀm, 150 × 4.6 mm) with a flow rate of 0.5 mL/min at ambient temperature
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and a gradient of Solvent A (0.01 M NH4OAc in CH3CNꢀWater 10:90) and Solvent B (0.01 M NH4OAc
in CH3CNꢀWater 90:10) holding at 35% solvent B for the first 12 min, then from 35% to 80% solvent B
over 3 min, and holding at 80% B for additional 5 min. The detection was by UV at 220nm. The
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retention times were 16.7 min for 15, 14.0 min for its (R)ꢀisomer. All H NMR and C NMR spectra
were recorded on a Bruker 400 MHz spectrometer using DMSOꢀd6 or CDCl3 as the solvents unless
otherwise specified. Codexis ketoreductase KREDꢀNADHꢀ110 (Lot# 2011608WW, 25 U/mg), Glucose
dehydrogenase (GDHꢀ102, Lot# 011005CL, 90 U/mg) and NAD (Free acid, MW 663.4, Boehringer
Mannheim, Lot83431124ꢀ72, Feb/97), and the ketone 2ꢀbromoꢀ4’ꢀcyanoacetophenone (CAS number
20099ꢀ89ꢀ2, from Aldrich, Lot 08528KH, chemical purity 96%) were used. The 0.2 M, pH 6.0 sodium
phosphate buffer was prepared and the recipe for 1 L buffer is Na2HPO4 24.6 mmol, FW 141.96, 3.492
g; NaH2PO4 175.4 mmol, FW 119.98, 21.044 g and DI water 1 L.
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(S)-4-(oxiran-2-yl)benzonitrile (15). To a 5ꢀL jacketed reactor were added 2 L of sodium
phosphate buffer (0.2 M, pH 6.0), 96 g of Dꢀglucose (1.2 equivalents), 4 g of NAD (βꢀNicotinamide
adenine dinucleotide), 8 kU of GDH (Glucose dehydrogenase), 500 mg of ketoreductase KREDꢀ
NADHꢀ110, followed by 0.5 L of additional sodium phosphate buffer (0.2 M, pH 6.0) to rinse the
different containers. The mixture was stirred to dissolve all solids and showed a pH of 5.5, which was
adjusted to 6.0 with 1 M NaOH (About 90 mL). After 30min, a solution of 2ꢀbromoꢀ4’ꢀ
cyanoacetophenone (8) (100 g, 446 mmol) in 250 mL DMSO was added, followed by an additional 50
mL DMSO, which was used to rinse the container. The mixture was stirred at 40 °C throughout the
reaction (5 h). The pH was maintained between 5.5 and 6.1 with 1 M NaOH (total consumption about
450 mL). The mixture was extracted with 2 L of MTBE. The aqueous layer was extracted a second time
with 1.6 L of MTBE. The combined MTBE extracts was washed with 0.5 L of 25% brine. The crude
bromohydrin 14 solution was treated with sodium tertꢀbutoxide solid (103 g, 2.4 equivalents,
portionwise) at ambient temperature for 1 h until no bromohydrin 14 remained (checked by HPLC). The
mixture was filtered through a pad of Celite. The cake was washed twice with 100 mL of MTBE. The
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combined filtrate and MTBE washing was washed with 0.5 L of sodium phosphate buffer (0.2 M, pH
6.0) and with 0.5 L of 25% brine. Removal of solvent from the MTBE solution on a rotary evaporator at
30 °C gave a solid, which was transferred into a dish and further dried in a vacuum oven at ambient
temperature for 3 days to give 60.4 g of the (S)ꢀepoxide 15 as a yellowish solid, yield 93.2%, AP 99.9%,
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ee 100%. H NMR (400MHz, CDCl3) δ ppm 7.75 ꢀ 7.58 (m, 2H), 7.47 ꢀ 7.35 (m, 2H), 3.91 (dd, J=4.0,
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2.4 Hz, 1H), 3.20 (dd, J=5.5, 4.0 Hz, 1H), 2.76 (dd, J=5.6, 2.5 Hz, 1H); MS m/e 146.1(M+H+); HPLC
(XBridge 5ꢀ C18 4.6x50 mm, 4 mL/min, Solvent A: 10 % MeOH/water with 0.2 % H3PO4, Solvent B:
90 % MeOH/water with 0.2 % H3PO4, gradient with 0ꢀ100 % B over 4 minutes): 1.16 minutes.
(S)-ethyl 1-((S)-2-(4-cyanophenyl)-2-hydroxyethyl)piperidine-3-carboxylate (10). (S)ꢀ4ꢀ
(oxiranꢀ2ꢀyl)benzonitrile (10.0 g, 68.9 mmol) was dissolved in IPA (100 mL),
4ꢀ
Dimethylaminopyridine (1.683 g, 13.78 mmol) was added, followed by (S)ꢀethyl piperidineꢀ3ꢀ
o
carboxylate (10.8 g, 68.9 mmol). The reaction mixture was heated up to 50 C overnight. The reaction
mixture was then cooled to RT. EtOAc (200 ml) was added, followed by 80 ml of H2O. The organic
layer was separated and concentrated under vacuum to give (S)ꢀethyl 1ꢀ((S)ꢀ2ꢀ(4ꢀcyanophenyl)ꢀ2ꢀ
hydroxyethyl)piperidineꢀ3ꢀcarboxylate (18.9 g, 59.9 mmol, 87 % yield). Recrystallization The crude
product was recrystallized from EtOH / EtOAc / H2O (3/2/2, v/v) (8ml / 1g crude) to give the desired
1
product 15g (80% recovery yield, 99% chemical purity, 99.6% EE) as a crystalline offꢀwhite solid. H
NMR (400MHz, CDCl3) δ 7.69 ꢀ 7.59 (m, 2H), 7.50 (d, J=7.9 Hz, 2H), 4.78 (dd, J=10.6, 3.3 Hz, 1H),
4.18 (q, J=7.0 Hz, 3H), 3.14 (d, J=9.5 Hz, 1H), 2.70 ꢀ 2.52 (m, 3H), 2.52 ꢀ 2.30 (m, 3H), 2.04 ꢀ 1.87 (m,
1H), 1.83 ꢀ 1.54 (m, 3H), 1.29 (t, J=7.2 Hz, 3H); 13C NMR (101MHz, CDCl3) δ 173.7, 147.7, 132.2 (s,
2C), 126.4 (s, 2C), 118.8, 111.2, 68.4, 65.9, 60.6, 54.5, 54.4, 41.7, 26.6, 24.6, 14.2; MS m/e
303.1(M+H+); HPLC (XBridge 5ꢀ C18 4.6x50 mm, 4 mL/min, Solvent A: 10 % MeOH/water with 0.2
% H3PO4, Solvent B: 90 % MeOH/water with 0.2 % H3PO4, gradient with 0ꢀ100 % B over 4 minutes):
0.95 minutes.
(S)-ethyl
1-((S)-2-(tert-butyldimethylsilyloxy)-2-(4-cyanophenyl)ethyl)
piperidine-3-
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carboxylate. To a mixture of (S)ꢀethyl 1ꢀ((S)ꢀ2ꢀ(4ꢀcyanophenyl)ꢀ2ꢀhydroxyethyl)piperidineꢀ3ꢀ
carboxylate (17.00 g, 56.2 mmol) and DIPEA (17.68 ml, 101 mmol) in DCM (187 ml) was added tertꢀ
butyldimethylsilyl trifluoromethaneꢀsulfonate (16 ml, 69.6 mmol) slowly. The reaction was complete in
2 hours by HPLC. The reaction mixture (a light brown solution) was quenched with ice water. The
aqueous was extracted with DCM. Organic phase was combined, dried with Na2SO4. After
concentration, the crude material was further purified by chromatography (330g silica, 10ꢀ30% ethyl
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acetate/hexane)
to
afford
(S)ꢀethyl
1ꢀ((S)ꢀ2ꢀ(tertꢀbutyldimethylsilyloxy)ꢀ2ꢀ(4ꢀ
1
cyanophenyl)ethyl)piperidineꢀ3ꢀcarboxylate (22.25 g, 53.4 mmol, 95 % yield) as a viscous film. H
NMR (400MHz, CDCl3) δ 7.60 (d, J=8.4 Hz, 2H), 7.46 (d, J=8.4 Hz, 2H), 4.78 (t, J=6.1 Hz, 1H), 4.22 ꢀ
4.05 (m, 2H), 2.91 ꢀ 2.83 (m, 1H), 2.81 ꢀ 2.73 (m, 1H), 2.59 (dd, J=13.3, 6.7 Hz, 1H), 2.53 ꢀ 2.44 (m,
1H), 2.39 (dd, J=13.2, 5.5 Hz, 1H), 2.36 ꢀ 2.30 (m, 1H), 2.19 ꢀ 2.08 (m, 1H), 1.96 ꢀ 1.84 (m, 1H), 1.74 ꢀ
1.63 (m, 1H), 1.53 ꢀ 1.38 (m, 2H), 1.25 (t, J=7.2 Hz, 3H), 0.89 (s, 9H), 0.09 (s, 3H), ꢀ0.07 (s, 3H);
HRMS (ESI) m/e 417.25765 [(M + H)+, calcd for C23H37N2O3Si 417.25680]. HPLC (XBridge 5ꢀ C18
4.6x50 mm, 4 mL/min, Solvent A: 10 % MeOH/water with 0.2 % H3PO4, Solvent B: 90 % MeOH/water
with 0.2 % H3PO4, gradient with 0ꢀ100 % B over 4 minutes): 3.05 minutes.
(S)-ethyl 1-((S)-2-(tert-butyldimethylsilyloxy)-2-(4-((Z)-N'-hydroxycarbamimidoyl)phenyl)
ethyl)piperidine-3-carboxylate (16).
(S)ꢀethylꢀ1ꢀ((S)ꢀ2ꢀ(tertꢀbutyldimethylsilyloxy)ꢀ2ꢀ(4ꢀ
cyanophenyl)ethyl)piperidineꢀ3ꢀcarboxylate (31.0 g, 74.4 mmol) was dissolved in EtOH (248 ml).
Hydroxylamine (50% aq.) (6.84 ml, 112 mmol) was added and stirred at RT overnight. Then, all
volatiles were removed in vacuum. The residue was purified by chromatography (330g silica, 0% ꢀ 50%
ethyl
acetate/hexane).
(S)ꢀethylꢀ1ꢀ((S)ꢀ2ꢀ(tertꢀbutyldimethylsilyloxy)ꢀ2ꢀ(4ꢀ((Z)ꢀN'ꢀ
hydroxycarbamimidoyl) phenyl) ethyl)piperidineꢀ3ꢀcarboxylate (31 g, 68.9 mmol, 93 % yield) was
obtained as a white foam. 1H NMR (400MHz, CDCl3) δ 8.39 (d, J=4.6 Hz, 1H), 7.57 (d, J=8.4 Hz, 2H),
7.36 (d, J=8.1 Hz, 2H), 4.88 (br. s., 2H), 4.84 ꢀ 4.76 (m, 1H), 4.18 ꢀ 4.06 (m, 2H), 2.97 (dd, J=11.1, 3.9
Hz, 1H), 2.83 (dt, J=7.4, 3.9 Hz, 1H), 2.61 (dd, J=13.4, 7.3 Hz, 1H), 2.57 ꢀ 2.48 (m, 1H), 2.42 (dd,
J=13.2, 4.6 Hz, 1H), 2.30 (t, J=10.7 Hz, 1H), 2.17 ꢀ 2.07 (m, 1H), 1.97 ꢀ 1.87 (m, 1H), 1.73 ꢀ 1.63 (m,
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1H), 1.62 ꢀ 1.50 (m, 1H), 1.48 ꢀ 1.34 (m, 1H), 1.25 (t, J=7.2 Hz, 3H), 0.89 (s, 9H), 0.08 (s, 3H), ꢀ0.09 (s,
3H); 13C NMR (101MHz, CDCl3) δ 174.3, 152.6, 146.5, 131.1, 126.4, 125.4, 73.0, 68.0, 60.3, 56.2,
54.7, 42.0, 26.8, 25.9 (s, 3C), 24.7, 18.3, 14.2, 0.0, ꢀ4.7 (d, J=4.6 Hz, 2C); HRMS (ESI) m/e 450.27922
[(M + H)+, calcd for C23H40O4N3Si 450.27826]; HPLC (XBridge 5ꢀ C18 4.6x50 mm, 4 mL/min,
Solvent A: 10 % MeOH/water with 0.2 % H3PO4, Solvent B: 90 % MeOH/water with 0.2 % H3PO4,
gradient with 0ꢀ100 % B over 4 minutes): 2.25 minutes.
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3-phenyl-4-(trifluoromethyl)isoxazole-5-carboxylic
acid
(3).
Ethyl
3ꢀphenylꢀ4ꢀ
ater
(trifluoromethyl)isoxazoleꢀ5ꢀcarboxylate (40g, 140 mmol) was dissolved in THF (240 mL) and
w
(24.01 mL), then cooled to 0°C (internal temperature). Subsequently, LiOH monohydrate (5.04 g, 210
mmol) was added in portions and monitored with HPLC. The reaction mixture was stirred overnight at
o
o
0 C. HPLC showed 17% AP of starting material. Then, 0.1eq (336mg) of LiOH was added at 0 C and
o
gradually warmed up to RT for 3 hours. The reaction mixture was reꢀcooled to 0 C, then diluted with
200mL ether and 200mL water. The aqueous phase was separated and washed with ether (2X50mL).
The organics were combined and extracted with water (3X30mL). The aqueous phases were combined,
o
cooled to 0 C, and then neutralized with 1N HCl and extracted with diethyl ether (3x200mL). Ether
layers were combined and dried with Na2SO4. After concentration, 3ꢀphenylꢀ4ꢀ
(trifluoromethyl)isoxazoleꢀ5ꢀcarboxylic acid (29.5 g, 114 mmol, 81 % yield) was obtained as pale solid
1
(purity 99%). HꢀNMR (CDCl3, 500 MHz) δ 7.44 ꢀ 7.68 (m, 5H), 6.2ꢀ7.0 (br, 1H); MS m/e
257.8(M+H+); HPLC (XBridge 5ꢀ C18 4.6x50 mm, 4 mL/min, Solvent A: 10 % MeOH/water with 0.2
% H3PO4, Solvent B: 90 % MeOH/water with 0.2 % H3PO4, gradient with 0ꢀ100 % B over 4 minutes):
2.45 minutes.
3-Phenyl-4-(trifluoromethyl)isoxazole-5-carbonyl
chloride
(7).
3ꢀphenylꢀ4ꢀ
(trifluoromethyl)isoxazoleꢀ5ꢀcarboxylic acid (29.2g, 114 mmol) was suspended in thionyl chloride (100
ml, 1370 mmol) at RT. Catalytic amount of DMF (a few drops) was added. The reaction mixture was
o
heated up to 90 C for 24 hours. The reaction was monitored with HPLC (Note: all HPLC samples were
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prepared by quenching an aliquot of reaction mixture with anhydrous methanol and monitoring for the
resulting methyl ester). HPLC at 24 hour indicated that the reaction was completed (peak to peak
conversion). The reaction mixture was cooled down to RT diluted with 100mL anhydrous toluene, then
concentrated in vacuum. The yellow oil residue was further pumped overnight under high vacuum to
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afford a gray solid (31.3g, 114mmol, 100% yield). HꢀNMR (CDCl3, 400 MHz) δ 7.50 ꢀ 7.63 (m, 5H).
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HPLC (XBridge 5ꢀ C18 4.6x50 mm, 4 mL/min, Solvent A: 10 % MeOH/water with 0.2 % H3PO4,
Solvent B: 90 % MeOH/water with 0.2 % H3PO4, gradient with 0ꢀ100 % B over 4 minutes): 3.29
minutes (methyl ester derivatized from MeOH quench).
(S)-ethyl
(trifluoromethyl)isoxazol-5-yl)-1,2,4-oxadiazol-3-yl)phenyl)ethyl)piperidine-3-carboxylate
(penultimate) (17) (S)ꢀethyl 1ꢀ((S)ꢀ2ꢀ(tertꢀbutyldimethylsilyloxy)ꢀ2ꢀ(4ꢀ((Z)ꢀN'ꢀ
hydroxycarbamimidoyl)phenyl)ethyl)piperidineꢀ3ꢀcarboxylate (32.6g, 72.5 mmol) was dissolved in
1-((S)-2-(tert-butyldimethylsilyloxy)-2-(4-(5-(3-phenyl-4-
o
anhydrous acetonitrile (145 ml) and cooled to ~3 C with ice bath. 3ꢀphenylꢀ4ꢀ
(trifluoromethyl)isoxazoleꢀ5ꢀcarbonyl chloride (19.98 g, 72.5 mmol) was dissolved in 50mL anhydrous
o
acetonitrile and added dropwise. The internal temperature was kept below 10 C during addition. Once
addition was completed, the reaction mixture was naturally warmed up to RT. At 30 minutes, HPLC
o
showed the 1st step was complete. The reaction mixture was reꢀcooled to below 10 C. DIPEA (18.99
o
ml, 109 mmol) was added slowly. After the addition, the reaction mixture was heated up to 55 C for 17
hours. HPLC/LCMS showed a complete conversion. All volatiles were removed in vacuum. The residue
was stirred in 250mL 20% ethyl acetate / hexane, DIPEA HCl salt was precipitated out, which was
removed via filtration. The filtrate was concentrated and purified by chromatography (3 X 330g silica,
0% to 50% ethyl acetate / hexane). (S)ꢀethyl 1ꢀ((S)ꢀ2ꢀ(tertꢀbutyldimethylsilyloxy)ꢀ2ꢀ(4ꢀ(5ꢀ(3ꢀphenylꢀ4ꢀ
(trifluoromethyl)isoxazolꢀ5ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)phenyl)ethyl)piperidineꢀ3ꢀcarboxylate (43g, 64.1
1
mmol, 88 % yield) was obtained a light yellow oil. H NMR (400MHz, CDCl3) δ 8.15 (d, J=8.4 Hz,
2H), 7.73 ꢀ 7.65 (m, 2H), 7.61 ꢀ 7.49 (m, 5H), 4.88 ꢀ 4.81 (m, 1H), 4.19 ꢀ 4.06 (m, 2H), 2.97 (dd,
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J=11.0, 3.7 Hz, 1H), 2.83 (d, J=11.4 Hz, 1H), 2.65 (dd, J=13.4, 7.0 Hz, 1H), 2.57 ꢀ 2.42 (m, 2H), 2.39 ꢀ
2.28 (m, 1H), 2.22 ꢀ 2.10 (m, 1H), 1.98 ꢀ 1.88 (m, 1H), 1.75 ꢀ 1.64 (m, 1H), 1.62 ꢀ 1.58 (m, 1H), 1.56 ꢀ
1.37 (m, 1H), 1.28 ꢀ 1.22 (m, 3H), 0.92 (s, 9H), 0.11 (s, 3H), ꢀ0.04 (s, 3H); 13C NMR (101MHz, CDCl3)
δ 174.2, 169.6, 163.7, 162.1, 148.9, 131.0, 129.31, 128.62, 127.5, 126.9, 125.9, 124.1, 73.0, 67.9, 60.3,
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56.3, 54.7, 42.1, 26.8, 25.9, 24.8, 18.3, 14.2, 0.0, ꢀ4.7; F NMR (376MHz, CDCl3) δ ꢀ55.04; HRMS
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(ESI) m/e 671.28867 [(M + H)+, calcd for C34H42O5N4F3Si 671.28711]; HPLC (XBridge 5ꢀ C18 4.6x50
mm, 4 mL/min, Solvent A: 10 % MeOH/water with 0.2 % H3PO4, Solvent B: 90 % MeOH/water with
0.2 % H3PO4, gradient with 0ꢀ100 % B over 4 minutes): 4.07 minutes
(S)-1-((S)-2-hydroxy-2-(4-(5-(3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)-1,2,4-oxadiazol-3-
yl)phenyl)ethyl)piperidine-3-carboxylic acid,
HCl (13, BMS-960) (S)ꢀethyl 1ꢀ((S)ꢀ2ꢀ(tertꢀ
butyldimethylsilyloxy)ꢀ2ꢀ(4ꢀ(5ꢀ(3ꢀphenylꢀ4ꢀ(trifluoromethyl)isoxazolꢀ5ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀ
yl)phenyl)ethyl)piperidineꢀ3ꢀcarboxylate (42g, 62.6 mmol) was dissolved in 1,4ꢀdioxane (150 ml) and
6N HCl (150 ml) was added (during addition, some cloudiness was observed which quickly disappeared
upon stirring. By the end of addition, the reaction mixture became cloudy again ). The reaction mixture
o
was heated to 65 C for 6 hours, and EtOH was distilled out to push equilibrium forward. 1,4ꢀDioxane
was removed in vacuum. The residue was reꢀdissolved in CH3CN/water (1:1 v/v) and lyophilized
overnight. The crude ~37g solid/foam was obtained with ~3% impurity.
Recrystallization After lyophilization, ~37g light yellow solid/film was transferred to 2 liter
o
threeꢀnecked flask with acetonitrile (740mL). The suspension was heated up to 60 C, and then 15mL
water was added gradually. A clear solution was observed, which was cooled to RT and concentrated to
a small volume (very viscous but no precipitates). To the above viscous oil, 740mL ethyl acetate was
o
added and vigorously stirred. A clear solution was observed. When heated up to 60 C, the ethyl acetate
solution gradually became cloudy, then precipitates were observed. The suspension was cooled to RT
for 1 hour. The solid was collected and dried to afford (S)ꢀ1ꢀ((S)ꢀ2ꢀhydroxyꢀ2ꢀ(4ꢀ(5ꢀ(3ꢀphenylꢀ4ꢀ
(trifluoromethyl)isoxazolꢀ5ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)phenyl)ethyl)piperidineꢀ3ꢀcarboxylic acid,
HCl
1
(BMSꢀ960) (30g, 87% yield, > 99% EE, 99.5% purity, no single impurity >0.5%). H NMR (400MHz,
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DMSOꢀd6) δ 12.88 (br. s, 1H), 10.5 (br. s, 1H), 8.14 (d, J=8.6 Hz, 2H), 7.72 (d, J=8.4 Hz, 2H), 7.69 ꢀ
7.57 (m, 5H), 6.43 (br. s., 1H), 5.37 (d, J=10.8 Hz, 1H), 3.89 ꢀ 3.60 (m, 2H), 3.50 ꢀ 2.82 (m, 6H), 2.14 ꢀ
1.99 (m, 1H), 1.97 ꢀ 1.75 (m, 1H), 1.63 ꢀ 1.35 (m, 1H); 13C NMR (101MHz, CDCl3)
δ 172.8, 168.5, 164.0, 161.6, 155.4, 156.2, 131.2, 129.0, 128.9, 127.4, 127.2, 125.5, 124.3, 120.2, 111.6,
66.6, 63.0, 52.9, 52.2, 38.8, 25.0, 21.7; 19F NMR (376MHz, DMSOꢀd6) δ ꢀ54.16; Anal. calcd for
C26H23F3N4O5.HCl: C, 54.71; H, 4.36; N, 9.80. Found: C, 54.76; H, 3.94; N, 9.76; HRMS (ESI) m/e
529.17040 [(M + H)+, calcd for C26 H24 N4 O5 F3 529.16933].
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ACKNOWLEDGEMENTS
We are grateful to Jun Dai, Mike Witkus, Yingru Zhang, Sarah Traeger, Bethanne Warrack, and David
WangꢀIverson for their dedicated analytical support, BMS Biocon Research Center in India for
supplying the intermediate isoxazoleꢀ5ꢀcarboxylic acid. We acknowledge the contributions of Peng Li
and DauhꢀRurng Wu for chiral separation. We thank James Kempson for reviewing the manuscript.
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