Design, Synthesis, and in Vitro and in Vivo Evaluation of an 18F-Labeled Sphingosine 1-Phosphate Receptor 1 (S1P1) PET Tracer

Article abstract of DOI:10.1021/acs.jmedchem.6b00390

Sphingosine 1-phosphate receptor 1 (S1P₁) plays a pivotal signaling role in inflammatory response; because S1P₁ modulation has been identified as a therapeutic target for various diseases, a PET tracer for S1P₁ would be a useful tool. Fourteen fluorine-containing analogues of S1P ligands were synthesized and their in vitro binding potency measured; four had high potency and selectivity for S1P₁ (S1P₁ IC₅₀ < 10 nM, >100-fold selectivity for S1P₁ over S1P₂ and S1P₃). The most potent ligand, 28c (IC₅₀ = 2.63 nM for S1P₁) was ¹⁸F-labeled and evaluated in a mouse model of LPS-induced acute liver injury to determine its S1P₁-binding specificity. The results from biodistribution, autoradiography, and microPET imaging showed higher [¹⁸F]28c accumulation in the liver of LPS-treated mice than controls. Increased expression of S1P₁ in the LPS model was confirmed by immunohistochemical analysis (IHC). These data suggest that [¹⁸F]28c is a S1P₁ PET tracer with high potential for imaging S1P₁ in vivo.

Full text of DOI:10.1021/acs.jmedchem.6b00390

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Article
Design, synthesis and in vitro and in vivo evaluation of an 18F-
labeled sphingosine 1-phosphate receptor 1 (S1P1) PET tracer
Adam J Rosenberg, Hui Liu, Hongjun Jin, Xuyi Yue, Sean Riley, Steven J Brown, and Zhude Tu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00390 • Publication Date (Web): 09 Jun 2016
Downloaded from http://pubs.acs.org on June 11, 2016
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Design, synthesis and in vitro and in vivo evaluation of an F-labeled sphingosine 1-
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phosphate receptor 1 (S1P ) PET tracer
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Adam J. Rosenberg , Hui Liu , Hongjun Jin , Xuyi Yue , Sean Riley , Steven J. Brown , and Zhude Tu *
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Department of Radiology, Washington University School of Medicine, 510 South Kingshighway Boulevard, St. Louis,
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Missouri 63110
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The Scripps Research Institute Molecular Screening Center, 10550 N. Torrey Pines Rd., La Jolla, CA 92037
ABSTRACT: Sphingosine 1ꢀphosphate receptor 1 (S1P ) plays a pivotal signaling role in inflammatory response;
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because S1P modulation has been identified as a therapeutic target for various diseases, a PET tracer for S1P would be a
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useful tool. Fourteen fluorineꢀcontaining analogues of S1P ligands were synthesized and their in vitro binding affinities
measured; four had high affinity and selectivity for S1P (S1P IC < 10 nM, > 100ꢀfold selectivity for S1P over S1P
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and S1P ). The most potent ligand, 28c (IC = 2.63 nM for S1P ) was Fꢀlabeled and evaluated in a mouse model of
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LPSꢀinduced acute liver injury to determine its S1P ꢀbinding specificity. The results from biodistribution,
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autoradiography, and microPET imaging showed higher [ F]28c accumulation in the liver of LPSꢀtreated mice than
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controls. Increased expression of S1P in the LPS model was confirmed by IHC. These data suggest that [ F]28c is a
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S1P ꢀ PET tracer with high potential for imaging S1P in vivo
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. INTRODUCTION
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Sphingosine 1ꢀphosphate (S1P, Fig. 1) receptors are a class of Gꢀproteinꢀcoupled receptors (GPCRs) with five distinct
1, 2
subtypes, denoted as S1P ꢀ . These receptors are regulated by S1P and have important regulatory functions in both
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normal biological processes and in disease; particularly in pathological processes involving the immune system, the CNS,
and the cardiovascular system. Because the S1P/S1Pꢀreceptor pathway is especially important in cancer and autoimmune
disorders including multiple sclerosis (MS), tremendous development efforts have focused on therapeutically targeted S1P
receptor ligands. These efforts discovered a promising compounds, 2ꢀaminoꢀ2ꢀ[2ꢀ(4ꢀoctylꢀphenyl)ꢀethyl]ꢀpropaneꢀ1,3ꢀdiol
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(FTY720, Fingolimod, Fig. 1), that was approved by the US Food and Drug Administration (FDA) for treatment of
relapse remitting multiple sclerosis in 2010. Compound 1 is phosphorylated in vivo by sphingosine kinase 2, this
stereospecific process generates the biologically active metabolite (S)ꢀFTY720ꢀPhosphate ((S)-1-P, Fig. 1), a potent S1P
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receptor agonist. (S)-1-P is a nonselective S1P receptor ligand, which binds to each of the S1P receptor subtypes except
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S1P . Since its primary mechanism of action in MS is believed to be through S1P , recent development has focused on
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ligands having high potency and selectivity for S1P with suitable pharmacological properties for therapeutic applications.
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Although rodent studies using S1P receptor ligands suggested that adverse cardiovascular effects were the result of S1P3
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agonism,
the Phase I study of a S1P selective therapeutic reported bradycardia associated with the first dose
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administered to human subjects. Because activity at S1P has clear effects on the vascular system and does not appear to
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contribute to therapeutic efficacy of these ligands in the immune system, our efforts have focused on identifying subtypeꢀ
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selective ligands. S1P ligands are also being evaluated in cancer,
rheumatoid arthritis, ulcerative colitis,
as well
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as liver and lung injury among other therapeutic uses.
Positron emission tomography (PET) is a widely utilized imaging modality which can be used to nonꢀinvasively quantify
molecular targets in vivo. PET is used in drug discovery to assess receptor occupancy, downstream functional changes,
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and impact on disease pathophysiology. A S1P specific PET tracer could greatly assist the evaluation of new
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therapeutics by measuring the relationship between drug exposure (dose administered or plasma concentration) and
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receptor occupancy, thus enabling accelerated dose selection during early clinical trials.
A successful PET ligand
should have a binding affinity < 10 nM and must be specific to the target versus neighboring nonꢀtarget tissues to allow
23, 24
for quantification of the regions of interests during the timeframe of clinical studies (0ꢀ2 hours).
To date, no S1P receptor imaging agent suitable for clinical studies has been reported, although since 2011, several groups
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have reported their efforts to develop a S1P receptor tracer.
The iodinated analogue, 2ꢀaminoꢀ2ꢀ[2ꢀ(2ꢀiodoꢀ4ꢀ
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octylphenyl)ethyl]ꢀ1,3ꢀpropandiol 2 (BZM055, Fig. 2), was Iꢀlabeled for single photon emission computed tomography
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SPECT) studies, and could be Iꢀlabeled for PET imaging. Although compound 2 showed pharmacokinetic trends
similar to compound 1, its lower rate of phosphorylation (required for receptor binding) compared to compound 1 limits
its effectiveness as an imaging agent. The long halfꢀlives of either iodineꢀ123 (t1/2 = 13.2 h) or iodineꢀ124 (t1/2 = 4.2 days)
would enable study of both the distribution kinetics of the parent ligand and its elimination kinetics. Haufe and coꢀworkers
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separately reported Fꢀlabeled PET imaging agents based on the only known S1P antagonist, [(3R)ꢀaminoꢀ4ꢀ[(3ꢀ
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hexylphenyl)amino]ꢀ4ꢀoxobutyl]ꢀphosphonic acid, mono(trifluoroacetate) (W146), and based on compound 1. (Fig. 2)
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The Fꢀlabeled W146 analog (R)ꢀ1ꢀ[[3ꢀ(6ꢀfluorohexyl)phenyl]aminoꢀ4ꢀoxobutyl]phosphonic acid, ([ F]3) was shown to
bind with S1P , and unlike 1 does not require a preliminary phosphorylation step for binding potency. However, despite
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its in vitro stability in mouse serum, PET imaging of [ F]3 in wildꢀtype mice showed rapid total body clearance as well as
bone uptake, which is a measure of in vivo defluorination. Their recent report describes the more favorable evaluation of
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Fꢀlabeled 2 analogues, with either an 8ꢀcarbon (2ꢀaminoꢀ2ꢀ[4ꢀ(8ꢀfluorooctyl)phenethyl]propaneꢀ1,3ꢀdiol, [ F]4a) or a 6ꢀ
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carbon (2ꢀaminoꢀ2ꢀ[4ꢀ(6ꢀfluorohexyl)phenethyl]propaneꢀ1,3ꢀdiol, [ F]4b) aliphatic tail (Fig. 2). The ligand with the
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shorter tail, [ F]4b, showed reduced in vitro activity, but both tracers demonstrated uptake in S1P target organs of wildꢀ
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type mice with no evidence of in vivo defluorination. Unlike [ F]3, the 1 analogues require phosphorylation before
binding to S1P receptors. While potentially useful tools, at this time it is unclear how rapidly and to what extent the
ligands undergo phosphorylation during the typical time frame for PET imaging (1ꢀ2 h postꢀinjection).
We have also used the wealth of knowledge in this field to investigate PET radiotracers for S1P receptors using rodent
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models of human disease.
Our group previously reported the radiosynthesis of the Cꢀlabeled S1P selective ligand 3ꢀ
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(2ꢀfluoroꢀ4ꢀ(5ꢀ(2'ꢀmethylꢀ2ꢀ(trifluoromethyl)ꢀ[1,1'ꢀbiphenyl]ꢀ4ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)benzyl)ꢀ(methyl)amino)ꢀ
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propanoic acid 5 ([ C]TZ3321, Fig. 2) and demonstrated the feasibility of PET imaging S1P as a measure of
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inflammatory response in both the femoral artery wireꢀinjury mouse model of restenosis and in the experimental
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autoimmune encephalomyelitis (EAE) rat model of multiple sclerosis (MS). Here, we present the synthesis and
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screening of fluorineꢀcontaining ligands based on reported compounds with either a benzoxazole core, or an oxadiazole
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core.
Compound 28c was identified as a potent and selective ligand for S1P over S1P or S1P in our competitive
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binding assay, so was Fꢀlabeled for biological evaluation in the mouse model of LPSꢀinduced liver injury.
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. RESULTS AND DISCUSSION
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The synthetic strategy is discussed in detail below, but the two lead compounds used for development of an Fꢀlabeled
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tracer were selected due to published data indicating that they are not prodrugs which require in vivo phosphorylation, and
31, 32
due to their favorable biological behavior.
Our screening focused on S1P selective ligands because the efficacy of 2
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in MS is attributed to its high binding potency toward to S1P1.
As shown in Table 2, 28c has a high potency (IC =
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2.63 ± 0.27 nM) and selectivity for S1P over S1P or S1P (IC > 1000 nM). The synthesis of [ F]28c was accomplished.
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the mouse model of LPSꢀinduced acute liver injury was used for the in vivo evaluation of [ F]28c. Biodistribution studies
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99m
carried out with both [ F]28c and the liver imaging agent [ Tc]ꢀNꢀ(3ꢀbromoꢀ2,4,6ꢀtrimethyacetanilide) iminodiacetic
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acid ([ Tc]mebrofenin) in LPS and sham mice. The absence of a significant difference between the two tracers in this
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model 60 min postꢀinjection suggested that the higher liver uptake of [ F]28c in LPSꢀtreated mice compared to control
mice was not caused by reduced hepatobiliary clearance in the liver injury model. MicroPET imaging in LPS and sham
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treated mice also showed increased retention of [ F]28c in the injured liver which correlated with increased S1P
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expression observed in IHC studies, while no difference in liver uptake of [ O]water was seen between LPSꢀtreated and
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sham mice,
suggesting the higher injured liver accumulation of [ F]28c is not due to a change in blood flow in injured
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liver. These data indicate liver uptake of [ F]28c correlates well with the expression of S1P in mouse liver.
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.1 Chemistry
Ligands with a benzoxazole core were prepared as shown in Schemes 1 & 2. The starting halogenated
trifluoromethylbenzoic acids were functionalized by either palladium catalyzed Suzuki coupling with the desired aryl
boronic acid to provide the biaryl moieties, or by nucleophilic aromatic substitution with the desired alcohol to give the
ethereal products. The appendant carboxylic acid was transformed to the corresponding acid chloride with oxalyl chloride
and catalytic DMF, which was then reacted with a functionalized 2ꢀhydroxyaniline under Schotten–Baumann conditions
to give amides 10. These amides were subsequently heated under acidic conditions to cyclize and provide the benzoxazole
cores. The installation of the polar head groups was accomplished by a variety of methods. βꢀAmino acids 15a and 15b,
were prepared by reduction of a methyl ester to an alcohol, and functional group transformation to a chloride, and
subsequently to the azetidine ester, which was unmasked to provide 15. Acid 16 was prepared by a Suzuki coupling with a
thiophene boronic acid. Palladiumꢀcatalyzed amination with methyl piperidineꢀ4ꢀcarboxylate gave the protected products,
which were subsequently saponified to give acids 18.
Ligands with an oxadiazole core were synthesized according to Scheme 3. The required carboxylic acids were prepared as
above, or from methyl 4ꢀhydroxybenzoate (22) by alkylation and saponification. The desired carboxylic acid was reacted
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with N'ꢀhydroxyꢀ4ꢀ(hydroxymethyl)benzimidamide (25) under standard peptide coupling conditions, and subsequently
thermally cyclized to give the oxadiazole cores. Oꢀ(Benzotriazolꢀ1ꢀyl)ꢀN,N,N′,N′ꢀtetramethyluronium tetrafluoroborate
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(
TBTU) was also used as a coupling agent, followed by thermal cyclization. The appendant benzyl alcohol was oxidized
under Swern conditions to give aldehydes 27. The βꢀamino acid head group was affixed by reductive amination with the
desired amine to provide the targeted ligands 28a-f. The lipophilicity of a radiotracer, expressed as logP, impacts
nonspecific binding and its ability to cross the BBB, which is a key physical constraint for targets in CNS and intracellular
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targets including enzymes. The calculated lipophilicity at a pH of 7.4 of the new compounds is reported as cLogD values
in Tables 1ꢀ2. The oxadiazole 28c had cLogD of 2.32 which is within the desired range for CNS tracers and, as discussed
below, a favorable in vitro binding profile, thus we elected to pursue its development as a radiotracer and synthesized
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precursors for radiolabeling. Precursor 32 for radiolabeling [ F]2ꢀfluoroethyltosylate was prepared from phenol 29, as
shown in Scheme 4, using a synthetic route similar to that used for the oxadiazole analogues. Formation of the oxadiazole
proceeded as expected, however the oxidation step required different conditions due to the presence of the unprotected
phenol. Oxidation with manganese (IV) oxide provided smooth conversion to desired aldehyde 31. Reductive amination
under buffered conditions afforded 32. Precursor 34 for direct labeling was also prepared as shown in Scheme 5. Starting
with aldehyde 31, the free phenol was alkylated with ethylene ditosylate to provide compound 33, which was subjected to
reductive amination to yield 34. The reductive amination conditions were modified to utilize sodium
triacetoxyborohydride, because harsher reducing agents tended to reduce the alkyl tosylate.
2
.2 In Vitro Binding Assays
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Competitive inhibition of the binding of radiolabeled [ P]S1P to the new analogues was measured to determine the
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affinity of the ligands for S1P , S1P , and S1P . The results are reported in Tables 1 and 2. The benzoxazole series of
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ligands displayed nanomolar activity in the functional assay (EC ), however the ligands displayed lower binding affinity
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for S1P in the competitive binding assay (IC ). Although the reason for this discrepancy in the assay results is not clear,
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similar results have observed with other S1P receptor ligands. Additionally while functional assays can be extremely
useful in the preliminary stages of imaging agent development, the binding affinity of a ligand cannot be determined
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without a competitive binding assay. While none of the ligands in this series had sufficient binding affinity (IC < 10
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nM) to be pursued as a PET tracer, structureꢀactivity relationships (SAR) became apparent. The residues and shape of the
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S1P binding pocket have been well documented with a published crystal structure. The binding pocket consists of two
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major components, a charged head portion and a lipophilic tail area. The strong recognition for ligand binding is thought
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to be derived from interactions between the positively charged Arg and the phosphate in S1P, and interactions between
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the negatively charged Glu and the ammonium in S1P. The lipophilic part of the binding pocket appears to be quite
tolerant of a variety of functional groups, with the noted exception of the orthoꢀsubstituted fluoroethyl ether 18b (IC =
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94 ± 367 nM).
The oxadiazole ligand series showed much more promise than the benzoxazole series, as seen in Table 2. We explored the
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effects of modifying the lipophilic sideꢀchain of 28a (IC = 8.53 ± 3.14 nM). Addition of a fluorine (28b, IC = 9.94 ±
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.03 nM) had no significant change on the binding affinity, while an orthoꢀtrifluoromethyl group noticeably improved the
affinity to 2.63 ± 0.27 nM (28c). Next we turned to the polar headꢀgroup. While some S1P ligands without a polar headꢀ
1
group have a high degree of potency, we elected to maintain this property in our explorations. Changing the azetidine ring
of 28c to a piperidine (28d) gave a 20ꢀfold decrease in binding affinity to 45.4 ± 2.7 nM. Bioisosteric substitution of the
acid to a tetrazole resulted significant loss of activity of over 100ꢀfold to 509 ± 167 nM (28e), while shortening the twoꢀ
carbon tail to a methyl group adversely impacted the binding affinity giving an IC > 100 nM (28f). Finally, the benzyl
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alcohol 26c was synthesized to determine how critical the amino acid head group was, surprisingly this head group was
not required as 26c was quite potent with an IC of 6.67 ± 0.70 nM. All compounds with a binding affinity under 50 nM
50
were screened for selectivity versus S1P and S1P ; no tested compounds showed detectable binding with S1P or S1P .
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8c had a high potency for S1P (IC = 2.63 ± 0.27 nM) with no measurable potency for S1P (IC >1000 nM) or S1P
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(IC >1000 nM) with a cLogD of 2.32, thus we elected to pursue 28c as our lead compound for Fꢀlabeling.
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.3 Radiochemistry
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The radiosynthesis of 28c was initially attempted using the versatile indirect approach of labeling with [ F]2ꢀ
fluoroethyltosylate. The precursor 32 was prepared as shown in Scheme 4. Unfortunately attempts to label 32 indirectly
resulted only in extremely low yields under a variety of conditions (< 5% radiochemical yield for the alkylation step),
likely due to the low nucleophilicity of the electronꢀdeficient phenol. Due to this concern, as well as a desire to avoid a
threeꢀstep, twoꢀpot radiosynthesis, we subsequently pursued a direct labeling approach starting with the tosylate precursor
(
34) as shown in Scheme 6.
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The tosylate precursor (34) was reacted with Kryptofix 222 (K222), and [ F]KF in acetonitrile for 15 min at 110 °C to
install the fluoroethyl tail. The carboxylic acid head group was unmasked by saponification with sodium hydroxide. The
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resulting crude product was purified by high performance liquid chromatography (HPLC) to give [ F]28c in 25.7 ± 4.6%
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Journal of Medicinal Chemistry
(
(
n = 10, decay corrected to start of synthesis), with >98% chemical and radiochemical purity, and a high specific activity
1.43 ± 0.12 Ci/µmol).
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.4 Biological Studies
.4.1 Biodistribution in normal rats
18
To investigate the tissue distribution of [ F]28c in living subjects, a biodistribution study was performed in adult male
SpragueꢀDawley (SD) rats. As shown in Table 3, the results showed significant uptake and retention in the liver, without
evidence of in vivo defluorination (no increased accumulation in bone was observed from 5 to 120 min postꢀinjection).
Relatively high uptake was observed for the heart, lungs, pancreas, and spleen; all organs known to have high S1P1
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expression. The surprisingly low brain uptake of [ F]28c as shown in Table 3, may potential limit its utility for CNS
applications.
2.4.2 In vitro autoradiography and IHC in LPS-treated mice
We utilized the mouse model of LPSꢀinduced liver injury and inflammation, in which inflammation caused by LPS is
18
known to cause increased liver expression of S1P after ~ 24 h to evaluate [ F]28c in tissue from an animal model of
1
44, 45
18
inflammatory disease.
In vitro autoradiographic data showed increased uptake of [ F]28c in the liver of LPSꢀtreated
18
mice, compared with the sham liver shown in Fig. 3A. The specificity of [ F]28c for S1P was demonstrated by
1
incubation of adjacent mouse liver slices with the tracer in presence of either the native ligand (S1P) or the S1P selective
1
4
6
compound, SEW2871. The upregulation of S1P in the liver at the 24 h time point following LPS treatment was further
1
confirmed by IHC staining shown in Fig. 3B. These blocking studies in conjunction with the IHC results suggest that the
18
binding of [ F]28c in the liver of LPSꢀtreated mice is the result of specific binding to the receptor and is increased when
S1P expression is increased.
1
1
8
15
2
.4.3 MicroPET imaging of LPS-treated mice with [ F]28c and [ O]water
Subsequent microPET imaging in the LPS model of liver inflammation also clearly showed increased accumulation of
1
8
[ F]28c in the liver of LPSꢀtreated mice compared to sham controls (Fig. 4A). Timeꢀactivity curves (TAC) confirmed
higher tracer uptake in the liver of LPS preꢀtreated mice compared with the sham controls (Fig. 4B). Quantification of the
liver region of interest (ROI) in the microPET scan from 100ꢀ120 min shows a 61% increase (P < 0.001, n = 4) (Fig. 4C).
These results were confirmed by euthanasia of the mice post PET for an acute biodistribution study. The liver uptake of
1
8
[
F]28c in the LPS treated mouse at 2 h was 1.95ꢀfold higher than that in the sham mice (12.5 vs. 24.4 % ID/gram, P <
1
8
0
.001, n = 4).To further demonstrate that the increased uptake of [ F]28c in the liver was not attributed to changes in
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blood flow in the LPSꢀinduced model of liver injury, additional microPET studies were performed with an injection of
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47, 48
[
O]water 30 min prior to injection of [ F]28c. [ O]Water is a widely radiotracer for assessing blood flow.
As shown
15
in Fig. 4D, the liver TAC for [ O]water in LPS treated mice showed no significant change compared to that in the sham
1
8
mice. This suggested the increase in vivo uptake of [ F]28c in the liver of LPSꢀtreated mice is the result of increased S1P
1
expression and not increased blood flow.
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99m
2
.4.3 Biodistribution of [ F]28c and [ Tc]Mebrofenin in LPS-treated mice
18
To confirm that the uptake of [ F]28c was associated with S1P expression, we utilized a mouse model of LPSꢀinduced
1
liver injury and inflammation, in which inflammation caused by LPS is known to cause increased liver expression of S1P1
44, 45
18
after ~ 24 h.
Biodistribution studies comparing of [ F]28c uptake in salineꢀtreated sham controls vs. the LPSꢀtreated
mouse model demonstrated a large increase in liver uptake (Fig. 5). In order to demonstrate that tracer retention in the
liver was not due to impaired hepatobiliary clearance in the liver injury model, an additional study was carried out with
99m
this model using a liver imaging agent. [ Tc]Mebrofenin is used to detect if the hepatobiliary transport is impaired.
99m
Increased liver uptake of [ Tc]mebrofenin indicates the potential for nonꢀspecific retention of drugs (in this case,
18
[
F]28c) in the liver due to impaired clearance, rather than uptake specific to the pathological increase in S1P at the
1
36, 49, 50 32, 33
99m
molecular level.
We hypothesized that increased liver uptake of [ Tc]mebrofenin in LPSꢀtreated mice versus
the sham control mice at 60 min would suggest that increased uptake of the S1P tracer resulted from severely damaged
1
99m
liver, however if the liver uptake of [ Tc]mebrofenin in LPSꢀtreated mice versus the sham mice was similar at 60 min
3
6, 49
(
as we observed), the increase should reflect an increase in S1P rather than impaired clearance.
To confirm that this
1
18
increase in uptake of [ F]28c resulted from the increase expression of S1P receptor response to liver injury, the
1
99m
biodistribution study of [ Tc]mebrofenin was performed in the LPSꢀinduced liver injury models. Although increased
9
9m
[
Tc]mebrofenin was observed in the liver of LPSꢀtreated mice at 30 min p.i. compared with sham mice, at 60 min p.i.
1
8
there was no significant difference between the sham and LPS study groups. The uptake of [ F]28c showed a significant
increase in the LPSꢀtreated mice compared to the sham control mice at both 30 min and 60 min (Fig. 5). The
1
8
biodistribution data demonstrated that the increased hepatobiliary uptake of [ F]28c in LPSꢀtreated mice is mainly caused
by the upregulation of S1P expression in the liver.
1
3
. CONCLUSIONS
We synthesized fourteen fluorineꢀcontaining analogues based on two lead pharmacophores and determined their affinity
for S1P . Potent compounds (IC50 < 50 nM for S1P ) were subsequently screened for their S1P and S1P binding
1
1
2
3
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Journal of Medicinal Chemistry
affinities. Seven compounds were found to have IC values < 100 nM while three were very potent with S1P IC values
5
0
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50
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<
10 nM. We explored the SAR studies on the oxadiazole containing pharmacophore by optimizing the lipophilic tail
binding portion of the binding pocket. The oxadiazole 28c had a high potency (2.63 nM for S1P ) and selectivity (>100ꢀ
1
fold for S1P versus S1P ), and was successfully radiolabeled in high yield and high specific activity. Biodistribution in
1
2/3
normal rats showed no evidence of defluorination, so the a mouse model of liver inflammation was used for subsequent
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biological evaluation of [ F]28c as a PET tracer for imaging S1P . In vitro autoradiography and microPET imaging
1
18
studies showed increased increased binding in vitro and in vivo retention of [ F]28c in the liver of LPSꢀtreated vs. control
mice. IHC staining studies confirmed that S1P expression was increased in the liver of LPSꢀtreated mice. Additional
1
1
5
37, 38
microPET imaging showed no difference in liver uptake of [ O]water between LPSꢀtreated and control mice,
further
1
8
supporting the hypothesis that the higher uptake and retention of [ F]28c in the liver of LPSꢀtreated mice was a reflection
of increased S1P due to inflammation and not due to a change in blood flow. Parallel acute biodistribution studies in the
1
18
99m
36
LPS model were carried out with both [ F]28c and the liver imaging agent [ Tc]mebrofenin. The absence of a
99m
significant difference in liver uptake of [ Tc]mebrofenin in LPSꢀtreated vs. control liver uptake 60 min postꢀinjection
18
suggested that the increased liver uptake of [ F]28c in observed in the PET studies of LPSꢀtreated mice was not the result
1
8
of reduced hepatobiliary clearance due to liver injury. Together these data indicate uptake of [ F]28c reflects the
18
expression of S1P and suggest that [ F]28c is a S1P specific radiotracer with potential for use in quantifying S1P
1
1
1
receptor expression in response to inflammation.
4
4
. EXPERIMENTAL SECTION
.1 Chemistry Materials and Methods
Unless otherwise indicated, all reactions were conducted in ovenꢀdried (140 °C) glassware. Stainless steel syringes or
cannulae that had been ovenꢀdried (140 °C) and cooled under a nitrogen atmosphere or in a desiccator were used to
1
transfer airꢀ and moistureꢀsensitive liquids. Yields refer to chromatographically and spectroscopically ( H NMR)
homogeneous materials, unless otherwise stated. Reactions were monitored by thinꢀlayer chromatography (TLC) carried
out on precoated glass plates of silica gel (0.25 mm) 60 F254 from EMD Chemicals Inc. Visualization was accomplished
with ultraviolet light (UV 254 nm), or by shaking the plate in a sealed jar containing silica gel and Iodine. Alternatively,
plates were treated with one of the following solutions (this was accomplished by holding the edge of the TLC plate with
forceps or tweezers and immersing the plate into a wideꢀmouth jar containing the desired staining solution) and carefully
heating with a hotꢀair gun (450 °C) for approximately 1ꢀ2 min (NOTE: excess stain was removed by resting the TLC on a
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paper towel prior to heating): 10% phosphomolybdic acid in ethanol, 1% potassium permanganate/7% potassium
carbonate/0.5% sodium hydroxide aqueous solution, and/or anisaldehyde in ethanol with 10% sulfuric acid. Flash column
chromatography was performed using Silia Flash® P60 silica gel (40ꢀ63 ꢁm) from Silicycle. All workꢀup and purification
procedures were carried out with reagent grade solvents in air.
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1
H NMR spectra were recorded on Varian 400 MHz instrument. Chemical shifts are reported in parts per million (ppm)
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0
and are calibrated using residual undeuterated solvent as an internal reference (CDCl : δ 7.26 ppm; MeODꢀd4: δ 3.31
3
ppm; DMSOꢀd6: δ 2.50 ppm). Data are reported as follows: chemical shift, multiplicity, coupling constants (Hz), and
integration. The following abbreviations or combinations thereof were used to explain the multiplicities: s = singlet, d =
doublet, t = triplet, q = quartet, pent = pentet, sext = sextet, sept = septet, m = multiplet, at = apparent triplet, aq = apparent
1
3
quartet, b = broad. C NMR spectra were recorded on a Varian instrument (100 MHz) spectrometer with complete proton
decoupling. Chemical shifts are reported in ppm and are calibrated using residual undeuterated solvent as an internal
reference (CDCl : δ 77.16 ppm; MeODꢀd4: 49.00 ppm; DMSOꢀd6: 39.52 ppm). Elemental compositions (C, H, N) are
3
within ±0.4% of the calculated values, and were determined by Atlantic Microlab, Inc. Lipophilicity values as cLogD are
reported as the calculated Log P value at pH = 7.4, and were obtained using ACD/IꢀLab ver. 7.0 (Advanced Chemistry
Development, Inc. Toronto, Ontario, Canada). All the final compounds for which the biologic activity was determined by
analytical HPLC method with purity ≥ 95%.
4.1.1 Synthesis
General Procedure A: Suzuki Coupling. To an ovenꢀdried roundꢀbottom flask equipped with a stirbar was added
Pd(OAc) (2 mol %), 2ꢀdicyclohexylphosphinoꢀ2',6'ꢀdimethoxybiphenyl (SPhos, 5 mol %), commercially available 4ꢀ
2
bromoꢀ3ꢀ(trifluoromethyl)benzoic acid (6) (1 equiv), the boronic acid (1.1 equiv), and a fluoride base (3 equiv). 1,4ꢀ
dioxane (0.37 M) and degassed H O (0.37 M) were added to the reaction mixture and the reaction was degassed by
2
splurging with N2(g) for 10 min, at which time it was equipped with a condenser and placed in a preꢀheated 110 °C oilꢀ
bath. The reaction mixture was stirred for the time indicated, cooled to room temperature (r.t.) and poured into a 1:1
mixture of ethyl acetate and 1N HCl (aq). The quenched reaction mixture was stirred for 20 min, and the layers were then
separated. The aqueous layer was extracted with ethyl acetate (×2). The combined organic layers were dried over MgSO4,
concentrated in vacuo and purified as specified.
General Experimental B: Amide Formation. To an ovenꢀdried roundꢀbottom flask equipped with a stirbar was added
the carboxylic acid (1 equiv) followed by CH Cl (0.1 M). Five drops of N,Nꢀdimethylformamide was added via pipette
2
2
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followed by a slow addition of oxalyl chloride (2.3 equiv) via syringe. The reaction was then stirred at r.t. for 2 h, at
which time it was concentrated in vacuo. The crude acid chloride was then dissolved in toluene (0.15 M) and 10%
NaHCO3(aq) (0.3 M) was added. The aniline 9a or commercially available 2ꢀaminoꢀ4ꢀbromophenol (9b) (1 equiv) was
added and the reaction was stirred overnight. Upon completion of the reaction, the resulting precipitate was filtered, and
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washed with H O, toluene, and hexanes to give the desired amide.
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General Experimental C: Benzoxazole Formation. To a roundꢀbottom flask equipped with a stirbar was added the
amide (1 equiv), toluene (0.1 M), and pꢀtoluenesulfonic acid (2.1 equiv) or pyridinium pꢀtoluenesulfonate (2.1 equiv). The
reaction was equipped with a reflux condenser and lowered into a preꢀheated 130 °C oilꢀbath. The reaction mixture was
stirred for the specified time, cooled to r.t., diluted with EtOAc, and washed with: sat. NaHCO3(aq) (×2), and brine. The
organic layer was dried over MgSO and concentrated in vacuo to give the desired benzoxazole.
4
General Procedure D: Buchwald-Hartwig Coupling. To an ovenꢀdried flask (pressureꢀvessel or Schlenk tube)
equipped with a stirbar was added Pd (dba) ·CHCl (2 mol %), XPhos (8 mol %), sodium tertꢀbutoxide (1.4 equiv), and
2
3
3
the aryl bromide (1 equiv). Toluene (0.5 M) was added followed by the amine (1.2 equiv) and the reaction mixture was
degassed by splurging with N2(g) for 10 min. The reaction vessel was sealed with a Teflon screwꢀcap, and lowered into a
preꢀheated 110 °C oilꢀbath. After 18ꢀ20 h, the reaction mixture was allowed to cool to r.t. and diluted with ethyl acetate.
The reaction mixture was then concentrated in vacuo, and purified as specified.
General Procedure E: Saponification. To a 16 × 150 mm test tube equipped with a stirbar was added the ester (1 equiv),
THF (0.24 M), H O (1.2 M), and lithium hydroxide (2 equiv). The reaction mixture was stirred at r.t. for 16ꢀ18 h, at which
2
time it was acidified to pH 1 with 1M HCl(aq). The reaction mixture was extracted with ethyl acetate (×4). The combined
organic layers were washed with brine and concentrated in vacuo to give the desired products.
General Procedure F: Oxadiazole Formation. To an ovenꢀdried roundꢀbottom flask equipped with a stirbar was added
the acid (1 equiv), EDC·HCl (1 equiv), HOBt (1 equiv), and DMF (0.8 M). The reaction mixture was stirred at r.t. for 30
min, at which time the amidoxime (1 equiv) was added. A reflux condenser was equipped and the reaction vessel was
placed in a preꢀheated 120ꢀ140 °C oilꢀbath and stirred overnight (14ꢀ18 h). The reaction mixture was cooled to r.t., diluted
with EtOAc and water. The layers were separated, and the aqueous layer was extracted with EtOAc (×1). The combined
organic layers were washed with water, 1M HCl(aq), water, brine, dried over MgSO , and concentrated in vacuo to give the
4
desired product.
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General Procedure G: Reductive Amination. To a roundꢀbottom flask equipped with a stirbar was added the aldehyde
1 equiv), amine (1.05 equiv), methanol (0.07 M), and acetic acid (2 M). The reaction mixture was stirred for 30 min at
1
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(
which time sodium cyanoborohydride (0.5 equiv) was added as a solution in methanol (0.25 M). The reaction mixture was
stirred for 1ꢀ2 h, at which time the solids were filtered and washed with methanol to give the desired product.
4-(2-Fluoroethoxy)-3-(trifluoromethyl)benzoic acid (7a) To an ovenꢀdried 500 mL roundꢀbottom flask equipped with a
stirbar was added 4ꢀfluoroꢀ3ꢀ(trifluoromethyl)benzoic acid (8)(8.32 g, 40.0 mmol), 100 mL DMSO and 2ꢀfluoroethanol
0
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0
(
3.52 mL, 60.0 mmol). Sodium hydride (3.60 g, 90.0 mmol) was added portionwise, and the reaction mixture was stirred
for 16 h. The reaction mixture was poured into water, and acidified with 12 M HCl(aq) to pH = 1. The resulting precipitate
1
was filtered, washed with water and hexanes to give a tan solid (9.8 g, 97% yield). H NMR (400 MHz, DMSOꢀd6) δ =
1
2.18 (s, 1H), 8.18 (dd, J = 8.8 Hz, 2.0 Hz, 1H), 8.11 (d, J = 2.0 Hz, 1H), 7.39 (d, J = 8.8 Hz, 1H), 4.85 – 4.40 (m, 4H)
C NMR (101 MHz, DMSOꢀd6) δ = 166.0, 159.44, 135.6, 128.0 (q, JCꢀF = 4.9 Hz), 123.2 (q, JCꢀF = 274 Hz), 123.2, 117.4
q, JCꢀF = 31.1 Hz), 113.9, 81.8 (d, JCꢀF = 168 Hz), 68.6 (d, JCꢀF = 19.1 Hz) MP: 131ꢀ133 °C HRMS (EIꢀTOF) m/z calcd
13
(
for C H F O [MꢀH] 251.0337. Found [MꢀH] 251.0289.
1
0
7
4
3
4
'-Fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-carboxylic acid (7b). Following general experimental procedure A:
Pd(OAc) (67 mg, 0.30 mmol), SPhos (306 mg, 0.75 mmol), 6 (4.0 g, 14.9 mmol), 4ꢀfluorophenylboronic acid (2.29 mg,
2
1
6.4 mmol), cesium fluoride (6.79 g, 44.7 mmol), 1,4ꢀdioxane (40 mL), and degassed H O (40 mL) were combined. The
2
1
reaction mixture was stirred for 18 h, to give 3.0 g of a tan solid which was used without further purification. H NMR
(
400 MHz, DMSOꢀd6) δ = 13.56 (br s, 1H), 8.28 (s, 1H), 8.23 (d, J = 8 Hz, 1H), 7.57 (d, J = 8 Hz, 1H), 7.43 – 7.38 (m,
2
H), 7.35 – 7.29 (m, 2H).
2
'-Hydroxy-2-(trifluoromethyl)-[1,1'-biphenyl]-4-carboxylic acid (7c). Following general experimental procedure A:
Pd(OAc) (84 mg, 0.37 mmol), SPhos (382 mg, 0.93 mmol), 6 (5.0 g, 18.6 mmol), 2ꢀhydroxyphenylboronic acid (2.82 g,
2
2
0.4 mmol), cesium fluoride (8.48 g, 55.8 mmol), 1,4ꢀdioxane (50 mL) and degassed H O (50 mL) were combined. The
2
reaction mixture was stirred for 20 h, and purified on a silica gel column, eluting with 5% MeOH/ CH Cl , to provide 5.2
2
2
1
g of a yellow semiꢀsolid. H NMR (400 MHz, DMSOꢀd6) δ = 9.54 (s, 1H), 8.25 (s, 1H), 8.18 (d, J = 8 Hz, 1H), 7.47 (d, J
8 Hz, 1H), 7.23 (td, J = 8.8 Hz, 1.6 Hz, 1H), 7.04 (d, J = 7.2 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 6.85 (t, J = 7.2 Hz, 1H).
'-Hydroxy-2-(trifluoromethyl)-[1,1'-biphenyl]-4-carboxylic acid (7d). Following general experimental procedure A:
Pd(OAc) (67 mg, 0.30 mmol), SPhos (306 mg, 0.75 mmol), 6 (4.0 g, 14.9 mmol), 4ꢀhydroxyphenylboronic acid (2.46
=
4
2
mg, 17.8 mmol), cesium fluoride (6.79 g, 44.7 mmol), 1,4ꢀdioxane (40 mL) and degassed H O (40 mL). The reaction
2
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mixture was stirred for 20 h, and purified on a silica gel column, eluting with 5% MeOH / CH Cl , to provide 4.2 g of a
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yellow semiꢀsolid. H NMR (400 MHz, DMSOꢀd6) δ = 9.73 (s, 1H), 8.25 (s, 1H), 8.18 (d, J = 8 Hz, 1H), 7.52 (d, J = 8
Hz, 1H), 7.16 (d, J = 8.4 Hz, 2H), 6.83(d, J = 8.8 Hz, 2H).
2
,4'-Bis(trifluoromethyl)-[1,1'-biphenyl]-4-carboxylic acid (7e). Following general experimental procedure A:
Pd(OAc) (42 mg, 0.19 mmol), SPhos (191 mg, 0.47 mmol), 6 (2.5 g, 9.3 mmol), 4ꢀ(trifluorofluoromethyl)phenylboronic
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acid (2.12 g, 11.2 mmol), potassium fluoride (4.24 g, 27.9 mmol), 1,4ꢀdioxane (25 mL), and degassed H O (25 mL). The
2
1
reaction mixture was stirred for 22 h, to give 2.35 g of a yellow solid, which was used without further purification. H
NMR (400 MHz, DMSOꢀd6) δ = 12.67 (br s, 1H), 8.31 (d, J = 1.2 Hz, 1H), 8.26 (d, J = 8 Hz, 1H), 7.85 (d, J = 8.4 Hz,
2
H), 7.61 (t, J = 7.6 Hz, 3H).
4
-(6-Fluoropyridin-3-yl)-3-(trifluoromethyl)benzoic acid (7f). Following general experimental procedure A: Pd(OAc)2
(50 mg, 0.22 mmol), SPhos (230 mg, 0.56 mmol), 6 (3.0 g, 11.15 mmol), (6ꢀfluoropyridinꢀ3ꢀyl)boronic acid (1.89 g, 13.4
mmol), cesium fluoride (5.08 g, 33.5 mmol), 30 mL 1,4ꢀdioxane, and 30 mL degassed H O. The reaction mixture was
2
1
stirred for 18h, to give a yellow solid, which was used without further purification (3.0 g, 94% yield). H NMR (400 MHz,
DMOSꢀd6) δ = 13.63 (br s, 1H), 8.31 (s, 1H), 8.30 – 8.24 (m, 2H), 8.09 – 7.98 (m, 1H), 7.66 (d, J = 7.6 Hz), 7.34 (dd, J =
8
.4 Hz, 2.8 Hz, 1H).
3
'-Methoxy-2-(trifluoromethyl)-[1,1'-biphenyl]-4-carboxylic acid (7g). Following general experimental procedure A:
Pd(OAc) (50 mg, 0.22 mmol), SPhos (230 mg, 0.56 mmol), 6 (3.0 g, 11.15 mmol), 3ꢀmethoxyphenylboronic acid (2.04
2
g, 13.4 mmol), cesium fluoride (5.08 g, 33.5 mmol), 30 mL 1,4ꢀdioxane, and 30 mL degassed H O. The reaction mixture
2
1
was stirred for 18 h, to give a yellow solid, which was used without further purification (3.12 g, 95% yield). H NMR (400
MHz, DMSOꢀd6) δ = 13.22 (br s, 1H), 8.28 (d, J = 1.2 Hz, 1H), 8.22 (dd, J = 8.0 Hz, 1.6 Hz, 1H), 7.57 (d, J = 8.0 Hz,
1
H), 7.38 (t, J = 8.4 Hz, 1H), 7.06 – 7.01 (m, 1H), 6.93 – 6.86 (m, 2H), 3.78 (s, 3H).
Methyl 3-amino-4-hydroxybenzoate (9). To a 250 mL roundꢀbottom flask equipped with a stirbar was added methyl 4ꢀ
hydroxyꢀ3ꢀnitrobenzoate (5.0 g, 25.4 mmol), 120 mL EtOH, and 540 mg 10% Pd/C. One balloon of H2(g) was bubbled
through the reaction mixture, and then it was stirred under 1 atm of H2(g) overnight (16 h). At this time the reaction was
filtered through a Celite plug, washing with EtOH, and concentrated in vacuo to give a brown solid (4.17 g, 98% yield).
1
H NMR (400 MHz, CDCl ) δ = 7.45 (d, J = 2.0 Hz, 1H), 7.42 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 3.87
3
(
s, 3H).
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Journal of Medicinal Chemistry
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Methyl 3-(4-(2-fluoroethoxy)-3-(trifluoromethyl)benzamido)-4-hydroxybenzoate (10a). To an ovenꢀdried 100 mL
roundꢀbottom flask equipped with a stirbar was added acid 7a (2.50 g, 9.91 mmol), 50 mL CH Cl and 10 drops of DMF.
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Oxalyl chloride (1.68 mL, 19.8 mmol) was added carefully and the reaction mixture was stirred for 2h at r.t. The reaction
was concentrated in vacuo and dissolved in 33 mL CH Cl . Triethylamine (5.52 mL, 39.6 mmol) was added followed by
2
2
DMAP (120 mg, 0.99 mmol), and aniline 9a (1.66 g, 9.91 mmol). The reaction mixture was stirred overnight and diluted
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with MTBE, at which point a precipitate had formed. The precipitate was filtered, washed with MTBE and hexanes to
1
give a white solid which was used in the next step without further purification. H NMR (400 MHz, DMSOꢀd6) δ = 10.75
(
br s, 1H), 9.83 (s, 1H), 8.27 (app q, J = 2.4 Hz, 2H), 8.23 (d, J = 2.4 Hz, 1H), 7.70 (dd, J = 8.4 Hz, 2.4 Hz, 1H), 7.43 (d, J
9.2 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 4.87 – 4.68 (m, 2H), 4.57 – 4.42 (m, 2H).
N-(5-Bromo-2-hydroxyphenyl)-4'-fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-carboxamide
general experimental procedure B: Carboxylic acid 7b (720 mg, 2.53 mmol), 25.3 mL CH Cl , oxalyl chloride (0.49 mL,
=
(10b).
Following
2
2
5
.82 mmol), 17 mL toluene, 8.5 mL of 10% NaHCO3(aq), 9b (476 mg, 2.53 mmol) were combined to give amide 10b as a
1
tan solid (1.11 g, 99% yield). H NMR (400 MHz, DMSOꢀd6) δ = 8.30 (s, 1H), 8.19 (d, J = 8 Hz, 1H), 7.99 (s, 1H), 7.51
(
d, J = 8 Hz, 1H), 7.35 – 7.25 (m, 4H), 7.08 (d, J = 8 Hz, 1H), 6.76 (d, J = 8 Hz, 1H).
N-(5-Bromo-2-hydroxyphenyl)-2'-hydroxy-2-(trifluoromethyl)-[1,1'-biphenyl]-4-carboxamide (10c). Following
general experimental procedure B: Carboxylic acid 7c (3.5 g, 12.4 mmol), 124 mL CH Cl , oxalyl chloride (2.41 mL, 28.5
2
2
mmol) , 82 mL toluene, 41 mL of 10% NaHCO3(aq), and 9b (2.33 g, 12.4 mmol) were combined to give amide 10c as a
1
brown solid (1.15 g, 20% yield). H NMR (400 MHz, DMSOꢀd6) δ = 8.32 (s, 1H), 8.19 (d, J = 8.0 Hz, 1H), 7.87 (d, J =
2
.4 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.28 – 7.17 (m, 2H), 7.05 (d, J = 6.8 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 6.89 – 6.82
m, 2H).
N-(5-Bromo-2-hydroxyphenyl)-4'-hydroxy-2-(trifluoromethyl)-[1,1'-biphenyl]-4-carboxamide (10d). Following
general experimental procedure B: Carboxylic acid 7d (4.2 g, 14.9 mmol), 149 mL CH Cl , oxalyl chloride (2.90 mL,
(
2
2
34.3 mmol), 100 mL toluene, 50 mL of 10% NaHCO3(aq), and 9b (2.80 g, 14.9 mmol) were combined to give amide 10d
as a brown solid which was used in the next step without further purification (3.4 g, 50% yield).
N-(5-Bromo-2-hydroxyphenyl)-2,4'-bis(trifluoromethyl)-[1,1'-biphenyl]-4-carboxamide (10e). Following general
experimental procedure B: Acid 7e (2.27 g, 6.79 mmol), 68 mL CH Cl oxalyl chloride (1.21 mL, 14.3 mmol), 46 mL
2
2,
toluene, 23 mL 10% NaHCO3(aq), and 9b (1.28 g, 6.79 mmol) were combined to give a light brown solid (2.81 g, 82%
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Journal of Medicinal Chemistry
1
yield). H NMR (400 MHz, DMSOꢀd6) δ = 10.09 (br s, 1H), 8.42 (s, 1H), 8.29 (d, J = 8.0 Hz, 1H), 7.92 – 7.84 (m, 3H),
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7
.65 – 7.56 (m, 3H), 7.23 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 6.89 (d, J = 8.8 Hz, 1H).
N-(5-Bromo-2-hydroxyphenyl)-4-(6-fluoropyridin-3-yl)-3-(trifluoromethyl)benzamide (10f). Following general
experimental procedure B: Acid 7f (1.00 g, 3.51 mmol), 35 mL CH Cl , oxalyl chloride (0.68 mL, 8.1 mmol), 24 mL
2
2
1
toluene, 12 mL 10% NaHCO3(aq), and 9b (660 mg, 3.51 mmol) were combined to give a tan solid (1.0 g, 63% yield). H
NMR (400 MHz, DMSOꢀd6) δ = 10.32 (br s, 1H), 8.40 (s, 1H), 8.35 – 8.18 (m, 2H), 8.03 (t, J = 8.4 Hz, 1H), 7.89 (d, J =
0
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.6 Hz, 1H), 7.64 (d, J = 7.6 Hz, 1H), 7.35 (d, J = 6.4 Hz, 1H), 7.19 (d, J = 8.4 Hz, 1H), 6.87 (d, J = 8.8 Hz, 1H).
Methyl 3-(4-(6-fluoropyridin-3-yl)-3-(trifluoromethyl)benzamido)-4-hydroxybenzoate (10g). To an ovenꢀdried 100
mL roundꢀbottom flask equipped with a stirbar was added acid 7f (1.50 g, 5.26 mmol), 53 mL CH Cl and ten drops of
2
2
DMF. Oxalyl chloride (0.89 mL, 10.5 mmol) was added carefully, and the reaction was stirred for 2h, where upon it was
concentrated in vacuo to give a yellow semiꢀsolid. The crude acid chloride was suspended in 26 mL CH Cl and
2
2
triethylamine (2.93 mL, 21.0 mmol) was added, followed by DMAP (64 mg, 0.53 mmol). The reaction was stirred for 5
min at which time aniline 9a (880 mg, 5.26 mmol) was added. The reaction mixture was stirred overnight (18 h),
quenched with 1M HCl(aq) and diluted with CH Cl , causing a suspension to form in the organic layer. The layers were
2
2
separated, and the aqueous layer was extracted with CH Cl (×3). The combined organic layers were diluted with hexanes
2
2
1
and filtered to give a brown solid (1.02 g, 45% yield). H NMR (400 MHz, DMSOꢀd6) δ = 10.78 (br s, 1H), 10.05 (br s,
1H), 8.45 (s, 1H), 8.33 (d, J = 7.6 Hz, 1H), 8.29 (s, 2H), 8.05 (t, J = 6.4 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.68 (d, J = 8.0
Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H), 3.82 (s, 3H).
N-(5-Bromo-2-hydroxyphenyl)-3'-methoxy-2-(trifluoromethyl)-[1,1'-biphenyl]-4-carboxamide (10h). Following
general experimental procedure B: Acid 7g (3.1 g, 10.5 mmol), 105 mL CH Cl , oxalyl chloride (2.04 mL, 24.2 mmol), 70
2
2
mL toluene, 35 mL 10% NaHCO3(aq), and 9b (1.97 g, 10.5 mmol) were combined to give a brown solid which was used in
the next step without further purification (3.96 g, 81% yield).
Methyl 2-(4-(2-fluoroethoxy)-3-(trifluoromethyl)phenyl)benzo[d]oxazole-5-carboxylate (11a). Following general
experimental procedure C: Amide 10a (crude from previous step), 99 mL toluene and pꢀtoluenesulfonic acid (3.77 g, 19.8
1
mmol) were combined to give a tan solid (1.1 g, 29% yield over two steps). H NMR (400 MHz, CDCl ) δ = 8.56 – 8.49
3
(
m, 1H), 8.45 (d, J = 1.6 Hz, 1H), 8.41 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 8.12 (dd, J = 8.4 Hz, 1.6 Hz, 1H), 7.62 (d, J = 8.4 Hz,
1
H), 7.17 (d, J = 8.4 Hz, 1H), 4.93 – 4.75 (m, 2H), 4.48 – 4.37 (m, 2H), 3.97 (s, 3H).
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5
-Bromo-2-(4'-fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)benzo[d]oxazole (11b). Following general experimental
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procedure C: Amide 10b (980 mg, 2.17 mmol), 21.7 mL toluene, and pꢀtoluenesulfonic acid (867 mg, 4.56 mmol) were
1
combined to give oxazole 11b as a red solid (760 mg, 80% yield). H NMR (400 MHz, DMSOꢀd6) δ = 8.52 (s, 1H), 8.48
(
d, J = 8 Hz, 1H), 8.13 (d, J = 2 Hz, 1H), 7. 86 (d, J = 8.8 Hz, 1H), 7.69 (d, J = 8 Hz, 1H), 7.66 (dd, J = 8.8 Hz, 2 Hz, 1H),
7.50 – 7.41 (m, 2H), 7.35 (t, J = 8.8 Hz, 2H).
-Bromo-2-(2'-(2-fluoroethoxy)-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)benzo[d]oxazole (11c). Following general
0
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experimental procedure C: Amide 10c (1.06 g, 2.34 mmol), 23.4 mL toluene, and pꢀtoluenesulfonic acid (0.94 g, 4.92
mmol) were combined to give the crude product which was applied to a silica gel column, eluted with 20% EtOAc/
1
hexanes, to provide the phenol intermediate as a red solid (820 mg, 80% yield). H NMR (400 MHz, DMSOꢀd6) δ = 9.64
(
s, 1H), 8.49 (s, 1H), 8.43 (d, J = 8.0 Hz, 1H), 8.13 (d, J = 1.6 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.65 (dd, J = 8.8 Hz, 2.0
Hz, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.26 (t, J = 7.6 1H), 7.09 (d, J = 7.2 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.87 (t, J = 7.2
Hz, 1H).
To an ovenꢀdried 16 × 125 mm test tube equipped with a stirbar was added the phenol prepared above (290 mg, 0.67
mmol), cesium carbonate (437 mg, 1.34 mmol) and 2.2 mL N,Nꢀdimethylformamide. 2ꢀFluoroethyl 4ꢀ
methylbenzenesulfonate (160 mg, 0.73 mmol) was added via syringe and the reaction vessel was placed in a preꢀheated 70
°C oilꢀbath and stirred for 4 h. The reaction mixture was cooled to r.t., diluted with EtOAc and H O, and the layers were
2
separated. The organic layer was washed with H O (×5) and brine, dried over sodium sulfate, and concentrated in vacuo to
2
1
give a light brown solid, which was used without further purification (310 mg, 97% yield). H NMR (400 MHz, CDCl ) δ
3
=
8.58 (s, 1H), 8.34 (d, J = 8.4 Hz, 1H), 7.89 (s, 1H), 7.53 – 7.43 (m, 3H), 7.38 (t, J = 7.6 Hz, 1H), 7.20 (d, J = 7.2 Hz,
1
H), 7.04 (t, J = 7.6 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 4.52 (dt, J = 47.2 Hz, 4 Hz, 2H), 4.31 – 4.03 (m, 2H).
5
-Bromo-2-(4'-(2-fluoroethoxy)-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)benzo[d]oxazole (11d). Following general
experimental procedure C: Amide 10d (3.4 g, 7.52 mmol), 75 mL toluene, and pꢀtoluenesulfonic acid (3.0 g, 15.8 mmol)
were combined to give the crude product, which was applied to a silica gel column, eluted with 20% EtOAc/ hexanes, to
1
provide the phenol intermediate as a red solid (1.21 g, 37% yield). H NMR (400 MHz, DMSOꢀd6) δ = 9.77 (s, 1H), 8.46
(
s, 1H), 8.41 (d, J = 8 Hz, 1H), 8.10 (d, J = 1.6 Hz, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.63 (d, J = 8 Hz, 1H), 7.66 (dd, J = 8.8
Hz, 2 Hz, 1H), 7.20 (d, J = 8.4 2H), 7.86 (d, J = 8.4 Hz, 2H).
To an ovenꢀdried 16 × 125 mm test tube equipped with a stirbar was added the phenol prepared above (217 mg, 0.50
mmol), cesium carbonate (326 mg, 1.0 mmol) and 1.7 mL N,Nꢀdimethylformamide. 2ꢀFluoroethyl 4ꢀ
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Journal of Medicinal Chemistry
methylbenzenesulfonate (120 mg, 0.55 mmol) was added via syringe and the reaction vessel was placed in a preꢀheated 70
C oilꢀbath and stirred for 4 h. The reaction mixture was cooled to r.t., diluted with EtOAc and H O, and the layers were
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°
2
separated. The organic layer was washed with H O (×5) and brine, dried over sodium sulfate, and concentrated in vacuo to
2
1
give 232 mg of a light brown solid, which was used without further purification. H NMR (400 MHz, DMSOꢀd6) δ = 8.51
(s, 1H), 8.46 (d, J = 8 Hz, 1H), 8.13 (d, J = 2 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.70 – 7.62 (m, 2H), 7.34 (d, J = 8.4 Hz,
0
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8
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2
H), 7.09 (d, J = 8.8 Hz, 2H), 4.87 – 4.70 (m, 2H), 4.37 – 4.25 (m, 2H).
2
-(2,4'-Bis(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-5-bromobenzo[d]oxazole (11e). Following general experimental
procedure C: Amide 10e (2.70 g, 5.35 mmol), 54 mL toluene, and pꢀtoluenesulfonic acid monohydrate (2.14 g, 11.2
1
mmol) were combined to give a light red solid (2.39 g, 92 % yield). H NMR (400 MHz, DMSOꢀd6) δ = 8.55 (s, 1H),
8
.52 (d, J = 8.0 Hz, 1H), 8.15 (d, J = 2.0 Hz, 1H), 7.92 – 7.85 (m, 3H), 7.75 (d, J = 8.4 Hz, 1H), 7.69 – 7.61 (m, 3H).
5-Bromo-2-(4-(6-fluoropyridin-3-yl)-3-(trifluoromethyl)phenyl)benzo[d]oxazole (11f). To an ovenꢀdried 25 mL
roundꢀbottom flask equipped with a stirbar was added amide 10f (935 mg, 2.05 mmol) and 8.2 mL THF.
Triphenylphosphine (1.19 g, 4.52 mmol) was added followed by diisopropylazodicarboxylate (0.89 mL, 4.52 mmol). The
reaction mixture was stirred at r.t. for 22 h, and concentrated in vacuo. The crude product was diluted with diethyl ether,
and filtered through a Celite plug, washing with ether. The ether mixture was concentrated in vacuo and applied to a silica
1
gel column, eluting with 15% EtOAc/ hexanes to give a pink solid (500 mg, 56% yield). H NMR (400 MHz, CDCl ) δ =
3
8.66 (d, J = 1.2 Hz, 1H), 8.46 (dd, J = 8.0 Hz, 1.6 Hz, 1H), 8.24 (d, J = 2.0 Hz, 1H), 7.82 (td, J = 8.0 Hz, 2.4 Hz, 1H),
7
.55 – 7.47 (m, 3H), 7.04 (dd, J = 8.4 Hz, 2.8 Hz, 1H).
Methyl 2-(4-(6-fluoropyridin-3-yl)-3-(trifluoromethyl)phenyl)benzo[d]oxazole-5-carboxylate (11g). Following
general experimental procedure C: Amide 10g (500 mg, 1.15 mmol), 11.5 mL toluene, and pyridinium pꢀtoluenesulfonate
1
(
(
607 mg, 2.42 mmol) were combined to give a yellow solid (415 mg, 87% yield). H NMR (400 MHz, CDCl ) δ = 8.64
3
d, J = 1.2 Hz, 1H), 8.48 – 8.42 (m, 2H), 8.22 (d, J = 2.0 Hz, 1H), 8.13 (dd, J = 8.4 Hz, 1.6 Hz, 1H), 7.81 (td, J = 8.0 Hz,
2.4 Hz, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.03 (dd, J = 8.4 Hz, 2.8 Hz, 1H), 3.96 (s, 3H).
-Bromo-2-(3'-methoxy-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)benzo[d]oxazole (11h). Following
5
general
experimental procedure C: Amide 10h (3.96 g, 8.49 mmol), 85 mL toluene, and pꢀtoluenesulfonic acid (3.39 g, 17.8
1
mmol) were combined to give a red solid (3.24 g, 85% yield). H NMR (400 MHz, CDCl ) δ = 8.61 (d, J = 1.6 Hz, 1H),
3
8
.39 (dd, J = 8.0 Hz, 1.6 Hz, 1H), 7.96 – 7.93 (m, 1H), 7.57 – 7.49 (m, 3H), 7.36 (t, J = 7.6 Hz, 1H), 7.02 – 6.90 (m, 3H),
3
.85 (s, 3H).
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Journal of Medicinal Chemistry
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(
2-(4-(6-Fluoropyridin-3-yl)-3-(trifluoromethyl)phenyl)benzo[d]oxazol-5-yl)methanol (12a). To a 25 mL roundꢀ
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bottom flash equipped with a stirbar was added ester 11g (415 mg, 0.99 mmol) and 5 mL THF. The reaction was cooled in
a ꢀ 78°C cooling bath (CO2(s) / acetone) and DIBAlꢀH (2.99 mL, 2.99 mmol, 1.0 M in hexanes) was added slowly. The
reaction was allowed to warm in the bath to ꢀ 15°C for 1 h, at which time TLC indicated consumption of the starting ester.
The reaction was quenched with 2 mL EtOAc and 5 mL saturated Rochelle’s salt solution and stirred 30 min. The reaction
mixture was diluted with EtOAc, and the layers separated. The aqueous layer was extracted with EtOAc (×2). The
combined organic layers were washed with H O, brine, dried over MgSO , and concentrated in vacuo to give a white solid
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(
350 mg, 91% yield). H NMR (400 MHz, CDCl ) δ = 8.69 (s, 1H), 8.48 (dd, J = 8.0 Hz, 1.6 Hz, 1H), 8.24 (d, J = 2.0 Hz,
3
1
H), 7.86 – 7.78 (m, 2H), 7.62 (d, J = 8.4 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.45 (dd, J = 8.4 Hz, 1.6 Hz, 1H), 7.04 (dd, J
8.4 Hz, 3.2 Hz, 1H), 4.86 (s, 2H).
=
(2-(4-(2-Fluoroethoxy)-3-(trifluoromethyl)phenyl)benzo[d]oxazol-5-yl)methanol (12b). To an ovenꢀdried 50 mL
roundꢀbottom flask equipped with a stirbar was added ester 11a (844 mg, 2.2 mmol) and 11 mL 2ꢀmethyltetrahydrofuran.
The reaction mixture was cooled to ꢀ78 °C (CO2(s)/acetone bath) and DIBAlꢀH (1.0 M solution in hexanes, 6.6 mL, 6.6
mmol) was added carefully. The reaction mixture was allowed to warm to ꢀ5 °C in the bath over 1 h, at which time TLC
showed consumption of the ester. The reaction was quenched with EtOAc and sat. Rochelle’s salt solution(aq), and stirred
for 30 min when a precipitate had formed. The reaction mixture was filtered through a Celite plug, washing with EtOAc
and water. The layers were separated and the aqueous layer was extracted with EtOAc (×1). The combined organic layers
were washed with water, brine, dried over MgSO , and concentrated in vacuo to give a white solid (743 mg, 95% yield).
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H NMR (400 MHz, CDCl ) δ = 8.48 (d, J = 2.0 Hz, 1H), 8.37 (dd, J = 8.8 Hz, 2.0 Hz, 1H), 7.74 (s, 1H), 7.55 (d, J = 8.4
3
Hz, 1H), 7.40 – 7.35 (m, 1H), 7.14 (d, J = 8.8 Hz, 1H), 4.90 – 4.74 (m, 2H), 4.82 (s, 2H), 4.47 – 4.35 (m, 2H).
5
-(Chloromethyl)-2-(4-(6-fluoropyridin-3-yl)-3-(trifluoromethyl)phenyl)benzo[d]oxazole (13a). To an ovenꢀdried 10
mL roundꢀbottom flask equipped with a stirbar was added 0.11 mL DMF, followed by cyanuric chloride (194 mg, 1.05
mmol). The reaction was stirred for 30 min, at which time TLC analysis showed consumption of the cyanuric chloride. 2.5
mL CH Cl was added, followed by alcohol 12a (388 mg, 1.00 mmol). The reaction was stirred for 1 h, at which time
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TLC analysis showed consumption of the alcohol. The reaction was quenched with H O and diluted with CH Cl . The
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layers were separated, and the aqueous layer was extracted with CH Cl (× 1). The combined organic layers were washed
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with saturated sodium carbonate solution, 1M HCl(aq), brine, dried over MgSO and concentrated in vacuo to give 398 mg
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of an offꢀwhite solid. H NMR (400 MHz, CDCl ) δ = 8.68 (s, 1H), 8.48 (d, J = 8.0 Hz, 1H), 8.24 (d, J = 2.0 Hz, 1H), 7.86
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Page 19 of 58
Journal of Medicinal Chemistry
–
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7.77 (m, 2H), 7.63 (d, J = 8.4 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.47 (dd, J = 8.4 Hz, 1.6 Hz, 1H), 7.04 (dd, J = 8.4 Hz,
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.4 Hz, 1H), 4.75 (s, 2H).
-(Chloromethyl)-2-(4-(2-fluoroethoxy)-3-(trifluoromethyl)phenyl)benzo[d]oxazole (13b). To an ovenꢀdried 50 mL
roundꢀbottom flask equipped with a stirbar was added 0.23 mL DMF and cyanuric chloride (218 mg, 1.18 mmol). The
reaction mixture was stirred for 1 h at which time 3 mL CH Cl and alcohol 12b (400 mg, 1.13 mmol) were added. The
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reaction mixture was stirred for 18 h, diluted with CH Cl and water. The layers were separated and the aqueous layer was
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extracted with CH Cl (×2). The combined organic layers were washed with sat. Na CO3(aq), 1M HCl (aq), brine; dried over
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MgSO , and concentrated in vacuo to give a tan solid (250 mg, 59% yield). H NMR (400 MHz, CDCl ) δ = 8.47 (s, 1H),
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.38 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 7.76 (s, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.40 (dd, J = 8.4 Hz, 1.6 Hz, 1H), 7.15 (d, J = 8.8
Hz, 1H), 4.92 – 4.74 (m, 2H), 4.73 (s, 2H), 4.48 – 4.36 (m, 2H).
Methyl 1-((2-(4-(6-fluoropyridin-3-yl)-3-(trifluoromethyl)phenyl)benzo[d]oxazol-5-yl)methyl)azetidine-3-carboxy-
late (14a). To a 10 mL roundꢀbottom flask equipped with a stirbar was added chloride 13a (260 mg, 0.64 mmol), methyl
azetidineꢀ3ꢀcarboxylate hydrochloride (146 mg, 0.96 mmol), 2.1 mL acetonitrile and potassium carbonate (265 mg, 1.92
mmol). The reaction was stirred 3h, at which time no progress was observed by TLC. iPr NEt (0.33 mL, 1.92 mmol) was
2
added and the reaction stirred 20 h at r.t. The reaction was diluted with water and EtOAc, and the layers separated. The
aqueous layer was extracted with EtOAc (× 1). The combined organic layers were washed with water (×2), brine, dried
over MgSO and concentrated in vacuo to give a yellow semiꢀsolid. The crude product was then purified on a silica gel
4
1
column, eluting with EtOAc to give a white solid (110 mg, 35% yield). H NMR (400 MHz, CDCl ) δ = 8.63 (d, J = 1.2
3
Hz, 1H), 8.43 (dd, J = 8 Hz, 1.6 Hz, 1H), 8.21 (d, J = 2.0 Hz, 1H), 7.80 (td, J = 8.4 Hz, 2.4 Hz, 1H), 7.69 (d, J = 0.8 Hz,
1
H), 7.54 (d, J = 8.4 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.33 (dd, J = 8.4 Hz, 1.6 Hz, 1H), 7.01 (dd, J = 8.4 Hz, 3.2 Hz,
1
H), 3.73 (s, 2H), 3.70 (s, 3H), 3.57 – 3.52 (m, 2H), 3.40 – 3.30 (m, 3H).
Methyl
1-((2-(4-(2-fluoroethoxy)-3-(trifluoromethyl)phenyl)benzo[d]oxazol-5-yl)methyl)azetidine-3-carboxylate
(14b). To a 10 mL roundꢀbottom flask equipped with a stirbar was added chloride 13b (187 mg, 0.5 mmol), methyl
azetidineꢀ3ꢀcarboxylate hydrochloride (114 mg, 0.75 mmol), 1.7 mL MeCN, and DIPEA (0.26 mL, 1.5 mmol). The
reaction was placed in a preꢀheated 60 °C oilꢀbath and stirred overnight (~18 h). The reaction mixture was allowed to cool
to r.t., diluted with water and EtOAc. The layers were separated, aqueous layer extracted with EtOAc (× 2). The combined
organic layers were washed with water (× 3), brine, dried over MgSO , and concentrated in vacuo to give a tan solid (150
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mg, 66% yield). H NMR (400 MHz, CDCl ) δ = 8.41 (s, 1H), 8.29 (d, J = 8.8 Hz, 1H), 7.60 (s, 1H), 7.44 (d, J = 8.4 Hz,
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Journal of Medicinal Chemistry
Page 20 of 58
1
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H), 7.23 (d, J = 8.0 Hz, 1H), 7.07 (d, J = 8.8 Hz, 1H), 4.85 – 4.67 (m, 2H), 4.40 – 4.28 (m, 2H), 3.75 – 3.65 (m, 4H),
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.55 – 3.45 (m, 2H), 3.36 – 3.25 (m, 2H).
-((2-(4-(6-Fluoropyridin-3-yl)-3-(trifluoromethyl)phenyl)benzo[d]oxazol-5-yl)methyl)azetidine-3-carboxylic acid
(
15a). To a 13 × 100 mm testꢀtube equipped with a stirbar was added ester 14a (70 mg, 0.14 mmol), 1.1 mL THF, and
0.21 2M LiOH(aq). The reaction was stirred overnight at r.t., and quenched with 1M HCl(aq) to give a white precipitate. The
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precipitate was removed by filtration and dried under reduced pressure to provide 30 mg of the product. H NMR (400
MHz, MeODꢀd4) δ = 8.69 (s, 1H), 8.57 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 8.23 (d, J = 2.0 Hz, 1H), 8.07 – 7.97 (m, 2H), 7.86
(
d, J = 8.4 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.22 (dd, J = 8.4 Hz, 2.8 Hz, 1H), 4.63 (d, J = 22.4
Hz, 2H), 4.48 (t, J = 10.4 Hz, 1H), 4.39 – 4.32 (m, 3H), 3.83 – 3.71 (m, 1H).
-((2-(4-(2-Fluoroethoxy)-3-(trifluoromethyl)phenyl)benzo[d]oxazol-5-yl)methyl)azetidine-3-carboxylic acid (15b).
1
To a 10 ml roundꢀbottom flask equipped with a stirbar and containing ester 14b (120 mg, 0.27 mmol) was added 2.1 ml
THF and 0.4 ml 2M LiOH(aq). The reaction mixture was stirred overnight (18 h) and quenched with 1M HCl(aq). The
reaction mixture was neutralized with sat. NaHCO3(aq) which caused the formation of a precipitate. This precipitate was
1
filtered and washed with Et O to give a white solid (41 mg, 35% yield). H NMR (400 MHz, MeODꢀd4) δ = 8.50 – 8.43
2
(
m, 2H), 7.90 (s, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.45 (d, J = 9.6 Hz, 1H), 4.86 – 4.71 (m, 2H),
.56 (d, J = 8.0 Hz, 2H), 4.54 – 4.25 (m, 6H), 3.72 (pent, J = 9.2 Hz, 1H).
4
5-(2-(4-(6-Fluoropyridin-3-yl)-3-(trifluoromethyl)phenyl)benzo[d]oxazol-5-yl)thiophene-2-carboxylic acid (16). To
an ovenꢀdried 25 mL Schlenk tube equipped with a stirbar was added palladium acetate (2.2 mg, 0.01 mmol), bromide 11f
(
219 mg, 0.5 mmol), 2ꢀcarboxythiopheneꢀ5ꢀboronic acid (95 mg, 0.55 mmol), cesium fluoride (228 mg, 1.5 mmol), 1.35
mL 1,4ꢀdioxane, and 1.35 mL degassed H O. The reaction vessel was equipped with a coldꢀfinger, placed in a preꢀheated
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10 °C oilꢀbath, and heated overnight (~18 h). The reaction was cooled to r.t. and poured into a 1:1 mixture of EtOAc/ 1M
HCl(aq). The layers were separated, and the aqueous layer was extracted with EtOAc (×3). The combined organic layers
1
were washed with brine, dried over Na SO , and concentrated in vacuo to give a brown solid (223 mg, 92% yield). H
2
4
NMR (400 MHz, DMSOꢀd6) δ = 13.19 (br s, 1H), 8.57 (s, 1H), 8.54 (d, J = 8.0 Hz, 1H), 8.31 (d, J = 2.4 Hz, 1H), 8.26 (d,
J = 2.0 Hz, 1H), 8.09 (td, J = 8.0 Hz, 2.4 Hz, 1H), 7.95 (d, J = 8.8 Hz, 1H), 7.86 (dd, J = 8.8 Hz, 2.0 Hz, 1H), 7.79 (d, J =
8
.0 Hz, 1H), 7.75 (d, J = 3.6 Hz, 1H), 7.68 (d, J = 4.0 Hz, 1H), 7.37 (dd, J = 8.8 Hz, 2.8 Hz, 1H).
Methyl 1-(2-(4'-fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)benzo[d]oxazol-5-yl)piperidine-4-carboxylate (17a).
Following general experimental procedure D: Pd (dba) ·CHCl (10 mg, 0.01 mmol), XPhos (19 mg, 0.040 mmol), sodium
2
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Journal of Medicinal Chemistry
tertꢀbutoxide (67 mg, 0.70 mmol), oxazole 11b (218 mg, 0.5 mmol), toluene (1 mL), methyl piperidineꢀ4ꢀcarboxylate (81
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µL, 0.60 mmol) were combined to give the crude product which was applied to a silica gel column, eluting with 5ꢀ15%
1
ethyl acetate in hexanes to give ester 17a as a yellow solid (165 mg, 66% yield). H NMR (400 MHz, CDCl ) δ = 8.59 (s,
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1
H), 8.36 (d, J = 8.0 Hz, 1H), 7.47 (d, J = 6.0 Hz, 1H), 7.44 (d, J = 4.4 Hz, 1H), 7.37 – 7.27 (m, 3H), 7.11 (t, J = 8.4 Hz,
2H), 7.07 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 3.71 (s, 3H), 3.61 (dt, J = 8.8 Hz, 3.2 Hz, 2H), 2.82 (td, J = 12 Hz, 2.0 Hz, 2H),
.47 (tt, J = 11.2 Hz, 4 Hz, 1H), 2.13 – 2.02 (m, 2H), 2.01 – 1.87 (m, 2H).
Methyl 1-(2-(2'-(2-fluoroethoxy)-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)benzo[d]oxazol-5-yl)piperidine-4-
carboxylate (17b). Following general experimental procedure D: Pd (dba) ·CHCl (6.2 mg, 0.006 mmol), XPhos (11.4
0
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mg, 0.024 mmol), sodium tertꢀbutoxide (40 mg, 0.42 mmol), oxazole 11c (144 mg, 0.30 mmol), toluene (0.6 mL), and
methyl piperidineꢀ4ꢀcarboxylate (48 µL, 0.36 mmol) were combined to give the crude product which was applied to a
1
silica gel column, eluting with 20% ethyl acetate in hexanes to give ester 17b as a yellow solid (90 mg, 56% yield). H
NMR (400 MHz, CDCl ) δ = 8.60 (s, 1H), 8.37 (d, J = 8.0 Hz, 1H), 7.47 (dd, J = 9.2 Hz, 4.0 Hz, 2H), 7.39 (t, J = 7.6 Hz,
3
1H), 7.32 (br s, 1H), 7.21 (d, J = 7.2 Hz, 1H), 7.13 – 7.01 (m, 2H), 6.97 (d, J = 8.4 Hz, 1H), 4.54 (dt, J = 47.6 Hz, 4.0 Hz,
2H), 4.29 – 4.04 (m, 2H), 3.72 (s, 3H), 3.62 (d, J = 12.4 Hz, 2H), 2.84 (t, J = 11.2 Hz, 2H), 2.53 – 2.44 (m, 1H), 2.14 –
2.04 (m, 2H), 2.03 – 1.85 (m, 2H).
Methyl 1-(2-(4'-(2-fluoroethoxy)-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)benzo[d]oxazol-5-yl)piperidine-4-carboxy-
late (17c). Following general experimental procedure D: Pd (dba) ·CHCl (7.2 mg, 0.007 mmol), XPhos (13 mg, 0.028
2
3
3
mmol), sodium tertꢀbutoxide (47 mg, 0.49 mmol), oxazole 11d (168 mg, 0.35 mmol), toluene (0.7 mL), and methyl
piperidineꢀ4ꢀcarboxylate (57 µL, 0.42 mmol) were combined to give the crude product, applied to a silica gel column,
1
eluting with 20% ethyl acetate in hexanes to give ester 17c as a yellow solid (95 mg, 50% yield). H NMR (400 MHz,
CDCl ) δ = 8.59 (s, 1H), 8.36 (d, J = 8.0 Hz, 1H), 7.47 (d, J = 6.0 Hz, 1H), 7.44 (d, J = 4.4 Hz, 1H), 7.37 – 7.27 (m, 3H),
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.11 (t, J = 8.4 Hz, 2H), 7.07 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 3.71 (s, 3H), 3.61 (dt, J = 8.8 Hz, 3.2 Hz, 2H), 2.82 (td, J = 12
Hz, 2.0 Hz, 2H), 2.47 (tt, J = 11.2 Hz, 4 Hz, 1H), 2.13 – 2.02 (m, 2H), 2.01 – 1.87 (m, 2H).
Methyl 1-(2-(2,4'-bis(trifluoromethyl)-[1,1'-biphenyl]-4-yl)benzo[d]oxazol-5-yl)piperidine-4-carboxylate (17d).
Following general experimental procedure D: Pd (dba) ·CHCl (21 mg, 0.02 mmol), XPhos (38 mg, 0.08 mmol), sodium
2
3
3
tertꢀbutoxide (135 mg, 1.4 mmol), bromide 11e (486 mg, 1.0 mmol), and 2 mL toluene were combined to provide the
crude product. This was applied to a silica gel column, eluting with 20% EtOAc/ hexanes to provide a yellow powder
1
(
265 mg, 48% yield). H NMR (400 MHz, CDCl ) δ = 8.63 (s, 1H), 8.41 (d, J = 8.0 Hz, 1H), 7.70 (d, J = 8.0 Hz, 2H),
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7
.52 – 7.45 (m, 4H), 7.31 (d, J = 2.4 Hz, 1H), 7.10 (dd, J = 8.8 Hz, 2.0 Hz, 1H), 3.73 (s, 3H), 3.63 (dt, J = 12.4 Hz, 3.2
Hz, 2H), 2.84 (td, J = 11.6 Hz, 2.4 Hz, 2H), 2.47 (tt, J = 10.8 Hz, 4.0 Hz, 1H), 2.13 – 2.04 (m, 2H), 2.02 – 1.87 (m, 2H).
Methyl 1-(2-(3'-methoxy-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)benzo[d]oxazol-5-yl)piperidine-4-carboxylate
17e). Following general experimental procedure D: Pd (dba) ·CHCl (21 mg, 0.02 mmol), XPhos (38 mg, 0.08 mmol),
1
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(
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sodium tertꢀbutoxide (135 mg, 1.4 mmol), bromide 11h (447 mg, 1.0 mmol), and 2 mL toluene were combined to provide
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the crude product. This was applied to a silica gel column, eluting with 20% EtOAc / hexanes to provide a yellow powder
1
(
93 mg, 18% yield). H NMR (400 MHz, CDCl ) δ = 8.60 (s, 1H), 8.36 (d, J = 8.0 Hz, 1H), 7.49 (t, J = 8.0 Hz, 2H), 7.38
3
–
7.29 (m, 2H), 7.08 (d, J = 8.4 Hz, 1H), 7.00 – 6.87 (m, 3H), 3.84 (s, 3H), 3.72 (s, 3H), 3.62 (d, J = 12.4 Hz, 2H), 2.84 (t,
J = 10.8 Hz, 2H), 2.53 – 2.42 (m, 1H), 2.13 – 2.04 (m, 2H), 2.03 – 1.86 (m, 2H).
-(2-(4'-Fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)benzo[d]oxazol-5-yl)piperidine-4-carboxylic acid (18a).
1
Following general experimental procedure E: Ester 17a (150 mg, 0.30 mmol), 1.25 mL tetrahydrofuran, 0.25 mL H O,
2
and lithium hydroxide (14 mg, 0.60 mmol) were combined to give carboxylic acid 18a as an offꢀwhite pearly solid (129
1
mg, 89% yield). H NMR (400 MHz, MeODꢀd4) δ = 8.67 (s, 1H), 8.54 (d, J = 8.0 Hz, 1H), 8.18 (d, J = 2.0 Hz, 1H), 7.98
(
d, J = 9.2 Hz, 1H), 7.78 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 7.65 (d, J = 8 Hz, 1H), 7.41 (dd, J = 8.4 Hz, 5.2 Hz, 2H), 7.22 (t, J
8.8 Hz, 2H), 3.93 – 3.74 (m, 4 H), 2.93 – 2.82 (m, 1H), 2.41 (dd, J = 15.2 Hz, 3.6 Hz, 2H), 2.32 – 2.16 (m, 2H).
-(2-(2'-(2-Fluoroethoxy)-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)benzo[d]oxazol-5-yl)piperidine-4-carboxylic acid
=
1
(18b). Following general experimental procedure E: Ester 17b (90 mg, 0.17 mmol), 0.70 mL tetrahydrofuran, 0.13 mL
H O, and lithium hydroxide (8.0 mg, 0.332 mmol were combined to give carboxylic acid 18b as an offꢀwhite pearly solid
2
1
(
82 mg, 94% yield). H NMR (400 MHz, MeODꢀd4) δ = 8.52 (s, 1H), 8.35 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 8.4 Hz, 1H),
7
7
1
.56 (s, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.45 – 7.31 (m, 2H), 7.16 (d, J = 7.2 Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 7.03 (t, J =
.2 Hz, 1H), 4.51 (d, J = 48 Hz, 2H), 3.31 – 3.05 (m, 2H), 3.68 (d, J = 12 Hz, 2H), 3.14 (t, J = 10.4 Hz, 2H), 2.59 (t, J =
0.4 Hz, 1H), 2.26 – 2.10 (m, 2H), 2.09 – 1.92 (m, 2H).
1-(2-(4'-(2-Fluoroethoxy)-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)benzo[d]oxazol-5-yl)piperidine-4-carboxylic acid
18c). Following general experimental procedure E: Ester 17c (90 mg, 0.166 mmol), 0.7 mL tetrahydrofuran, 0.13 mL
(
H O, and lithium hydroxide (8.0 mg, 0.332 mmol) were combined to give carboxylic acid 18c as a light tan solid (87 mg,
2
1
9
9% yield). H NMR (400 MHz, MeODꢀd4) δ = 8.64 (s, 1H), 8.50 (d, J = 7.6 Hz, 1H), 8.29 (s, 1H), 7.96 (d, J = 8.8 Hz,
H), 7.83 (dd, J = 8.8 Hz, 2.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.32 (d, J = 8.4 Hz, 2H), 7.06 (d, J = 8.8 Hz, 2H), 4.77
1
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Journal of Medicinal Chemistry
(
dt, J = 48 Hz, 4 Hz, 2H), 4.29 (dt, J = 28.8 Hz, 3.6 Hz, 2H), 3.90 – 3.74 (m, 4H), 2.93 – 2.83 (m, 1H), 2.49 – 2.35 (m,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
H), 2.35 – 2.20 (m, 2H).
1
-(2-(2,4'-Bis(trifluoromethyl)-[1,1'-biphenyl]-4-yl)benzo[d]oxazol-5-yl)piperidine-4-carboxylic
acid
(18d).
Following general experimental procedure E: Ester 17d (102 mg, 0.19 mmol), 0.79 mL THF, and 0.16 mL H O. LiOH
2
1
(8.9 mg, 0.37 mmol) were combined to give an offꢀwhite solid. (101 mg, 99% yield). H NMR (400 MHz, MeODꢀd4) δ =
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
7
–
1
.70 (s, 1H), 8.58 (d, J = 8.0 Hz, 1H), 8.28 (d, J = 2.0 Hz, 1H), 7.99 (d, J = 9.2 Hz, 1H), 7.89 (dd, J = 8.8 Hz, 2.4 Hz, 1H),
.80 (d, J = 8.4 Hz, 2H), 7.69 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 8.0 Hz, 2H), 3.92 – 3.81 (m, 4H), 2.96 – 2.85 (m, 1H), 2.47
2.25 (m, 4H).
-(2-(3'-Methoxy-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)benzo[d]oxazol-5-yl)piperidine-4-carboxylic acid (18e).
Following general experimental procedure E: Ester 17e (92 mg, 0.18 mmol), 0.9 mL THF, and 0.2 mL H O. LiOH (8.6
2
1
mg, 0.36 mmol) were combined to give an offꢀwhite solid (72 mg, 81% yield). H NMR (400 MHz, MeODꢀd4) δ = 8.62
(
(
s, 1H), 8.47 (d, J = 8.0 Hz, 1H), 7.91 (s, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.65 – 7.56 (m, 2H), 7.37 (t, J = 8.0 Hz, 1H), 7.02
dd, J = 8.4 Hz, 2.8 Hz, 1H), 6.95 – 6.89 (m, 2H), 3.83 (s, 3H), 3.84 – 3.73 (m, 2H), 3.58 – 3.43 (m, 2H), 2.76 (br s, 1H),
2
.35 – 2.25 (m, 2H), 2.21 – 2.16 (m, 2H).
N-(Cyanomethyl)benzamide (19). To an ovenꢀdried 250 mL roundꢀbottom flask equipped with a stirbar was added
aminoacetonitrile hydrochloride (8.0 g, 86.5 mmol). Pyridine (50 mL) was carefully added dropwise via addition funnel.
Benzoyl chloride (10.5 mL, 90 mmol) was added via addition funnel dropwise over 30 min, and the reaction was stirred
overnight (20 h). 70 mL of H O added to dissolve the pyridinium hydrochloride and precipitate the product. The new
2
precipitate was filtered, washed with H O, dried, and recrystallized from 95% ethanol to give a white crystalline solid (9.5
2
1
g, 69% yield). H NMR (400 MHz, DMSOꢀd6) δ = 9.22 (t, J = 5.2 Hz, 1H), 7.86 (dd, J = 8.0 Hz, 0.8 Hz, 2H), 7.58 (tt, J =
7
.2 Hz, 1.2 Hz, 1H), 7.50 (t, J = 7.6 Hz, 2H), 4.31 (d, J = 5.6 Hz, 2H).
N-((1H-Tetrazol-5-yl)methyl)benzamide (20). To a 100 mL roundꢀbottom flask equipped with a stirbar was added
nitrile 19 (5.9 g, 36.8 mmol), 32 mL DMF, sodium azide (2.51 g, 38.7 mmol) and ammonium chloride (2.17 g, 40.5
mmol). The reaction vessel was lowered into a preꢀheated 125°C oilꢀbath and stirred overnight (18h). The reaction was
cooled to r.t., diluted with 85 mL 2M HCl(aq) causing the product to precipitate. The precipitate was filtered, washed with
1
copious amounts of H O and air dried to give a white fluffy solid (6.89 g, 92% yield). H NMR (400 MHz, DMSOꢀd6) δ =
2
9
.27 (t, J = 5.6 Hz, 1H), 7.89 (dd, J = 8.4 Hz, 1.2 Hz, 2H), 7.57 (tt, J = 7.2 Hz, 2.4 Hz, 1H), 7.49 (t, J = 7.2 Hz, 2H), 4.76
(
d, J = 5.6 Hz, 2H).
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Journal of Medicinal Chemistry
Page 24 of 58
(
1H-Tetrazol-5-yl)methanamine hydrochloride (21). To a 250 mL roundꢀbottom flask equipped with a stirbar was
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
added amide 20 (6.89 g, 33.9 mmol) and 50.6 mL 12M HCl(aq). The reaction was equipped with a reflux condenser, placed
in a preꢀheated 110 °C oilꢀbath, and stirred at reflux overnight (18 h). The reaction was cooled to r.t., then to 0 °C causing
the formation of a precipitate. The reaction mixture was filtered and the filtrate was washed with Et O (×2), and
2
concentrated in vacuo to give a white solid. This solid was triturated with ethanol and dried under vacuum to give a pale
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
yellow solid (3.59 g, 78% yield). H NMR (400 MHz, D O) δ = 4.59 (s, 2H).
2
Methyl 4-(2-fluoroethoxy)benzoate (23). To a 500 mL roundꢀbottom flask equipped with a stirbar was added methyl 4ꢀ
hydroxybenzoate (22) (5.0 g, 32.9 mmol), potassium carbonate (13.6 g, 98.6 mmol), and 219 mL acetone. The reaction
vessel was heated to reflux in a preꢀheated 70 °C oilꢀbath, and stirred for 30 min. 1ꢀBromoꢀ2ꢀfluoroethane (7.34 mL, 98.6
mmol) was added via syringe, and the reaction mixture was stirred for 20 h. The reaction mixture was cooled to r.t. and
concentrated in vacuo. The resulting solid was dissolved in EtOAc and H O. The layers were separated and the aqueous
2
layer was extracted with EtOAc (×1). The combined organic layers were washed with brine, dried over MgSO , and
4
1
concentrated in vacuo to give a white solid (6.25 g, 96% yield). H NMR (400 MHz, CDCl ) δ = 8.00 (d, J = 8.8 Hz, 2H),
3
6
3
4
.94 (d, J = 9.2 Hz, 2H), 4.78 (ddd, J = 47.2 Hz, 5.6 Hz, 4.0 Hz, 2H), 4.27 (ddd, J = 27.6 Hz, 5.6 Hz, 4.4 Hz, 2H), 3.89 (s,
H).
-(2-Fluoroethoxy)benzoic acid (24). To a 500 mL roundꢀbottom flask equipped with a stirbar was added ester 23 (5.84
g, 29.5 mmol), followed by 98 mL methanol and 49 mL H O. NaOH (4.71 g, 118 mmol) was added and the reaction
2
mixture was stirred for 16 h at r.t. The reaction was acidified with conc. HCl(aq) to pH = 1 and diluted with H O. The
2
1
resulting precipitate was filtered, washed with H O and hexanes, and airꢀdried to give a white solid (5.41 g, 99%). H
2
NMR (400 MHz, CDCl ) δ = 8.05 (d, J = 9.2 Hz, 2H), 6.97 (d, J = 8.8 Hz, 2H), 4.86 – 4.72 (m, 2H), 4.34 – 4.23 (m, 2H).
3
3
3
N'ꢀHydroxyꢀ4ꢀ(hydroxymethyl)benzimidamide (25). To a 500 mL roundꢀbottom flask equipped with a stirbar was added
4
ꢀ(hydroxymethyl)benzonitrile (13.3 g, 100 mmol), hydroxyamine hydrochloride (11.1 g, 160 mmol), sodium bicarbonate
(26.9 g, 320 mmol), and 167 mL methanol. The reaction was equipped with a reflux condenser and lowered into a preꢀ
heated 70 °C oilꢀbath. The reaction mixture was stirred for 5 h and then cooled to r.t. The precipitate was filtered and
1
washed with methanol. The filtrate was concentrated in vacuo to give a white solid (16.5 g, 99% yield). H NMR (400
MHz, MeODꢀd ) matches literature values.
4
3
3
(
4-(5-(4-Ethoxyphenyl)-1,2,4-oxadiazol-3-yl)phenyl)methanol (26a). Following general experimental procedure F: 4ꢀ
Ethoxybenzoic acid (2.49 g, 15.0 mmol), EDC·HCl (2.88 g, 15.0 mmol), HOBt (2.03 g, 15.0 mmol), 19 mL DMF, and
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Page 25 of 58
Journal of Medicinal Chemistry
1
amidine 25 (2.49 g, 15.0 mmol were combined to give an offꢀwhite solid (2.2 g, 50% yield). H NMR (400 MHz, CDCl )
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
δ = 8.18 – 8.11 (m, 4H), 7.50 (d, J = 8.0 Hz, 2H), 7.02 (d, J = 8.8 Hz, 2H), 4.79 (s, 2H), 4.13 (q, J = 7.2 Hz, 2H), 1.47 (t, J
=
6.8 Hz, 3H).
(
4-(5-(4-(2-Fluoroethoxy)phenyl)-1,2,4-oxadiazol-3-yl)phenyl)methanol (26b). Following general experimental
procedure F: Carboxcyclic acid 24 (2.76 g, 15.0 mmol), EDC·HCl (2.88 g, 15.0 mmol), HOBt (2.03 g, 15.0 mmol), 19
mL DMF, and amidine 25 (2.49 g, 15.0 mmol) were combined to provide the crude product, which was used without
further purification for the next step.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
4-(5-(4-(2-Fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)phenyl)methanol (26c) Following general
experimental procedure F: Acid 7a (3.78 g, 15.0 mmol), EDC•HCl (2.88 g, 15.0 mmol), HOBt (2.54 g, 15.0 mmol), 19
1
mL DMF, and amidine 25 (2.49 g, 15.0 mmol) were combined to give an offꢀwhite solid (3.12 g, 55% yield). H NMR
(400 MHz, CDCl ) δ = 8.45 (d, J = 2.0 Hz, 1H), 8.33 (dd, J = 8.8 Hz, 2.0 Hz, 1H), 8.13 (d, J = 8.0 Hz, 2H), 7.50 (d, J =
3
1
3
8
1
1
.0 Hz, 2H), 7.16 (d, J = 8.4 Hz, 1H), 4.90 – 4.74 (m, 4H), 4.48 – 4.35 (m, 2H) C NMR (101 MHz, CDCl ) δ = 174.4,
3
68.9, 159.7, 144.4, 133.4, 127.8, 127.8 (q, JCꢀF = 5.2 Hz), 127.3, 122.9, (q, JCꢀF = 274 Hz), 120.4 (q, JCꢀF = 32.1 Hz),
17.2, 113.5, 81.4 (d, JCꢀF = 173 Hz), 68.5 (d, JCꢀF = 21.1 Hz), 64.9 MP: 146ꢀ147 °C HRMS (ESI) calcd for C H F N O
10
14
4
2
3
+
+
[
M+H ] 383.1013. Found [M+H ] 383.1011.
4-(5-(4-Methoxy-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)phenyl)methanol (26d). To a 100 ml roundꢀbottom
(
flask equipped with a stirbar was added 4ꢀmethoxyꢀ3ꢀ(trifluoromethyl)benzoic acid (2.2 g, 10.0 mmol), HOBt (0.39 g, 2.0
mmol), TBTU (3.21 g, 10.0 mmol), 20 ml DMF, and DIPEA (5.23 ml, 30.0 mmol). The reaction was stirred at r.t. for 30
min, and N'ꢀhydroxyꢀ4ꢀ(hydroxymethyl)benzimidamide (1.66 g, 10.0 mmol) was added. The reaction was stirred at r.t. for
1
h, heated in a 120 °C oilꢀbath for 3 h, and cooled to r.t. The reaction mixture was diluted with EtOAc and water. The
layers were separated, and the aqueous layer was extracted with EtOAc (×1). The combined organic layers were washed
with 1M HCl(aq), water, sat. NaHCO3(aq), water (×3), and brine, dried over MgSO , and concentrated in vacuo to give a tan
4
1
solid (2.6 g, 74% yield). H NMR (400 MHz, CDCl ) δ = 8.45 (d, J = 2.0 Hz, 1H), 8.35 (dd, J = 8.8 Hz, 2.0 Hz, 1H), 8.15
3
(
d, J = 8.0 Hz, 2H), 7.51 (d, J = 8.0 Hz, 2H), 7.16 (d, J = 8.8 Hz, 1H), 4.80 (s, 2H), 4.02 (s, 3H).
33
4
-(5-(4-Ethoxyphenyl)-1,2,4-oxadiazol-3-yl)benzaldehyde (27a) . To an ovenꢀdried 100 mL roundꢀbottom flask
equipped with a stirbar was added 32 mL CH Cl and dimethylsulfoxide (1.27 mL, 17.9 mmol). The reaction mixture was
2
2
cooled in a ꢀ78 °C bath (CO2(s) / Acetone). Oxalyl chloride (1.03 mL, 12.1 mmol) was added carefully, and the reaction
stirred for 30 min. Alcohol 26a (1.71 g, 5.77 mmol) was added and the reaction mixture was stirred 30 min. Triethylamine
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Journal of Medicinal Chemistry
Page 26 of 58
(
6.43 mL, 46.2 mmol) was added and the reaction was allowed to warm to r.t. over 2 h. The reaction mixture was
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
concentrated in vacuo, and partitioned between EtOAc & 1M HCl(aq). The layers were separated and the aqueous layer
was extracted with EtOAc(×1). The combined organic layers were washed with sat. NaHCO3(aq), water, brine, dried over
1
MgSO , and concentrated in vacuo to give an offꢀwhite solid (930 mg, 55% yield). H NMR (400 MHz, CDCl ) δ = 10.11
4
3
(s, 1H), 8.34 (d, J = 8.4 Hz, 2H), 8.15 (d, J = 8.8 Hz, 2H), 8.02 (d, J = 8.4 Hz, 2H), 7.03 (d, J = 8.8 Hz, 2H), 4.14 (q, J =
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
.8 Hz, 2H), 1.47 (t, J = 7.2 Hz, 3H).
4
-(5-(4-(2-Fluoroethoxy)phenyl)-1,2,4-oxadiazol-3-yl)benzaldehyde (27b). To an ovenꢀdried 250 mL roundꢀbottom
flask equipped with a stirbar was added 53 mL CH Cl and dimethylsulfoxide (2.1 mL, 29.6 mmol). The reaction mixture
2
2
was cooled in a ꢀ78 °C bath (CO2(s) / Acetone). Oxalyl chloride (1.7 mL, 20.0 mmol) was added carefully, reaction stirred
0 min. Alcohol 26b (3.00 g, 9.54 mmol) was added and the reaction mixture was stirred 30 min. Triethylamine (10.6 mL,
3
76.3 mmol) was added and the reaction was allowed to warm to r.t. over 2 h. The reaction mixture was concentrated in
vacuo, and partitioned between EtOAc and 1M HCl(aq). The layers were separated and the aqueous layer was extracted
with EtOAc(×1). The combined organic layers were washed with sat. NaHCO3(aq), water, brine, dried over MgSO , and
4
1
concentrated in vacuo to give an offꢀwhite solid (1.31 g, 44% yield). H NMR (400 MHz, CDCl ) δ = 10.11 (s, 1H), 8.34
3
(
d, J = 8.4 Hz, 2H), 8.18 (d, J = 9.2 Hz, 2H), 8.02 (d, J = 8.4 Hz, 2H), 7.09 (d, J = 8.8 Hz, 2H), 4.89 – 4.70 (m, 2H), 4.38
4.25 (m, 2H).
–
4-(5-(4-(2-Fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzaldehyde (27c) To an ovenꢀdried 100
ml roundꢀbottom flask equipped with a stirbar was added 29 mL CH Cl and DMSO (1.15 mL, 16.2 mmol). The reaction
2
2
mixture was cooled to – 78 °C (acetone / CO2(s)) and oxalyl chloride (0.93 mL, 11.0 mmol) was added carefully. The
reaction mixture was stirred for 30 min at which time alcohol 26c (2.0 g, 5.23 mmol) was added. The reaction mixture
was stirred for 30 min at which time Et N (5.83 mL, 41.8 mmol) was added. The cooling bath was removed and the
3
reaction was allowed to warm to r.t. over 2 h. The reaction mixture was concentrated in vacuo and partitioned between
EtOAc and 1M HCl(aq). The layers were separated and the aqueous layer was extracted with EtOAc (×2). The combined
organic layers were washed with 1M HCl(aq), sat. NaHCO3(aq), brine, dried over MgSO , concentrated in vacuo, and
4
1
triturated with MTBE to give a paleꢀyellow solid (1.75 g, 88% yield). H NMR (400 MHz, CDCl ) δ = 10.11 (s, 1H), 8.47
3
(
d, J = 1.6 Hz, 1H), 8.39 – 8.31 (m, 3H), 8.02 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 8.8 Hz, 1H), 4.93 – 4.75 (m 2H), 4.49 –
1
3
4
.35 (m, 2H) C NMR (101 MHz, CDCl ) δ = 191.7, 174.9, 168.3, 159.9, 138.2, 133.5, 132.2, 130.2, 128.3, 127.9 (q, J
3
CꢀF
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Journal of Medicinal Chemistry
=
5.4 Hz), 122.9 (q, JCꢀF = 274 Hz), 120.5 (q, JCꢀF = 32.3 Hz), 116.9, 113.6, 81.4 (d, JCꢀF = 173 Hz), 68.6 (d, JCꢀF = 21.1
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
+
Hz) MP: 149ꢀ150 °C HRMS (ESI) calcd for C H F N O [M+H ] 381.0857. Found [M+H ] 381.0857.
18
13
4
2
3
4
-(5-(4-Methoxy-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzaldehyde (27d). To an ovenꢀdried 100 ml
roundꢀbottom flask equipped with a stirbar was added 32 mL CH Cl and DMSO (1.26 mL, 17.7 mmol). The reaction
2
2
mixture was cooled to – 78 °C (acetone / CO2(s)) and oxalyl chloride (1.01 mL, 12.0 mmol) was added carefully. The
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
reaction mixture was stirred for 30 min at which time alcohol 26d (2.0 g, 5.71 mmol) was added. The reaction mixture
was stirred for 30 min at which time Et N (6.37 mL, 45.7 mmol) was added. The cooling bath was removed and the
3
reaction was allowed to warm to r.t. over 2 h. The reaction mixture was concentrated in vacuo and partitioned between
EtOAc and 1M HCl(aq). The layers were separated and the aqueous layer was extracted with EtOAc (×2). The combined
organic layers were washed with 1M HCl(aq), sat. NaHCO3(aq), brine, dried over MgSO , and concentrated in vacuo to give
4
1
a paleꢀyellow solid (1.51 g, 76% yield). H NMR (400 MHz, CDCl ) δ = 10.11 (s, 1H), 8.46 (s, 1H), 8.40 – 8.32 (m, 3H),
3
8
.03 (d, J = 8.0 Hz, 2H), 7.19 (d, J = 8.8 Hz, 1H), 4.04 (s, 3H).
33
1
-(4-(5-(4-Ethoxyphenyl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid (28a).
Following general
experimental procedure G: Aldehyde 27a (500 mg, 1.70 mmol), azetidineꢀ3ꢀcarboxylic acid (180 mg, 1.79 mmol), 24 mL
methanol, 0.85 mL acetic acid, and NaBH CN (53 mg, 0.85 mmol) in 3.4 mL were combined to give a white solid (331
3
1
mg, 51% yield). H NMR (400 MHz, DMSOꢀd6) δ = 8.11 (d, J = 8.8 Hz, 2H), 8.01 (d, J = 8.0 Hz, 2H), 7.47 (d, J = 8.4
Hz, 2H), 7.17 (d, J = 8.8 Hz, 2H), 4.16 (q, J = 7.2 Hz, 2H), 3.64 (s, 2H), 3.45 – 3.38 (m, 1H), 3.28 – 3.15 (m, 4H), 1.37 (t,
J = 6.8 Hz, 3H).
1
-(4-(5-(4-(2-Fluoroethoxy)phenyl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid (28b). Following general
experimental procedure G: Aldehyde 27b (500 mg, 1.60 mmol), azetidineꢀ3ꢀcarboxylic acid (170 mg, 1.68 mmol),23 mL
methanol, 0.8 mL acetic acid, and NaBH CN (50 mg, 0.80 mmol) in 3.2 mL methanol were combined to give 400 mg of
3
1
1
8
5% pure acetate salt H NMR. H NMR (400 MHz, MeODꢀd4) δ = 8.21 (d, J = 8.0 Hz, 2H), 8.18 (d, J = 8.8 Hz, 2H),
7.62 (d, J = 8.0 Hz, 2H), 7.19 (d, J = 8.8 Hz, 2H), 4.85 – 4.69 (m, 2H), 4.42 – 4.30 (m, 2H), 4.37 (s, 2H), 4.19 – 4.09 (m,
4
H), 3.41 (pent, J = 8.4 Hz, 1H), 1.96 (s, 3H).
1
-(4-(5-(4-(2-Fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic
acid
(
28c) Following general experimental procedure G: Aldehyde 27c (500 mg, 1.31 mmol), azetidineꢀ3ꢀcarboxylic acid (139
mg, 1.38 mmol), 19 mL MeOH, 0.70 mL AcOH, and NaBH CN (41 mg, 0.70 mmol) in 2.6 mL MeOH were combined to
3
1
give an offꢀwhite solid (317 mg, 52% yield). H NMR (400 MHz, MeODꢀd4) δ = 8.45 – 8.38 (m, 2H), 8.23 (d, J = 8.0 Hz,
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Journal of Medicinal Chemistry
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2
H), 7.64 (d, J = 8.0 Hz, 2H), 7.46 (d, J = 9.6 Hz, 1H), 4.87 – 4.71 (m, 2H), 4.54 – 4.43 (m, 2H), 4.43 (s, 2H), 4.25 – 4.15
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
(
m, 4H), 3.44 (pent, J = 8.8 Hz, 1H) C NMR (101 MHz, MeODꢀd4) δ = 166.3, 160.5, 151.8, 137.2, 125.3, 119.0, 118.8,
1
18.7, 118.6 (q, JCꢀF = 5.45 Hz), 117.3, 114.9 (q, JCꢀF = 273 Hz), 111.3 (q, JCꢀF = 32.0 Hz), 108.4, 105.8, 105.8, 73.1 (d, JCꢀ
70.4 Hz), 60.7, 60.5, 55.2 (d, JCꢀF = 11.0 Hz), 55.1 MP: 146ꢀ148 °C Anal. Calcd for C H F N O •2H O: C, 52.70; H,
=
F
22 19
4
3
4
2
+
+
4.62; N, 8.38. Found: C, 52.93; H, 4.46; N, 8.37 HRMS (ESI) calcd for C H F N O [M+H ] 466.1384. Found [M+H ]
2
2
20
4
3
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
66.1383.
-(4-(5-(4-(2-Fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzyl)piperidine-4-carboxylic
1
acid
(
28d). Following general experimental procedure G: Aldehyde 27c (380 mg, 1.0 mmol), piperidineꢀ4ꢀcarboxylic acid (136
mg, 1.05 mmol), 14.3 mL MeOH, 0.5 mL AcOH, and NaBH CN (31 mg, 0.5 mmol) in 2 mL methanol were combined to
3
1
give a white solid (193 mg, 39% yield). H NMR (400 MHz, DMSOꢀd6) δ = 8.45 (dd, J = 8.8 Hz, 1.6 Hz, 1H), 8.33 (d, J
= 1.6 Hz, 1H), 8.05 (d, J = 8.0 Hz, 2H), 7.58 (d, J = 8.8 Hz, 1H), 7.51 (d, J = 8.0 Hz, 2H), 4.75 – 4.50 (m, 4H), 3.54 (s,
2
H), 3.25 – 1.51 (m, 9H).
N-((1H-Tetrazol-5-yl)methyl)-1-(4-(5-(4-(2-fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-
yl)phenyl)methanamine (28e). Following general experimental procedure G: Aldehyde 27c (380 mg, 1.0 mmol), 21 (142
mg, 1.05 mmol), 14.3 mL MeOH, 0.5 mL AcOH, and NaBH CN (31 mg, 0.5 mmol) in 2 mL methanol were combined.
3
The reaction mixture was stirred for 3 h, diluted with MTBE to give a precipitate. The precipitate was filtered to give an
1
offꢀwhite solid (145 mg, 31% yield). H NMR (400 MHz, MeODꢀd4) δ = 8.46 – 8.42 (m, 2H), 8.24 (d, J = 8.4 Hz, 2H),
7
.70 (d, J = 8.0 Hz, 2H), 7.48 (d, J = 8.4 Hz, 1H), 4.83 – 4.72 (m, 2H), 4.54 – 4.44 (m, 2H), 4.50 (s, 2H), 4.40 (s, 2H).
-(4-(5-(4-Methoxy-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid (28f).
1
Following general experimental procedure G: Aldehyde 27d (500 mg, 1.44 mmol), azetidineꢀ3ꢀcarboxylic acid (152 mg,
1
.51 mmol), 21 mL methanol, 0.72 mL acetic acid, and NaBH CN (45 mg, 0.72 mmol) in 2.9 mL methanol were
3
1
combined to give a white solid (300 mg, 48% yield). H NMR (400 MHz, DMSOꢀd6) δ = 8.43 (d, J = 8.4 Hz, 1H), 8.30
(s, 1H), 8.02 (d, J = 7.2 Hz, 2H), 7.60 – 7.42 (m, 3H), 4.03 (s, 3H), 3.64 (br s, 2H), 3.23 (br s, 3H).
4
-(3-(4-(Hydroxymethyl)phenyl)-1,2,4-oxadiazol-5-yl)-2-(trifluoromethyl)phenol (30) To a 100 ml roundꢀbottom flask
equipped with a stirbar was added 4ꢀhydroxyꢀ3ꢀ(trifluoromethyl)benzoic acid (29) (2.06 g, 10.0 mmol), HOBt (0.39 g, 2.0
mmol), TBTU (3.21 g, 10.0 mmol), 20 ml DMF, and DIPEA (5.23 ml, 30.0 mmol). The reaction was stirred at r.t. for 30
min, and 25 (1.66 g, 10.0 mmol) was added. The reaction was stirred at r.t. for 1 h, heated in a 120 °C oilꢀbath for 3 h, and
cooled to r.t. The reaction mixture was diluted with EtOAc and water. The layers were separated, and the aqueous layer
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Journal of Medicinal Chemistry
was extracted with EtOAc (×1). The combined organic layers were washed with 1M HCl(aq), water, sat. NaHCO3(aq), water
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
(
×3), and brine, dried over MgSO , and concentrated in vacuo to give a tan solid (1.7 g, 51% yield). H NMR (400 MHz,
4
DMSOꢀd6) δ = 11.83 (br s, 1H), 8.29 – 8.22 (m, 2H), 8.04 (d, J = 8.0 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 8.4
13
Hz, 1H), 5.38 (br s, 1H), 4.60 (s, 2H) C NMR (101 MHz, DMSOꢀd6) δ = 174.3, 168.1, 160.0, 146.4, 133.5, 126.9, 126.8
(q, JCꢀF = 5.6 Hz), 124.5, 123.3 (q, JCꢀF = 274 Hz), 118.2, 118.1, 116.4 (q, JCꢀF = 31.0 Hz), 114.0, 62.5 MP: > 200 °C
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
HRMS (ESI, m/z) calcd for C H F N O [M+H ] 337.0795. Found [M+H ] 337.0789.
16
11
3
2
3
4
-(5-(4-Hydroxy-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzaldehyde (31) To a 250 mL roundꢀbottom flask
equipped with a stirbar was added alcohol 30 (1.0 g, 2.97 mmol), 59 mL 1,4ꢀdioxane, and 10 g MnO . A reflux condenser
2
was added then the reaction was heated in an 80 °C oilꢀbath and stirred 4 h at which point TLC showed consumption of
the alcohol. The reaction mixture was cooled to r.t. and filtered through a Celite plug, washing with EtOAc. The filtrate
1
was concentrated in vacuo to give a tan solid (620 mg, 62% yield). H NMR (400 MHz, DMSOꢀd6) δ = 10.11 (s, 1H),
1
3
8
1
3
.34 – 8.22 (m, 4H), 8.10 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.4 Hz, 1H) C NMR (101 MHz, DMSOꢀd6) δ = 192.7, 174.9,
67.5, 160.7, 138.0, 133.7, 131.2, 130.2, 127.8, 126.9 (q, JCꢀF = 5.66 Hz), 123.3 (q, JCꢀF = 274 Hz), 118.3, 116.4 (q, JCꢀF
=
0.8), 113.3, MP: > 200 °C HRMS (ESI, m/z) calcd for C H F N O [MꢀH] 333.0493. Found [MꢀH] 333.0400.
1
6
8
3
2
3
Methyl 1-(4-(5-(4-hydroxy-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylate (32) To a
5 ml roundꢀbottom flask equipped with a stirbar was added aldehyde 31 (300 mg, 0.90 mmol), 4.1 ml CH Cl , methyl
2
2
2
azetidineꢀ3ꢀcarboxylate hydrochloride (204 mg, 1.35 mmol), AcOH (0.21 ml, 3.6 mmol), DIPEA (0.24 ml, 1.35 mmol),
and 4.1 ml MeOH. The reaction mixture was stirred for 30 min and sodium cyanoborohydride (57 mg, 0.90 mmol) was
added. The reaction mixture was stirred for 2 h at which time TLC showed consumption of the aldehyde. The reaction
mixture was concentrated in vacuo, dissolved in EtOAc and quenched with sat. NaHCO3(aq). The layers were separated
and the aqueous layer was extracted with EtOAc (×2). The combined organic layers were washed with water and brine,
dried over MgSO , and concentrated in vacuo. The crude product was purified on a silica gel column eluted with 75 –
4
1
100% EtOAc/ hexanes to give a white crystalline solid (193 mg, 49% yield). H NMR (400 MHz, MeODꢀd4) δ = 8.14 (d,
J = 1.2 Hz, 1H), 8.00 (d, J = 8.8 Hz, 1.6 Hz, 1H), 7.91 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 6.95 (d, J = 8.8 Hz,
13
1
H), 3.72 – 3.62 (m, 5H), 3.56 (t, J = 8.0 Hz, 2H), 3.45 (t, J = 6.8 Hz, 2H), 3.34 (pent, J = 7.6 Hz, 1H) C NMR (101
MHz, MeODꢀd4) δ = 174.8, 173.1, 168.1, 161.4, 139.6, 132.7, 129.0, 127.1, 126.9 (q, JCꢀF = 5.15 Hz), 126.0, 123.5 (q, JCꢀ
273 Hz), 117.8, 117.4 (q, JCꢀF = 31.1 Hz), 113.5, 61.8, 56.1, 51.2, 33.4 MP: 125 °C (decomposition) HRMS (ESI, m/z)
=
F
+
+
calcd for C H F N O [M+H ] 434.1322. Found [M+H ] 434.1320.
22 18
3
3
4
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