Potent and Selective Agonists of Sphingosine 1-Phosphate 1 (S1P1

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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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Journal of Medicinal Chemistry

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 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-1Phosphate 1 (S1P1): The Discovery and SAR of a 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 Bristol-Myers Squibb Research and Development, P.O. Box 4000, Princeton, New Jersey 08543

RECEIVED DATE *

To

whom

correspondence

should

be

addressed.

Phone:

609-252-6778.

E-mail:

[email protected]. Note: The authors declare no competing financial interest.

a

Abbreviations: S1P, Sphingosine-1-phosphate; S1P1-5, Sphingosine-1-phosphate receptors 1-5; GTPγS,

Guanosine-5’-O-[gamma-thio]triphosphate; PK, pharmacokinetic; SAR, structure-activity relationship; SFC, super critical fluid chromatography; hERG, human ether-a-go-go-related gene.


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ABSTRACT: Sphingosine-1-phosphate (S1P) is the endogenous ligand for the sphingosine-1-phophate receptors (S1P1-5) 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.


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Introduction Sphingosine-1-phosphate (S1P) is a lysophopholipid, endogenous mediator that functions as a highaffinity ligand for five members of the endothelial differentiation gene class of G-protein-coupled receptors, S1P1-5. 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.1 Levels of S1P in various tissues are regulated through the coordinated activities of biosynthetic kinases and biodegradative phosphatases and lyases.2 Specifically, S1P is derived from the phosphorylation of sphingosine by two kinases, S1Pkinase-1 and S1Pkinase-2. Conversely, S1P is degraded by S1P lyase and lysophospholipid phosphatases. The S1P1 receptor is the predominant receptor expressed on lymphocytes and plays a crucial role in lymphocyte egress from the thymus and lymph nodes to systemic circulation and ultimately to sites of tissue inflammation.3 The S1P concentration gradient differential between blood plasma and tissue is sensed by the lymphocytes through the interaction of the S1P1 receptor with circulating S1P, resulting in egress.2,4 In support of this, conditional deletion of the S1P1 receptors expressed on lymphocytes in mice resulted in internalization of cell-membrane expressed receptors.3,5,6 The subsequent decrease of S1P1 receptor activity correlated with sequestration of lymphocytes in the lymph nodes.3,5,6 This was further supported in studies with conditional S1Pkinase-deficient mice in which the lack of S1P in the plasma and lymph nodes resulted in a significant reduction in circulating lymphocytes.2,6 Fingolimod (1) was shown in vivo to induce a significant, but reversible, reduction in circulating lymphocytes,7 and as a result, 1 was found to be efficacious in several animal models of inflammation. Although 1 is inactive toward the S1P1-5 receptors, a phosphorylated

metabolic derivative 2

(fingolimod-P) generated through the stereospecific action of S1Pkinase-2, analogous to S1P, was found to act as a potent, full agonist of the S1P1,3,4,5 receptors with no activity toward S1P2.7 Additionally, unlike S1P, fingolimod-P (2) agonism of S1P1 resulted in prolonged internalization and degradation of the receptor, thus acting as a “functional antagonist.”7 With this mechanism of action, lymphocytes are no longer able to recognize and respond to changes in the S1P gradient, thus preventing their migration ACS Paragon Plus Environment

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into circulation and tissue. With positive Phase III clinical results, fingolimod (1) was approved in 2010 as the first oral disease-modifying treatment for relapsing-remitting multiple sclerosis (RRMS).7 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 half-life resulting in a prolonged pharmacologic effect after drug discontinuation.8 Initially, the cardiovascular effects were linked to the agonism of S1P3, as supported by rodent studies,9 but more recent clinical data suggest that there is no solid correlation between rodent and human. In particular, clinical studies with S1P3 sparing agonists have revealed that the cardiovascular effects seen in humans are independent of S1P3 receptor agonism.10 In fact, recent evidence suggests that the bradycardia in humans may be triggered by S1P1 receptor activation.10,11,13d With fingolimod’s clinical success, there has been an enormous effort to identify direct-acting, full agonists of the S1P1 receptor that do not require phosphorylation for pharmacological activity.12,13 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 half-life 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 S1P1 receptor over the S1P3 receptor was

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, S1P3 receptor agonism does not appear to contribute to the efficacy, so a desire for selectivity has remained a primary emphasis of most research efforts, including our own. The key structural features of 2, and other typical S1P1 receptor agonists, include a polar head group, a rigid linker, and a lipophilic tail group. Merck was the first to report on results aimed at identifying direct-acting agonists of the S1P receptors, ultimately leading to the identification of compounds with an azetidine-3-carboxylic polar group utilized in conjunction with a 3,5-diaryl-1,2,4ACS Paragon Plus Environment

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oxadiazole linker, as exemplified by compound 3.14 In our efforts to identify novel lipophilic fragments, the azetidine-3-carboxylic acid substituted linker 4, derived from 3, provided a valuable combination for identifying agonists of the S1P1 receptor when combined with our fragments.15,16 This effort ultimately led to the identification of two highly potent, selective isoxazole-based full agonists of the S1P1 receptor, as depicted in structures 5 and 6 shown in Figure 3. As discussed in this article, further optimization led to the identification of isoxazole 6d (BMS-520).16 Chemistry The synthetic pathways utilized in the preparation of the isoxazole analogs presented in Tables 1 and 2 are provided in Schemes 1 – 9.16 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 4-

cyanobenzaldehyde and the tert-butyl ester protected azetidine-3-carboxylic acid. Subsequent treatment with hydroxylamine afforded the amidoxime intermediate 10. Intermediate 10 was then coupled with the substituted isoxazole-3-carboxylic acids 13a-n or isoxazole-5-carboxylic 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. Isoxazole-3-carboxylic 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 and dimethyl nitromalonate was either heated in mesitylene in a sealed tube at 150oC overnight or heated in acetonitrile under microwave conditions at 170oC in the presence of an ionic liquid, 1-butyl-3methylimidazoliumhexafluorophosphate, to give ethers 12b-j and 12l-m. Subsequent hydrolysis with either lithium hydroxide or sodium hydroxide afforded the desired isoxazole-2-carboxylic acids 13b-j and 13l-m in good yield. The C4 trifluoromethyl-isoxazoles acids 5k and 5n were prepared in high yield as outlined in Scheme 3. Isoxazole ester 14 was iodinated at C4 with N-iodosuccinimide and trifluoroacectic acid to give intermediate 15 in 97% yield. Trifluoromethylation at C4 was carried out ACS Paragon Plus Environment

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with methyl 2,2-difluoro-2-(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 5-isobutyl-4-(trifluoromethyl)isoxazole-3-carboxylic acid 21 was prepared in an analogous manner in high yield. The C4-difluoroethyl isoxazole 5l was prepared as shown in Scheme 4. The synthetic routes used to prepare the C5 linked isoxazoles 6a-d are outlined in Schemes 5 – 9. Isoxazole-5-carboxylic acids 29a-c were either commercially available or prepared as outlined in Scheme 5. A mixture of the N-hydroxybenzimidoyl chloride 2617 and substituted ethyl bromo-enoates 27b 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 1,3-dipolar cycloaddition reaction between ethyl trifluorobut-2-ynoate and a dipole generated in situ from N-hydroxybenzimidoyl chloride favoring the formation of the undesired isomer 31 in a ratio of 85:15.18 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 trifluorobut-2-ynoate with the trifluorobut-2-yn-1-ol, prepared as shown in Scheme 8.19 As expected, slow addition of triethylamine to the mixture of N-hydroxybenzimidoyl chloride 2617

and

trifluorobutyne alcohol 32 at 70 oC 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 3-phenyl-4-(trifluoromethyl)isoxazole-5-carboxylic 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 ACS Paragon Plus Environment

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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. Isoxazole agonists presented in Tables 1 and 2 were evaluated in a functional GTPγS assay looking at both human S1P1 and S1P3 receptor agonism. While it is important to maintain selectivity against the S1P2 and S1P3 receptors, activity against the S1P4 and S1P5 receptors was less of a concern and was 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 structure-activity relationship (SAR) for agonists 5 derived from isoxazole-3carboxylic acids. The more facile synthesis of these examples allowed for a broader exploration of SAR, with a particular focus on exploring substitution at R2 while maintaining R1 as a phenyl substituent (5a-l). Isoxazole 5a, with no C4 substitution, provided modest agonist activity against the S1P1 receptor with an EC50 of 44 nM.

The addition of a methyl group (5b) provided a 9-fold

improvement in potency, as well as enhanced selectivity for the S1P1 receptor relative to the S1P3 receptor. In general, increasing the length of the alkyl chain (5c-f) resulted in improvements in activity, with butyl and iso-butyl substitution providing potent agonist activity against the S1P1 receptor with EC50 values of 0.27 nM and 0.56 nM, respectively. On the other hand, shorter, branched alkyl groups (5g-i) were less potent compared with the longer, linear side chains (e.g. 5i, EC50 = 5.3 nM vs. 5d, EC50 = 0.27 nM). In the BLR PD/PK assay, the lymphocyte reduction at 4 and 24 h time points with an oral ACS Paragon Plus Environment

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dose of 1 mg/kg was consistent with the agonist activity trends observed for the C4 alkyl isoxazoles 5bf 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 S1P1 receptor but selectivity against the S1P3 receptor was reduced relative to 5b-i. Although the C4 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 (ED50 of 0.98 nM; S1P3/S1P1 selectivity: 4,000-fold). 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 peak-to-through 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 isoxazole-5-carloxylic acids highlighted in Table 2 followed the same trends observed with the C3-linked 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 4h/24h peak-to-through ratio of only 3. ACS Paragon Plus Environment

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The ability of 6d to drive receptor internalization was investigated using CHO cells transfected with human S1P1 fused with GFP. In this assay, 6d induced S1P1 internalization with an EC50 of 0.27 ±0.004 nM (n=3), similar to fingolimod-P (2, EC50: 0.28 ±0.1 nM). The maximal signal of S1P1 receptor internalization was similar between 4d and S1P treatment. As seen in Figure 4, S1P resulted in >90% receptor internalization. Following a washout period, S1P1 receptors reappeared on the cell surface by 4 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 S1P1 receptor degradation similar to that reported for fingolimod-P (2).7 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 half-lives 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 half-life of only 3 h, consequently leading to a larger peak-to-through 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. In a rat adjuvant arthritis study, a preclinical model of human rheumatoid arthritis,20 arthritis was induced by the subcutaneous injection of Complete Freund’s Adjuvant (CFA) containing heat-killed 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 ACS Paragon Plus Environment

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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 ED50 was determined to be 0.05 mg/kg. At the end of the 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 dose-dependent delay in disease onset and a dose-dependent 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 ED50 for 6d was determined to be 0.02 mg/kg. At the end of the 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 S1P1 receptor with selectivity over the S1P2 and S1P3 receptors has been identified. Through this effort, we identified isoxazole 6d (BMS520) 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 ACS Paragon Plus Environment

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rheumatoid arthritis, 6d demonstrated robust efficacy with an ED50 of 0.05 mg/kg. In a mouse EAE 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 (BMS-520) was selected as a development candidate. Experimental Proton magnetic resonance (1H) 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 SCL-10A liquid chromatograph and a SPD UV-Vis detector at 220 or 254 nm with the MS detection performed on a Waters Micromass ZQ spectrometer. Preparative reverse-phase 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 µm). 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. ACS Paragon Plus Environment

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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. Method C. A linear gradient using 10% methanol, 90% water, and 0.2% H3PO4 (Solvent A) and 90% methanol, 10% water, and 0.2% H3PO4 (Solvent B); t = 0 min., 0% B, t = 4 min., 100% B (5 min.) was 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% H3PO4 (Solvent A) and 90% methanol, 10% water, and 0.2% H3PO4 (Solvent B); t = 0 min., 0% B, t = 4 min., 100% B (5 min.) was 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 5-phenylisoxazole-3-carboxylic acid (0.200 g, 1.057 mmol) and pyridine (0.103 mL, 1.269 mmol) in dichloromethane (10 mL) at room temperature was added 2,4,6-trifluoro1,3,5-triazine (0.107 mL, 1.269 mmol). The reaction mixture was stirred at room temperature for 2 h. The heterogeneous reaction mixture was diluted with dichloromethane (25 mL), washed with an icecold solution of 0.5N aqueous hydrochloric acid (2 x 10 mL), and the organic layer was collected. The aqueous layer was back-extracted with dichloromethane (25 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated to afford 5-phenylisoxazole-3-carbonyl fluoride (0.201 g, 1.05 mmol, 99%) as a white solid. HPLC tr = 1.97 min., corresponding to the methyl ester (Method C). A mixture of 5-phenylisoxazole-3-carbonyl fluoride (0.201 g, 1.05 mmol), tert-butyl 1-(4-(N'hydroxycarbamimidoyl)benzyl)azetidine-3-carboxylate (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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Page 14 of 67

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 tert-butyl 1-(4-(5-(5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylate (0.253 g, 0.552 mmol, 53%) as a tan solid (two crops which were combined). HPLC tr = 2.69 min. (Method C); LCMS (ESI) m/z Calcd for C26H26N4O4 [M + H]+ 459.2. Found: 459.3. A mixture of tert-butyl 1-(4-(5-(5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3carboxylate (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 portion-wise 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 re-adjust 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 1-(4-(5-(5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid (5a, 0.150 g, 0.369 mmol, 67%) as a white solid. HPLC purity 99.6%; tr = 6.80 min. (Method A); 99.1%; tr = 8.05 min. (Method B); LCMS (ESI) m/z Calcd for C22H18N4O4 [M + H]+ 417.2. Found: 403.1.

1

H-

NMR (500 MHz, DMSO-d6) δ ppm 8.06 (4H, d, J=8.05 Hz), 7.92 (1H, s), 7.59 - 7.62 (3H, m), 7.53 (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 prop-1-ynylbenzene (1.08 mL, 8.61 mmol) and dimethyl 2-nitromalonate (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 ACS Paragon Plus Environment

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silica gel chromatography (2% - 5% ethyl acetate in hexane) to give methyl 4-methyl-5phenylisoxazole-3-carboxylate (47 mg, 0.216 mmol, 28%) as a white solid. HPLC tr = 2.42 min. (Method C); LCMS (ESI) m/z Calcd for C12H11NO3 [M + H]+ 218.1. Found: 218.1. A mixture of methyl 4-methyl-5-phenylisoxazole-3-carboxylate (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 4-methyl-5-

phenylisoxazole-3-carboxylic acid, lithium salt (0.045 g, 0.214 mmol, 99%) as a white solid. HPLC tr = 2.08 min. (Method C); LCMS (ESI) m/z Calcd for C12H11NO3 [M + H]+ 204.1. Found: 204.0. 1H-NMR (500 MHz, DMSO-d6) δ ppm 2.25 (s, 3H), 7.44 - 7.49 (m, 1H), 7.53 (t, J=7.70 Hz, 2H), and 7.69 (d, J=7.70 Hz, 2H). A mixture of 4-methyl-5-phenylisoxazole-3-carboxylic acid, lithium salt (0.021 g, 0.100 mmol), tert-butyl 1-(4-(N'-hydroxycarbamimidoyl)benzyl)azetidine-3-carboxylate (10, 0.031 g, 0.100 mmol), N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine

hydrochloride

(0.019

g,

0.100

mmol), and HOBt (0.015 g, 0.100 mmol) in N,N-dimethylformamide (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 reverse-phase preparative HPLC to give 1-(4-(5-(4-methyl-5-phenylisoxazol3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid, TFA (5b, 0.026 g, 0.048 mmol, 48%) as a white solid. HPLC purity 97.0%; tr = 7.16 min. (Method A); 97.0%; tr = 8.35 min. (Method B); LCMS (ESI) m/z Calcd for C23H20N4O4 [M + H]+ 417.2. Found: 417.1. 1H-NMR (500 MHz, DMSO-d6) 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., 1H). Preparation of 1-(4-(5-(4-ethyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3carboxylic acid (5c).16b

A solution of but-1-ynylbenzene (404 mg, 3.11 mmol) and dimethyl ACS Paragon Plus Environment

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Page 16 of 67

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 4-ethyl-5-phenylisoxazole-3-carboxylate (194 mg, 0.791 mmol, 28%). HPLC tr = 3.25 min. (Method D); LCMS (ESI) m/z Calcd for C13H13NO3 [M + H]+ 232.1. Found: 232+. A solution of methyl 4-ethyl-5-phenylisoxazole-3-carboxylate (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 4-ethyl-5-phenylisoxazole-3carboxylic acid (148 mg, 0.735 mmol, 87%). HPLC tr = 2.93 min. (Method D); LCMS (ESI) m/z Calcd for C12H11NO3 [M + H]+ 218.1. Found: 218+. To a solution of 4-ethyl-5-phenylisoxazole-3-carboxylic 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 N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine hydrochloride (45.6 mg, 0.238 mmol) and tert-butyl 1-(4-(N'-hydroxycarbamimidoyl)benzyl)azetidine-3-carboxylate (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

tert-butyl

1-(4-(5-(4-ethyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-

carboxylate. A

solution

of

tert-butyl

1-(4-(5-(4-ethyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-

yl)benzyl)azetidine-3-carboxylate 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

1-(4-(5-(4-ethyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic ACS Paragon Plus Environment

15

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acid (5c, 32 mg, 0.074 mmol, 73% over two steps). HPLC purity 99.1%; tr = 7.46 min. (Method A); 99.5%; tr = 8.83 min. (Method B); LCMS (ESI) m/z Calcd for C24H22N4O4 [M + H]+ 431.2. Found: 431+.

1

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). 1-(4-(5-(5-phenyl-4-propylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid (5d).16b A solution of pent-1-ynylbenzene (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; 10:1) to yield methyl 5-phenyl-4-propylisoxazole-3-carboxylate (138 mg, 0.564 mmol, 41%). A solution of methyl 5-phenyl-4-propylisoxazole-3-carboxylate (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 5-phenyl-4-propylisoxazole-3-carboxylic acid (56 mg, 0.260 mmol, 46%).

HPLC tr = 2.88 min. (Method D); LCMS (ESI) m/z Calcd for

C13H13NO3 [M + H]+ 232.1. Found: 232+. To a solution of 5-phenyl-4-propylisoxazole-3-carboxylic 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 N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine hydrochloride (44.8 mg, 0.234 mmol) and tert-butyl 1-(4-(N'-hydroxycarbamimidoyl)benzyl)azetidine-3-carboxylate (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 tert-butyl 1-(4-(5(5-phenyl-4-propylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylate. ACS Paragon Plus Environment

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A

solution

of

tert-butyl

Page 18 of 67

1-(4-(5-(5-phenyl-4-propylisoxazol-3-yl)-1,2,4-oxadiazol-3-

yl)benzyl)azetidine-3-carboxylatein 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

1-(4-(5-(5-phenyl-4-propylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-

carboxylic acid (5d, 33 mg, 0.074 mmol, 75% over two steps). HPLC purity 95.7%; tr = 7.72 min. (Method A); 95.3%; tr = 8.91 min. (Method B); LCMS (ESI) m/z Calcd for C25H24N4O4 [M + H]+ 445.2. Found: 445+. 1H 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). 1-(4-(5-(4-Butyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid (5e).16b A solution of hex-1-ynylbenzene (0.274 mL, 1.56 mmol), dimethyl 2-nitromalonate (0.421 mL, 3.12 mmol), 1-butyl-3-methylimidazoliumhexafluorophosphate (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 4-butyl-5-phenylisoxazole-3-carboxylate (0.142 g, 0.531 mmol, 34%) as a clear, colorless oil. HPLC purity 97%; tr = 3.11 min. (Method C); LCMS (ESI) m/z Calcd for C15H17NO3 [M + H]+ 260.1. Found: 260+. A mixture of methyl 4-butyl-5-phenylisoxazole-3-carboxylate (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 4-butyl-5-

phenylisoxazole-3-carboxylic acid, lithium salt (0.134 g, 0.531 mmol, 97%) as a white solid. HPLC tr = 2.82 min. (Method C); LCMS (ESI) m/z Calcd for C14H15NO3 [M + H]+ 246.1. Found: 246.2. 1H-NMR (500 MHz, DMSO-d6) δ ppm 0.84 (t, J=7.42 Hz, 3H), 1.23 - 1.32 (m, 2H), 1.44 - 1.53 (m, 2H), 2.66 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 4-butyl-5-phenylisoxazole-3-carboxylic acid, lithium salt (0.132 g, 0.523 mmol), tertbutyl 1-(4-(N'-hydroxycarbamimidoyl)benzyl)azetidine-3-carboxylate (10, 0.160 g, 0.523 mmol), N1ACS Paragon Plus Environment

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((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine hydrochloride (0.100 g, 0.523 mmol), and HOBt (0.080 g, 0.523 mmol) in N,N-dimethylformamide (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

tert-butyl

1-(4-(5-(4-butyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-

yl)benzyl)azetidine-3-carboxylate (0.156 g, 0.303 mmol, 58%) as a pale yellow solid. HPLC purity >99%; tr = 3.40 min. (Method C); LCMS (ESI) m/z Calcd for C30H34N4O4 [M + H]+ 515.3. Found: 515.0+. The

tert-butyl

1-(4-(5-(4-butyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)-azetidine-3-

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 1-(4-(5-(4-butyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid (5e, 0.102 g, 0.222 mmol, 74%) as a white solid. 99.0%; tr = 8.75 min. (Method B); LCMS (ESI) m/z Calcd for C26H26N4O4 [M + H]+ 459.2. Found: 459.1. 1H-NMR (500 MHz, DMSO-d6) δ ppm 0.91 (t, 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 acid (5f).16b

A solution of (4-methylpent-1-ynyl)benzene (0.247 g, 1.56 mmol), dimethyl 2-

nitromalonate (0.421 mL, 3.12 mmol), 1-butyl-3-methylimidazoliumhexafluorophosphate (0.032 mL, ACS Paragon Plus Environment

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Page 20 of 67

0.156 mmol) in toluene (5 mL) was subjected to the microwave conditions at 170°C for 500 min. The 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 4-isobutyl-5phenylisoxazole-3-carboxylate (0.122 g, 0.470 mmol, 30% yield) as a clear, colorless oil. HPLC tr = 2.96 min. (Method C); LCMS (ESI) m/z Calcd for C15H17NO3 [M + H]+ 260.1. Found: 259.9. 1H-NMR (500 MHz, CDCl3) 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 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 4-isobutyl-5-phenylisoxazole-3-carboxylate (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 4-isobutyl-5-

phenylisoxazole-3-carboxylic acid, lithium salt (0.119 g, 0.472 mmol, 100 % yield) as a white solid. HPLC tr = 2.68 min. (Method C); LCMS (ESI) m/z Calcd for C14H15NO3 [M + H]+ 245.1. Found: 245.8. A mixture of 4-isobutyl-5-phenylisoxazole-3-carboxylic acid, lithium salt (0.119 g, 0.472 mmol), tert-butyl 1-(4-(N'-hydroxycarbamimidoyl)benzyl)azetidine-3-carboxylate (10, 0.144 g, 0.472 mmol), N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine

hydrochloride

(0.090

g,

0.472

mmol), and HOBt (0.072 g, 0.472 mmol) in N,N-dimethylformamide (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 tert-butyl 1-(4-(5-(4isobutyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylate (0.179 g, 0.348 mmol, 74%) as a pale yellow solid. HPLC purity 98%; tr = 3.27 min. (Method C); LCMS (ESI) m/z Calcd for C30H34N4O4 [M + H]+ 515.3. Found: 515.4.


19

Page 21 of 67

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The tert-butyl 1-(4-(5-(4-isobutyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)-azetidine-3carboxylate 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

1-(4-(5-(4-isobutyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-

yl)benzyl)azetidine-3-carboxylic acid (5f, 0.074 g, 0.161 mmol, 34% yield) as a white solid. HPLC purity 98.4%; tr = 8.12 min. (Method A); 97.4%; tr = 8.60 min. (Method B); LCMS (ESI) m/z Calcd for C26H26N4O4 [M + H]+ 459.2. Found: 459.3. 1H-NMR (500 MHz, DMSO-d6) δ ppm 0.87 (d, J=6.60 Hz, 6H), 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).16b 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,N-dimethylformamide (20 mL) was added 3methylbut-1-yne (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 (3methylbut-1-ynyl)benzene (225 mg). HPLC tr = 3.58 min. (Method D). A solution of (3-methylbut-1-ynyl)benzene (225 mg, 1.56 mmol), dimethyl nitromalonate (0.527 mL, 3.90 mmol), and 1-butyl-3-methylimidazolium 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 4-


20


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Page 22 of 67

isopropyl-5-phenylisoxazole-3-carboxylate (136 mg, 0.554 mmol, 36%). HPLC tr = 3.37 min. (Method D); LCMS (ESI) m/z Calcd for C14H15NO3 [M + H]+ 246.1. Found: 246+. A solution of methyl 4-isopropyl-5-phenylisoxazole-3-carboxylate (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 4-isopropyl-5phenylisoxazole-3-carboxylic acid (23 mg, 0.099 mmol, >99%). HPLC tr = 3.01 min. (Method D); LCMS (ESI) m/z Calcd for C13H13NO3 [M + H]+ 232.1. Found: 232+. To a solution of 4-isopropyl-5-phenylisoxazole-3-carboxylic 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 N1((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine hydrochloride (45.2 mg, 0.236 mmol) and tert-butyl 1-(4-(N'-hydroxycarbamimidoyl)benzyl)azetidine-3-carboxylate (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 tertbutyl 1-(4-(5-(4-isopropyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)-azetidine-3-carboxylate. A

solution

of

tert-butyl

1-(4-(5-(4-isopropyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-

yl)benzyl)azetidine-3-carboxylate in dichloromethane (0.5 mL) and trifluoroacetic acid (0.5 mL) was stirred at room temperature for 30 minutes. Concentration under reduce pressure afforded 1-(4-(5-(4isopropyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid (5g, 35.1 mg, 0.079 mmol, 80% over two steps). HPLC purity 97.2%; tr = 7.88 min. (Method A); 98%; tr = 8.40 min. (Method B); LCMS (ESI) m/z Calcd for C25H24N4O4 [M + H]+ 445.2. Found: 445+.

1

H-NMR (500

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)-

azetidine-3-carboxylic acid (5h).16b To a degassed solution of of iodobenzene (0.549 mL, 4.90 mmol), ACS Paragon Plus Environment

21

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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,N-dimethylformamide (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 (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 tr = 3.40 min. (Method 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 4-cyclopropyl-5-phenylisoxazole-3-carboxylate. The compound was repurified by reverse phase preparative HPLC to provide methyl 4-cyclopropyl-5-phenylisoxazole-3carboxylate (170 mg, 0.699 mmol, 33%). HPLC tr = 2.77 min. (Method D); LCMS (ESI) m/z Calcd for C14H13NO3 [M + H]+ 244.1. Found: 244+. A solution of methyl 4-cyclopropyl-5-phenylisoxazole-3-carboxylate (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 4-cyclopropyl-5phenylisoxazole-3-carboxylic acid (124 mg, 0.541 mmol, 77%). HPLC tr = 2.81 min. (Method D); LCMS (ESI) m/z Calcd for C13H11NO3 [M + H]+ 230.1. Found: 230+. To a solution of 4-cyclopropyl-5-phenylisoxazole-3-carboxylic 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 N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine hydrochloride (43.2 mg, 0.226 mmol) and tert-butyl 1-(4-(N'-hydroxycarbamimidoyl)benzyl)azetidine-3-carboxylate (10, 29.3 mg, ACS Paragon Plus Environment

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Page 24 of 67

0.096 mmol). The reaction mixture was stirred at 80°C for 2 h. The reaction mixture was concentrated, and the residue was purified by reverse phase preparative HPLC to give tert-butyl 1-(4-(5-(4cyclopropyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylate. A

solution

of

tert-butyl

1-(4-(5-(4-cyclopropyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-

yl)benzyl)azetidine-3-carboxylate in dichloromethane (0.5 mL) and trifluoroacetic acid (0.5 mL) was stirred at room temperature for 30 minutes. Concentration under reduced pressure afforded 1-(4-(5-(4cyclopropyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-carboxylic acid (5h, 26.2 mg, 0.059 mmol, 62% over two steps). HPLC purity 97%; tr = 3.06 min. (Method D); LCMS (ESI) m/z Calcd for C25H22N4O4 [M + H]+ 443.2. Found: 443+. 1H-NMR (400 MHz, CD3OD) δ ppm 0.41 - 0.47 (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), 4.53 (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, 2H). 1-(4-(5-(4-tert-butyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)-azetidine-3-carboxylic acid

(5i).16b

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,N-dimethylformamide (20 mL) was added 3,3-dimethylbut-1-yne (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 (3,3dimethylbut-1-ynyl)benzene (740 mg). HPLC tr = 3.78 min. (Method D). A solution of (3,3-dimethylbut-1-ynyl)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


23

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yield methyl 4-tert-butyl-5-phenylisoxazole-3-carboxylate (101 mg, 0.389 mmol, 15%). HPLC tr = 3.39 min. (Method D); LCMS (ESI) m/z Calcd for C15H17NO3 [M + H]+ 260.1. Found: 260+. A solution of methyl 4-tert-butyl-5-phenylisoxazole-3-carboxylate (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 4-tert-butyl-5phenylisoxazole-3-carboxylic acid, sodium salt (25 mg, 0.100 mmol, >99%). HPLC tr = 2.97 min. (Method D); LCMS (ESI) m/z Calcd for C14H15NO3 [M + H]+ 246.1. Found: 246+. To a solution of 4-tert-butyl-5-phenylisoxazole-3-carboxylic 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 N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine hydrochloride (45.9 mg, 0.240 mmol) and tert-butyl 1-(4-(N'-hydroxycarbamimidoyl)benzyl)azetidine-3-carboxylate (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 tert-butyl 1-(4-(5-(4-tert-butyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylate. A

solution

of

tert-butyl

1-(4-(5-(4-tert-butyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-

yl)benzyl)azetidine-3-carboxylate in dichloromethane (0.5 mL) and trifluoroacetic acid (0.5 mL) was stirred at room temperature for 30 minutes. Concentration under reduced pressure afforded 1-(4-(5-(4tert-butyl-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic

acid,

2,2,2-

trifluoroacetic acid salt (5i, 31 mg, 0.068 mmol, 67% over two steps). HPLC purity 97.0%; tr = 7.95 min. (Method A); 97.9%; tr = 8.53 min. (Method B); LCMS (ESI) m/z Calcd for C26H26N4O4 [M + H]+ 459.2. Found: 459+.

1

H-NMR (500 MHz, CD3OD) δ ppm 1.27 (s, 9H), 3.68 - 3.76 (m, 1H), 4.39 (d,

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). ACS Paragon Plus Environment

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1-(4-(5-(4,5-diphenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic

Page 26 of 67

acid

(5j).16b A mixture of 1,2-diphenylethyne (1.53 g, 8.6 mmol) and dimethyl 2-nitromalonate (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 ( 0-10%) afforded methyl 4,5-diphenylisoxazole-3-carboxylate (307 mg, 1.1 mmol, 14%) as a light yellow solid. HPLC tr = 1.85 min. (Method D); LCMS (ESI) m/z Calcd for C17H13NO3 [M + H]+ 280.1. Found: 280.1. 1H-NMR (400 MHz, CDCl3) δ ppm 3.87 (s, 3 H) 7.29 - 7.40 (m, 5 H) 7.43 (m, 3H) 7.47 - 7.53 (m, 2 H). To a thick suspension of methyl 4,5-diphenylisoxazole-3-carboxylate (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 4,5-diphenylisoxazole-3-carboxylic 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 C16H11NO3 [M + H]+ 266.1. Found: 266.1. A mixture of 4,5-diphenylisoxazole-3-carboxylic acid (26.5 mg, 0.1 mmol), tert-butyl 1-(4-(N'hydroxycarbamimidoyl)benzyl)azetidine-3-carboxylate, 10, (30.5 mg, 0.100 mmol), HOBt (16.85 mg, 0.110 mmol) and N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine hydrochloride (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


25

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concentrated to give a yellow solid which was purified by flash silica gel chromatography eluting with a 0-60% mixture of ethyl acetate and hexane to afford tert-butyl 1-(4-(5-(4,5-diphenylisoxazol-3-yl)1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylate (37 mg, 0.069 mmol, 69% yield) as a white solid. HPLC tr = 1.90 min. (Method D); LCMS (ESI) m/z Calcd for C32H30N4O4 [M + H]+ 535.2. Found: 535.2. A solution of tert-butyl 1-(4-(5-(4,5-diphenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3carboxylate (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

1-(4-(5-(4,5-diphenylisoxazol-3-yl)-1,2,4-oxadiazol-3-

yl)benzyl)azetidine-3-carboxylic acid, trifluoroacetic acid salt (5j, 1 mg, 0.052 mmol, 76%) as a white solid. HPLC purity 95.9%; tr = 9.17 min. (Method A); 95.0%; tr = 9.94 min. (Method B); LCMS (ESI) m/z Calcd for C28H22N4O4 [M + H]+ 479.2. Found: 479.1.

1

H-NMR (500 MHz, CD3OD) δ ppm 3.70

(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) 8.09 (d, J=8.3 Hz, 2 H). 1-(4-(5-(5-phenyl-4-(trifluoromethyl)isoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3carboxylic acid (5k).16b To a mixture of (Z)-ethyl 2-chloro-2-(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 (0-12%) to afford ethyl 5-phenylisoxazole-3-carboxylate (14, 3.06 g, 14.1 mmol, 70%) as a white solid. HPLC tr = 2.99 min. (Method D); LCMS (ESI) m/z Calcd for C12H11NO3 [M + H]+ 218.1. Found: 218.1.

1

H-NMR (400 MHz, CDCl3) δ ppm 1.45 (t, J=7.3Hz, 3H), 4.48 (q, J=7.3,

2H), 6.93 (s, 1H), 7.45 - 7.53 (m, 3H), and 7.77 - 7.85 (m, 2H). A mixture of ethyl 5-phenylisoxazole-3-carboxylate (14, 3.05 g, 14.0 mmol) and N-iodosuccinimide (3.79 g, 16.9 mmol) in trifluoroacetic acid (78 mL) was stirred at room temperature for 3.5 h. The ACS Paragon Plus Environment

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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 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 4-iodo-5-phenylisoxazole-3-carboxylate (15, 4.69 g, 13.7 mmol, 97%) as a light yellow oil. HPLC tr = 3.36 min. (Method D); LCMS (ESI) m/z Calcd for C12H10INO3 [M + H]+ 344.0. Found: 344.0. 1H-NMR (400 MHz, CDCl3) δ ppm 1.47 (t, J=7.1 Hz, 3H), 4.50 (q, J=7.0Hz, 2H), 7.52 - 7.56 (m, 3H), and 8.05 (m, 2H). To a solution of ethyl 4-iodo-5-phenylisoxazole-3-carboxylate (4.62 g, 13.5 mmol) and copper(I) iodide (0.513 g, 2.69 mmol) in N,N-dimethylformamide (59.8 mL) and HMPA (7.48 mL) at room temperature was added methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (6.86 mL, 53.9 mmol) at once. The reaction mixture was immediately immersed in an oil bath at 75-80 °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 (0-10%) afforded ethyl 5phenyl-4-(trifluoromethyl)isoxazole-3-carboxylate (16, 3.6 g, 12.6 mmol, 94% yield) as a colorless oil. HPLC tr = 3.44 min. (Method D); LCMS (EI) m/z Calcd for C13H10INO3 [M + H]+ 286.1. Found: 286.0. 1

H-NMR (400 MHz, CDCl3) δ ppm 1.45 (t, J=7.2 Hz, 3H), 4.51 (q, J=7.3 Hz, 2H), 7.52 - 7.62 (m, 3H),

and 7.69 (d, J=7.5 Hz, 2H). To a solution of ethyl 5-phenyl-4-(trifluoromethyl)isoxazole-3-carboxylate (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 ACS Paragon Plus Environment

27

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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 99%). HPLC tr = 2.39 min. (Method D); LCMS (ESI) m/z Calcd for C12H9F2NO3 [M + H]+ 254.1. Found: 254+. A solution of 4-(1,1-difluoroethyl)-5-phenylisoxazole-3-carboxylic acid (58.7 mg, 0.232 mmol), HOBt (64.0 mg, 0.418 mmol), and diisopropylethylamine (0.162 mL, 0.928 mmol) in acetonitrile (2 mL) was added N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine hydrochloride (105 mg, 0.545 mmol) and tert-butyl 1-(4-(N'-hydroxycarbamimidoyl)benzyl)azetidine-3-carboxylate (10, 70.8 mg, 0.232 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 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 gave tertbutyl

1-(4-(5-(4-(1,1-difluoroethyl)-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-

carboxylate. A solution of tert-butyl 1-(4-(5-(4-(1,1-difluoroethyl)-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3yl)benzyl)azetidine-3-carboxylatein dichloromethane (0.5 mL) and trifluoroacetic acid (0.5 mL) was stirred at room temperature for 30 minutes. Concentration under reduced pressure afforded 1-(4-(5-(4(1,1-Difluoroethyl)-5-phenylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)-azetidine-3-carboxylic acid (5l, 19 mg, 0.041 mmol, 18% over two steps). HPLC purity 99.0%; tr = 7.40 min. (Method A); 99.0%; tr = 8.12 min. (Method B); LCMS (ESI) m/z Calcd for C24H20F2N4O4 [M + H]+ 467.2. Found: 467+.

1

H-

NMR (500 MHz, CD3OD) δ ppm 2.24 (t, J=18.56 Hz, 3H) 3.64 - 3.73 (m, 1H), 4.35 - 4.39 (m, 4H),


31

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4.52 (s, 2H), 7.58 - 7.67 (m, 3H), 7.70 (d, J=8.25 Hz, 2H), 7.76 (d, J=7.15 Hz, 2H), and 8.29 (d, J=8.25 Hz, 2H). 1-(4-(5-(5-isobutyl-4-propylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid (5m).16b To a solution of 4-methylpent-1-yne (0.588 mL, 5.00 mmol) in tetrahydrofuran at -78 °C was added butyl lithium, 2.5 M in hexanes (2.20 mL, 5.50 mmol). After stirring for 15 minutes at -78 °C, the reaction mixture was allowed to warm to room temperature. 1-Iodopropane (0.488 mL, 5.00 mmol) was added, and the reaction mixture was stirred at room temperature for 3 days. The reaction mixture was quenched with water (50 mL), and the resulting mixture was transferred to a separatory funnel. The mixture was extracted with ether (75 mL), and the organic layer was washed with brine (50 mL), dried over anhydrous magnesium sulfate, and concentrated to afford 2-methyloct-4-yne (555 mg, 4.47 mmol, 89%) as a light yellow liquid. 1H-NMR (400 MHz, CDCl3) δ ppm 0.91-0.99 (m, 9 H) 1.451.53 (m, 2 H) 1.70-1.81 (m, 1 H) 2.03 (dt, J=6.5, 2.4 Hz, 2 H) 2.12 (tt, J=7.0, 2.4 Hz, 2 H). A mixture of 2-methyloct-4-yne (248 mg, 2.00 mmol), dimethyl 2-nitromalonate (567 mg, 3.20 mmol) and 1-Butyl-3-methylimidazolium hexafluorophosphate (0.041 mL, 0.020 mmol) in toluene (4 mL) was heated to 170 °C under microwave conditions for 1 h. After cooling to room temperature, the reaction mixture was partitioned between ethyl acetate (50 mL) and water (50 mL), and the organic layer was collected, washed with brine, dried over magnesium sulfate, and concentrated to afford an orange oil that was chromatographed on a 24 gm ISCO silica gel cartridge (0-5% ethyl acetate/hexane). The pure fractions were concentrated under reduced pressure to afford a 1:1 mixture of methyl 5isobutyl-4-propylisoxazole-3-carboxylate and methyl 4-isobutyl-5-propylisoxazole-3-carboxylate (164 mg, 0.728 mmol, 36%) as a light yellow oil. These isomers were not readily separable by normal or reverse phase chromatography and this material was used without further purification in the next step. To a mixture of methyl 5-isobutyl-4-propylisoxazole-3-carboxylate and methyl 4-isobutyl-5propylisoxazole-3-carboxylate (164 mg, 0.728 mmol) in methanol (8 mL) and water (2 mL) was added lithium hydroxide, monohydrate (15.3 mg, 0.364 mmol) and the mixture was allowed to stir overnight at room temperature. An additional amount of lithium hydroxide, monohydrate (15.3 mg, 0.364 mmol) ACS Paragon Plus Environment

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was added, and the reaction mixture was stirred at room temperature for 2 h. The methanol was removed under reduced pressure, and the remaining aqueous layer was acidified to pH ~1 with 1N aqueous hydrochloric acid. The mixture was extracted with ethyl acetate (20 mL), and the organic layer was washed with brine (20 mL), dried over anhydrous sodium sulfate, and concentrated to afford 144 mg, (0.682 mmol, 94 % yield) of the product mixture which was approximately a 1:1 mixture of 5isobutyl-4-propylisoxazole-3-carboxylic acid and 4-isobutyl-5-propylisoxazole-3-carboxylic acid as a light yellow oil (Compounds were not readily separable by normal or reverse phase chromatography and were used without further purification in the next step). HPLC tr = 1.72 min. (Method D); LCMS (ESI) m/z Calcd for C11H17NO3 [M + H]+ 212.1. Found: 212.2. A mixture of 5-isobutyl-4-propylisoxazole-3-carboxylic acid and 4-isobutyl-5-propylisoxazole-3carboxylic acid (42.3 mg, 0.2 mmol), tert-butyl 1-(4-(N'-hydroxycarbamimidoyl) benzyl)azetidine-3carboxylate (10, 61.1 mg, 0.200 mmol), HOBt (38.3 mg, 0.250 mmol), diisopropylethylamine (0.070 mL, 0.400 mmol) and N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine hydrochloride (47.9 mg, 0.250 mmol) in dimethylformamide (1 mL) was stirred at room temperature for 18 h. Additional dimethylformamide (1 mL) was added, and the reaction mixture was warmed to 50°C and stirred for 8 h. The reaction mixture was partitioned between ethyl acetate (30 mL) and a saturated sodium bicarbonate solution (30 mL), and the organic layer was washed with water (2 x 30 mL), washed with brine (30 mL), and dried over anhydrous magnesium sulfate. Concentration followed by purification by flash silica gel chromatography (0 - 100% ethyl acetate/hexane) afforded a mixture of tert-butyl-1-(4-(5-(5-isobutyl-4-propylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3carboxylate (32 mg, 0.067 mmol, 66%) and tert-butyl 1-(4-(5-(4-isobutyl-5-propylisoxazol-3-yl)-1,2,4oxadiazol-3-yl)benzyl)azetidine-3-carboxylate (32 mg, 0.067 mmol, 67% yield) as a colorless oil. LCMS (ESI) m/z Calcd for C27H36N4O4 [M + H]+ 481.3. Found: 481.3. The mixture was separated on a Berger Prep SFC MGIII Unit on a Chiral AD-H 30 X 3 cm ID, 5µm column, eluting with 70/30 CO2/(MeOH, 0.1 % diethylamine) and a flow rate of 88 mL/min, monitored at 220 nm. The fractions from the second peak to elute were concentrated to afford tert-butyl 1-(4-(5ACS Paragon Plus Environment

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(5-isobutyl-4-propylisoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylate (24 mg; 50%) as a colorless oil. 1H-NMR (500 MHz, CDCl3) δ ppm 0.95-1.01 (m, 9 H) 1.44 (s, 9 H) 1.57-1.65 (m, 2 H) 2.10-2.19 (m, 1 H) 2.67 (d, J=7.4 Hz, 2 H) 2.69-2.73 (m, 2 H) 3.22-3.30 (m, 3 H) 3.50-3.56 (m, 2 H) 3.67 (s, 2 H) 7.42 (d, J=8.1 Hz, 2 H) 8.10 (d, J=8.1 Hz, 2 H). A

solution

of

tert-butyl

1-(4-(5-(5-isobutyl-4-propylisoxazol-3-yl)-1,2,4-oxadiazol-3-

yl)benzyl)azetidine-3-carboxylate (23 mg, 0.048 mmol) in trifluoroacetic acid (0.5 mL) was allowed to stand at room temperature for 2.5 h. The solvent was removed under reduced pressure, and the residue was triturated with ether and dried to afford 1-(4-(5-(5-isobutyl-4-propylisoxazol-3-yl)-1,2,4-oxadiazol3-yl)benzyl)azetidine-3-carboxylic acid, trifluoroacetic acid salt (24 mg, 0.044 mmol, 92%) as a light yellow glassy solid. HPLC purity 99.5%; tr = 9.44 min. (Method A); 99.6%; tr = 9.80 min. (Method B); LCMS (ESI) m/z Calcd for C23H28N4O4 [M + H]+ 425.2. Found: 425.2. 1H-NMR (400 MHz, MeOD) δ ppm 1.01 (m, 9 H) 1.60-1.71 (m, 2 H) 2.14 (m, 1 H) 2.77 (m, 4 H) 3.62-3.73 (m, 1 H) 4.30-4.40 (m, 4 H) 4.51 (s, 2 H) 7.68 (d, J=7.8 Hz, 2 H) 8.24 (d, J=7.8 Hz, 2 H). 1-(4-(5-(5-isobutyl-4-(trifluoromethyl)isoxazol-3-yl)-1,2,4-oxadiazol-3-yl)benzyl)-

azetidine-3-

carboxylic acid (5n).16b A mixture of methyl 5-isobutylisoxazole-3-carboxylate (18, 0.923 g, 5.04 mmol) and N-iodosuccinimide (1.25 g, 5.54 mmol) in trifluoroacetic acid (25 mL) was stirred at room temperature overnight. The trifluoroacetic acid was removed under reduced pressure, and the residue was diluted with dichloromethane (100 mL), washed with a saturated aqueous solution of sodium bicarbonate (2 x 25 mL), washed with a 2.5% aqueous solution of sodium bisulfite (25 mL), and dried over anhydrous sodium sulfate. Concentration under reduced pressure followed by purification by flash silica gel chromatography using a 5% mixture of ethyl acetate in hexane afforded methyl 4-iodo-5isobutylisoxazole-3-carboxylate (19, 1.21 g, 3.91 mmol, 78%) as a pale yellow oil. HPLC tr = 2.40 min. (Method C); LCMS (ESI) m/z Calcd for C13H11F2NO3 [M + H]+ 310.0. Found: 310.1. To a solution of methyl 4-iodo-5-isobutylisoxazole-3-carboxylate (19, 1.21 g, 3.91 mmol), copper(I) iodide (0.149 g, 0.783 mmol), and HMPA (2.59 mL) in N,N-dimethylformamide (19 mL) was added methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (1.993 mL, 15.66 mmol) over 1 min.

The reaction


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mixture was immediately immersed in an oil bath at 75°C and was stirred overnight. The clear, orange reaction mixture was cooled to room temperature and diluted with ether (100 mL), washed with a saturated aqueous solution of ammonium chloride (2 x 100 mL), washed with a 10% aqueous solution of lithium chloride (2 x 50 mL), and washed with brine (50 mL). The aqueous layer was back-extracted with ether (100 mL + 50 mL), and the combined organic layers were dried over anhydrous sodium sulfate.

Concentration under reduced pressure followed by purification by flash silica gel

chromatography using a 5% mixture of ethyl acetate in hexane provided methyl 5-isobutyl-4(trifluoromethyl)isoxazole-3-carboxylate (20, 0.819 g, 3.26 mmol, 83%) as a clear, colorless oil. HPLC tr = 2.52 min. (Method C). 1H-NMR (500 MHz, CDCl3) δ ppm 0.99 (s, 3H), 1.00 (s, 3H), 2.09 - 2.20 (m, 1H), 2.86 (dd, J=7.21, 1.11 Hz, 2H), and 4.01 (s, 3H). A mixture of methyl 5-isobutyl-4-(trifluoromethyl)isoxazole-3-carboxylate (20, 0.816 g, 3.25 mmol) and lithium hydroxide hydrate (0.136 g, 3.25 mmol) in methanol (18 mL) and water (9.00 mL) was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in 1N aqueous hydrochloric acid and extracted with ether. The organic layer was collected and dried over anhydrous sodium sulfate. Concentration under reduced pressure afforded 5-isobutyl-4-(trifluoromethyl)isoxazole-3-carboxylic acid (21, 0.746 g, 3.15 mmol, 97%) as an off-white solid. HPLC tr = 2.00 min. (Method C). 1H-NMR (400 MHz, DMSO-d6) δ ppm 0.91 (s, 3H), 0.93 (s, 3H), 1.97 - 2.09 (m, 1H), and 2.89 (d, J=7.28 Hz, 2H). To a mixture of 3-isobutyl-4-(trifluoromethyl)isoxazole-5-carboxylic acid (21, 0.200 g, 0.843 mmol) and pyridine (0.082 mL, 1.01 mmol) in dichloromethane (8 mL) at room temperature was added 2,4,6trifluoro-1,3,5-triazine (cyanuric fluoride) (0.085 mL, 1.01 mmol). The reaction mixture was stirred at room temperature overnight. The heterogeneous reaction was diluted with dichloromethane, washed with an ice-cold solution of 0.5N aqueous hydrochloric acid (2x), and the organic layer was collected. The aqueous layer was back-extracted with dichloromethane, and the combined organic layers were dried over anhydrous sodium sulfate and concentrated to afford 3-isobutyl-4-(trifluoromethyl)isoxazole-


35

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5-carbonyl fluoride (0.130 g, 0.544 mmol, 65%) as a yellow solid. HPLC tr = 2.51 min., corresponding to the methyl ester (Method C). A mixture of 5-isobutyl-4-(trifluoromethyl)isoxazole-3-carbonyl fluoride (0.130 g, 0.544 mmol), tert-butyl 1-(4-(N'-hydroxycarbamimidoyl)benzyl)azetidine-3-carboxylate (10, 0.166 g, 0.544 mmol), and Hunig's Base (0.123 mL, 0.707 mmol) in acetonitrile (2 mL) was stirred at room temperature for 2 days. HPLC analysis indicated that there was ~7% of the uncyclized intermediate remaining. The reaction mixture was gently heated with a heat gun for ~15 min., resulting in further cyclization. The homogenous mixture was diluted with dichloromethane and washed with a saturated aqueous solution of sodium bicarbonate. The aqueous layer was extracted with dichloromethane, and the organic layers were combined and dried over anhydrous sodium sulfate.

Concentration under reduced pressure

followed by purification by flash silica gel chromatography using a 1% mixture of methanol in dichloromethane

afforded

tert-butyl

1-(4-(5-(5-isobutyl-4-(trifluoromethyl)isoxazol-3-yl)-1,2,4-

oxadiazol-3-yl)benzyl)azetidine-3-carboxylate (0.219 g, 0.432 mmol, 80%) as a clear, viscous yellow oil. HPLC tr = 3.03 min. (Method C); LCMS (ESI) m/z Calcd for C25H29F3N4O4 [M + H]+ 507.2. Found: 507.4. A mixture of tert-butyl 1-(4-(5-(5-isobutyl-4-(trifluoromethyl)isoxazol-3-yl)-1,2,4-oxadiazol-3yl)benzyl)azetidine-3-carboxylate (0.219 g, 0.432 mmol) and trifluoroacetic acid (2.496 mL, 32.4 mmol) was stirred at room temperature for 45 min. The trifluoroacetic acid was removed under reduced pressure, and the residue was suspended in ~ 6 mL of water with sonication. The product gummed up and did not disperse. The pH was adjusted to ~4.5 with 1N aqueous sodium hydroxide, and the product still remained largely clumped on the bottom. Dichloromethane (~8 mL) was added, and the mixture was stirred vigorously overnight. The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated.

The oily residue was dissolved in methanol and left standing overnight.

The

methanol solution was passed through a Whatman 0.45 um PTFE w/GMF syringe filter and concentrated to give the product (0.195 g, 0.424 mmol, 98%) as a pale, yellow oil.


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A portion of the compound was purified by flash silica gel chromatography using a mixture of methanol, dichloromethane, and ammonium hydroxide (10:90:0 - 20:80:1). The desired fractions were concentrated, diluted with dichloromethane and water, and the pH was adjusted to 4.5 with 1.0 N aqueous hydrochloric acid. After shaking, the resulting emulsion was left to settle for 30 min. The organic layer was collected, and the aqueous layer/emulsion was back-extracted with dichloromethane (2x). The cloudy organic layer was dried over anhydrous sodium sulfate and concentrated to give a white solid residue which was redissolved in dichloromethane and filtered through a Whatman 0.45 um PTFE w/GMF syringe filter and concentrated to give the product as a white solid (118 mg). The solid was suspended in methanol with sonication and concentrated (2x). After the third resuspension, the solid was collected by vacuum filtration to give 1-(4-(5-(5-isobutyl-4-(trifluoromethyl)isoxazol-3-yl)1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid (5n, 0.011 g) as a white solid. HPLC purity 99.2%; tr = 7.51 min. (Method A); 99.4%; tr = 8.44 min. (Method B); LCMS (ESI) m/z Calcd for C21H21F3N4O4 [M + H]+ 451.2. Found: 451.1. 1H-NMR (500 MHz, CDCl3) δ ppm 1.02 (s, 3H), 1.03 (s, 3H), 2.15 - 2.25 (m, 1H), 2.93 (d, J=7.21 Hz, 2H), 3.39 (br. s., 1H), 3.93 (br. s., 2H), 4.15 (br. s., 4H), 7.55 (d, J=7.77 Hz, 2H), and 8.14 (d, J=7.77 Hz, 2H). 1-(4-(5-(3-phenylisoxazol-5-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid (6a). To a homogeneous mixture of 3-phenylisoxazole-5-carboxylic acid (0.200 g, 1.06 mmol) and pyridine (0.103 mL, 1.27 mmol) in dichloromethane (10 mL) at room temperature was added 2,4,6-trifluoro-1,3,5triazine (0.11 mL, 1.27 mmol). The reaction mixture was stirred at room temperature overnight. The heterogeneous reaction mixture was diluted with dichloromethane (25 mL), washed with an ice-cold aqueous solution of 0.5N aqueous hydrochloric acid (2 x 10 mL), and the organic layer was collected. The aqueous layer was back-extracted with dichloromethane (25 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated to afford 3-phenylisoxazole-5-carbonyl fluoride (0.193 g, 1.01 mmol, 95%) as a white solid. A mixture of 3-phenylisoxazole-5-carbonyl fluoride (0.193 g, 1.010 mmol), tert-butyl 1-(4-(N'hydroxycarbamimidoyl)benzyl)azetidine-3-carboxylate (10, 0.308 g, 1.01 mmol), and Hunig's Base ACS Paragon Plus Environment

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(0.229 mL, 1.31 mmol) in acetonitrile (2.0 mL) was stirred at room temperature for 30 min. The solidified reaction mixture was immersed in an oil bath at 60°C, stirred for 5 h, and then stirred at room temperature overnight. The solidified reaction mixture was diluted with 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 tert-butyl 1-(4-(5(3-phenylisoxazol-5-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylate as a white solid (0.276 g, 0.602 mmol, 60%). HPLC tr = 2.68 min. (Method C); LCMS (ESI) m/z Calcd for C26H26N4O4 [M + H]+ 459.2. Found: 459.2. A mixture of tert-butyl 1-(4-(5-(3-phenylisoxazol-5-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3carboxylate (0.276 g, 0.602 mmol) and trifluoroacetic acid (3.29 ml, 42.7 mmol) was stirred at room temperature for 1.0 h. The solvent was removed under reduced pressure, and the residue was suspended in water with sonication. To the resulting white suspension was added a 1N aqueous solution of sodium hydroxide portion-wise until the pH was ~9 with sonication. The pH was then adjusted to ~5 with 1N aqueous hydrochloric acid, and the suspension was sonicated for 20 min. The solid was collected by vacuum filtration (a significant amount passed through the filter), washed with water several times, and dried under reduced pressure overnight. The solid was suspended in methanol and sonicated for 20 min. During the sonication, the solution became very thick. The product was collected by vacuum filtration, washed with methanol, and dried under reduced pressure to give 1-(4-(5-(3-phenylisoxazol-5-yl)-1,2,4oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid (6a, 0.112 g, 0.272 mmol, 45%) as a white solid. HPLC purity 98.4%; tr = 6.74 min. (Method A); 97.7%; tr = 8.02 min. (Method B); LCMS (ESI) m/z Calcd for C22H18N4O4 [M + H]+ 403.1. Found: 403.2. 1H-NMR (400 MHz, DMSO-d6) δ 3.55 - 3.66 (m, 1H), 4.11 - 4.22 (m, 4H), 4.41 - 4.48 (m, 2H), 7.56 - 7.62 (m, 3H), 7.67 - 7.73 (m, 2H), 8.03 - 8.08 (m, 2H), 8.14 - 8.21 (m, 2H), 8.32 - 8.37 (m, 1H), 10.26 - 10.72 (m, 1H), and 12.95 - 13.32 (m, 1H).


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1-(4-(5-(4-methyl-3-phenylisoxazol-5-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid (6b). To a solution of methyl 4-methyl-3-phenylisoxazole-5-carboxylate (100 mg, 0.460 mmol) in methanol (4 mL) and water (0.8 mL) at room temperature was added lithium hydroxide, monohydrate (19.3 mg, 0.460 mmol), and the reaction mixture was stirred at room temperature for 1 h. The volatiles were removed under reduced pressure, and the residue was co-evaporated from ethyl acetate/heptane several times to afford lithium 4-methyl-3-phenylisoxazole-5-carboxylate (96 mg, 0.502 mmol, 100%) as a white foamy solid. HPLC tr = 1.45 min. (Method D); LCMS (ESI) m/z Calcd for C11H9NO3 [M + H]+ 204.1. Found: 204.1. A mixture of lithium 4-methyl-3-phenylisoxazole-5-carboxylate (21 mg, 0.100 mmol), tert-butyl 1(4-(N'-hydroxycarbamimidoyl)benzyl)azetidine-3-carboxylate (10, 30.5 mg, 0.1 mmol), HOBt (24.50 mg, 0.160 mmol), N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine hydrochloride (45.0 mg, 0.235 mmol), and Hunig'sBase (0.070 mL, 0.400 mmol) in dimethylfomamide (1 mL) was allowed to stir at room temperature for 3 days. The reaction mixture was warmed to 60°C for 4.5 h was then partitioned between ethyl acetate (30 mL) and a saturated aqueous solution of sodium bicarbonate (30 mL). The organic layer was washed with water (2 x 30 mL), washed with brine (20 mL), and dried over anhydrous magnesium sulfate.

Concentration followed by purification by flash silica gel

chromatography (0-70% ethyl acetate/hexane) afforded tert-butyl 1-(4-(5-(4-methyl-3-phenylisoxazol5-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylate (28 mg, 0.059 mmol, 59%) as a colorless solid. HPLC tr = 3.16 min. (Method D); LCMS (ESI) m/z Calcd for C27H28N4O4 [M + H]+ 473.2. Found: 473.1. A

solution

of

tert-butyl

1-(4-(5-(4-methyl-3-phenylisoxazol-5-yl)-1,2,4-oxadiazol-3-

yl)benzyl)azetidine-3-carboxylate (28 mg, 0.059 mmol) in trifluoroacetic acid (0.75 mL) was allowed to stand at room temperature for 1 h. Concentration followed by purification by reverse phase preparative HPLC

afforded

1-(4-(5-(4-methyl-3-phenylisoxazol-5-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-

carboxylic acid, trifluoroacetic acid salt (6b, 25 mg, 0.047 mmol, 80%) as a white solid. HPLC purity 99.2%; tr = 8.15 min. (Method A); 98.8%; tr = 8.82 min. (Method B); LCMS (ESI) m/z Calcd for ACS Paragon Plus Environment

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C23H20N4O4 [M + H]+ 417.2. Found: 417.1. 1H-NMR (CD3OD) δ 8.31 (d, J= 8.0 Hz, 2H), 7.77 (dd, J= 6.5, 2.8 Hz, 2H), 7.70 (d, J= 8.3 Hz, 2H) 7.64 (m, 3H), 4.54 (s, 2H), 4.43-4.34 (m, 4H), 3.76-3.68 (m, 1H), 2.63 (s, 3H). 1-(4-(5-(3-phenyl-4-propylisoxazol-5-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid (6c).16b To a suspension of sodium hydride, 60% (0.800 g, 20.0 mmol) in tetrahydrofuran (40 mL),

cooled

in

an

adiabatic

cooling

bath,

was

added

portionwise

over

15

minutes,

triethylphosphonoacetate (4.00 mL, 20 mmol). The reaction mixture was allowed to stir at room temperature for 45 min. Bromine (1.03 mL, 20.0 mmol) was added dropwise over 15 minutes. The resulting suspension was warmed to 40 °C, stirred for 10 minutes, and then at room temperature for 1 h. The reaction mixture was cooled to 10°C, sodium hydride (60%, 0.800 g, 20.0 mmol) was added in one portion, and the reaction mixture was allowed to warm to room temperature and stir for 45 minutes. Butyraldehyde (1.80 mL, 20.0 mmol) was then added over 2 minutes, and the reaction mixture was stirred for 18 h. The reaction mixture was partitioned between ether (200 mL) and water (100 mL). The organic layer was washed with a saturated aqueous solution of sodium bicarbonate (150 mL), washed with water (100 mL), washed with brine (100 mL), and dried over anhydrous magnesium sulfate. Concentration followed by purification by flash silica gel chromatography (0 - 5% ethyl acetate/hexane) ethyl 2-bromohex-2-enoate (3.61 g, 16.3 mmol, 82%) as a colorless liquid. HPLC tr = 1.81 min. (Method D); LCMS (ESI) m/z Calcd for C8H13BrO2 [M + H]+ 221.1 and 223.1. Found: 221.1 and 223.1 (Note: The product is a 3:1 mixture of olefin isomers). To a solution of N-hydroxybenzimidoyl chloride (770 mg, 4.95 mmol) and ethyl 2-bromohex-2enoate (1.09 g, 4.95 mmol) in dichloromethane (15 mL) was added triethylamine (2.07 mL, 14.9 mmol) over 5 min. The reaction mixture was stirred overnight at room temperature and was then partitioned between ether (100 mL) and water (100 mL). The organic layer was washed with brine (100 mL) and dried over anhydrous magnesium sulfate. Concentration followed by purification by flash silica gel crhomatorgraphy (0 - 1% ethyl acetate/hexane afforded ethyl 3-phenyl-4-propylisoxazole-5-carboxylate (34 mg, 0.131 mmol, 3%) as a colorless oil. HPLC tr = 1.91 min. (Method D); LCMS (ESI) m/z Calcd ACS Paragon Plus Environment

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for C15H17NO3 [M + H]+ 260.1. Found: 260.2. 1H-NMR (500 MHz, CDCl3) δ ppm 0.90 (t, J= 7.4 Hz, 3H), 1.45 (t, J= 7.2 Hz, 3H), 1.53 (m, 2H), 2.80 (m, 2H), 4.46 (q, J= 7.2 Hz, 2H), 7.45 (m, 1H), 7.50 (m, 2H), 7.59 (m, 2H). To a mixture of ethyl 3-phenyl-4-propylisoxazole-5-carboxylate (27 mg, 0.104 mmol) in methanol (0.8 mL) and water (0.2 mL) was added lithium hydroxide, monohydrate (4.37 mg, 0.104 mmol), and the reaction mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure to afford 3-phenyl-4-propylisoxazole-5-carboxylic acid, lithium salt (24 mg, 0.101 mmol, 97%) as a colorless oil, which was used without further purification. HPLC tr = 1.66 min. (Method D); LCMS (ESI) m/z Calcd for C13H13NO3 [M + H]+ 232.1. Found: 232.2. A mixture of tert-butyl 1-(4-(N'-hydroxycarbamimidoyl)benzyl)azetidine-3-carboxylate (10, 30.5 mg, 0.1 mmol), HOBt (16.85 mg, 0.110 mmol), diisopropylethylamine (0.044 mL, 0.250 mmol), and N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine hydrochloride (23.0 mg, 0.120 mmol) was stirred in dimethylformamide (1 mL) at room temperature for 18 h.

Additional N1-

((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine hydrochloride (23.0 mg, 0.120 mmol) was added, and the reaction mixture was stirred at 60°C for 5 h. The reaction mixture was partitioned between ethyl acetate (30 mL) and a saturated aqueous solution of sodium bicarbonate (30 mL). The organic layer was washed with water (2 x 25 mL), washed with brine (25 mL), and dried over anhydrous sodium sulfate. Concentration followed by purification by flash silica gel chromatography (0 - 50% ethyl acetate/hexane) afforded tert-butyl 1-(4-(5-(3-phenyl-4-propylisoxazol-5-yl)-1,2,4oxadiazol-3-yl)benzyl)azetidine-3-carboxylate (22 mg, 0.044 mmol, 44%) as a colorless oil. HPLC tr = 1.84 min. (Method D); LCMS (ESI) m/z Calcd for C29H32N4O4 [M + H]+ 501.3. Found: 501.2. A

solution

of

tert-butyl

1-(4-(5-(3-phenyl-4-propylisoxazol-5-yl)-1,2,4-oxadiazol-3-

yl)benzyl)azetidine-3-carboxylate (22 mg, 0.044 mmol) in trifluoroacetic acid (0.5 mL) was allowed to stand at room temerature for 2.5 h. The solvent was removed under reduced pressure, and the residue was triturated with ether and dried to afford 1-(4-(5-(3-phenyl-4-propylisoxazol-5-yl)-1,2,4-oxadiazol-3yl)benzyl)azetidine-3-carboxylic acid, trifluoroacetic acid salt (6c, 16 mg, 0.028 mmol, 63%) as an offACS Paragon Plus Environment

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white solid. HPLC purity 97.4%; tr = 8.97 min. (Method A); 96.7%; tr = 9.61 min. (Method B); LCMS (ESI) m/z Calcd for C25H24N4O4 [M + H]+ 445.2. Found: 445.2. 1H NMR (400 MHz, CD3OD) δ ppm 0.97 (t, J= 7.4 Hz, 3H), 1.66 (m, 2H), 3.09 (t, J= 7.8 Hz, 2H), 3.71 (m, 1H), 4.39 (m, 4H), 4.54 (s, 2H), 7.61 (m, 3H), 7.72 (m, 4H), and 8.31 (d, J= 8.3 Hz, 2H). 1-(4-(5-(3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3carboxylic acid (6d).16b To a solution of diisopropylamine (24.7 mL, 176 mmol) in ether (100 mL) at 78°C was added a 10M solution of butyllithium in ether (17.6 mL, 176 mmol) over 5 min. After 10 min. at -78°C, 2-bromo-3,3,3-trifluoroprop-1-ene (14.0 g, 80 mmol) was added to the pale yellow solution. After an additional 10 min., paraformaldehyde (2.40 g, 80 mmol) was added, the dry-ice bath was removed, and the reaction mixture was stirred at room temperature overnight. As the reaction mixture approached room temperature, it became dark in color. The reaction was quenched with a 1N aqueous solution of hydrochloric acid (100 mL), diluted with ether (500 mL), washed with a 1N aqueous solution of hydrochloric acid (2 x 100 mL), washed with brine 100 mL, and dried over anhydrous sodium sulfate. Concentration under reduced pressure afforded a dark liquid which was distilled under low-vacuum (~50 Torr, ~50°C) to give 4,4,4-trifluorobut-2-yn-1-ol (32, 7.10 g, 57.2 mmol, 72%) as a pale yellow liquid. 1H-NMR (500 MHz, CDCl3) δ ppm 2.31 (br. s., 1H) and 4.38 4.42 (m, 2H). To a pale yellow, homogeneous mixture of N-hydroxybenzimidoyl chloride (5.50 g, 35.4 mmol,)17 and 4,4,4-trifluorobut-2-yn-1-ol (32, 5.46 g, 39.6 mmol) in dichloroethane (85 mL) in a 250 mL round bottom flask at 70°C was added triethylamine (9.85 mL, 70.7 mmol) in 22 mL of dichloroethane over 2.5 h via an addition funnel (the first ~50% over 2 h and the remaining 50% over 0.5 h). After the addition, the reaction mixture was complete based on HPLC analysis (total time at 70°C was 3 h). The reaction mixture was diluted with dichloromethane (100 mL), washed with water (100 mL), and the organic layer was collected. The aqueous layer was extracted with dichloromethane (2 x 50 mL), and the combined organic layers were dried over anhydrous sodium sulfate. The solvent was removed under


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reduced pressure. Analysis indicated that the product mixture was composed of a 9:1 mixture of the desired regioisomer, (3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)methanol (33), and the undesired regioisomer, (3-phenyl-5-(trifluoromethyl)isoxazol-4-yl)methanol (34). The mixture was purified by silica gel chromatography using a mixture of ethyl acetate and hexane (1% to pack and load - 5% - 9% 12%) to afford (3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)methanol (33, 5.34 g, 22.0 mmol, 62%) as a pale yellow oil. HPLC tr = 1.91 min. (Method C); LCMS (ESI) m/z Calcd for C11H8F3NO2 [M + H]+ 244.1. Found: 244.2. 1H-NMR (500 MHz, CDCl3) δ ppm 2.21 (br. s., 1H), 4.97 (s, 2H), 7.47 - 7.56 (m, 3H), and 7.65 (d, J=6.60 Hz, 2H). To a solution of (3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)methanol (33, 5.24 g, 21.6 mmol) in acetone (75 mL) at room temperature (immersed in a water bath) was slowly added Jones' Reagent (43.8 mL, 53.9 mmol) via addition funnel over 1.5 h. The dark reaction mixture was stirred at room temperature overnight. HPLC analysis indicated that the reaction was 93% complete. An additional 0.5 equivalents (9 mL) of the Jones' Reagent was added. After 1 h, the reaction was 95% complete. After an additional 3h, the reaction was 96% complete. An additional 0.5 equivalents (9 mL) of the Jones' Reagent was added. The reaction mixture was stirred for an additional 2.5 h and was determined to be 97% complete. Isopropyl alcohol (6 mL) was added, and the mixture was stirred for 90 min, resulting in a dark green precipitate. The mixture was diluted with ether (600 mL), washed with a 2% aqueous solution of sodium hydrogen sulfite (5 x 100 mL), and the organic layer was collected. The aqueous layer was back-extracted with ether (2 x 100 mL). The combined organic layers were washed with water (100 mL), washed with a saturated aqueous solution of brine (100 mL), and dried over anhydrous sodium sulfate. The aqueous layer was back-extracted with ether (100 mL), and the organic layer was added to the previous organic layers. The solution was concentration under reduced pressure to give 3phenyl-4-(trifluoromethyl)isoxazole-5-carboxylic acid as an off-white solid. The solid was diluted with dichloromethane (200 mL), washed with a 2% aqueous solution of sodium hydrogen sulfite, washed with brine, and dried over anhydrous sodium sulfate. Concentration under reduced pressure afforded 3phenyl-4-(trifluoromethyl)isoxazole-5-carboxylic acid (3.84 g, 14.9 mmol, 70%) as a pale yellow solid. ACS Paragon Plus Environment

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The sodium hydrogen sulfite aqueous layer still contained a significant amount of product. The brine layer contained no additional product and was discarded. The aqueous layer was saturated with sodium chloride, the pH was adjusted to ~3.5, and the solution was extracted with ether (3 x 100 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated to afford additional 3phenyl-4-(trifluoromethyl)isoxazole-5-carboxylic acid (1.12 g, 4.36 mmol, 20%) as a white solid. The

products

were

combined

to

give

4.96

g

(35,

90%

yield)

of

3-phenyl-4-

(trifluoromethyl)isoxazole-5-carboxylic acid. HPLC tr = 1.60 min. (Method C); LCMS (ESI) m/z Calcd for C11H6F3NO3 [M + H]+ 258.0. Found: 258.2. 1H-NMR (500 MHz, DMSO-d6) δ ppm 7.55 - 7.63 (m, 5H). A mixture of (3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)methanol (33, 2.1 g, 8.64 mmol), TEMPO (0.094 g, 0.604 mmol), and a sodium phosphate buffer (0.67M) (32.2 mL, 21.59 mmol) was heated to 35°C. A solution of sodium phosphate buffer (40 mL, pH ~6.5), consisting of a 1:1 solution of NaH2PO4 (20 mL, 0.67M) and Na2HPO4 (20 mL, 0.67M), was prepared in acetonitrile (30 mL) prior to use. Solutions of sodium chlorite (3.91 g, 34.5 mmol) in water (4.5 mL) and bleach (4.3 mL, 6% wt.) were added simultaneously over 40 min. The reaction was monitored by HPLC, and after 2 h, ~30% of the starting material remained. After 6 h, 10% remained. Additional bleach (100 µL) was added, and the reaction mixture was left at room temperature overnight. Additional bleach (100 µL) was added, and the resulting mixture was allowed to stir at 35°C for additional 2 h. HPLC indicated complete conversion. The reaction was quenched by the slow addition of a solution of sodium sulfite (2.07 mL, 43.2 mmol) in water (90 mL) at 0°C, resulting in the disappearance of the brown reaction color. The solvent was removed under reduced pressure, and the remaining aqueous residue was extracted with ethyl acetate (3 x 40 mL). The organic layers were combined, washed with water (8 mL), washed with brine (8 mL), and dried over anhydrous sodium sulfate.

Concentration under reduced pressure afforded 3-phenyl-4-(trifluoromethyl)isoxazole-5-

carboxylic acid (35, 2.2 g, 8.55 mmol, 99 % yield) as a pale yellow solid.


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To a mixture of 3-phenyl-4-(trifluoromethyl)isoxazole-5-carboxylic acid (35, 3.00 g, 11.7 mmol) and pyridine (1.13 mL, 14.0 mmol) in dichloromethane (100 mL) at room temperature was added 2,4,6trifluoro-1,3,5-triazine (cyanuric fluoride) (1.18 mL, 14.0 mmol). The reaction mixture was stirred at room temperature overnight, diluted with dichloromethane (300 mL), washed with an ice-cold solution of 0.5N aqueous hydrochloric acid (2 x 100 mL), and the organic layer was collected. The aqueous layer was back-extracted with dichloromethane (200 mL), and the combined organic layers were dried anhydrous sodium sulfate and concentrated to afford 3-phenyl-4-(trifluoromethyl)isoxazole-5-carbonyl fluoride (36, 2.91 g, 11.2 mmol, 96%) as a yellow, viscous oil. The product was found to react readily with methanol and during analysis was characterized as the methyl ester, which had an HPLC tr = 2.56 min. (Method C); LCMS (ESI) [M + H]+: 272.3 (methyl ester). A suspension of 3-phenyl-4-(trifluoromethyl)isoxazole-5-carbonyl fluoride (36, 2.91 g, 11.2 mmol), tert-butyl 1-(4-(N'-hydroxycarbamimidoyl)benzyl)azetidine-3-carboxylate (10, 3.43 g, 11.2 mmol), and Hunig's Base (2.55 mL, 14.6 mmol) in acetonitrile (20 mL) was stirred at room temperature over the weekend. The completely solidified reaction mixture was diluted with dichloromethane and washed with a saturated aqueous solution of sodium bicarbonate (150 mL). The organic layer was collected, and the aqueous layer was extracted with dichloromethane (2 x 100 mL). The combined organic layers were dried over anhydrous sodium sulfate. Concentration under reduced pressure afforded a tan solid which was purified by flash silica gel chromatography using a mixture of ethyl acetate in hexane (050%) to afford tert-butyl 1-(4-(5-(3-phenyl-4-(trifluoromethyl) isoxazol-5-yl)-1,2,4-oxadiazol-3yl)benzyl)azetidine-3-carboxylate (4.60 g; 78%) as a white solid. The material was suspended in methanol (~75 mL) and was sonicated for 5 minutes. The MeOH was removed under reduce pressure, and the residue was re-suspended in methanol (~50 mL) with sonication. Vacuum filtration and drying afforded

tert-butyl

1-(4-(5-(3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)-1,2,4-oxadiazol-3-

yl)benzyl)azetidine-3-carboxylate (37, 4.04 g, 7.67 mmol, 68%) as a white solid. The methanol filtrate was concentrated to afford additional tert-butyl 1-(4-(5-(3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylate (33, 570 mg; 10%) as a slightly off-white solid. ACS Paragon Plus Environment

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HPLC tr = 3.12 min. (Method C); LCMS (ESI) m/z Calcd for C27H25F3N4O4 [M + H]+ 527.2. Found: 527.1. 1H-NMR (500 MHz, CDCl3) δ ppm 1.47 (s, 9H) 3.28 - 3.37 (m, 3H), 3.60 (br. s., 2H), 3.74 (br. s., 2H), 7.49 (d, J=7.70 Hz, 2H), 7.53 - 7.62 (m, 3H), 7.69 (d, J=7.15 Hz, 2H), and 8.16 (d, J=7.70 Hz, 2H). A mixture of tert-butyl 1-(4-(5-(3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)-1,2,4-oxadiazol-3yl)benzyl)azetidine-3-carboxylate (37, 6.12 g, 11.6 mmol) and trifluoroacetic acid (50.1 mL, 651 mmol) was stirred at room temperature for 1.5 h. The solvent was removed under reduced pressure, and the oily residue was diluted with water (100 mL) and sonicated for 5 min. The resulting suspension was stirred for an additional 10 min until a consistent white suspension was observed. A 1N aqueous solution of sodium hydroxide was added portion-wise until the pH was ~4.5 (42 mL of 1N NaOH). Over time, the pH drifted back down to 3-4, and additional 1N aqueous sodium hydroxide had to be added. The suspension was stirred overnight at room temperature. Several drops of 1N aqueous sodium hydroxide were added to re-adjust the pH to 4.5, and after 60 min., the pH appeared to be stable. 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 (110 mL) in a 150 mL round bottom flask and sonicated for 15 min. During the sonication, the solution became very thick. An additional 25 mL of methanol was added, and the suspension was stirred overnight. The product was collected by vacuum filtration, washed with methanol (~50 mL), and dried under reduced pressure. The solid was transferred to a 250 mL round bottom flask, re-suspended in methanol (115 mL), sonicated for 5 min., and stirred for 60 min. The solid was collected by vacuum filtration, washed with methanol (~50 mL), and dried over well under reduced pressure to give 1-(4-(5-(3-phenyl4-(trifluoromethyl)isoxazol-5-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid (6d, 5.06 g, 10.7 mmol, 92%) as a crystalline, white solid. HPLC purity 99.8%; tr = 7.62 min. (Method A); 99.9%; tr = 8.45 min. (Method B); LCMS (ESI) m/z Calcd for C23H17F3N4O4 [M + H]+ 445.2. Found: 471.3. 1

H-NMR (500 MHz, DMSO-d6) δ ppm 3.20 - 3.46 (m, 5H), 3.66 (s, 2H), 7.53 (d, J=8.25 Hz, 2H), 7.60 -


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Page 48 of 67

7.70 (m, 5H), and 8.06 (d, J=7.70 Hz, 2H). Anal. Calcd for C23H17N4O4F3, 0.44% water: C, 58.42; H, 3.70; N, 11.83. Found: C, 58.52; H, 3.43; N, 11.86. tert-Butyl 1-(4-(N'-hydroxycarbamimidoyl)-benzyl)azetidine-3-carboxylate (10).16b

To a

solution of azetidine-3-carboxylic acid (88 g, 0.871 mol) and sodium bicarbonate (161 g, 1.92 mol) in water (1.75 L) at room temperature was added a solution of benzyl 2,5-dioxopyrrolidin-1-ylcarbonate (239 g, 0.959 mol) in tetrahydrofuran (3.5 L). The reaction mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure, and the aqueous layer was washed with ethyl acetate (2 x 500 mL). The aqueous layer was acidified with a 1.0 N aqueous hydrochloric acid solution and was then extracted with ethyl acetate (3 x 750 mL). The organic layer was washed with water, washed with brine, and dried over anhydrous sodium sulfate. Concentration under reduced pressure afforded 1-(benzyloxycarbonyl) azetidine-3-carboxylic acid as colorless oil (7, 202 g, 99% yield). HPLC tr = 2.27 min. (Method D); LCMS (ESI) m/z Calcd for C12H13NO4 [M + H]+ 236.1. Found: 236.2. 1H-NMR (400 MHz, CDCl3) δ ppm 3.39 – 3.49 (m, 1H), 4.22 (d, J=7.28 Hz, 4H), 5.11 (s, 2H), and 7.29 – 7.39 (m, 5H). To a solution of 1-(benzyloxycarbonyl)azetidine-3-carboxylic acid (7, 200 g, 0.851 mol) in dichloromethane (6.0 L) at 0°C was added tert-butanol (158 g, 2.13 mol), DMAP (52.0 g, 0.425 mol), and N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine hydrochloride (163 g, 0.853 mol). The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated, and the residue was dissolved in ethyl acetate. The organic layer washed with 10% aqueous citric acid, washed with 10 % aqueous sodium bicarbonate solution, and washed with brine. Subsequent drying over anhydrous sodium sulfate followed by concentration under reduced pressure afforded 1-benzyl-3-tert butyl-azetidine-1,3-dicarboxylate (200 g, 81% yield) as a colorless oil. HPLC tr = 3.27 min. (Method D); LCMS (ESI) m/z Calcd for C16H21NO4 [M + H]+ 292.2. Found: 292.2. 1HNMR (400 MHz, CDCl3) δ ppm 1.46 (s, 9H), 3.24 – 3.33 (m, 1H), 4.14 (d, J=7.53 Hz, 4H), 5.10 (s, 2H), and 7.30 – 7.39 (m, 5H).


47

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A mixture of 1-benzyl-3-tert-butyl-azetidine-1,3-dicarboxylate (140 g, 0.480 mol) and 10% palladium on carbon (28.0 g) in ethyl acetate (1.40 L) was placed in an autoclave under 3.0 kg/cm2 of hydrogen pressure overnight. The reaction mixture was filtered through Celite®, and the Celite® bed was washed with ethyl acetate. Acetic acid (28.9 g, 0.480 mol) was added to the filtrate, and it was concentrated under reduced pressure maintaining the temperature below 50°C to give tert-butyl azetidine-3-carboxylate acetic acid salt (8, 96 g, 92% yield) as a colorless oil.

1

H-NMR (400 MHz,

CDCl3) δ ppm 1.47 (s, 9H), 2.02 (s, 3H), 3.52 – 3.63 (m, 1H), and 4.00 – 4.10 (m, 4H). To a solution of tert-butyl azetidine-3-carboxylate acetic acid salt (8, 92.0 g, 0.423 mol) in methanol (1.0 L) at room temperature was added 4-formylbenzonitrile (50.8 g, 0.381 mol). The reaction mixture was cooled to 0°C, and sodium cyanoborohydride (28.8 g, 0.458 mol) was added portion-wise. The reaction mixture was allowed to warm to room temperature and stirring continued overnight. After the reaction mixture was concentrated under reduced pressure, the residue was diluted with a 10% aqueous sodium bicarbonate solution and extracted with ethyl acetate. The organic layer was collected, washed with brine, and dried over anhydrous sodium sulfate. Concentration under reduced pressure followed by purification by flash silica gel chromatography using 20% ethyl acetate in petroleum ether afforded tertbutyl 1-(4-cyanobenzyl)azetidine-3-carboxylate (9, 89%). LCMS (ESI) m/z Calcd for C16H20N2O2 [M + H]+ 273.2. Found: 273.2. 1H-NMR (400 MHz, CDCl3) δ ppm 1.46 (s, 9H), 3.22 – 3.31 (m, 3H), 3.48 – 3.56 (m, 2H), 3.66 (s, 2H), 7.39 (d, J=8.28 Hz, 2H), and 7.60 (d, J=8.28 Hz, 2H). To tert-butyl-1-(4-cynaobenzyl)azetidine-3-carboxylate (9, 89.0 g, 0.326 mol) in tert-butanol (1.30 L) was added sodium bicarbonate (109.8 g, 1.31 mol) and hydroxylamine hydrochloride (45.5 g, 0.654 mol). The reaction was heated at reflux for 7 h and then cooled to room temperature and stirred overnight. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was collected, washed with water, washed with brine, then dried over anhydrous sodium sulfate. Concentration followed by purification by flash silica gel chromatography using 2.5% methanol in chloroform

containing

0.2%

triethylamine

as

eluent

afforded

(Z)-tert-butyl

1-(4-(N'-


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Page 50 of 67

hydroxycarbamimidoyl)benzyl)azetidine-3-carboxylate (10, 64 g, 0.210 mol, 55% yield over 2 steps). HPLC tr = 7.03 min. (Method B); LCMS (ESI) m/z Calcd for C16H23N3O3 [M + H]+ 306.2. Found: 306.2. 1H-NMR (400 MHz, CDCl3) δ ppm 1.45 (s, 9H), 3.23 – 3.30 (m, 3H), 3.49 – 3.57 (m, 2H), 3.63 (s, 2H), 4.85 (s, 2H), 7.31 (d, J=8.28 Hz, 2H), and 7.57 (d, J=8.28 Hz, 2H). Receptor [35S] GTPγγS Binding Assays Compounds were loaded in a 384 Falcon v-bottom plate (0.5 µl/well in a 3-fold dilution). Membranes prepared from S1P1/CHO cells or EDG3-Ga15-bla HEK293T cells were added to the compound plate (40 µl/well, final protein 3 µg/well) with multidrop liquid handler (Thermo Scientific, Waltham, MA). [35S]GTP (1250 Ci/mmol, Perkin Elmer) was diluted in assay buffer: 20 mM HEPES, pH7.5, 10 mM MgCl2, 150 mM NaCl, 1 mM EGTA, 1 mM DTT, 10 µM GDP, 0.1% fatty acid free BSA, and 10 µg/ml Saponin to 0.4 nM. 40 µl of the [35S] GTP solution was added to the compound plate with a final concentration of 0.2 nM. The reaction was kept at room temperature for 45 min. At the end of incubation, all the mixtures in the compound plate were transferred to a 384 well FB filter plates via GPCR robot system. The filter plate was washed with water 4 times by using the modified manifold Embla plate washer and dried at 60°C for 45 min. 30 µl of MicroScint 20 scintillation fluid was added to each well for counting at Packard TopCount. EC50 is defined as the agonist concentration that corresponds to 50% of the Ymax (maximal response) obtained for each individual compound tested. S1P-1 Internalization and Recycling Assay (high content based) hS1P-1 internalization assay: For quantification of S1P-1 expression using a high content system, GFP was fused to hS1P-1 at its C-terminus and a stable CHO cell line expressing hS1P-1/GFP was established. Cells were suspended in assay medium (F12 medium with 5 % Charcoal-Dextran treated FBS, 20mM HEPES, and 1X Pen/Strep) at 7.5x104 cells/ml and plated into 384 well plates (in 20 µl, 1,500 cells/well final) with a multidrop liquid handler (Thermo Scientific, Waltham, MA). Cell plates were incubated at 37 ºC for 48 hr. Titrated S1P and compounds of interest were dispensed into the cell ACS Paragon Plus Environment

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plates (concentration range from 100 µM to 0.005 µM) and the plates were further incubated at 37 ºC for 50 min. Cells were fixed by adding 10 µl of fixation buffer (7.4% v/v formaldehyde containing 15 µM Draq5 in DPBS) directly onto the assay medium and incubated for 15 min at RT followed by two washes with DPBS. This was followed by the addition of 50 µl DPBS to the cell layer and the plates were sealed with a transparent plate seal (Perkin Elmer #6005185). Imaging was carried out using the Evotec Opera High Content Confocal System (Perkin Elmer, Boston, MA) equipped with 3 diode lasers, peltier cooled CCD camera detector and a Nipkow spinning disk. Cell images were quantified using a membrane fluorescent scoring algorithm. Data was analyzed using a customized HTS data analysis software package. EC50 values were determined using XL-Fit software program. hS1P-1 recycling assay: After 50 min stimulation with compounds to drive internalization, cells were washed 8 times with 100 µl DPBS followed by the addition of F12 medium containing 2.5 % Charcol-Dextran treated serum to each well. Cells were incubated at 37 ºC for different time points (1 hr, 4 hr, 8 hr, and 16 hr). Cell plates were then fixed, stained and scanned as described in the internalization assay protocol. In both receptor internalization and receptor recycling assays, the buffer control is considered as 100% S1P-1/GFP present on the cell membrane; 1 µM 3 (reference compound) is used as the reference for 100 % S1P-1/GFP internalized. All procedures involving animals were reviewed and approved by the Institutional Animal Care Use Committee. Blood Lymphocyte Reduction Assay (BLR) in Rat: Lewis rats were dosed orally with test article (as a solution or suspension in the vehicle) or vehicle alone (polyethylene glycol 300, “PEG300”). Blood was drawn at 4hr and 24h by retro-orbital bleeding. Blood lymphocyte counts were determined on an ADVIA 120 Hematology Analyzer (Siemens Healthcare Diagnostics). The results were measured as a reduction in the percentage of circulating lymphocytes as compared to the vehicle treated group at the 4 hr and 24 hr measurement. The results represent the average results of all animals within each treatment group (n = 3-4). ACS Paragon Plus Environment

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Rat Adjuvant Induced Arthritis Assay (AA) Male Lewis rats (200-225 g; Harlan, n=8 treatment group) were immunized at the base of the tail with 100 µl of 10 mg/ml freshly ground Mycobacterium butyricum (Difco Laboratories) in incomplete Freund’s adjuvant (sigma). Animals were dosed once daily with the test article (as a solution or suspension in the vehicle) or vehicle alone (PEG300) starting from the day of immunization. The volumes of their hind paws were measured in a water displacement plethysmometer (Ugo Basile, Italy). The baseline paw measurements were taken before onset of the disease (between day 7 to day 10). The paw measurements were then taken three times a week until the end of the study on day 20. Mouse Experimental Autoimmune Encephalomyelitis Assay (EAE) Mice (C57BL/6 female, 6-8 weeks of age, Charles River, n=10-12 treatment group) were immunized subcutaneously with 150 µg MOG35-55 peptide emulsified 1:1 with incomplete Freund’s adjuvant (sigma) supplemented with 150 µg Mycobacterium tuberculosis H37RA (Difco Laboratories). 400 ng of pertussis toxin (CalBiochem) was injected intraperitoneally on the day of immunization and two days later. Animals were dosed once daily with the test article (as a solution or suspension in the vehicle) or vehicle alone (PEG300) starting from 1 day after immunization. Clinical scoring and body weight were taken 3 times per week until the end of the study on day 24. Clinical scoring system: 0.5: partial tail weakness; 1: limp tail or waddling gait with tail tonicity; 1.5: waddling gait with partial tail weakness; 2: waddling gait with limp tail (ataxia); 2.5: ataxia with partial limb paralysis; 3: full paralysis of one limb; 3.5: full paralysis of one limbs with partial paralysis of a second limb; 4: full paralysis of two limbs; 4.5: moribund; 5: death. References (1)

(a) Chun, J.; Hartung, H. Mechanism of action of oral fingolimod (FTY720) in multiple schlerosis. Clinical Neuropharmacology 2010, 33, 91-101. (b) Mandala, S.; Hajdu, R.; Bergstrom, J.; Quackenbush, E.; Xie, J.; Milligan, J.; Thornton, R.; Shei, G-J.; Card, D.; Keohane, C.; Rosenbach, M.; Hale, J.; Lynch, C. L.; Rupprecht, K.; Parsons, W.; Rosen, H.


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Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists. Science 2002, 296, 346-348. (c) Brinkmann, V.; Davis, M. D.; Heise, C. E.; Albert, R.; Cottens, S.; Hof, R.; Bruns, C.; Prieschl, E.; Baumruker, T.; Hiestand, P.; Foster, C. A.; Zollinger, M.; Lynch, K. R. The immune modulator FTY720 targets sphingosine 1-phosphate receptors. J. Biol. Chem. 2002, 277, 21453-21457. (2)

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

(3)

Matloublan, M, m.; Lo, C. G.; Cinamon, G.; Lesneski, M. J.; Xu, Y.; Brinkmann, V.; Allende, M. L.; Prola, R. L.; Cyster, J. G. Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 2004, 427, 355-360.

(4) (a) Hla, T.; Venkataraman, K.; Michaud, J. The vascular S1P gradient: cellular sources and biological significance. Biochim. Biophys. Acta 2008, 1781, 477-482. (b) Schwab, S. R.; Cyster, J. G. Finding a way out: lymphocyte egress from lymphoid organs. Nat. Immunol. 2007, 8, 1295-1301. (c) Rosen, H.; Goetzl, E. J. Sphingosine 1-phosphate and its receptors: an autocrine and paracrine network. Nat. Rev. Immunol. 2005, 5, 560-570. (5)

Allende, M. L.; Dreier, J. L.; Mandala, S.; Proia, R. L. Expression of the sphingosine 1phosphate receptor, S1P1, on T-cells controls thymic emigration. J. Biol, Chem. 2004, 279, 15396-15401.

(6) Pham, T. H. Okada, T., Matloubian, M.; Lo, C. G.; Cyster, J. G. S1P1 receptor singling overrides retention mediated by Gαi-coupled receptors to promote T-cell egress. Immunity 2008, 28, 122133. (7)

(a) Brinkmann, V.; Billich, A.; Baumruker, T.; Heining, P.; Schmouder, R.; Francis, G.; Aradhye, S.; Burtin, P. Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis. Nature Reviews Drug Discovery. 2010, 9, 883-897 and references therein. (b) Chun, J.; Brinkmann, V. A mechanistically novel, first oral therapy for multiple ACS Paragon Plus Environment

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sclerosis: The development of fingolimod (FTY720, Gilenya). Discovery Med. 2011, 12, 213228. (c) Chun, J.; Hartung, H.-P. Mechanism of action of oral fingolimod (FTY720) in multiple sclerosis. Clinical Neuropharmacology 2010, 33, 91-101. (8)

(a) Pelletier, D.; Hafler, D. A. Fingolimod for multiple sclerosis. N. Engl. J. Med. 2012, 366, 339-347. (b) Kappos, L.; Antel, J.; Comi, G.; Montalban, X.; O’Connor, P.; Polman, C. H.; Haas, T.; Korn, A. A.; Karlsson, G.; Radue, E. W. Oral fingolimod (FTY720) for relapsing multiple sclerosis. N. Engl. J. Med. 2006, 355, 1124-1140.

(9)

(a) Forrest, M.; Sun, S-Y.; Bergstrom, J.; Card, D.; Doherty, G.; Hale, J.; Keohane, C.; Meyers, C.; Milligan, J.; Mills, S.; Nomura, N.; Rosen, H.; Rosenbach, M.; Shei, G-J.; Singer, I. I.; Tian, M.; West, S.; White, V.; Xie, J.; Proia, R. L.; Mandala, S. Immune cell regulation and cardiovascular effects of sphingosine 1-phosphate receptor agonists in rodents are mediated via distinct receptor subtypes J. Pharmacol. Exp. Ther. 2004, 309, 758-768. (b) Li, Z.; Chen, W.; Hale, J. J.; Lynch, C. L.; Mills, S. G.; Hajdu, T.; Keohane, C. A.; Rosenbach, M. J.; Milligan, J. A.; Shei, G.-J.; Chrebet, G.; Parent, S. A.; Bergstrom, J.; Card, D.; Forrest, M.; Quackenbush, E. J.; Wickham, L. A.; Vargas, H.; Evans, R. M.; Rosen, H.; Mandala, S. Discovery of Potent 3,5Diphenyl-1,2,4-oxadiazole

Sphingosine-1-phosphate-1

(S1P1)

Receptor

Agonists

with

Exceptional Selectivity against S1P2 and S1P3. J. Med. Chem. 2005, 48, 6169-6173. (10) Gergely, P.; Nuesslein-Hildesheim, B.; Guerini, D.; Brinkmann, V.; Traebert, M.; Bruns, C.; Pan, S.; Gray, N. S.; Hinterding, K.; Cooke, N. G.; Groenewegen, A.; Vitaliti, A.; Sing, T.; Luttringer, O.; Yang, J.; Gardin, A.; Wang, N.; Crumb, W. J. Jr.; Saltzman, M.; Rosenberg, M.; Wallstrom, E. The selective sphingosine 1-phosphate receptor modulator BAF312 redirects lymphocyte distribution and has species-specific effects on heart rate. Br. J. Pharm. 2012, 167, 1035-1047. (11) (a) Brossard, P.; Hofmann, S.; Cavallaro, M.; Halabi, A.; Dingemanse, J. Entry-into-Humans Study with ACT-128800, a Selective S1P1 Receptor Agonist: Tolerability, Safety, Pharmacokinetics, and Pharmacodynamics. Presented at ASCPT 2009, Annual Meeting of the ACS Paragon Plus Environment

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American Society for Clinical Pharmacology and Therapeutics, Washington DC, USA, March 18–21, 2009; PII-87; Clin. Pharmcol. Ther. 2009, S63– S64. (b) Gergely, P.; Wallström, E.; Nuesslein-Hildesheim, B.; Bruns, C.; Zécri, F. J.; Cooke, N. G.; Traebert, M.; Tuntland, T.; Rosenberg, M.; Saltzman, M. Phase I Study with the Selective S1P1/S1P5 Receptor Modulator BAF312 Indicates That S1P1 Rather Than S1P3 Mediates Transient Heart Rate Reduction in Humans. Presented at the 25th Congress of the European Committe for Treatment and Research in Multiple Sclerosis (ECTRIMS), Düsseldorf, Germany, 2009; P437. (c) Wallström, E.; Gergely, P.; Nuesslein-Hildesheim, B.; Zécri, F. J.; Cooke, N. G.; Bruns, C.; Luttringer, O.; Sing, T.; Groenewegen, A.; Rosenberg, M.; Saltzman, M. BAF312, a Selective S1P1/S1P5 Receptor Modulator, Effectively Reduces Absolute Lymphocyte Counts in Human Volunteers and Demonstrates the Relevance of S1P1 in Mediating a Transient Heart Rate Reduction. Presented at the 62nd Annual Meeting of the American Academy of Neurology, Toronto, Ontario, Canada, 2010; PO5.052. (12) Dyckman, A. J. Recent Advances in the Discovery and Development of Sphingosine-1Phosphate-1 Receptor Agonists Ann. Rep. Med. Chem. 2012 195-207 and references cited therein. (13) (a) Buzard, D. J.; Schrader, O.; Zhu, X.; Lehmann, J.; Johnson, B.; Kasem, M.; Kim, S.-H.; Kawasaki, A.; Lopez, L.; Moody, J.; Han, S.; Gao, Y.; Edwards, J.; Barden, J.; Thatte, J.; Gatlin, J.; Jones, R. M. Design and synthesis of new trycyclic indoles as potent modulators of the S1P1 receptor. Bioorg. Med. Chem. Lett. 2015, 25, 659-663. (b) Buzard, D. J.; Lopez, L.; Moody, J.; Kawasaki, A.; Schrader, T. O.; Kasem, M.; Johnson, B.; Zhu, X.; Thoresen, L.; Kim, S. H.; Gharbaoui, T.; Sengupta, D.; Calvano, L.; Krishnan, A.; Gao, Y.; Semple, G.; Edwards, J.; Barden, J.; Morgan, M.; Usmani, K.; Chen, C.; Sadeque, A.; Chen, W.; Christopher, R. J.; Thatte, J.; Fu, L.; Solomon, M.; Whelan, K.; Al-Shamma, H.; Gatlin, J.; Gaidarov, I.; Anthony, T.; Le, M.; Unett, D. J.; Stirn, S.; Blackburn, A.; Behan, D. P.; Jones, R. M. (7-Benzyloxy-2,3dihydro-1H-pyrrolo[1,2-a]indol-1-yl)acetic acids as S1P1 functional agonists. Med. Chem. Lett. ACS Paragon Plus Environment

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2014, 5, 1334-1339. (c) Skidmore, J.; Heer, J., Jhonson, C. N.; Norton, D.; Redshaw, S.; Sweeting, J.; Hurst, D.; Cridland, A.; Vesey, D.; Wall, I.; Ahmed, M.; Rivers, D.; Myatt, J.; Giblin, G.; Philpott, K.; Kumar, U.; Stevens, A.; Bit, R. A.; Haynes, A.; Taylor, S.; Watson, R.; Witherington, J.; Demont, E.; Heightman, T. D. Optimization of Sphingosine-1-phosphate receptor agonists: effects of acidic, basic, and zwitterionic chemotypes on pharmacokinetic and pharmacodynamics profiles. J. Med. Chem. 2014, 57, 10424-10442. (d) Bolli, M. H.; Abele, S.; Birker, M.; Brave, R.; Bur, D.; de Kanter, R.; Kohl, C.; Grimont, J.; Hess, P.; Lescop, C.; Mathys, B.; Muller, C.; Nayler, O.; Rey, M.; Scherz, M.; Schmidt, G.; Seifert, J.; Steiner, B.; Velker, J.; Weller, T. Novel S1P1 receptor agonists – Part 3: From thiophenes to pyridines. J. Med. Chem. 2014, 57, 110-130. (e) Bolli, M. H.; Velker, J.; Muller, C.; Mathys, B.; Birker, M.; Bravo, R.; Bur, D.; de Kanter, R.; Hess, P.; Kohl, C.; Lehmann, D.; Meyer, S.; Nayler, O.; Rey, M; Scherz, M.; Steiner, B. Novel S1P1 receptor agonists – Part 2: From bicylco[3.1.0]hexanefused thiophenes to isobutyl substituted thiophenes. J. Med. Chem. 2014, 57, 78-97. (f) Tian, Y.; Jin, J.; Wang, X.; Hu, J.; Xiao, Q.; Zhou, W.; Chen, X.; Yin, D. Discovery of oxazole and triazole derivatives as potent and selective S1P1 agonists through pharmacophore-guided design. Eur. J. Med. Chem. 2014, 85, 1-15. (g) Horan, J. C.; Sanyal, S.; Choi, Y.; Hill-Drzewi, M.; Patnaude, L.; Anderson, S.; Fogal, S.; Mao, C.; Cook, B. N.; Gueneva-Boucheva, K.; Fisher, M. B.; Hickey, E.; Pack, E.; Bannen, L. C.; Chan, D. S.; Mac, M. B.; Ng, S. M.; Wang, Y.; Xu, W.; Modis, L. K.; Lemieux, R. M. Piperazinyl-oxadiazoles as selective sphingosine-1-phosphate receptor agonists. Bioorg. Med. Chem. Lett. 2014, 24, 4807-4811. (14) Hale, J. J.; Lynch, C. L.; Neway, W.; Mills, S. G.; Hajdu, R.; Keohane, C. A.; Rosenbach, M. J.; Milligan, J. A.; Shei, G.-J.; Parent, S. A.; Chrebet, G.; Bergstrom, J.; Card, D.; Ferrer, M.; Hodder, P.; Strulovici, B.; Rosen, H.; Mandala, S. A rational utilization of high-throughput screening affords selective, orally bioavailable 1-benyl-3-carboxyazetidine sphingosine-1phosphate-1 receptor agonists. J. Med. Chem. 2004, 47, 6662-6665.


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(15) Dyckman, A. J.; Li, L.; Watterson, S. H.; Das, J.; Li, T.; Banas, D.; Cvijic, M. E.; Liu, R.; Suchard, S.; Shen, D. R.; Yarde, M.; Gillooly, K.; McIntyre, K.; Shuster, D.; Taylor, T.; Yang, X.; Cornelius, G.; D'Arienzo, C.; Marino, A.; Balimane, P.; Salter-Cid, L.; McKinnon, M.; Barrish, J. C.; Carter, P. H.; Pitts, W. J.; Xie, J. Identification of potent and selective pyrazole based agonists of sphingosine-1-phosphate 1 (S1P1) Abstracts of Papers, 244th ACS National Meeting & Exposition, Philadelphia, PA, United States, August 19-23, 2012, MEDI-72. (16) (a) Watterson, S. H.; Guo, J.; Spergel, S. H.; Langevine, C. M.; Moquin, R. V.; Kempson, J.; Shen, D. R.; Yarde, M.; Cvijic, M. E.; Banas, D.; Liu, R.; Suchard, S. J.; Gillooly, K.; Taylor, T.; RexRabe, S.; Shuster, D. J.; McIntyre, K. W.; Cornelius, G.; Darienzo, C.; Marino, A.; Balimane, P.; Saltercid, L.; McKinnon, M.; Barrish, J. C.; Carter, P. C.; Pitts, W. J.; Xie, J.; Dyckman, A. J. Potent and selective agonists of sphingosine-1-phosphate 1 (S1P1): The discovery and SAR of a novel isoxazole based series. Abstracts of Papers, 241st ACS National Meeting & Exposition, Anaheim, CA, United States, March 27-31, 2011, MEDI-96. (b) Watterson, S. H.; Dyckman, A. J.; Pitts, W. J.; Spergel, S. H. Substituted isoxazole derivatives as S1P agonists in the treatment of autoimmune and inflammatory diseases and their preparation. WO 2010085581; Chem. Abstr. 2010, 153, 232588 (US Patent 8354398 2013). (17) Liu, K.-C.; Shelton, B. R.; How, R. K. A particularly convenient preparation of benzohydroximinoyl chlorides (nitrile oxide precursors). J. Org. Chem. 1980, 45, 3916-1918. (18) Hamper, B. C.; Leschinshy, K. L. Reaction of benzohydroximinoyl chlorides and Βtrifluoromethyl)-acetylenic

esters:

Synthesis

of

regioisomeric

(trifluoromethyl)-

isoxazolecarboxylate esters and oxime addition products. J. Heterocyclic Chem. 2003, 40, 575583. (19) Jiang, Z.-H.; Qin, Y.-Y.; Qing, F.-L. Asymmetric synthesis of both enantiomers of anti-4,4,4trifluorothreonine and 2-amino-4,4,4-trifluorobutanoic acid. J. Org. Chem. 2003, 68, 7544-7547. (20) Bendele, A.; McComb, J.; Gould, T.; McAbee, T.; Sennello, G.; Chlipala, E.; Guy, M. Animal models of arthritis: relevance to human disease. Toxicol. Pathol. 1999, 27, 134-142. ACS Paragon Plus Environment

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Figure 1. Structures of sphingosine and sphingosine1-phospate. OH OH NH2 Sphingosine Sphingosine kinases OH

NH2

O P OH O OH

Sphingosine1-phosphate (S1P)

Figure 2. Structures of fingolimod (1) and fingolimod-P (2). Me

NH2 OH OH 1 (Fingolimod)

Sphingosine Kinases

Me

NH2 OH O 2 (Fingolimod-P)

P OH O OH


57

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Figure 3. Discovery of isoxazole based agonists of S1P1.

Figure 4. Quantification of S1P1 on the Cell Membrane.

130.0

%S1P1-GFP Signal Reappearance on Membrane

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


+ 1.2 µM S1P

115.0

S-1P (rapid recycling)

+ 4.6 nM 6d

100.0 85.0 70.0 55.0

Isoxazole 6d

40.0 25.0

Wash off

10.0 -5.0 -3

-1

1

3

5

7

9

11

13

15

17

19

Time (hr)


58


Figure 5a. (A) Paw swelling of 6d (0.03, 0.1, 0.5, and 3.0 mg/kg) and 1 (0.3 mg/kg) vs. vehicle in a rat adjuvant arthritis model. (B) Paw histology of 6d (0.03, 0.1, 0.5, and 3.0 mg/kg) and 1 (0.3 mg/kg) vs. vehicle in a Rat Adjuvant Arthritis model. 5A 1.75

Paw Swelling (in mL)

1.50 1.25

Vehicle 6d-0.03mg/kg 6d-0.1mg/kg 6d-0.5mg/kg 6d-3.0mg/kg 1-0.3mg/kg

1.00 0.75

*

0.50 0.25 0.00 7

* * 9

11

13

* *

* * 15

17

* 19

21

Study Day

5B 10 8

Histologic Score

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 60 of 67

6 4 2

*

*

0 Inflammation

*

* ** Bone resorption

*

Total Score

a Rats (n=8-10/group) were administered 6d and 1 PO/QD starting on Day 0 at the time of immunization. Vehicle: PEG300. *p value < 0.05 compared to vehicle treatment group.


59

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Figure 6a. (A) Efficacy of 6d (0.0, 0.2, and 2.0 mg/kg) and 1 (1.0 mg/kg) vs. vehicle in a mouse experimental autoimmune encephalomyelitis model (EAE). (B) Spinal cord histology of 6d (0.0, 0.2, and 2.0 mg/kg) and 1 (1.0 mg/kg) vs. vehicle in a mouse experimental autoimmune encephalomyelitis model (EAE). 6A 4.0 3.5 3.0

Vehicle

2.5

* * *

1.5 1.0 0.5 0.0 10

* *

* * *

*

6d-0.02mg/kg 6d-0.2mg/kg

* * * *

2.0

6d-2mg/kg 1-1mg/kg

* 12

14

16

18

20

22

Study Day 6B

5

Histologic Score

Mean Clinical Score

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4 3

*

2 1

*

*

*

*

*

0 Inflammation

Demyelination

Total Score

a

Mice (n=10/group) were administered 3d and 1 PO/QD starting on Day 0 at the time of immunization with MOG-peptide (myelin oligodendrocyte glycoprotein). Vehicle: PEG300. *p value < 0.05 compared to vehicle treatment group.


60


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Scheme 1a. Preparation of isoxazoles 5 and 6.

a

Reagents and conditions: (a) t-BuOH, DMAP, EDCI, CH2Cl2, 81%; (b) 10% Pd/C, EtOAc, 92%; (c) 4-cyanobenzaldehyde, Na(CN)BH3; MeOH, 89%; (d) HONH2-HCl; NaHCO3, t-BuOH, 55%; (e) 13, 25, or 29, EDCI, HOBt, DMF, 53-86%; (f) TFA.

Scheme 2a. Preparation of isoxazole-3-carboxylic acids.

a Reagents and conditions: (a) 1-butyl-3-methyl-1H-imidazol-3-ium hexafluorophosphate, toluene, microwave, 170oC; (b) mesitylene, 150oC; (c) LiOH-H2O, MeOH/H2O; (d) 1N aqueous NaOH, MeOH, microwave, 100oC.


61

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 3a. Preparation of 4-trifluoromethyl-isoxazole-3-carboxylic acids.

a Reagents and conditions: (a) NIS, TFA, 97%; (b) methyl 2,2-difluoro-2-(fluorosulfonyl)acetate, CuI, HMPA, DMF, 94%; LiOH-H2O, MeOH, H2O, 96%.

a Reagents and conditions: (a) NIS, TFA,78%; (b) methyl 2,2-difluoro-2-(fluorosulfonyl)acetate, CuI, HMPA, DMF, 83%; LiOH-H2O, MeOH, H2O, 97%.

Scheme 4a. Preparation of isoxazole-3-carboxylic acid 25.


62


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a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Reagents and conditions: (a) NBS, 5% fuming HNO3 in AcOH; (b) tributyl(1-ethoxyvinyl)tin, dioxane, 100oC; (c) deoxofluor, ethanol, 85oC; (d) 1N aq/ NaOH, methanol.

Scheme 5a. Preparation of isoxazole-5-carboxylic acids.

a

Reagents and conditions: (a) Et3N, CH2Cl2, 95%; (b) Hunig's base, MeCN, 80%; (c) TFA, neutralization, >90%


64


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Page 66 of 67

Table 1 In vitro and in vivo potency of C3-linked isoxazoles (5). O N 5 1 R N 3

4

R2

N

O N 5

In Vitro Activity Cmpd

5a 5b

a

R1 Ph Ph

R2 H Me

hS1P1 GTPγS EC50 nMa 44b 5.0

b

CO2H Rat Blood Lymphocyte Reduction Assay

hS1P3 GTPγS EC50 nMa

S1P3/ S1P1 Sel.

6,300b

150x

22%

140

0%

--

ND

>6300x

68%

260

0%

6.4

41

7,500x

76%

320

52%

29

11

>31

b b

Reduction 4h (1.0 mpk)

Plasma Conc. (nM)

Reduction 24 h (1.0 mpk)

Plasma Conc. (nM)

4h/24h ratio

5c

Ph

Et

1.6±0.05

12,000

5d

Ph

Pr

0.44±0.16

1,300±410

2,900x

85%

250

60%

7.9

31

5e

Ph

Bu

0.27±0.03

680±20

250x

82%

81

60%

5.0

16

5f

Ph

iBu

0.56±0.12

730±450

1,300x

80%

190

82%

30

6

5g

Ph

iPr

1.3±0.60

5,100±3,900

4,000x

ND

ND

ND

ND

ND

5h

Ph

cycPr

3.2±2.2

1,800±280

540x

ND

ND

ND

ND

ND

5i

Ph

tBu

5.3±1.7

2,600 (n=2)

500x

73%

520

52%

55

9

5j

Ph

Ph

0.99±0.41

130±34

130x

ND

ND

ND

ND

ND

5k

Ph

CF3

0.98±0.05

4,100±710

4,100x

84%

1,100

85%

180

6

5l

Ph

CF2Me

0.54±0.25

1,900 (n=2)

3,500x

81%

1,400

82%

100

14

5m

iBu

Pr

0.30±0.08

850 (n=2)

2,900x

86%

1,300

78%

41

33

5n

iBu

CF3

0.39±0.21

6,200±1,400

16,000x

88%

1,600

83%

280

6

EC50 values are shown as mean values of at least three determinations; b EC50 values are shown as a single determination; ND = Not Determined

Table 2 In vitro and in vivo potency of C5-linked isoxazoles (6).

In Vitro Activity Cmpd

a

R1

R2

hS1P1 GTPγS EC50 nM

Rat Blood Lymphocyte Reduction Assay

hS1P3 GTPγS EC50 nM

S1P3/ S1P1 Sel.

Reduction 4h (1.0 mpk)

Plasma Conc. (nM)

Reduction 24 h (1.0 mpk)

Plasma Conc. (nM)

4h/24h ratio

6a

Ph

H

25b

ND

ND

ND

ND

ND

ND

ND

6b

Ph

Me

1.7 (n=2)

9,200±6,200

5500x

ND

ND

ND

ND

ND

6c

Ph

Pr

0.44 (n=2)

1,000±440

2300x

85%

170

82%

11

15

6d

Ph

CF3

0.47±0.25

1,600±320

3400x

78%

2,100

91%

700

3

b

EC50 values are shown as mean values of at least three determinations; EC50 values are shown as a single determination; ND = Not Determined


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Table 3 Partial in vitro profiling data for Compound 6d.

Parameter

Result

Human S1P2/S1P1 selectivity Human S1P4/S1P1 selectivity Human S1P5/S1P1 selectivity Protein Binding (bound)

a

> 10,000x 5.2x 2.7x 99.7% human 99.9% mounse 99.4% rat 99.0% dog 99.3% monkey Mutagenicity Ames negative hERG (Patch Clamp) IC50 = 18 µM Na+ (Patch Clamp) 40 µM 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4 PAMPA permeability 1032 nm/s @ pH 7.4 Caco2 Permeabilityb 104 nm/s pKa 3.7 and 7.9 Log D (pH 6.5) 2.68 CYP = cytochrome P450; b Apical to basolateral

Table 4 In vivo potency of isoxazole 6d in the rat blood lymphocyte reduction assay. Dose (mg/kg)

Reduction 4h

Plasma Conc. (nM)

Reduction 24 h

Plasma Conc. (nM)

4h/24h ratio

1.00

78%

2100

91%

700

3

0.30

82%

180

73%

25

7

0.03

75%

20

45%

4.0

5


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Page 68 of 67

Table 5 Pharmacokinetic Parameters for Compound 6d. Parameter

Mouse b

Rata

Cynob

b

po dose (mg/kg) 10 5 5 iv dose (mg/kg) 2 2 2 Cmax (µM), PO 31 3.9 4.0 Tmax (µM), PO 3.0 3.3 1.0 AUC (µM*h), PO 250 44 25 T1/2 (h), iv 9.8 10 3.0 MRT (h), iv 14 14 2.2 CL (mL/min/kg),iv 0.30 1.7 5.2 CL (%HBF)c 0.30 2.4 12 Vss (L/kg), iv 0.20 1.4 0.60 Fpo (%) 71 52 70 Peak/Trough ratio 2 6 37 a Average of three animals; b cynomologus monkey; c % hepatic blood flow

Doga 1b 1 1.3 8.9 11 7.4 8.9 1.6 5.2 0.70 45 9

TOC Graphic


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Potent and Selective Agonists of Sphingosine 1-Phosphate 1 (S1P1

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