Potent and Selective Agonists of Sphingosine 1-Phosphate 1 (S1P1): Discovery and SAR of a Novel Isoxazole Based Series

Article abstract of DOI:10.1021/acs.jmedchem.6b00089

Sphingosine 1-phosphate (S1P) is the endogenous ligand for the sphingosine 1-phosphate receptors (S1P_1-5) and evokes a variety of cellular responses through their stimulation. The interaction of S1P with the S1P receptors plays a fundamental physiological role in a number of processes including vascular development and stabilization, lymphocyte migration, and proliferation. Agonism of S1P₁, in particular, has been shown to play a significant role in lymphocyte trafficking from the thymus and secondary lymphoid organs, resulting in immunosuppression. This article will detail the discovery and SAR of a potent and selective series of isoxazole based full agonists of S1P₁. Isoxazole 6d demonstrated impressive efficacy when administered orally in a rat model of arthritis and in a mouse experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis.

Full text of DOI:10.1021/acs.jmedchem.6b00089

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Article
Potent and Selective Agonists of Sphingosine-1-Phosphate 1
S1P1): The Discovery and SAR of a Novel Isoxazole Based Series
(
Scott H. Watterson, Junqing Guo, Steven H. Spergel, Charles M Langevine, Robert V Moquin, Ding
Ren Shen, Melissa Yarde, Mary Ellen Cvijic, Dana Banas, Richard Liu, Suzanne J Suchard, Kathleen
M. Gillooly, Tracy Taylor, Sandra Rex-Rabe, David J. Shuster, Kim W. McIntyre, Georgia Cornelius,
Celia J. D´Arienzo, Anthony Marino, Praveen V. Balimane, Bethanne Warrack, Luisa Salter-Cid,
Murray McKinnon, Joel C. Barrish, Percy H Carter, William J Pitts, Jenny Xie, and Alaric J. Dyckman
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00089 • Publication Date (Web): 28 Feb 2016
Downloaded from http://pubs.acs.org on March 1, 2016
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Balimane, Praveen; Bristol-Myers Squibb Pharmaceutical Research and
Development
Warrack, Bethanne; Bristol-Myers Squibb Pharmaceutical Research and
Development
Salter-Cid, Luisa; Bristol-Myers Squibb Pharmaceutical Research and
Development
McKinnon, Murray; Bristol-Myers Squibb Pharmaceutical Research and
Development
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Barrish, Joel; Bristol-Myers Squibb Pharmaceutical Research and
Development
Carter, Percy; Bristol-Myers Squibb Pharmaceutical Research and
Development
Pitts, William; Bristol-Myers Squibb Pharmaceutical Research and
Development
Xie, Jenny; Bristol-Myers Squibb Pharmaceutical Research and
Development
Dyckman, Alaric; Bristol-Myers Squibb Pharmaceutical Research and
Development
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Potent and Selective Agonists of Sphingosine-1-
Phosphate 1 (S1P ): The Discovery and SAR of a
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Novel Isoxazole Based Series
Scott H. Watterson,* Junqing Guo, Steve H. Spergel, Charles M. Langevine, Robert V. Moquin, Ding
Ren Shen, Melissa Yarde, Mary Ellen Cvijic, Dana Banas, Richard Liu, Suzanne J. Suchard, Kathleen
Gillooly, Tracy Taylor, Sandra Rex-Rabe, David J. Shuster, Kim W. McIntyre, Georgia Cornelius, Celia
D’Arienzo, Anthony Marino, Praveen Balimane, Bethanne Warrack, Luisa Salter-Cid, Murray
McKinnon, Joel C. Barrish, Percy H. Carter, William J. Pitts, Jenny Xie, and Alaric J. Dyckman
BristolꢀMyers Squibb Research and Development, P.O. Box 4000, Princeton, New Jersey 08543
RECEIVED DATE
*
To whom correspondence should be addressed. Phone: 609ꢀ252ꢀ6778. Eꢀmail:
scott.watterson@bms.com.
Note: The authors declare no competing financial interest.
a
Abbreviations: S1P, Sphingosineꢀ1ꢀphosphate; S1P , Sphingosineꢀ1ꢀphosphate receptors 1ꢀ5; GTPγS,
1
ꢀ5
Guanosineꢀ5’ꢀOꢀ[gammaꢀthio]triphosphate; PK, pharmacokinetic; SAR, structureꢀactivity relationship;
SFC, super critical fluid chromatography; hERG, human etherꢀaꢀgoꢀgoꢀrelated gene.
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ABSTRACT: Sphingosineꢀ1ꢀphosphate (S1P) is the endogenous ligand for the sphingosineꢀ1ꢀphophate
receptors (S1P ) and evokes a variety of cellular responses through their stimulation. The interaction
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of S1P with the S1P receptors plays a fundamental physiological role in a number of processes
including vascular development and stabilization, lymphocyte migration, and proliferation. Agonism of
S1P , in particular, has been shown to play a significant role in lymphocyte trafficking from the thymus
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and secondary lymphoid organs, resulting in immunosuppression. This article will detail the discovery
and SAR of a potent and selective series of isoxazole based full agonists of S1P . Isoxazole 6d
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demonstrated impressive efficacy when administered orally in a rat model of arthritis and in a mouse
experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis.
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Introduction
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Sphingosineꢀ1ꢀphosphate (S1P) is a lysophopholipid, endogenous mediator that functions as a highꢀ
affinity ligand for five members of the endothelial differentiation gene class of Gꢀproteinꢀcoupled
receptors, S1P . The interaction of S1P with the S1P receptors plays a fundamental physiological role
1
ꢀ5
in a number of processes including vascular development and stabilization, lymphocyte migration, and
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proliferation. Levels of S1P in various tissues are regulated through the coordinated activities of
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biosynthetic kinases and biodegradative phosphatases and lyases. Specifically, S1P is derived from the
phosphorylation of sphingosine by two kinases, S1Pkinaseꢀ1 and S1Pkinaseꢀ2. Conversely, S1P is
degraded by S1P lyase and lysophospholipid phosphatases.
The S1P receptor is the predominant receptor expressed on lymphocytes and plays a crucial role in
1
lymphocyte egress from the thymus and lymph nodes to systemic circulation and ultimately to sites of
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tissue inflammation. The S1P concentration gradient differential between blood plasma and tissue is
sensed by the lymphocytes through the interaction of the S1P receptor with circulating S1P, resulting in
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,4
egress.
In support of this, conditional deletion of the S1P receptors expressed on lymphocytes in
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3,5,6
mice resulted in internalization of cellꢀmembrane expressed receptors.
The subsequent decrease of
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,5,6
S1P receptor activity correlated with sequestration of lymphocytes in the lymph nodes.
This was
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further supported in studies with conditional S1Pkinaseꢀdeficient mice in which the lack of S1P in the
2,6
plasma and lymph nodes resulted in a significant reduction in circulating lymphocytes.
Fingolimod (1) was shown in vivo to induce a significant, but reversible, reduction in circulating
7
lymphocytes, and as a result, 1 was found to be efficacious in several animal models of inflammation.
Although 1 is inactive toward the S1P1ꢀ5 receptors, a phosphorylated metabolic derivative 2
(fingolimodꢀP) generated through the stereospecific action of S1Pkinaseꢀ2, analogous to S1P, was found
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to act as a potent, full agonist of the S1P1,3,4,5 receptors with no activity toward S1P2. Additionally,
unlike S1P, fingolimodꢀP (2) agonism of S1P resulted in prolonged internalization and degradation of
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the receptor, thus acting as a “functional antagonist.” With this mechanism of action, lymphocytes are
no longer able to recognize and respond to changes in the S1P gradient, thus preventing their migration
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into circulation and tissue. With positive Phase III clinical results, fingolimod (1) was approved in 2010
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as the first oral diseaseꢀmodifying treatment for relapsingꢀremitting multiple sclerosis (RRMS).
Although fingolimod (1) has proven to be very effective in the treatment of RRMS, several concerns
emerged during clinical trials including cardiovascular effects (bradycardia and blood pressure
elevation), macular edema, decline in pulmonary function, and an extended halfꢀlife resulting in a
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prolonged pharmacologic effect after drug discontinuation. Initially, the cardiovascular effects were
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linked to the agonism of S1P , as supported by rodent studies, but more recent clinical data suggest that
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there is no solid correlation between rodent and human. In particular, clinical studies with S1P sparing
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agonists have revealed that the cardiovascular effects seen in humans are independent of S1P receptor
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1
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agonism. In fact, recent evidence suggests that the bradycardia in humans may be triggered by S1P1
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receptor activation.
With fingolimod’s clinical success, there has been an enormous effort to identify directꢀacting, full
1
2,13
agonists of the S1P receptor that do not require phosphorylation for pharmacological activity.
1
Additionally, most research efforts have focused on improving both the pharmacokinetic profile and the
receptor selectivity of fingolimod (1), including the work discussed in this article. Identification of a
compound with a shorter pharmacokinetic halfꢀlife would reduce concerns over the prolonged recovery
from lymphocyte suppression observed with 1 upon drug discontinuation, which could potentially
hinder recovery from an infection. Selectivity for the S1P receptor over the S1P receptor was
1
3
originally anticipated to improve the cardiovascular profile, but with a better understanding of the
cardiovascular effects in humans, this is no longer expected to have a significant benefit. With that
being said, S1P receptor agonism does not appear to contribute to the efficacy, so a desire for
3
selectivity has remained a primary emphasis of most research efforts, including our own.
The key structural features of 2, and other typical S1P receptor agonists, include a polar head
1
group, a rigid linker, and a lipophilic tail group. Merck was the first to report on results aimed at
identifying directꢀacting agonists of the S1P receptors, ultimately leading to the identification of
compounds with an azetidineꢀ3ꢀcarboxylic polar group utilized in conjunction with a 3,5ꢀdiarylꢀ1,2,4ꢀ
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1
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oxadiazole linker, as exemplified by compound 3. In our efforts to identify novel lipophilic fragments,
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the azetidineꢀ3ꢀcarboxylic acid substituted linker 4, derived from 3, provided a valuable combination for
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identifying agonists of the S1P receptor when combined with our fragments.
This effort ultimately
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led to the identification of two highly potent, selective isoxazoleꢀbased full agonists of the S1P1
receptor, as depicted in structures 5 and 6 shown in Figure 3. As discussed in this article, further
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optimization led to the identification of isoxazole 6d (BMSꢀ520).
Chemistry
The synthetic pathways utilized in the preparation of the isoxazole analogs presented in Tables 1 and
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2
are provided in Schemes 1 – 9. Table 1 outlines isoxazole derivatives appended to the linker at C3
with substitution at C4 and C5. Table 2 outlines derivatives appended to the linker at C5 with
substitution at C3 and C4. Compounds presented in Tables 1 and 2 were prepared as depicted in
Scheme 1. Intermediate 9 was prepared through a reductive amination reaction between 4ꢀ
cyanobenzaldehyde and the tertꢀbutyl ester protected azetidineꢀ3ꢀcarboxylic acid. Subsequent treatment
with hydroxylamine afforded the amidoxime intermediate 10. Intermediate 10 was then coupled with
the substituted isoxazoleꢀ3ꢀcarboxylic acids 13a-n or isoxazoleꢀ5ꢀcarboxylic acids 22a-c, utilizing
EDCI and HOBt in dimethyl formamide, followed by deprotection of the azetidine carboxylic acid with
trifluoroacetic acid to provide the desired analogs 5a-n or 6a-c in good yield.
Isoxazoleꢀ3ꢀcarboxylic acids used for the preparation of 5a-j and 5l-m were either commercially
available or were prepared as shown in Scheme 2. A mixture of the appropriately substituted alkyne
o
and dimethyl nitromalonate was either heated in mesitylene in a sealed tube at 150 C overnight or
o
heated in acetonitrile under microwave conditions at 170 C in the presence of an ionic liquid, 1ꢀbutylꢀ3ꢀ
methylimidazoliumhexafluorophosphate, to give ethers 12b-j and 12l-m. Subsequent hydrolysis with
either lithium hydroxide or sodium hydroxide afforded the desired isoxazoleꢀ2ꢀcarboxylic acids 13b-j
and 13l-m in good yield. The C4 trifluoromethylꢀisoxazoles acids 5k and 5n were prepared in high
yield as outlined in Scheme 3. Isoxazole ester 14 was iodinated at C4 with Nꢀiodosuccinimide and
trifluoroacectic acid to give intermediate 15 in 97% yield. Trifluoromethylation at C4 was carried out
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with methyl 2,2ꢀdifluoroꢀ2ꢀ(fluorosulfonyl)acetate in the presence of copper iodide and HMPA to give
isoxazole ester 16 in 94% yield. Subsequent hydrolysis with lithium hydroxide provided isoxazole acid
17 in near quantitative yield. The related 5ꢀisobutylꢀ4ꢀ(trifluoromethyl)isoxazoleꢀ3ꢀcarboxylic acid 21
was prepared in an analogous manner in high yield. The C4ꢀdifluoroethyl isoxazole 5l was prepared as
shown in Scheme 4.
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The synthetic routes used to prepare the C5 linked isoxazoles 6a-d are outlined in Schemes 5 – 9.
Isoxazoleꢀ5ꢀcarboxylic acids 29a-c were either commercially available or prepared as outlined in
1
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Scheme 5. A mixture of the Nꢀhydroxybenzimidoyl chloride 26 and substituted ethyl bromoꢀenoates
2
7b and 27c in dichloromethane were stirred at room temperature in the presence of triethylamine to
give esters 28b and 28c. Subsequent hydrolysis with lithium hydroxide afforded the desired acids 29b
and 29c.
Preparation of C4 trifluoromethyl isoxazole acid 35 proved to be a challenge. An initial survey of
the literature revealed that the only reported method for the preparation of ester 30 occurred through a
1,3ꢀdipolar cycloaddition reaction between ethyl trifluorobutꢀ2ꢀynoate and a dipole generated in situ
from Nꢀhydroxybenzimidoyl chloride favoring the formation of the undesired isomer 31 in a ratio of
18
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5:15.
In our hands, the reaction provided 30, with a ratio consistent with that reported in the
literature, in ~7% yield after chromatographic separation (Scheme 6). To optimize the [3+2]ꢀ
cycloaddition reaction, the electronic pairing of the dipolarophile was inverted by replacing ethyl
1
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trifluorobutꢀ2ꢀynoate with the trifluorobutꢀ2ꢀynꢀ1ꢀol, prepared as shown in Scheme 8. As expected,
1
7
slow addition of triethylamine to the mixture of Nꢀhydroxybenzimidoyl chloride 26
and
o
trifluorobutyne alcohol 32 at 70 C resulted in a mixture of isomers 33 and 34, but in a ratio of 9:1
favoring the desired isomer 33, with a typical isolated yield of ~65% after chromatographic separation
(Scheme 7). The desired alcohol isomer 33 was oxidized with either Jones’ reagent or TEMPO/bleach
to give 3ꢀphenylꢀ4ꢀ(trifluoromethyl)isoxazoleꢀ5ꢀcarboxylic acid 35 in >90% yield.
An optimized, high yielding procedure for the preparation of isoxazole 6d is outlined in Scheme 9.
This approach relies on the coupling of acid fluoride 36, derived from the treatment of acid 35 with
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cyanuric fluoride, with amidoxime intermediate 10 in the presence of Hunig’s Base to generate
oxadiazole 37 in 80% yield. Subsequent deprotection of the azetidine carboxylic acid with
trifluoroacetic acid afforded 6d in >90% yield. This improved sequence was applicable for the
preparation of the other analogs described in this article, especially 5k and 5n.
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Isoxazole agonists presented in Tables 1 and 2 were evaluated in a functional GTPγS assay looking
at both human S1P and S1P receptor agonism. While it is important to maintain selectivity against the
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3
S1P and S1P receptors, activity against the S1P and S1P receptors was less of a concern and was
2
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4
5
only monitored periodically. In parallel with the functional assays, liability profiling was utilized to
facilitate the progression of compounds into a rat blood lymphocyte reduction (BLR)
pharmacodynamic/pharmacokinetic (PD/PK) assay. In this assay, healthy rats were dosed orally with a
1
.0 mg/kg dose of each compound. Blood samples were drawn at 4 hour and 24 hour time points to
assess the reduction in circulating lymphocytes relative to control and to evaluate plasma concentration
for each compound. Selected compounds with appropriate in vivo activity were further evaluated in full
PK studies and ultimately animal efficacy models of rheumatoid arthritis and multiple sclerosis.
Results and Discussion
Table 1 highlights the structureꢀactivity relationship (SAR) for agonists 5 derived from isoxazoleꢀ3ꢀ
carboxylic acids. The more facile synthesis of these examples allowed for a broader exploration of
2
1
SAR, with a particular focus on exploring substitution at R while maintaining R as a phenyl
substituent (5a-l). Isoxazole 5a, with no C4 substitution, provided modest agonist activity against the
S1P receptor with an EC50 of 44 nM. The addition of a methyl group (5b) provided a 9ꢀfold
1
improvement in potency, as well as enhanced selectivity for the S1P receptor relative to the S1P
1
3
receptor. In general, increasing the length of the alkyl chain (5c-f) resulted in improvements in activity,
with butyl and isoꢀbutyl substitution providing potent agonist activity against the S1P receptor with
1
EC values of 0.27 nM and 0.56 nM, respectively. On the other hand, shorter, branched alkyl groups
50
(
5g-i) were less potent compared with the longer, linear side chains (e.g. 5i, EC = 5.3 nM vs. 5d, EC50
50
=
0.27 nM). In the BLR PD/PK assay, the lymphocyte reduction at 4 and 24 h time points with an oral
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dose of 1 mg/kg was consistent with the agonist activity trends observed for the C4 alkyl isoxazoles 5b-
f and 5i, with the longer alkyl substituents 5d-f providing a higher level of lymphopenia at 4 h with a
sustained PD effect out to 24 h. When the C5 phenyl in 5d was replaced with an isobutyl group (5m),
activity and selectivity were both maintained, but significantly higher plasma concentrations were
observed in the BLR assay. Interestingly, phenyl substitution at C4 (5j) retained potency against the
S1P receptor but selectivity against the S1P receptor was reduced relative to 5b-i. Although the C4
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0
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3
substitution outlined in compounds 5b-j,m provided improvements in both activity and selectivity
relative to 5a, these modifications introduced hERG, sodium, and calcium channel concerns, based on in
vitro liability profiling (data not shown).
Substitution of C4 with a trifluoromethyl group (5k), on the other hand, provided a very clean
liability profile while maintaining potent agonist activity and selectivity (ED of 0.98 nM; S1P /S1P
1
5
0
3
selectivity: 4,000ꢀfold). In the BLR PD/PK assay, a 1 mg/kg oral dose of 5k resulted in an 84%
reduction in lymphocyte counts at 4h with a sustained PD effect providing an 85% reduction at 24 h.
Compound plasma concentrations at both time points for 5k were considerably higher when compared
to those observed for 5a-j. Additionally, 5k had a 4h/24h peakꢀtoꢀthrough exposure ratio of 6,
suggesting that this compound has a low rate of clearance. Other fluorinated variations 5l and 5n were
also promising, with 5n providing significant enhancements in selectivity while demonstrating a similar
PD/PK effect as 5k, but with higher overall compound plasma levels.
The SAR of agonists (6a-d) derived from isoxazoleꢀ5ꢀcarloxylic acids highlighted in Table 2
followed the same trends observed with the C3ꢀlinked isoxazoles (5). The more synthetically
challenging nature of this series resulted in comparably limited exploration of substitution. Consistent
with the data presented in Table 1, C4 trifluoromethyl substitution (6d) provided a highly desirable
activity and selectivity profile. In the rat BLR PD/PK assay, 6d showed robust lymphopenia at an 1
mg/kg oral dose at both 4h and 24 time points, 78% and 91% respectively. Additionally, the plasma
concentrations of 6d were higher than those observed for 5k (~1.9x at 4 h and ~3.9x at 24 h) with a
4
h/24h peakꢀtoꢀthrough ratio of only 3.
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The ability of 6d to drive receptor internalization was investigated using CHO cells transfected with
human S1P fused with GFP. In this assay, 6d induced S1P internalization with an EC of 0.27 ±0.004
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1
50
nM (n=3), similar to fingolimodꢀP (2, EC : 0.28 ±0.1 nM). The maximal signal of S1P receptor
50
1
internalization was similar between 4d and S1P treatment. As seen in Figure 4, S1P resulted in >90%
receptor internalization. Following a washout period, S1P receptors reappeared on the cell surface by 4
0
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h and 16 h time points with levels of 60% and 100%, respectively. However, 6d which demonstrated a
similar level of receptor internalization showed receptor recycling levels of only 20% at 4 h and 40% at
16 h, compared with baseline levels prior to stimulation. It is worth noting that newly synthesized
protein may have contributed to the total receptor count, since protein synthesis inhibitors were not used
in this assay. As a result, 6d is likely to induce S1P receptor degradation similar to that reported for
1
7
fingolimodꢀP (2).
With a highly desirable activity and selectivity profile and a very clean liability profile (Table 3), 6d
was further evaluated in vivo. As shown in Table 4, 6d demonstrated dose dependent lymphopenia in
the rat BLR assay, demonstrating a significant reduction in circulating lymphocytes at 24 h with an oral
dose of only 0.03 mg/kg with compound plasma concentrations of 4 nM at the 24 h time point. In
pharmacokinetic (PK) studies in mice, rat, cyno, and dog (Table 5), bioavailabilities were around 50%
or higher across all species with low clearance and desirable halfꢀlives in the range of 7.4 ꢀ 10 h in
mouse, rat, and dog. Interestingly, cyno proved to be an outlier with a more moderate rate of clearance
resulting in a shorter halfꢀlife of only 3 h, consequently leading to a larger peakꢀtoꢀthrough ratio. With
an excellent dose response in the rat BLR PD/PK assay, an overall acceptable liability profile, and a
desirable PK profile differentiated from that of fingolimod (1), 6d was advanced into animal models of
human autoimmune disease.
20
In a rat adjuvant arthritis study, a preclinical model of human rheumatoid arthritis, arthritis was
induced by the subcutaneous injection of Complete Freund’s Adjuvant (CFA) containing heatꢀkilled
mycobacterium butyricum into the base of the tail of male Lewis rats. Isoxazole 6d was administered at
doses of 0.03. 0.1, 0.5, and 3 mg/kg PO/QD, along with fingolimod (1) at 1 mg/kg PO/QD, for 21 days
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Journal of Medicinal Chemistry
starting at day 0 (Figure 5A). Baseline measurements of hind paw volume were obtained by water
displacement plethysmometry. Additional paw volume measurements were performed over the next
three weeks, and the increases in paw volume above baseline were calculated. Although a dose of 0.03
mg/kg had little impact on disease development, a dose of 0.1 mg/kg provided >50% suppression of
paw swelling, and the higher doses of 0.5 and 3.0 mg/kg resulted in robust inhibition. On the basis of
the paw swelling observed in this study, the ED was determined to be 0.05 mg/kg. At the end of the
1
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0
study, histological analysis of the paws confirmed that 6d provided inhibition of inflammation and bone
resorption in a dose dependent fashion, as depicted in Figure 5B.
With success in the rat adjuvant arthritis model, 6d was evaluated in a mouse experimental
autoimmune encephalomyelitis study (EAE), a preclinical model of multiple sclerosis. Disease was
induced by subcutaneous immunization with MOG (myelin oligodendrocyte glycoprotein)/CFA and two
doses of pertussis toxin in C57BL/6 mice (see experimental section for details). Mice were dosed
orally, QD with 6d beginning on the day of immunization. Figure 6A presents the mean clinical scores
of groups of mice treated with vehicle or varying dose levels of 6d . Isoxazole 6d treatment provided a
doseꢀdependent delay in disease onset and a doseꢀdependent reduction in the overall severity of disease
compared with the vehicle treatment group. In this study, 6d at a dose of 0.2 mg/kg QD provided a
degree of efficacy comparable to that of fingolimod (1) at a 1.0 mg/kg dose QD. On the basis of the
efficacy observed in this study, the ED for 6d was determined to be 0.02 mg/kg. At the end of the
5
0
study, histological analysis of the spinal cords confirmed that 6d provided inhibition of inflammation
and demyelination in a dose dependent fashion, as shown in Figure 6B.
Conclusion
In summary, a novel series of isoxazole based full agonists of the S1P receptor with selectivity over
1
the S1P and S1P receptors has been identified. Through this effort, we identified isoxazole 6d (BMSꢀ
2
3
5
20) as our lead compound. With a potent and selective in vitro activity profile, an acceptable PK
profile, robust potency in the rat BLR assay, and a clean liability profile, 6d was advanced into rodent
models of human autoimmune disease. In a rat adjuvant arthritis study, a preclinical model of human
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rheumatoid arthritis, 6d demonstrated robust efficacy with an ED50 of 0.05 mg/kg. In a mouse EAE
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study, a preclinical model of multiple sclerosis, treatment with 6d resulted in significant reduction of
disease with an ED50 of 0.02 mg/kg. As a result, 6d (BMSꢀ520) was selected as a development
candidate.
Experimental
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1
Proton magnetic resonance ( H) spectra were recorded on either a Bruker Avance 400 or a JEOL
Eclipse 500 spectrometer and are reported in ppm relative to the reference solvent of the sample in
which they were run. HPLC and LCMS analyses were conducted using a Shimadzu SCLꢀ10A liquid
chromatograph and a SPD UVꢀVis detector at 220 or 254 nm with the MS detection performed on a
Waters Micromass ZQ spectrometer. Preparative reverseꢀphase HPLC purifications were performed
using the following conditions: YMC S5 ODS 20 x 100 mm column with a binary solvent system where
solvent A = 10% methanol, 90% water, 0.1% trifluoroacetic acid and solvent B = 90% methanol, 10%
water, and 0.1% trifluoroacetic acid, flow rate = 20 mL/min, linear gradient time = 10 min, start %B =
20, final %B = 100. Fractions containing the product were concentrated in vacuo to remove the
methanol and neutralized with aqueous sodium bicarbonate. The pH was adjusted to ~4.5 – 5.0, and the
aqueous layer was extracted with organic solvents. The organic layer was collected, dried over
anhydrous sodium sulfate or magnesium sulfate, and concentrated under reduced pressure. All flash
column chromatography was performed on EM Science silica gel 60 (particle size of 40 – 60 ꢀm). All
reagents were purchased from commercial sources and used without further purification unless
otherwise noted. All reactions were performed under an inert atmosphere.
HPLC analyses were performed using the following conditions. All final compounds had an HPLC
purity of ≥ 95%.
Method A: A linear gradient using 5% acetonitrile, 95% water, and 0.05% TFA (Solvent A) and 95%
acetonitrile, 5% water, and 0.05% TFA (Solvent B); t = 0 min., 10% B, t = 15 min., 100% B (20 min.)
was employed on a SunFire C18 3.5u 4.6 x 150 mm column. Flow rate was 1.0 ml/min and UV
detection was set to 220/254 nm. The LC column was maintained at ambient temperature.
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Method B: A linear gradient using 5% acetonitrile, 95% water, and 0.05% TFA (Solvent A) and 95%
acetonitrile, 5% water, and 0.05% TFA (Solvent B); t = 0 min., 10% B, t = 15 min., 100% B (20 min.)
was employed on a XBridge Ph 3.5u 4.6 x 150 mm column. Flow rate was 1.0 ml/min and UV
detection was set to 220/254 nm. The LC column was maintained at ambient temperature.
1
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9
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5
6
Method C. A linear gradient using 10% methanol, 90% water, and 0.2% H PO (Solvent A) and 90%
0
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4
methanol, 10% water, and 0.2% H PO (Solvent B); t = 0 min., 0% B, t = 4 min., 100% B (5 min.) was
3
4
employed on a Chromolith SpeedROD 4.6 x 50 mm column. Flow rate was 4.0 ml/min and UV
detection was set to 220 nm. The LC column was maintained at ambient temperature.
Method D. A linear gradient using 10% methanol, 90% water, and 0.2% H PO (Solvent A) and 90%
3
4
methanol, 10% water, and 0.2% H PO (Solvent B); t = 0 min., 0% B, t = 4 min., 100% B (5 min.) was
3
4
employed on a YMC S5 CombiScreen 4.6 x 50 mm column. Flow rate was 4.0 ml/min and UV
detection was set to 220 or 254 nm. The LC column was maintained at ambient temperature.
1
-(4-(5-(5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid (5a). To
a homogeneous mixture of 5ꢀphenylisoxazoleꢀ3ꢀcarboxylic acid (0.200 g, 1.057 mmol) and pyridine
0.103 mL, 1.269 mmol) in dichloromethane (10 mL) at room temperature was added 2,4,6ꢀtrifluoroꢀ
,3,5ꢀtriazine (0.107 mL, 1.269 mmol). The reaction mixture was stirred at room temperature for 2 h.
(
1
The heterogeneous reaction mixture was diluted with dichloromethane (25 mL), washed with an iceꢀ
cold solution of 0.5N aqueous hydrochloric acid (2 x 10 mL), and the organic layer was collected. The
aqueous layer was backꢀextracted with dichloromethane (25 mL). The combined organic layers were
dried over anhydrous sodium sulfate and concentrated to afford 5ꢀphenylisoxazoleꢀ3ꢀcarbonyl fluoride
(
0.201 g, 1.05 mmol, 99%) as a white solid. HPLC t = 1.97 min., corresponding to the methyl ester
r
(Method C).
A mixture of 5ꢀphenylisoxazoleꢀ3ꢀcarbonyl fluoride (0.201 g, 1.05 mmol), tertꢀbutyl 1ꢀ(4ꢀ(N'ꢀ
hydroxycarbamimidoyl)benzyl)azetidineꢀ3ꢀcarboxylate (10, 0.321 g, 1.05 mmol), and Hunig's Base
0.239 mL, 1.37 mmol) in acetonitrile (2.0 mL) was stirred at room temperature over the weekend. The
(
reaction mixture was heated at 50°C for 4 h. The solidified reaction mixture was diluted with
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dichloromethane (30 mL), and washed with a saturated aqueous solution of sodium bicarbonate (10
mL). The aqueous layer was extracted with dichloromethane (30 mL), and the organic layers were
combined and dried over anhydrous sodium sulfate. Concentration under reduced pressure afforded a
tan solid which was triturated with methanol with sonication and filtered under reduced pressure to give
tertꢀbutyl 1ꢀ(4ꢀ(5ꢀ(5ꢀphenylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)benzyl)azetidineꢀ3ꢀcarboxylate (0.253 g,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
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9
0
0
.552 mmol, 53%) as a tan solid (two crops which were combined). HPLC t = 2.69 min. (Method C);
r
+
LCMS (ESI) m/z Calcd for C H N O [M + H] 459.2. Found: 459.3.
2
6
26
4
4
A mixture of tertꢀbutyl 1ꢀ(4ꢀ(5ꢀ(5ꢀphenylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)benzyl)azetidineꢀ3ꢀ
carboxylate (0.253 g, 0.552 mmol) and trifluoroacetic acid (3.02 ml, 39.2 mmol) was stirred at room
temperature for 2.0 h. The trifluoroacetic acid was removed under reduced pressure, and the oily
residue was suspended in water with sonication. To the resulting white suspension was added a 1N
aqueous solution of sodium hydroxide portionꢀwise until the pH was ~5 (~2.9 mL of 1N NaOH). The
suspension was stirred overnight at room temperature. Several drops of 1N aqueous hydrochloric acid
were needed to reꢀadjust the pH to 5. The solid was collected by vacuum filtration, washed with water
several times, and dried under reduced pressure for 5 h. The solid was then suspended in methanol with
sonication and collected by vacuum filtration, washed with methanol, and dried under reduced pressure
to give 1ꢀ(4ꢀ(5ꢀ(5ꢀphenylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)benzyl)azetidineꢀ3ꢀcarboxylic acid (5a,
0
.150 g, 0.369 mmol, 67%) as a white solid. HPLC purity 99.6%; t = 6.80 min. (Method A); 99.1%; t
r r
+
1
= 8.05 min. (Method B); LCMS (ESI) m/z Calcd for C H N O [M + H] 417.2. Found: 403.1. Hꢀ
2
2
18
4
4
NMR (500 MHz, DMSOꢀd ) δ ppm 8.06 (4H, d, J=8.05 Hz), 7.92 (1H, s), 7.59 ꢀ 7.62 (3H, m), 7.53
6
(2H, s), 6.96 ꢀ 6.98 (1H, m), 3.66 (2H, s), 3.44 (2H, s), and 3.23 ꢀ 3.26 (3H, m).
1-(4-(5-(4-methyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)-azetidine-3-carboxylic
acid (5b). A mixture of propꢀ1ꢀynylbenzene (1.08 mL, 8.61 mmol) and dimethyl 2ꢀnitromalonate (1.06
mL, 7.83 mmol) in mesitylene (10.0 mL, 72.0 mmol) in a sealed tube was heated in an oil bath at 150°C
overnight. The reaction mixture was concentrated under reduced pressure to give 1.52 g of the product
mixture as an orange, viscous oil. Approximately 200 mg of the product mixture was purified by flash
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silica gel chromatography (2% ꢀ 5% ethyl acetate in hexane) to give methyl 4ꢀmethylꢀ5ꢀ
1
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7
8
9
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1
1
1
1
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5
5
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5
5
6
phenylisoxazoleꢀ3ꢀcarboxylate (47 mg, 0.216 mmol, 28%) as a white solid. HPLC t = 2.42 min.
r
+
(Method C); LCMS (ESI) m/z Calcd for C H NO [M + H] 218.1. Found: 218.1.
1
2
11
3
A mixture of methyl 4ꢀmethylꢀ5ꢀphenylisoxazoleꢀ3ꢀcarboxylate (0.047 g, 0.216 mmol) and lithium
hydroxide hydrate (9.08 mg, 0.216 mmol) in methanol (1.0 mL) and water (0.5 mL) was stirred at room
temperature overnight. The reaction mixture was concentrated to dryness to give 4ꢀmethylꢀ5ꢀ
0
1
2
3
4
5
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0
phenylisoxazoleꢀ3ꢀcarboxylic acid, lithium salt (0.045 g, 0.214 mmol, 99%) as a white solid. HPLC t =
r
+
1
2.08 min. (Method C); LCMS (ESI) m/z Calcd for C H NO [M + H] 204.1. Found: 204.0. HꢀNMR
1
2
11
3
(
500 MHz, DMSOꢀd ) δ ppm 2.25 (s, 3H), 7.44 ꢀ 7.49 (m, 1H), 7.53 (t, J=7.70 Hz, 2H), and 7.69 (d,
6
J=7.70 Hz, 2H).
A mixture of 4ꢀmethylꢀ5ꢀphenylisoxazoleꢀ3ꢀcarboxylic acid, lithium salt (0.021 g, 0.100 mmol),
tertꢀbutyl 1ꢀ(4ꢀ(N'ꢀhydroxycarbamimidoyl)benzyl)azetidineꢀ3ꢀcarboxylate (10, 0.031 g, 0.100 mmol),
N1ꢀ((ethylimino)methylene)ꢀN3,N3ꢀdimethylpropaneꢀ1,3ꢀdiamine hydrochloride (0.019 g, 0.100
mmol), and HOBt (0.015 g, 0.100 mmol) in N,Nꢀdimethylformamide (1.0 mL) was shaken on a shaker
block for 60 min. The homogeneous reaction mixture was then shaken at 50°C overnight. The solvent
was removed under reduced pressure. Trifluoroacetic acid (0.501 mL, 6.50 mmol) was added, and the
homogeneous reaction mixture was shaken at room temperature for 30 min., concentrated under reduced
pressure, and purified by reverseꢀphase preparative HPLC to give 1ꢀ(4ꢀ(5ꢀ(4ꢀmethylꢀ5ꢀphenylisoxazolꢀ
3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)benzyl)azetidineꢀ3ꢀcarboxylic acid, TFA (5b, 0.026 g, 0.048 mmol, 48%) as
a white solid. HPLC purity 97.0%; t = 7.16 min. (Method A); 97.0%; t = 8.35 min. (Method B);
r
r
+
1
LCMS (ESI) m/z Calcd for C H N O [M + H] 417.2. Found: 417.1. HꢀNMR (500 MHz, DMSOꢀd )
2
3
20
4
4
6
δ ppm 2.60 (s, 3H), 3.58 ꢀ 3.67 (m, 1H), 4.19 (br. s., 4H), 4.47 (br. s., 2H), 7.61 ꢀ 7.68 (m, 3H), 7.71 (d,
J=7.70 Hz, 2H), 7.87 (d, J=7.15 Hz, 2H), 8.21 (d, J=8.25 Hz, 2H), 10.51 (br. s., 1H), and 13.13 (br. s.,
1
H).
Preparation of 1-(4-(5-(4-ethyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-
16b
carboxylic acid (5c).
A solution of butꢀ1ꢀynylbenzene (404 mg, 3.11 mmol) and dimethyl
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nitromalonate (0.381 mL, 2.82 mmol) in mesitylene (5 mL) was heated to 150°C overnight. The
reaction mixture was concentrated, and the residue purified by flash silica gel chromatography
(hexane/ethyl acetate; 10:1) to afford methyl 4ꢀethylꢀ5ꢀphenylisoxazoleꢀ3ꢀcarboxylate (194 mg, 0.791
1
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5
5
5
6
+
mmol, 28%). HPLC t = 3.25 min. (Method D); LCMS (ESI) m/z Calcd for C H NO [M + H] 232.1.
r
13 13
3
+
Found: 232 .
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A solution of methyl 4ꢀethylꢀ5ꢀphenylisoxazoleꢀ3ꢀcarboxylate (194 mg, 0.839 mmol) and 1N
aqueous sodium hydroxide (1.26 mL, 1.26 mmol) in methanol (3 mL) was heated to 100°C for 10
minutes via microwave. The reaction mixture was acidified with acetic acid until the pH was about 4.
The mixture was concentrated, and the residue was suspended in water (2 mL) and stirred for 20
minutes. The solid was collected by vacuum filtration and dried to give 4ꢀethylꢀ5ꢀphenylisoxazoleꢀ3ꢀ
carboxylic acid (148 mg, 0.735 mmol, 87%). HPLC t = 2.93 min. (Method D); LCMS (ESI) m/z Calcd
r
+
+
for C H NO [M + H] 218.1. Found: 218 .
1
2
11
3
To a solution of 4ꢀethylꢀ5ꢀphenylisoxazoleꢀ3ꢀcarboxylic acid (22 mg, 0.101 mmol), HOBt (27.9
mg, 0.182 mmol), and diisopropylethylamine (0.071 mL, 0.405 mmol) in acetonitrile (1 mL) was added
N1ꢀ((ethylimino)methylene)ꢀN3,N3ꢀdimethylpropaneꢀ1,3ꢀdiamine hydrochloride (45.6 mg, 0.238
mmol) and tertꢀbutyl 1ꢀ(4ꢀ(N'ꢀhydroxycarbamimidoyl)benzyl)azetidineꢀ3ꢀcarboxylate (10, 30.9 mg,
0
.101 mmol). The reaction mixture was stirred at 80°C for 2 h. and then concentrated. The residue was
diluted with ethyl acetate (3 mL), washed a saturated aqueous solution of sodium bicarbonate (1 mL),
washed with water (1 mL), washed with brine (1 mL), and dried over anhydrous sodium sulfate.
Concentration followed by purification by flash silica gel chromatography (hexanes/ethyl acetate; 1:1)
provided tertꢀbutyl 1ꢀ(4ꢀ(5ꢀ(4ꢀethylꢀ5ꢀphenylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)benzyl)azetidineꢀ3ꢀ
carboxylate.
A
solution
of
tertꢀbutyl
1ꢀ(4ꢀ(5ꢀ(4ꢀethylꢀ5ꢀphenylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀ
yl)benzyl)azetidineꢀ3ꢀcarboxylate in dichloromethane (0.5 mL) and trifluoroacetic acid (0.5 mL) was
stirred at room temperature for 30 minutes. Concentration followed by drying under reduced pressure
afforded
1ꢀ(4ꢀ(5ꢀ(4ꢀethylꢀ5ꢀphenylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)benzyl)azetidineꢀ3ꢀcarboxylic
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Page 17 of 67
Journal of Medicinal Chemistry
acid (5c, 32 mg, 0.074 mmol, 73% over two steps). HPLC purity 99.1%; t = 7.46 min. (Method A);
r
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
9
4
9.5%; t = 8.83 min. (Method B); LCMS (ESI) m/z Calcd for C H N O [M + H] 431.2. Found:
r
24 22
4
4
+
1
31 . H NMR (400 MHz, MeOD) δ ppm 1.39 (t, J=7.40 Hz, 3H), 3.07 ꢀ 3.15 (m, 2H), 3.71 (qd,
J=8.32, 8.16 Hz, 1H), 4.35 ꢀ 4.41 (m, 4H), 4.53 (s, 2 H), 7.56 ꢀ 7.65 (m, 3H), 7.70 (d, J=8.28 Hz, 2H),
7.81 ꢀ 7.85 (m, 2H), and 8.30 (d, J=8.53 Hz, 2H).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1-(4-(5-(5-phenyl-4-propylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic
1
6b
acid (5d).
A solution of pentꢀ1ꢀynylbenzene (0.221 mL, 1.39 mmol) and dimethyl nitromalonate
(0.374 mL, 2.77 mmol) in mesitylene (3 mL) was heated to 150°C for 16 h. The reaction mixture was
concentrated, and the residue was purified by flash silica gel chromatography (hexanes/ethyl acetate;
1
0:1) to yield methyl 5ꢀphenylꢀ4ꢀpropylisoxazoleꢀ3ꢀcarboxylate (138 mg, 0.564 mmol, 41%).
A solution of methyl 5ꢀphenylꢀ4ꢀpropylisoxazoleꢀ3ꢀcarboxylate (138 mg, 0.564 mmol) and 1N
aqueous sodium hydroxide (0.846 mL, 0.846 mmol) in methanol (2.5 mL) was heated to 100°C for 10
minutes under microwave conditions. The reaction mixture was concentrated, and the crude product
was suspended in water (2 mL), acidified with acetic acid until the pH was ~4, and stirred for 20
minutes. The solid was collected by vacuum filtration to give 5ꢀphenylꢀ4ꢀpropylisoxazoleꢀ3ꢀcarboxylic
acid (56 mg, 0.260 mmol, 46%). HPLC t = 2.88 min. (Method D); LCMS (ESI) m/z Calcd for
r
+
+
C H NO [M + H] 232.1. Found: 232 .
13
13
3
To a solution of 5ꢀphenylꢀ4ꢀpropylisoxazoleꢀ3ꢀcarboxylic acid (23 mg, 0.099 mmol), HOBt (27.4
mg, 0.179 mmol), and diisopropylethylamine (0.069 mL, 0.398 mmol) in acetonitrile (1 mL) was added
N1ꢀ((ethylimino)methylene)ꢀN3,N3ꢀdimethylpropaneꢀ1,3ꢀdiamine hydrochloride (44.8 mg, 0.234
mmol) and tertꢀbutyl 1ꢀ(4ꢀ(N'ꢀhydroxycarbamimidoyl)benzyl)azetidineꢀ3ꢀcarboxylate (10, 30.4 mg,
0
.099 mmol). The reaction mixture was stirred at 80°C for 2 h The reaction mixture was concentrated
to yield a crude product which was diluted with ethyl acetate (3 mL), washed with a saturated aqueous
solution of sodium bircarbonate (1 mL), washed with water (1 mL), washed with brine (1 mL), and
dried over anhydrous sodium sulfate. Concentration under reduced pressure provided tertꢀbutyl 1ꢀ(4ꢀ(5ꢀ
(5ꢀphenylꢀ4ꢀpropylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)benzyl)azetidineꢀ3ꢀcarboxylate.
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Journal of Medicinal Chemistry
Page 18 of 67
A
solution
of
tertꢀbutyl
1ꢀ(4ꢀ(5ꢀ(5ꢀphenylꢀ4ꢀpropylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀ
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
yl)benzyl)azetidineꢀ3ꢀcarboxylatein dichloromethane (0.5 mL) and trifluoroacetic acid (0.5 mL) was
stirred at room temperature for 30 minutes. Concentration followed by purification by preparative
HPLC afforded 1ꢀ(4ꢀ(5ꢀ(5ꢀphenylꢀ4ꢀpropylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)benzyl)azetidineꢀ3ꢀ
carboxylic acid (5d, 33 mg, 0.074 mmol, 75% over two steps). HPLC purity 95.7%; t = 7.72 min.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
r
+
(
Method A); 95.3%; t = 8.91 min. (Method B); LCMS (ESI) m/z Calcd for C H N O [M + H] 445.2.
r
25 24
4
4
+
1
Found: 445 . H NMR (400 MHz, MeOD) δ ppm 1.05 (t, 3H), 1.72 ꢀ 1.84 (m, 2H), 3.03 ꢀ 3.11 (m, 2H),
3.71 (t, J=8.28 Hz, 1H), 4.34 ꢀ 4.42 (m, 4H), 4.53 (s, 2H), 7.56 ꢀ 7.65 (m, 3H), 7.70 (d, J=8.28 Hz, 2H),
7
.80 ꢀ 7.85 (m, 2H), and 8.30 (d, J=8.28 Hz, 2H).
ꢀ(4-(5-(4-Butyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid
1
1
6b
(
5e).
A solution of hexꢀ1ꢀynylbenzene (0.274 mL, 1.56 mmol), dimethyl 2ꢀnitromalonate (0.421 mL,
3
.12 mmol), 1ꢀbutylꢀ3ꢀmethylimidazoliumhexafluorophosphate (0.032 mL, 0.156 mmol) in toluene (6
mL) was subjected to microwave conditions at 170°C for 150 min. The reaction mixture was
concentrated under reduced pressure, and the residue was purified by flash silica gel chromatography
using 5% ethyl acetate in hexane to give methyl 4ꢀbutylꢀ5ꢀphenylisoxazoleꢀ3ꢀcarboxylate (0.142 g,
0.531 mmol, 34%) as a clear, colorless oil. HPLC purity 97%; t = 3.11 min. (Method C); LCMS (ESI)
r
+
+
m/z Calcd for C H NO [M + H] 260.1. Found: 260 .
1
5
17
3
A mixture of methyl 4ꢀbutylꢀ5ꢀphenylisoxazoleꢀ3ꢀcarboxylate (0.142 g, 0.548 mmol) and lithium
hydroxide hydrate (0.023 g, 0.548 mmol) in methanol (3.0 mL) and water (1.5 mL) was stirred at room
temperature overnight. The reaction mixture was concentrated to dryness to give 4ꢀbutylꢀ5ꢀ
phenylisoxazoleꢀ3ꢀcarboxylic acid, lithium salt (0.134 g, 0.531 mmol, 97%) as a white solid. HPLC t =
r
+
1
2
.82 min. (Method C); LCMS (ESI) m/z Calcd for C H NO [M + H] 246.1. Found: 246.2. HꢀNMR
14 15 3
(
500 MHz, DMSOꢀd ) δ ppm 0.84 (t, J=7.42 Hz, 3H), 1.23 ꢀ 1.32 (m, 2H), 1.44 ꢀ 1.53 (m, 2H), 2.66 ꢀ
6
2.72 (m, 2H), 7.44 ꢀ 7.49 (m, 1H), 7.53 (t, J=7.42 Hz, 2H), and 7.64 (d, J=7.15 Hz, 2H).
A mixture of 4ꢀbutylꢀ5ꢀphenylisoxazoleꢀ3ꢀcarboxylic acid, lithium salt (0.132 g, 0.523 mmol), tertꢀ
butyl 1ꢀ(4ꢀ(N'ꢀhydroxycarbamimidoyl)benzyl)azetidineꢀ3ꢀcarboxylate (10, 0.160 g, 0.523 mmol), N1ꢀ
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Page 19 of 67
Journal of Medicinal Chemistry
(
(ethylimino)methylene)ꢀN3,N3ꢀdimethylpropaneꢀ1,3ꢀdiamine hydrochloride (0.100 g, 0.523 mmol),
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and HOBt (0.080 g, 0.523 mmol) in N,Nꢀdimethylformamide (4 mL) was stirred at room temperature
for 60 min and then heated at 60°C overnight. The solvent was removed under reduced pressure, and
the residue was purified by flash silica gel chromatography eluting with 1% methanol in
dichloromethane to give tertꢀbutyl 1ꢀ(4ꢀ(5ꢀ(4ꢀbutylꢀ5ꢀphenylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
yl)benzyl)azetidineꢀ3ꢀcarboxylate (0.156 g, 0.303 mmol, 58%) as a pale yellow solid. HPLC purity
+
>
99%; t = 3.40 min. (Method C); LCMS (ESI) m/z Calcd for C H N O [M + H] 515.3. Found:
r
30 34
4
4
+
515.0 .
The tertꢀbutyl 1ꢀ(4ꢀ(5ꢀ(4ꢀbutylꢀ5ꢀphenylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)benzyl)ꢀazetidineꢀ3ꢀ
carboxylate intermediate (0.156 g, 0.303 mmol) was stirred in trifluoroacetic acid (4.03 mL, 52.3 mmol)
for 30 min. and concentrated under reduced pressure to give the desired product as a yellow, viscous oil.
The oil was dissolved in dichloromethane and washed with a saturated aqueous solution of sodium
bicarbonate. The organic layer, which contained the product (presumably as the sodium salt) was
concentrated under reduced pressure. The white, solid residue was dissolved in water (pH ~9), and the
pH was adjusted to ~4.5 with concentrated hydrochloric acid. The resulting white precipitate was
collected by vacuum filtration and dried. The solid was suspended in methanol with sonication, and the
solid was collected by vacuum filtration, washed with methanol, and dried under reduced pressure to
give 1ꢀ(4ꢀ(5ꢀ(4ꢀbutylꢀ5ꢀphenylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)benzyl)azetidineꢀ3ꢀcarboxylic acid
(5e, 0.102 g, 0.222 mmol, 74%) as a white solid. 99.0%; t = 8.75 min. (Method B); LCMS (ESI) m/z
r
+
1
Calcd for C H N O [M + H] 459.2. Found: 459.1. HꢀNMR (500 MHz, DMSOꢀd ) δ ppm 0.91 (t,
2
6
26
4
4
6
J=7.42 Hz, 3H), 1.37 ꢀ 1.46 (m, 2H), 1.62 ꢀ 1.70 (m, 2H), 3.00 ꢀ 3.06 (m, 2H), 3.23 (br. s., 3H), 3.43 (br.
s., 2H), 3.65 (s, 2H), 7.52 (d, J=8.25 Hz, 2H), 7.59 ꢀ 7.68 (m, 3H), 7.83 (d, J=7.15 Hz, 2H), 8.06 (d,
J=8.25 Hz, 2H), and 12.27 (s, 1H).
1-(4-(5-(4-isobutyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)-azetidine-3-carboxylic
1
6b
acid (5f).
A solution of (4ꢀmethylpentꢀ1ꢀynyl)benzene (0.247 g, 1.56 mmol), dimethyl 2ꢀ
nitromalonate (0.421 mL, 3.12 mmol), 1ꢀbutylꢀ3ꢀmethylimidazoliumhexafluorophosphate (0.032 mL,
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Journal of Medicinal Chemistry
Page 20 of 67
0
.156 mmol) in toluene (5 mL) was subjected to the microwave conditions at 170°C for 500 min. The
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
toluene was decanted off leaving a dark gum which was triturated several times with ethyl acetate. The
combined organic layers were concentrated under reduced pressure. The residue was purified by flash
silica gel chromatography using 5% ethyl acetate in hexane to give methyl 4ꢀisobutylꢀ5ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
phenylisoxazoleꢀ3ꢀcarboxylate (0.122 g, 0.470 mmol, 30% yield) as a clear, colorless oil. HPLC t =
r
+
1
2
.96 min. (Method C); LCMS (ESI) m/z Calcd for C H NO [M + H] 260.1. Found: 259.9. HꢀNMR
15 17 3
(500 MHz, CDCl ) δ ppm 0.88 (s, 3H), 0.89 (s, 3H), 1.89 (dt, J=13.75, 6.87 Hz, 1H), 2.80 (d, J=7.15
3
Hz, 2 H), 4.00 (s, 3H), 7.45 ꢀ 7.53 (m, 3H), 7.73 (d, J=6.60 Hz, 2H).
A mixture of methyl 4ꢀisobutylꢀ5ꢀphenylisoxazoleꢀ3ꢀcarboxylate (0.122 g, 0.470 mmol) and lithium
hydroxide hydrate (0.020 g, 0.470 mmol) in methanol (2.5 mL) and water (1.25 mL) was stirred at room
temperature overnight. The reaction mixture was concentrated to dryness to give 4ꢀisobutylꢀ5ꢀ
phenylisoxazoleꢀ3ꢀcarboxylic acid, lithium salt (0.119 g, 0.472 mmol, 100 % yield) as a white solid.
+
HPLC t = 2.68 min. (Method C); LCMS (ESI) m/z Calcd for C H NO [M + H] 245.1. Found: 245.8.
r
14 15
3
A mixture of 4ꢀisobutylꢀ5ꢀphenylisoxazoleꢀ3ꢀcarboxylic acid, lithium salt (0.119 g, 0.472 mmol),
tertꢀbutyl 1ꢀ(4ꢀ(N'ꢀhydroxycarbamimidoyl)benzyl)azetidineꢀ3ꢀcarboxylate (10, 0.144 g, 0.472 mmol),
N1ꢀ((ethylimino)methylene)ꢀN3,N3ꢀdimethylpropaneꢀ1,3ꢀdiamine hydrochloride (0.090 g, 0.472
mmol), and HOBt (0.072 g, 0.472 mmol) in N,Nꢀdimethylformamide (3.6 mL) was stirred at room
temperature for 15 min and then heated at 60°C overnight. The reaction mixture was diluted with ethyl
acetate, washed with a saturated aqueous solution of sodium bicarbonate (2x), washed with a 10%
aqueous solution of lithium chloride (2x), and dried over anhydrous sodium sulfate. The product
mixture was concentrated under reduced pressure. The residue was purified by flash silica gel
chromatography using a 1% mixture of methanol in dichloromethane to give tertꢀbutyl 1ꢀ(4ꢀ(5ꢀ(4ꢀ
isobutylꢀ5ꢀphenylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)benzyl)azetidineꢀ3ꢀcarboxylate (0.179 g, 0.348
mmol, 74%) as a pale yellow solid. HPLC purity 98%; t = 3.27 min. (Method C); LCMS (ESI) m/z
r
+
Calcd for C H N O [M + H] 515.3. Found: 515.4.
3
0
34
4
4
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Page 21 of 67
Journal of Medicinal Chemistry
The tertꢀbutyl 1ꢀ(4ꢀ(5ꢀ(4ꢀisobutylꢀ5ꢀphenylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)benzyl)ꢀazetidineꢀ3ꢀ
1
2
carboxylate intermediate was stirred in trifluoroacetic acid (3.64 mL, 47.2 mmol) for 30 min. and
concentrated under reduced pressure to give the desired product as a pale yellow solid. The solid was
dispersed in water with sonication. The pH was adjusted to ~4, and the resulting solid was collected by
vacuum filtration, washed with water (3x), and dried. The white solid was triturated with methanol with
sonication and filtered to give 1ꢀ(4ꢀ(5ꢀ(4ꢀisobutylꢀ5ꢀphenylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀ
yl)benzyl)azetidineꢀ3ꢀcarboxylic acid (5f, 0.074 g, 0.161 mmol, 34% yield) as a white solid. HPLC
purity 98.4%; t = 8.12 min. (Method A); 97.4%; t = 8.60 min. (Method B); LCMS (ESI) m/z Calcd for
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
r
r
+
1
C H N O [M + H] 459.2. Found: 459.3. HꢀNMR (500 MHz, DMSOꢀd ) δ ppm 0.87 (d, J=6.60 Hz,
26
26
4
4
6
6
H), 1.93 (ddd, J=13.61, 6.74, 6.60 Hz, 1H), 2.99 (d, J=7.15 Hz, 2H), 3.24 (br. s., 3H), 3.43 (s, 2 H),
3.66 (s, 2H), 7.53 (d, J=7.70 Hz, 2H), 7.58 ꢀ 7.67 (m, 3H), 7.87 (d, J=6.60 Hz, 2H), and 8.06 (d, 2H).
Preparation of 1-(4-(5-(4-isopropyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)-
azetidine-3-carboxylic acid (5g).
1
6b
A degassed solution of iodobenzene (0.549 mL, 4.90 mmol),
bis(triphenylphosphine)palladium(II) chloride (0.206 g, 0.294 mmol), copper(I) iodide (0.047 g, 0.245
mmol) and diisopropylamine (2.79 mL, 19.6 mmol) in N,Nꢀdimethylformamide (20 mL) was added 3ꢀ
methylbutꢀ1ꢀyne (0.501 g, 7.35 mmol). The reaction mixture was heated to 75°C for 1 h. The reaction
mixture was then diluted with ethyl acetate (150 mL), washed with a 10% aqueous solution of lithium
chloride (2 x 100mL), washed with a 2M aqueous solution of ammonium hydroxide (100 mL), washed
with brine, (100 mL), and dried over anhydrous sodium sulfate. Concentration under reduce pressure
followed by purification by silica gel chromatography with hexane/dichloromethane (10/1) afforded (3ꢀ
methylbutꢀ1ꢀynyl)benzene (225 mg). HPLC t = 3.58 min. (Method D).
r
A solution of (3ꢀmethylbutꢀ1ꢀynyl)benzene (225 mg, 1.56 mmol), dimethyl nitromalonate (0.527
mL, 3.90 mmol), and 1ꢀbutylꢀ3ꢀmethylimidazolium hexafluorophosphate (0.032 mL, 0.156 mmol) in
toluene (6 mL) was heated to 170°C for 160 minutes in a sealed tube. The reaction mixture was
concentrated and purified by silica gel chromatography (hexanes/ethyl acetate; 10:1) to afford methyl 4ꢀ
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Page 22 of 67
isopropylꢀ5ꢀphenylisoxazoleꢀ3ꢀcarboxylate (136 mg, 0.554 mmol, 36%). HPLC t = 3.37 min. (Method
r
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
+
D); LCMS (ESI) m/z Calcd for C H NO [M + H] 246.1. Found: 246 .
1
4
15
3
A solution of methyl 4ꢀisopropylꢀ5ꢀphenylisoxazoleꢀ3ꢀcarboxylate (25 mg, 0.108 mmol) and 1N
aqueous sodium hydroxide (150 µL, 0.150 mmol) in methanol (1 mL) was heated at 100°C for 10
minutes under microwave conditions. The reaction mixture was concentrated to yield 4ꢀisopropylꢀ5ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
phenylisoxazoleꢀ3ꢀcarboxylic acid (23 mg, 0.099 mmol, >99%). HPLC t = 3.01 min. (Method D);
r
+
+
LCMS (ESI) m/z Calcd for C H NO [M + H] 232.1. Found: 232 .
1
3
13
3
To a solution of 4ꢀisopropylꢀ5ꢀphenylisoxazoleꢀ3ꢀcarboxylic acid (23 mg, 0.099 mmol), HOBt (27.7
mg, 0.181 mmol) and diisopropylamine (0.070 mL, 0.401 mmol) in acetonitrile (1 mL) was added N1ꢀ
((ethylimino)methylene)ꢀN3,N3ꢀdimethylpropaneꢀ1,3ꢀdiamine hydrochloride (45.2 mg, 0.236 mmol)
and tertꢀbutyl 1ꢀ(4ꢀ(N'ꢀhydroxycarbamimidoyl)benzyl)azetidineꢀ3ꢀcarboxylate (10, 31 mg, 0.099
mmol). The reaction mixture was stirred at 80°C for 5 h. The reaction mixture was then concentrated,
and the residue was diluted with ethyl acetate (3 mL), washed with a saturated aqueous solution of
sodium bicarbonate (1 mL), washed with water (1 mL), washed with brine (1 mL), and dried over
anhydrous sodium sulfate. Concentration followed by purification by preparative HPLC afforded tertꢀ
butyl 1ꢀ(4ꢀ(5ꢀ(4ꢀisopropylꢀ5ꢀphenylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)benzyl)ꢀazetidineꢀ3ꢀcarboxylate.
A
solution
of
tertꢀbutyl
1ꢀ(4ꢀ(5ꢀ(4ꢀisopropylꢀ5ꢀphenylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀ
yl)benzyl)azetidineꢀ3ꢀcarboxylate in dichloromethane (0.5 mL) and trifluoroacetic acid (0.5 mL) was
stirred at room temperature for 30 minutes. Concentration under reduce pressure afforded 1ꢀ(4ꢀ(5ꢀ(4ꢀ
isopropylꢀ5ꢀphenylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)benzyl)azetidineꢀ3ꢀcarboxylic acid (5g, 35.1 mg,
0
.079 mmol, 80% over two steps). HPLC purity 97.2%; t = 7.88 min. (Method A); 98%; t = 8.40 min.
r r
+
+
1
(Method B); LCMS (ESI) m/z Calcd for C H N O [M + H] 445.2. Found: 445 . HꢀNMR (500
2
5
24
4
4
MHz, MeOD) δ ppm 1.42 (s, 3H), 1.44 (s, 3H), 3.49 ꢀ 3.55 (m, 1H), 3.71 (s, 1H), 4.38 (d, J=7.15 Hz,
4H), 4.53 (s, 2H), 7.59 ꢀ 7.63 (m, 3H), 7.66 ꢀ 7.72 (m, 4H), and 8.30 (d, J=8.25 Hz, 2H).
Preparation of 1-(4-(5-(4-cyclopropyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)-
1
6b
azetidine-3-carboxylic acid (5h).
To a degassed solution of of iodobenzene (0.549 mL, 4.90 mmol),
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Journal of Medicinal Chemistry
dichlorobis(triphenylphosphine)ꢀpalladium(II) (0.206 g, 0.294 mmol), copper(I) iodide (0.047 g, 0.245
mmol) and diiopropylamine (3.49 mL, 24.5 mmol) in N,Nꢀdimethylformamide (20 mL) was added
cyclopropylacetylene (0.622 mL, 7.35 mmol). The reaction mixture was heated at 75°C for 45 minutes.
The reaction mixture was then diluted with ethyl acetate (150 mL), washed with a 10% aqueous
solution of lithium chloride (2 x 100mL), washed with a 2M aqueous solution of ammonium hydroxide
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(100 mL), washed with brine (100 mL), and dried over anhydrous sodium sulfate. Concentration
afforded the crude product which was purified by flash silica gel chromatography with hexanes and
dichloromethane (10/1) to give (cyclopropylethynyl)benzene (691 mg). HPLC t = 3.40 min. (Method
r
D).
A solution of (cyclopropylethynyl)benzene (300 mg, 2.11 mmol) and dimethyl nitromalonate (0.854
mL, 6.33 mmol) in mesitylene (4 mL) was heated to 150°C for 16 h. The reaction mixture was
concentrated to yield the crude product which was purified by silica gel chromatography (hexanes/ethyl
acetate; 10:1) to give methyl 4ꢀcyclopropylꢀ5ꢀphenylisoxazoleꢀ3ꢀcarboxylate. The compound was reꢀ
purified by reverse phase preparative HPLC to provide methyl 4ꢀcyclopropylꢀ5ꢀphenylisoxazoleꢀ3ꢀ
carboxylate (170 mg, 0.699 mmol, 33%). HPLC t = 2.77 min. (Method D); LCMS (ESI) m/z Calcd for
r
+
+
C H NO [M + H] 244.1. Found: 244 .
14 13
3
A solution of methyl 4ꢀcyclopropylꢀ5ꢀphenylisoxazoleꢀ3ꢀcarboxylate (170 mg, 0.699 mmol) and 1N
aqueous sodium hydroxide (1 mL, 1.05 mmol) in methanol (6 mL) was heated to 80°C in a sealed tube
for 1 hr. The reaction mixture was acidified with acetic acid until the pH was ~4. The mixture was
concentrated, and the residue was purified by preparative HPLC to give 4ꢀcyclopropylꢀ5ꢀ
phenylisoxazoleꢀ3ꢀcarboxylic acid (124 mg, 0.541 mmol, 77%). HPLC t = 2.81 min. (Method D);
r
+
+
LCMS (ESI) m/z Calcd for C H NO [M + H] 230.1. Found: 230 .
1
3
11
3
To a solution of 4ꢀcyclopropylꢀ5ꢀphenylisoxazoleꢀ3ꢀcarboxylic acid (22 mg, 0.096 mmol), HOBt
26.5 mg, 0.173 mmol), and diisopropylethylamine (0.067 mL, 0.384 mmol) in acetonitrile (1 mL) was
(
added N1ꢀ((ethylimino)methylene)ꢀN3,N3ꢀdimethylpropaneꢀ1,3ꢀdiamine hydrochloride (43.2 mg, 0.226
mmol) and tertꢀbutyl 1ꢀ(4ꢀ(N'ꢀhydroxycarbamimidoyl)benzyl)azetidineꢀ3ꢀcarboxylate (10, 29.3 mg,
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Journal of Medicinal Chemistry
Page 24 of 67
0
.096 mmol). The reaction mixture was stirred at 80°C for 2 h. The reaction mixture was concentrated,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and the residue was purified by reverse phase preparative HPLC to give tertꢀbutyl 1ꢀ(4ꢀ(5ꢀ(4ꢀ
cyclopropylꢀ5ꢀphenylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)benzyl)azetidineꢀ3ꢀcarboxylate.
A
solution of tertꢀbutyl 1ꢀ(4ꢀ(5ꢀ(4ꢀcyclopropylꢀ5ꢀphenylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀ
yl)benzyl)azetidineꢀ3ꢀcarboxylate in dichloromethane (0.5 mL) and trifluoroacetic acid (0.5 mL) was
stirred at room temperature for 30 minutes. Concentration under reduced pressure afforded 1ꢀ(4ꢀ(5ꢀ(4ꢀ
cyclopropylꢀ5ꢀphenylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)benzyl)azetidineꢀcarboxylic acid (5h, 26.2 mg,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.059 mmol, 62% over two steps). HPLC purity 97%; t = 3.06 min. (Method D); LCMS (ESI) m/z
r
+
+
1
Calcd for C H N O [M + H] 443.2. Found: 443 . HꢀNMR (400 MHz, CD OD) δ ppm 0.41 ꢀ 0.47
2
5
22
4
4
3
(m, 2H), 1.09 ꢀ 1.16 (m, 2H), 2.12 (tt, J=8.31, 5.36 Hz, 1H), 3.66 ꢀ 3.77 (m, 1H), 4.35 ꢀ 4.41 (m, 4H),
4
2
.53 (s, 2H), 7.56 ꢀ 7.64 (m, 3H), 7.70 (d, J=8.53 Hz, 2H), 7.95 ꢀ 8.00 (m, 2H), and 8.32 (d, J=8.28 Hz,
H).
1-(4-(5-(4-tert-butyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)-azetidine-3-carboxylic
1
6b
acid (5i).
To
a
degassed solution of iodobenzene (0.549 mL, 4.90 mmol),
dichlorobis(triphenylphosphine)ꢀpalladium(II) (0.206 g, 0.294 mmol), copper(I) iodide (0.047 g, 0.245
mmol), and diisopropylamine (3.49 mL, 24.5 mmol) in N,Nꢀdimethylformamide (20 mL) was added
3
,3ꢀdimethylbutꢀ1ꢀyne (0.896 mL, 7.35 mmol). The reaction mixture was and heated to 75°C for 45
minutes. The reaction mixture was then diluted with ethyl acetate (150 mL), washed with 10% aqueous
solution of lithium chloride (2 x 100mL), washed with a 2M aqueous solution of ammonium hydroxide,
washed with brine (100 mL), and dried over anhydrous sodium sulfate. Concentration followed by
purification by flash silica gel chromatography (hexanes/dichloromethane; 10:1) afforded (3,3ꢀ
dimethylbutꢀ1ꢀynyl)benzene (740 mg). HPLC t = 3.78 min. (Method D).
r
A solution of (3,3ꢀdimethylbutꢀ1ꢀynyl)benzene (400 mg, 2.53 mmol) and dimethyl nitromalonate
(0.853 mL, 6.32 mmol) in mesitylene (5 mL) was heated at 150°C for 48 h. The reaction mixture was
concentrated, and the residue was purified by silica gel chromatography ( hexanes/ethyl acetate; 10:1) to
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Journal of Medicinal Chemistry
yield methyl 4ꢀtertꢀbutylꢀ5ꢀphenylisoxazoleꢀ3ꢀcarboxylate (101 mg, 0.389 mmol, 15%). HPLC t = 3.39
r
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
+
min. (Method D); LCMS (ESI) m/z Calcd for C H NO [M + H] 260.1. Found: 260 .
1
5
17
3
A solution of methyl 4ꢀtertꢀbutylꢀ5ꢀphenylisoxazoleꢀ3ꢀcarboxylate (26 mg, 0.100 mmol) and 1N
aqueous sodium hydroxide (150 µL, 0.150 mmol) in methanol (1 mL) was heated at 100°C for 10
minutes under microwave conditions. The reaction mixture was concentrated to give 4ꢀtertꢀbutylꢀ5ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
phenylisoxazoleꢀ3ꢀcarboxylic acid, sodium salt (25 mg, 0.100 mmol, >99%). HPLC t = 2.97 min.
r
+
+
(
Method D); LCMS (ESI) m/z Calcd for C H NO [M + H] 246.1. Found: 246 .
14 15 3
To a solution of 4ꢀtertꢀbutylꢀ5ꢀphenylisoxazoleꢀ3ꢀcarboxylic acid (25 mg, 0.102 mmol), HOBt (28.1
mg, 0.183 mmol), and diisopropylethylamine (0.071 mL, 0.408 mmol) in acetonitrile (1 mL) was added
N1ꢀ((ethylimino)methylene)ꢀN3,N3ꢀdimethylpropaneꢀ1,3ꢀdiamine hydrochloride (45.9 mg, 0.240
mmol) and tertꢀbutyl 1ꢀ(4ꢀ(N'ꢀhydroxycarbamimidoyl)benzyl)azetidineꢀ3ꢀcarboxylate (10, 31.1 mg,
0
.102 mmol). The reaction mixture was stirred at 80°C for 5 h. The reaction mixture was
concentrated, and the residue was diluted with ethyl acetate (3 mL), washed with a saturated aqueous
solution of sodium bicarbonate (1 mL), washed with water (1 mL), washed with brine (1 mL), and dried
over anhydrous sodium sulfate. Concentration followed by purification by reverse phase preparative
HPLC afforded tertꢀbutyl 1ꢀ(4ꢀ(5ꢀ(4ꢀtertꢀbutylꢀ5ꢀphenylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)benzyl)ꢀ
azetidineꢀ3ꢀcarboxylate.
A
solution
of
tertꢀbutyl
1ꢀ(4ꢀ(5ꢀ(4ꢀtertꢀbutylꢀ5ꢀphenylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀ
yl)benzyl)azetidineꢀ3ꢀcarboxylate in dichloromethane (0.5 mL) and trifluoroacetic acid (0.5 mL) was
stirred at room temperature for 30 minutes. Concentration under reduced pressure afforded 1ꢀ(4ꢀ(5ꢀ(4ꢀ
tertꢀbutylꢀ5ꢀphenylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)benzyl)azetidineꢀ3ꢀcarboxylic
acid,
2,2,2ꢀ
trifluoroacetic acid salt (5i, 31 mg, 0.068 mmol, 67% over two steps). HPLC purity 97.0%; t = 7.95
r
+
min. (Method A); 97.9%; t = 8.53 min. (Method B); LCMS (ESI) m/z Calcd for C H N O [M + H]
r
26 26
4
4
+
1
459.2. Found: 459 . HꢀNMR (500 MHz, CD OD) δ ppm 1.27 (s, 9H), 3.68 ꢀ 3.76 (m, 1H), 4.39 (d,
3
J=7.70 Hz, 4H), 4.54 (s, 2H), 7.53 ꢀ 7.57 (m, 4H), 7.57 ꢀ 7.63 (m, 1H), 7.70 (d, J=8.25 Hz, 2H), and
8
.29 (d, J=8.25 Hz, 2H).
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1
-(4-(5-(4,5-diphenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic
acid
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
6b
(
5j).
A mixture of 1,2ꢀdiphenylethyne (1.53 g, 8.6 mmol) and dimethyl 2ꢀnitromalonate (1.05 mL,
7
.83 mmol) in mesitylene (11 ml) was heated to 150°C for 18 hr. Concentration under reduced pressure
followed by purification by flash silica gel chromatography with a mixutre of ethyl acetate and hexane (
0ꢀ10%) afforded methyl 4,5ꢀdiphenylisoxazoleꢀ3ꢀcarboxylate (307 mg, 1.1 mmol, 14%) as a light
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
yellow solid. HPLC t = 1.85 min. (Method D); LCMS (ESI) m/z Calcd for C H NO [M + H] 280.1.
r
17 13
3
1
Found: 280.1. HꢀNMR (400 MHz, CDCl ) δ ppm 3.87 (s, 3 H) 7.29 ꢀ 7.40 (m, 5 H) 7.43 (m, 3H) 7.47
3
ꢀ
7.53 (m, 2 H).
To a thick suspension of methyl 4,5ꢀdiphenylisoxazoleꢀ3ꢀcarboxylate (306 mg, 1.09 mmol) in
methanol (8 mL), tetrahydrofuran (2 mL) and water (2 mL) at room temperature was added lithium
hydroxide, monohydrate (46.0 mg, 1.09 mmol), and the reaction mixture was allowed to stir at room
temperature for 1 hr. During this time, the reaction became homogeneous. The solvents were removed
under reduced pressure, and the remaining aqueous mixture was acidified to pH ~1 with 1N aqueous
hydrochloric acid. The mixture was then extracted with ethyl acetate (30 mL), and the organic layer
was washed with brine (20 mL), dried over anhydrous magnesium sulfate and concentrated to afford
4
,5ꢀdiphenylisoxazoleꢀ3ꢀcarboxylic acid (290 mg, 1.09 mmol, 100 % yield) as a yellow solid. HPLC tr
+
=
1.72 min. (Method D); LCMS (ESI) m/z Calcd for C H NO [M + H] 266.1. Found: 266.1.
1
6
11
3
A mixture of 4,5ꢀdiphenylisoxazoleꢀ3ꢀcarboxylic acid (26.5 mg, 0.1 mmol), tertꢀbutyl 1ꢀ(4ꢀ(N'ꢀ
hydroxycarbamimidoyl)benzyl)azetidineꢀ3ꢀcarboxylate, 10, (30.5 mg, 0.100 mmol), HOBt (16.85 mg,
.110 mmol) and N1ꢀ((ethylimino)methylene)ꢀN3,N3ꢀdimethylpropaneꢀ1,3ꢀdiamine hydrochloride
0
(21.09 mg, 0.110 mmol) in dimethylformamide (1 mL) was stirred at room temperature for 18 hr. The
reaction mixture was heated at 60°C for 2 h and 70°C for 2 h. After cooling to room temperature
overnight, the reaction mixture was partitioned between ethyl acetate (30 mL) and washed with a
saturated sodium bicarbonate solution (30 mL). The organic layer was washed with water (2 x 30 mL),
washed with brine (20 mL), and dried over anhydrous magnesium sulfate. The organic layer was
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Journal of Medicinal Chemistry
concentrated to give a yellow solid which was purified by flash silica gel chromatography eluting with a
ꢀ60% mixture of ethyl acetate and hexane to afford tertꢀbutyl 1ꢀ(4ꢀ(5ꢀ(4,5ꢀdiphenylisoxazolꢀ3ꢀyl)ꢀ
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1,2,4ꢀoxadiazolꢀ3ꢀyl)benzyl)azetidineꢀ3ꢀcarboxylate (37 mg, 0.069 mmol, 69% yield) as a white solid.
+
HPLC t = 1.90 min. (Method D); LCMS (ESI) m/z Calcd for C H N O [M + H] 535.2. Found:
r
32 30
4
4
5
35.2.
A solution of tertꢀbutyl 1ꢀ(4ꢀ(5ꢀ(4,5ꢀdiphenylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)benzyl)azetidineꢀ3ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
carboxylate (37 mg, 0.069 mmol) in tetrahydrofuran (1 mL) was allowed to stand at room temperature
for 1 hr. The volatiles were removed under reduced pressure, and the residue was subjected to reverse
phase preparative HPLC to afford 1ꢀ(4ꢀ(5ꢀ(4,5ꢀdiphenylisoxazolꢀ3ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀ
yl)benzyl)azetidineꢀ3ꢀcarboxylic acid, trifluoroacetic acid salt (5j, 1 mg, 0.052 mmol, 76%) as a white
solid. HPLC purity 95.9%; t = 9.17 min. (Method A); 95.0%; t = 9.94 min. (Method B); LCMS (ESI)
r
r
+
1
m/z Calcd for C H N O [M + H] 479.2. Found: 479.1. HꢀNMR (500 MHz, CD OD) δ ppm 3.70
2
8
22
4
4
3
(m, 1 H) 4.31 ꢀ 4.38 (m, 4 H) 4.48 (s, 2 H) 7.37 ꢀ 7.43 (m, 2 H) 7.43 ꢀ 7.53 (m, 6 H) 7.56 ꢀ 7.65 (m, 4 H)
.09 (d, J=8.3 Hz, 2 H).
-(4-(5-(5-phenyl-4-(trifluoromethyl)isoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-
8
1
1
6b
carboxylic acid (5k).
To a mixture of (Z)ꢀethyl 2ꢀchloroꢀ2ꢀ(hydroxyimino)acetate (3.03 g, 20 mmol)
and ethynylbenzene (4.39 mL, 40 mmol) in ether (80 mL) at room temperature was added a solution of
triethylamine (5.58 mL, 40.0 mmol) in ether (20 mL) dropwise over 60 minutes. The reaction mixture
was stirred for 2 h at room temperature. The reaction mixture was filtered, and the filtrate was
concentrated to a yellow oil which was purified by flash silica gel chromatography using a mixture of
ethyl acetate in hexane (0ꢀ12%) to afford ethyl 5ꢀphenylisoxazoleꢀ3ꢀcarboxylate (14, 3.06 g, 14.1 mmol,
7
0%) as a white solid. HPLC t = 2.99 min. (Method D); LCMS (ESI) m/z Calcd for C H NO [M +
r 12 11 3
+
1
H] 218.1. Found: 218.1. HꢀNMR (400 MHz, CDCl ) δ ppm 1.45 (t, J=7.3Hz, 3H), 4.48 (q, J=7.3,
3
2H), 6.93 (s, 1H), 7.45 ꢀ 7.53 (m, 3H), and 7.77 ꢀ 7.85 (m, 2H).
A mixture of ethyl 5ꢀphenylisoxazoleꢀ3ꢀcarboxylate (14, 3.05 g, 14.0 mmol) and Nꢀiodosuccinimide
(3.79 g, 16.9 mmol) in trifluoroacetic acid (78 mL) was stirred at room temperature for 3.5 h. The
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Journal of Medicinal Chemistry
Page 28 of 67
volatiles were removed under reduced pressure, and the residue was partitioned between ethyl acetate
150 mL) and water (150 mL). The organic layer was washed with a 1N aqueous solution of sodium
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
hydroxide (150 mL), washed with a 3% aqueous solution of sodium bisulfite (2 x 150 mL), washed with
brine (150 mL), and dried over anhydrous magnesium sulfate. Concentration under reduced pressure
afforded ethyl 4ꢀiodoꢀ5ꢀphenylisoxazoleꢀ3ꢀcarboxylate (15, 4.69 g, 13.7 mmol, 97%) as a light yellow
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
oil. HPLC t = 3.36 min. (Method D); LCMS (ESI) m/z Calcd for C H INO [M + H] 344.0. Found:
r
12 10
3
1
3
44.0. HꢀNMR (400 MHz, CDCl ) δ ppm 1.47 (t, J=7.1 Hz, 3H), 4.50 (q, J=7.0Hz, 2H), 7.52 ꢀ 7.56
3
(m, 3H), and 8.05 (m, 2H).
To a solution of ethyl 4ꢀiodoꢀ5ꢀphenylisoxazoleꢀ3ꢀcarboxylate (4.62 g, 13.5 mmol) and copper(I)
iodide (0.513 g, 2.69 mmol) in N,Nꢀdimethylformamide (59.8 mL) and HMPA (7.48 mL) at room
temperature was added methyl 2,2ꢀdifluoroꢀ2ꢀ(fluorosulfonyl)acetate (6.86 mL, 53.9 mmol) at once.
The reaction mixture was immediately immersed in an oil bath at 75ꢀ80 °C. Stirring was continued at
this temperature for 3.5 h. After cooling to room temperature, the reaction mixture was cooled in an ice
bath. A saturated aqueous solution of ammonium chloride (~50 mL) was added slowly to quench the
reaction. The mixture was partitioned between ethyl ether (400 mL) and a saturated aqueous solution of
ammonium chloride (400 mL). The organic layer was washed with a saturated aqueous solution of
ammonium chloride (200 mL), washed with water (2 x 200 mL), washed with brine (50 mL), and dried
over anhydrous magnesium sulfate. Concentration under reduced pressure followed by purification by
flash silica gel chromatography using a mixture of ethyl acetate in hexane (0ꢀ10%) afforded ethyl 5ꢀ
phenylꢀ4ꢀ(trifluoromethyl)isoxazoleꢀ3ꢀcarboxylate (16, 3.6 g, 12.6 mmol, 94% yield) as a colorless oil.
+
HPLC t = 3.44 min. (Method D); LCMS (EI) m/z Calcd for C H INO [M + H] 286.1. Found: 286.0.
r
13 10
3
1
HꢀNMR (400 MHz, CDCl ) δ ppm 1.45 (t, J=7.2 Hz, 3H), 4.51 (q, J=7.3 Hz, 2H), 7.52 ꢀ 7.62 (m, 3H),
3
and 7.69 (d, J=7.5 Hz, 2H).
To a solution of ethyl 5ꢀphenylꢀ4ꢀ(trifluoromethyl)isoxazoleꢀ3ꢀcarboxylate (16, 3.6 g, 12.6 mmol) in
methanol (100 mL) and water (20 mL) at room temperature was added lithium hydroxide, monohydrate
(0.583 g, 13.9 mmol). The reaction mixture was stirred at room temperature for 30 minutes. The
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Journal of Medicinal Chemistry
methanol was removed under reduce pressure, and the residue was diluted with water (~100 mL). Ethyl
ether (200 mL) was added, and the pH of the aqueous layer was adjusted to <1 with a 1N aqueous
solution of hydrochloric acid. The mixture was transferred to a separatory funnel, and after agitation,
the layers were separated. The organic layer was washed with brine (100 mL), dried over anhydrous
magnesium sulfate, and concentrated to afford 5ꢀphenylꢀ4ꢀ(trifluoromethyl)isoxazoleꢀ3ꢀcarboxylic acid
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
17, 3.12 g, 12.13 mmol, 96% yield) as a white solid. HPLC t = 2.58 min. (Method D); LCMS (ESI)
r
+
1
m/z Calcd for C H F NO [M + H] 258.0. Found: 258.0. H NMR (400 MHz, CDCl ) δ ppm 7.53 ꢀ
1
1
6
3
3
3
7.64 (m, 3H), and 7.70 (d, J=7.5 Hz, 2H).
To a mixture of 5ꢀphenylꢀ4ꢀ(trifluoromethyl)isoxazoleꢀ3ꢀcarboxylic acid (17, 197 mg, 0.766 mmol)
and pyridine (0.074 mL, 0.919 mmol) in dichloromethane (5 mL) at room temperature was added
cyanuric fluoride (0.078 mL, 0.919 mmol). The reaction mixture was stirred at room temperature for 18
h. The mixture was diluted with dichloromethane (40 mL) and washed with an iceꢀcold 0.5N aqueous
solution of hydrochloric acid (20 mL). The aqueous layer was extracted with dichloromethane (20 mL),
and the combined organic layers were washed with iceꢀcold water (20 mL) and dried over anhydrous
magnesium sulfate.
Concentration under reduced pressure afforded 5ꢀphenylꢀ4ꢀ
(trifluoromethyl)isoxazoleꢀ3ꢀcarbonyl fluoride (199 mg, 0.768 mmol, 100%) as a pale yellow oil.
HPLC t = 2.53 min., corresponding to the methyl ester (Method D).
r
To a mixture of 5ꢀphenylꢀ4ꢀ(trifluoromethyl)isoxazoleꢀ3ꢀcarbonyl fluoride (100 mg, 0.386 mmol)
and tertꢀbutyl 1ꢀ(4ꢀ(N'ꢀhydroxycarbamimidoyl)benzyl)azetidineꢀ3ꢀcarboxylate (10, 118 mg, 0.386
mmol) in acetonitrile (2 mL) at room temperature was added Hunig's Base (0.081 mL, 0.463 mmol).
The reaction mixture was stirred at room temperature for 3 days. The volatiles were removed under
reduced pressure, and the residue was partitioned between dichloromethane (30 mL) and a saturated
aqueous solution of sodium bicarbonate (30 mL).
The aqueous layer was extracted with
dichloromethane (2 x 20 mL), and the combined organic layers were dried over anhydrous magnesium
sulfate and concentrated to afford a light yellow oil. The compound was purified by flash silica gel
chromatography using a mixture of ethyl acetate/hexane (0ꢀ70%) to give tertꢀbutyl 1ꢀ(4ꢀ(5ꢀ(5ꢀphenylꢀ4ꢀ
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