Structure-Activity Relationship Studies of Trisubstituted Isoxazoles as Selective Allosteric Ligands for the Retinoic-Acid-Receptor-Related Orphan Receptor γt

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Structure−Activity Relationship Studies of Trisubstituted Isoxazoles

as Selective Allosteric Ligands for the Retinoic-Acid-Receptor-

Related Orphan Receptor γt

Femke A. Meijer, Annet O.W.M. Saris, Richard G. Doveston, Guido J.M. Oerlemans,

Rens M.J.M. de Vries, Bente A. Somsen, Anke Unger, Bert Klebl, Christian Ottmann, Peter J. Cossar,

and Luc Brunsveld *

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ABSTRACT: The inhibition of the nuclear receptor retinoic-acid-

receptor-related orphan receptor γt (RORγt) is a promising

strategy in the treatment of autoimmune diseases. RORγt features

an allosteric binding site within its ligand-binding domain that

provides an opportunity to overcome drawbacks associated with

orthosteric modulators. Recently, trisubstituted isoxazoles were

identiﬁed as a novel class of allosteric RORγt inverse agonists. This

chemotype oﬀers new opportunities for optimization into selective

and eﬃcacious allosteric drug-like molecules. Here, we explore the

structure−activity relationship proﬁle of the isoxazole series

utilizing a combination of structure-based design, X-ray crystallog-

raphy, and biochemical assays. The initial lead isoxazole (FM26)

was optimized, resulting in compounds with a ∼10-fold increase in

potency (low nM), signiﬁcant cellular activity, promising pharmacokinetic properties, and a good selectivity proﬁle over the

peroxisome-proliferated-activated receptor γ and the farnesoid X receptor. We envisage that this work will serve as a platform for the

accelerated development of isoxazoles and other novel chemotypes for the eﬀective allosteric targeting of RORγt.

With the exception of RTA-1701 (Figure 1C),^18,19all of the

RORγt inverse agonists that have entered clinical trials thus far

likely target the highly conserved ligand-binding pocket,

1. INTRODUCTION

Nuclear receptors (NRs) are a family of ligand-dependent

transcription factors and attractive drug targets because of

termed the orthosteric-binding site, within the ligand-binding

their central role in several regulatory processes in the human

domain (LBD) of RORγt (Figure 1A).^5,14While orthosteric

body.¹⁻⁴Within the NR family, the retinoic-acid-receptor-

targeting has been highly successful, novel molecular modal-

ities with alternative modes of action, such as allosteric

modulators, oﬀer an interesting alternative for targeting NRs

related orphan receptor γt (RORγt) has received increased

attention because of its essential role in the immune system.⁵

+

̈

RORγt is a key regulator in the diﬀerentiation of naıve CD4

and could circumvent issues related to promiscuity between

NRs.²⁰⁻²³Recently, an allosteric binding site was identiﬁed in

the LBD of RORγt (formed by helices 3, 4, 11, and 12), at a

location that is topographically distinct to the orthosteric site

(Figure 1A).²⁴The indazole MRL-871 (1) (Figure 1C) was

identiﬁed as a prototypical allosteric ligand for RORγt, acting

as a potent inverse agonist and decreasing coactivator binding

with a similar eﬃcacy as orthosteric inverse agonists.^24,25The

T cells into T helper 17 (Th17) cells, and the production of

the pro-inﬂammatory cytokine IL-17a.⁵⁻⁷Elevated IL-17a

levels are associated with the development of autoimmune

diseases, including psoriasis, multiple sclerosis, and rheumatoid

arthritis.⁸⁻¹¹Disrupting the Th17/IL-17a pathway shows high

potential for the treatment of autoimmune disorders, which

has already been validated by the clinical success of anti-IL-17a

monoclonal antibodies.^8,12,13The inhibition of RORγt could

pose an attractive alternative strategy to decrease IL-17a

production in the treatment of autoimmune diseases.¹²Many

research groups have shown signiﬁcant interest in the

identiﬁcation of small-molecule RORγt inhibitors (or, more

speciﬁcally, inverse agonists), with several synthetic com-

pounds progressing into clinical trials.¹⁴⁻¹⁸

Received: March 16, 2021

Published: May 19, 2021

American Chemical Society

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J. Med. Chem. 2021, 64, 9238−9258

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Figure 1. (A) Co-crystal structure of the RORγt LBD in complex with allosteric ligand FM26 (shown as orange sticks) (PDB: 6SAL),²⁸where the

allosteric site (orange circle) and the orthosteric site (blue circle) are indicated; (B) enlarged view of FM26 (shown as orange sticks) in the

allosteric pocket, where H-bond interactions are indicated with orange dashes; (C) chemical structures of allosteric RORγt ligands FM26 (2),

MRL-871 (1), and RTA-1701; and (D) exploration of the hit-to-lead SAR proﬁle of FM26.

allosteric pocket of RORγt is unique within the NR family and

thought not to be the target of endogenous ligands.^24,26

Therapeutic compounds targeting the allosteric-binding

pocket potentially have a higher selectivity proﬁle and do

not act in competition, but in synergy, with endogenous

agonists.^20,21,27Therefore, these compounds would be greatly

beneﬁcial for drug discovery and chemical biology applica-

tions.

Despite the high potential of allosteric inverse agonists for

RORγt, the number of examples have remained limited to

compounds based on the indazole core of 1 or similar

chemotypes, and the previously mentioned allosteric ligand

RTA-1701 (which binds to RORγt via covalent attachment to

Cys476 of helix 11 and partly overlaps with the allosteric

site).^{19,24,25,29−34}Follow-up studies improved the pharmaco-

kinetic (PK) and selectivity proﬁle of 1 and its derivatives.

However, oﬀ-target eﬀects toward other NRs, most notably

toward the peroxisome proliferated-activated receptor γ

(PPARγ), and challenges regarding cytotoxicity and metabolic

stability have been observed.^24,29−32The scarcity and lack of

scaﬀold diversity of allosteric RORγt ligands, and the

limitations regarding speciﬁcity and drug-like properties,

necessitates further exploration of the allosteric pocket.

Speciﬁcally, establishing structure−activity relationship

(SAR) proﬁles for allosteric chemotypes distinct from the

indazole-analogous ligand classes is of great importance.

Recently, we used an in silico pharmacophore search

approach to identify a novel class of RORγt allosteric inverse

agonists, featuring a trisubstituted isoxazole core.^28,35SAR

studies around this novel chemotype resulted in the discovery

of FM26 (2) (Figure 1C), which shows sub-micromolar

potency as a RORγt inverse agonist and signiﬁcant inhibition

of cellular IL-17a expression levels. Initial SAR studies of 2

identiﬁed two key pharmacophore features, where the 2,6-

disubstituted phenyl ring at the C-3 position and a C-4

benzoic acid moiety were shown to be optimal (Figure

1D).^{24,25,28,30−33}Less knowledge has been garnered on the

linker at the C-4 position and the C-5 substituent. Regarding

the C-4 linker, an amine linker (as is used for 2) was found to

be preferred for potency over some more rigid linkers.²⁸

Additionally, the co-crystal structure (Figure 1B) showed that

the presence of a hydrogen bond donating N-heterocycle at

the C-5 isoxazole position (as is the case for the pyrrole in 2)

signiﬁcantly increased the potency toward RORγt by the

formation of an additional polar interaction with the backbone

carbonyls of residues Leu353 and Lys354.²⁸To identify

compounds suitable for focused lead optimization, crucial

explorations around the C-4 and C-5 positions are needed to

further explore the SAR, improve the potency of the isoxazole

series, increase the RORγt speciﬁcity, and direct PK

optimization.

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a

Table 1. SAR Studies around the C-4 Isoxazole Position

a

cLogP values, glide scores, TR-FRET IC₅₀values (μM) from coactivator recruitment and AlexaFluor-MRL-871 displacement assays, ΔT_mvalues

(°C) from thermal shift assays, and fold decrease in IL-17a mRNA expression levels relative to DMSO reverse transcriptase PCR (RT-PCR).

Abbreviations: n.d., not determined. TR-FRET and TSA data are recorded in triplicate; values are representative of ≥3 repeated experiments. RT-

PCR data are recorded in triplicate; values are representative of ≥2 repeated experiments. cLogP values were predicted using MarvinSketch

(20.10).

Utilizing X-ray crystallography to guide focused library

development, herein, we report on the synthesis of allosteric

RORγt inverse agonists diversiﬁed at the C-4 and C-5

isoxazole positions (Figure 1D). The structure-based SAR

study was speciﬁcally focused on improved RORγt activity,

mitigating oﬀ-target PPARγ activity, and a ﬁrst exploration of

PK proﬁles.

was docked into the allosteric site of RORγt (PDB: 4YPQ²⁴),

̈

using Glide, the molecular docking tool in Schrodinger (12.3,

2020-1).^36,37For each ligand, the docking pose was evaluated

and given a “glide score”, which is an empirical scoring

function that approximates the ligand binding free energy (the

more negative the values, the higher the expected potency) but

will not always translate into experimental IC₅₀values.^36,37

Table 1 shows the compounds that resulted in the most

promising glide scores, indicating that linkers responsible for

an increased clog P of the compounds [i.e., ether (3),

thioether (4), methylated amine (5), and alkene (6 and 7)]

would be beneﬁcial for aﬃnity. Additionally, the data suggest

that a ﬂuoro substitution at the ortho position of the benzoic

acid moiety would be well tolerated (8 and 9).

2. RESULTS AND DISCUSSION

2.1. In Silico Docking Studies Guide a SAR Study at

the Isoxazole C-4 Position. The initial investigation started

with expanding the SAR data around the C-4 position of the

isoxazole series guided by in silico docking studies. A virtual

library of derivatives around 2, containing diﬀerent C-4 linkers

and benzoic acid substituents, was designed with a focus on

diversity and synthetic feasibility (see Table S1). This library

2.2. Optimized Synthesis Route Allowed the Eﬃcient

Synthesis of C-4 Isoxazole Derivatives. Isoxazoles 2−9

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a

Scheme 1. Synthesis Route for Diﬀerent Trisubstituted Isoxazoles (C-4 Library)

a

Reagents and conditions: (a) N-Boc-pyrrole-B(pin), Pd(dppf)Cl₂, Cs₂CO₃, and DME, 85 °C, 8 h, 50%; (b) DIBAL and CH₂Cl₂, −78 °C, 3 h,

80%; (c) (i) (MeSO₂)₂O, Et₃N, and CH₂Cl₂, 0 °C → rt, 3 h, (ii) aniline or N-methylaniline, rt, 3 h, 36% (17) and 52% (18); (d) (i) DMP and

CH₂Cl₂, rt, 3 h, 44%, (ii) methyl 4-amino benzoate, MeOH, and AcOH, reﬂux, 24 h, 42%, (iii) NaBH₄and EtOH, reﬂux, 3 h, 17% (19); (e) LiOH,

EtOH, and H₂O, 95 °C, 3 h, 29−81%; (f) methyl 4-hydroxybenzoate or methyl 2-ﬂuoro-4-hydroxybenzoate, DIAD, PPh₃, Et₃N, and THF, reﬂux, 3

h, 55% (20), 25% (21); (g) methyl 4-mercaptobenzoate, DIAD, PPh₃, Et₃N, and THF, reﬂux, 3 h, 27%; (h) DMP and CH₂Cl₂, rt, 3 h, quant.; (i)

(4-(methoxycarbonyl)benzyl)triphenylphosphonium (see Experimental Section for the synthetic procedure of the triphenylphosphonium),

LiHMDS, and THF, −78 °C → rt, 24 h, 7% (24), 27% (25).

(Table 1) were synthesized (Scheme 1) in order to

biochemically evaluate the predictions from the in silico

docking experiments. Common intermediate 14 was synthe-

sized via a 1,3-dipolar cycloaddition using a nitrile oxide and

alkynyl bromide, as described previously (Scheme S1).²⁸In

order to prepare derivatives 2−9, it was important to obtain

core intermediate 16 with a N-Boc-protected pyrrole moiety,

instead of the free pyrrole, as was the case in the original

synthesis route for 2 (Scheme S2).²⁸This was achieved via a

Suzuki cross-coupling reaction, employing a dppf instead of a

tetrakis palladium catalyst (used in previous research), which

proved to be essential for maintaining the Boc-protected

pyrrole 15 in an acceptable yield (Scheme 1). DIBAL was then

used to selectively reduce the ethyl ester, yielding 16 without a

concomitant loss of the Boc group (Scheme 1), which was

previously observed with LiAlH₄(Scheme S1).²⁸

The primary alcohol of 16 was used as a functional handle

for the derivatization of the isoxazole C-4 position. Critical to

the synthesis of this series of isoxazoles was the optimization

of the reductive amination step, which provided sub-optimal

yields in the synthesis of 2 (Scheme S2).²⁸In order to improve

the total yield of the synthesis, the reductive amination was

substituted for a nucleophilic substitution reaction. Final

compounds 2 and 5 were accessed in an eﬃcient manner by

the mesylation of alcohol 16 with methane sulfonic anhydride

(monitored by NMR), the in situ addition of the substituted

aniline, followed by the hydrolysis of the benzoate ester. In

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Figure 2. Biochemical analysis and X-ray crystallography data for isoxazole compounds with C-4 modiﬁcations. (A) Dose−response curves from

the TR-FRET coactivator recruitment assay for 1, 2, 3, and 6; (B) dose−response curves from the ligand displacement TR-FRET assay using the

AlexaFluor MRL-871 probe for 1, 2, 3, and 6; (C) melting temperatures (ΔT_min °C), as measured in TSA for 1, 2, 3, and 6. Data were recorded

in ≥3 independent experiments, each recorded in triplicate (one representative dataset shown). Error bars represent the SD of the mean; (D,G)

tertiary co-crystal structure of RORγt in complex with 3 (PDB: 7NPC) (D) and 9 (PDB: 7NP5) (G) (stick representation). The ﬁnal 2Fo−Fc

electron density map of compounds is shown as an isomesh contoured at 1σ; (E,H) overlay of the co-crystal structure of RORγt in complex with

FM26 (2) (PDB: 6SAL) and RORγt bound to 3 (E), and an overlay of RORγt with 3 and 9 (H); and (F,I) enlarged view of the allosteric pocket

of RORγt showing the interactions between 3 (F)/9 (I) and the protein.

contrast, 8 (ﬂuoro substituent) was synthesized via the original

reductive amination route (Scheme 1).

3:1 mixture of the cis/trans isomers (Scheme 1). These were

separated via preparative high-performance liquid chromatog-

raphy/ultraviolet (HPLC-UV) to aﬀord 24 and 25, and

followed by ester hydrolysis, which aﬀorded ﬁnal compounds

6 (trans) and 7 (cis) (for which the stereochemistry was

determined based on the relative H NMR J-coupling values

between the alkene protons).

2.3. Biochemical Assays and X-ray Crystallography

Reveal a Positive Correlation between the C-4 Linker

Compounds with an ether and thioether linkage (3, 9, and

4) were synthesized from core intermediate 16 via a

Mitsunobu reaction with hydroxy- or mercaptobenzoate,

respectively, furnishing the products in acceptable yields

(Scheme 1). For the cis and trans alkene linker (6 and 7),

the alcohol of compound 16 was oxidized to the aldehyde,

which was subsequently used in a Wittig reaction, obtaining a

1

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Lipophilicity and Potency. The potency of the C-4

modiﬁed compounds for RORγt was investigated in a time-

resolved FRET (TR-FRET) coactivator recruitment assay

(Table 1, Figure 2A, see Figure S3A for a schematic

representation of the assay setup).³⁸RORγt is constitutively

active, which means that it shows a basic level of transcrip-

tional activity, thus allowing the partial recruitment of

coactivators in the absence of an agonist.³⁹Reference

compounds 1 and 2 showed potent inhibition of coactivator

binding, with IC₅₀values comparable to previous studies.^24,28

Interestingly, compound 3 (FM156) containing an ether

linkage demonstrated a 9-fold increase in potency compared to

2 with an amine linker (IC₅₀of 31 3 nM vs 270 20 nM,

respectively), resulting in an IC₅₀value in the same range as

indazole 1. The trans-alkene linker (6, FM260) resulted in a

low nM IC₅₀value as well (IC₅₀of 20 2 nM), in contrast to

the cis-alkene linker (7) which was 25 times less potent (IC₅₀

particularly high ΔT_mvalues (4.9 and 6.4 °C resp.). These

values are improved compared to that for 2, which provides

further indication that 3 and 6 have a high-binding aﬃnity for

RORγt.

Crystallization studies provided the co-crystal structures of

the RORγt LBD in complex with compounds 3 (ether linker)

and 9 (ﬂuoro substituent), with resolutions of 1.46 and 1.55 Å,

respectively (Tables S4 and S5, Figure S6). The co-crystal

structures of RORγt with 3 and 9 showed clear electron

density for the compounds in the allosteric binding site

between helices 3, 4, 11, and 12 (Figure 2D,G). In Figure 2E,

an overlay of RORγt in complex with 2 and with 3 is shown,

demonstrating a common binding pose, with the C-3, C-4, and

C-5 isoxazole substituents anchored at the same position. The

pyrrole moiety forms a hydrogen bond interaction with the

main chain carbonyls of residues Leu353 and Lys354 (Figure

2F). Additionally, the carboxylic acid moiety forms hydrogen

bond interactions with the side chain of Gln329 and backbone

nitrogens of Phe498 and Ala497, as is also the case for 1 and

2. Interestingly, the electron density for 3 and 2 would allow

two diﬀerent conformations of the C-4 linker,²⁸but the

preferred conformation for the ether linker in 3 is opposite to

the preferred conformation for the amine linker in 2 (Figure

2E). For compound 9, the electron density and binding mode

are highly similar to 3, with the same conformation for the

ether linker present (Figure 2G−I). The ortho-ﬂuoro

substituent on the benzoic acid ring does not signiﬁcantly

inﬂuence the conformation of the compound, except for the

disubstituted phenyl ring at the isoxazole C-3 position, which

is normally ﬁxed at the same position but is now slightly

shifted (Figure 2H).

The increased potency of ether 3 and alkene 6 could be the

result of increased hydrophobic eﬀects of these more lipophilic

compounds toward the hydrophobic allosteric-binding pocket

(see the protein−ligand interaction plot in Figure S5). The

diﬀerent preferred conformations of the ether linker for 3, as

seen in the co-crystal structure, might also be contributing to

the potency. In contrast, the compound with a thioether

linkage (4) shows a signiﬁcantly lower potency, which might

be caused by a slight change in the bond angle and length of

the linker. The docking pose shows that the isoxazole core and

pyrrole moiety are clearly shifted compared to the compound

with ether linker (3) (Figure S4A). The drop in potency for 5,

featuring a methylated amine linker, likely results from the

restricted rotation of the linker. The electron density for the

isoxazoles shows that two conformations of the linker are

present, but when the linker is more rotationally restricted, as

is the case for 5, only one of these linker conformations will

most likely be present, potentially resulting in a higher

entropic penalty and in turn a decrease in the binding aﬃnity.

The introduction of an ortho-ﬂuoro substituent at the benzoic

acid moiety leads to a slight decrease in potency (8 vs 2 and 9

vs 3). The co-crystal structure in complex with 9 shows that

the ﬂuoro substituent is placed in the same plane as the

carboxylic acid moiety (Figure 2I). This unfavorable

conformation is believed to be caused in order to both ﬁt

the ﬂuoro substituent in the pocket and also allow hydrogen

bond interactions between the carboxylic acid and the protein,

which could lead to charge repulsion between the two

moieties and a decreased potency.

of 490

contrast, a thioether linkage (4) resulted in a signiﬁcantly

decreased potency compared to 2 (IC₅₀of 6.6 0.8 μM),

30 nM), as predicted by the docking scores. In

whereas a methylated amine linker (5) resulted in a loss of

activity (IC₅₀> 100 μM). The introduction of a ﬂuorine

substituent at the ortho position of the benzoic acid moiety

proved detrimental to coactivator inhibition with a 7−13 fold

decrease in potency compared to the compound without a

substituent (8 vs 2 and 9 vs 3), which was also the case for an

isoxazole analogue in a previous SAR study.²⁸

To prove an allosteric mode of action, the compounds were

also tested in two other TR-FRET assay formats. First, a

previously described AlexaFluor 647-labeled MRL-871 probe²⁴

(Figure S1) was used, which upon binding to RORγt shows

FRET pairing with an anti-His terbium cryptate donor on the

protein (see Figure S3B).²⁴All compounds showed displace-

ment of the AlexaFluor-MRL allosteric probe, proving an

allosteric binding mode (Table 1, Figure 2B), with the IC₅₀

values correlating with the IC₅₀values from the TR-FRET

coactivator recruitment assay (Table 1). In particular, ether 3

and trans-alkene 6 demonstrated eﬃcient displacement of the

probe, with IC₅₀values of 12

1 and 7.4

0.9 nM,

respectively, signiﬁcantly increased as compared to 2 (IC₅₀

value of 100 10 nM).

The most potent ligands 3 and 6 were also measured in a

competitive TR-FRET coactivator recruitment assay, where

the compounds were titrated in the presence of cholesterol,

which is an orthosteric agonist (activator) for RORγt⁴⁰

(Figure S2, see Figure S3C for a schematic representation of

the assay setup). The binding curves for compounds 3 and 6

show that the IC₅₀values did not decrease upon increasing

concentrations of cholesterol, demonstrating that their binding

mode is independent to that of cholesterol, supporting their

allosteric-binding mode. In fact, the presence of cholesterol

slightly enhanced the potency of both 3 and 6, suggesting a

cooperative behavior between orthosteric and allosteric ligand

binding, as was observed previously.^27,28

A thermal shift assay (TSA) was performed as an orthogonal

assay to investigate the eﬀect of the compounds on the

thermal denaturation of the RORγt protein (Table 1, Figure

2C). Ligand binding typically improves the thermal stability of

a protein by stabilizing the protein fold, as indicated by the

change in the melting temperature ΔT_m.⁴¹⁻⁴³The C-4

isoxazole derivatives 3−9 showed a thermal stabilization eﬀect

according to their potency, as observed in the TR-FRET

assays. The most potent compounds 3 and 6 showed

2.4. SAR Studies Show the Necessity of a H-Bond

Donor Moiety at the Isoxazole C-5 Position. The SAR

was further explored at the isoxazole C-5 position to

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a

Table 2. SAR Studies around the C-5 Isoxazole Position

a

Glide scores, TR-FRET IC₅₀values (μM) from the coactivator recruitment and AlexaFluor-MRL-871 displacement assays, ΔT_mvalues (°C) from

TSA, and fold decrease in IL-17a expression levels relative to DMSO (RT-PCR) are shown. Abbreviations: n.d., not determined. TR-FRET and

TSA data are recorded in triplicate; values are representative of ≥3 repeated experiments. RT-PCR data are recorded in triplicate; values are

representative of ≥2 repeated experiments.

a

Scheme 2. Synthesis Route for Diﬀerent Trisubstituted Isoxazoles (C-5 Library)

a

Reagents and conditions: (a) N-Boc-pyrrole-B(pin), N-THP-pyrazole-B(pin) or 5-methyl N-Boc-pyrrole-B(pin) (see Experimental Section for the

synthetic procedure of this pinacol ester), Pd(dppf)Cl₂, Cs₂CO₃, and DME, 85 °C, 8 h, 49% (26), 34% (27), and 24% (29); (b) N-methyl-pyrrole-

B(pin), Pd(PPh₃)₄, Na₂CO₃, DME, and H₂O, 85 °C, 8 h, 55% (28); (c) DIBAL and CH₂Cl_2,−78 °C, 3 h, 69% (30), 71% (31), and 58% (33); (d)

LiAlH₄and THF, 0 °C → rt, 2 h, 76% (32); (e) (i) methyl 4-hydroxybenzoate, DIAD, PPh₃, and THF, reﬂux, 3 h, 21%, (ii) LiOH, EtOH, and

H₂O, 95 °C, 3 h, 41% (10); (f) (i) methyl 4-hydroxybenzoate, DIAD, PPh₃, Et₃N, and THF, reﬂux, 3 h, 44−45%, (ii) LiOH, EtOH, and H₂O, 95

°C, 3 h, 86% (12), 83% (13); (g) (i) methyl 4-hydroxybenzoate, DIAD, PPh₃, Et₃N, and THF, reﬂux, 3 h, 49%, (ii) TFA and CH₂Cl₂, 40 °C, 2 h,

87%, (iii) LiOH, EtOH, and H₂O, 95 °C, 3 h, 89% (11).

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Figure 3. Biochemical analysis and X-ray crystallography data for isoxazole compounds with C-5 modiﬁcations. (A) Dose−response curves from

the TR-FRET coactivator recruitment assay for 2, 3, 10, and 11; (B) dose−response curves from the ligand displacement TR-FRET assay using

the AlexaFluor MRL-871 probe for 2, 3, 10, and 11; (C) melting temperatures (ΔT_min °C), as measured in TSA for 2, 3, 10, and 11. Data were

recorded in ≥3 independent experiments, each recorded in triplicate (one representative dataset shown). Error bars represent the SD of the mean.

(D,G) Tertiary co-crystal structure of RORγt in complex with 10 (D) (PDB: 7NEC) and 11 (G) (PDB: 7NP6) (stick representation). The ﬁnal

2Fo−Fc electron density map of the compounds is shown as an isomesh contoured at 1σ; (E,H) overlay of the crystal structure of RORγt in

complex with 3 and RORγt bound to 10 (E) or 11 (H); and (F,I) enlarged view of the allosteric pocket of RORγt showing the interactions

between 10 (F)/11 (I) and the protein.

investigate the hydrogen bonding character of the pyrrole

moiety of 2. Compound 3 with the ether linkage was chosen

as a core scaﬀold, based on the combination of its high

potency, availability of the co-crystal structure, and synthetic

feasibility. A focused library of C-5 derivatives (compounds

10−13, Table 2) was designed and synthesized (the full library

used for docking is shown in Table S2). Speciﬁcally, the eﬀect

of the nitrogen position (10), pK_a(11) (pK_a= 10.17 for 11 vs

14.99 for 3) and substitution (12) was investigated, as well as

the hydrophobic space around the pyrrole (13).

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Compounds 10−13 were synthesized using an analogous

synthesis route as shown previously (Scheme 2). Because the

C-5 moiety is incorporated early in the synthesis, it was not

possible to diversify from a late-stage intermediate, as for the

C-4 modiﬁcations. Bromide 14 was subjected to a Suzuki

reaction with the associated pinacol ester, containing a Boc-,

THP-, or methyl-protected heterocycle, followed by ester

hydrolysis with DIBAL to yield 30−33 (Scheme 2). For the

N-methyl pyrrole, the original conditions were used (Scheme

S2) (the tetrakis palladium catalyst in the Suzuki reaction and

LiAlH₄for reduction) because no labile-protecting group was

used in this synthesis route. Next, a Mitsunobu reaction with

methyl 4-hydroxybenzoate and ester hydrolysis combined with

heterocycle deprotection were conducted to aﬀord the ﬁnal

compounds 10−13.

To assess the SAR around the C-5 position, analogues 10−

13 were evaluated in the TR-FRET coactivator recruitment

assay (Figure 3A, Table 2). A nitrogen shift (2- instead of 3-

position) in the pyrrole ring (10) resulted in a 4.5-fold

decrease in potency (IC₅₀= 140 10 nM) compared to 3.

Pyrazole 11 (FM257) showed a slight drop in potency, with

an IC₅₀value of 110 10 nM. For compound 12 (containing

a methylated pyrrole) and compound 13 (methylation of the

pyrrole at the 5-position), a lower activity was observed with

still active on RORγt with an IC₅₀value < 150 nM, despite the

lack of the characteristic C-5 H-bond interaction. For the

pyrazole (11, FM257), the 3.5-fold lower potency indicates

that the lower pK_aof the pyrazole is not beneﬁcial for H-bond

formation, although other factors might also be involved, for

example, the existence of the pyrazole in two tautomeric forms

for which only one can establish the hydrogen bond

interaction with the protein. The much lower potency for

compound 12 (methylated pyrrole) suggests that the space

around the pyrrole is limited, which is also supported by the

low potency of the methyl substitution of the pyrrole at the 5-

positon (13). The docking score predicts compound 12 to be

highly active, based on the docking pose where the pyrrole

ring is rotated and the methyl substituent could point toward a

small cavity (Figure S4B). Together, based on the biochemical

data, the methylated pyrrole does not appear to have enough

space for this rotation in the allosteric pocket. Combined,

these data show the need for an H-bond donor at the right

position of the heterocycle at the C-5 position of the isoxazole

scaﬀold, while additional substituents at the ring appear to be

too bulky to be tolerated.

2.5. The New Isoxazole Series Show the Inhibition of

IL-17a Expression in EL4 Cells. A selection of the novel

isoxazole compounds was tested in a quantitative RT-PCR

assay to investigate their functional eﬀect. EL4 cells were

treated with 10 μM of the compound or dimethyl sulfoxide

(DMSO), after which the IL-17a mRNA levels were measured

(Figure 4). Hit compound 2 showed a 9.3-fold reduction, in

accordance with our previous reports.²⁸The optimized

compounds 3 and 6 (IC₅₀values of 31 and 20 nM,

respectively) showed a 15- and 16-fold reduction of IL-17a

levels compared to DMSO, which is in line with their high

biochemical potency and indicates good cellular uptake and

an IC₅₀value of 3.3

0.3 and 2.9

0.2 μM, respectively.

Additionally, the TR-FRET AlexaFluor-MRL assay was

performed, validating an allosteric binding mode for all

compounds (Figure 3B, Table 2). Analysis of the IC₅₀values

for this series showed that these were in line with the IC₅₀

values from the TR-FRET coactivator assay (Table 2). In the

TSA, compounds 10 and 11 induced thermal stabilization with

ΔT_mvalues of 2.8 and 2.7 °C respectively, which is lower than

for 3 (ΔT_m= 4.9 °C) but slightly higher than for 2 (ΔT_m=

2.4 °C). Compounds 12 and 13 did not show any response

(Figure 3C, Table 2).

Crystallization studies provided the co-crystal structures of

the RORγt LBD in complex with C-5-modiﬁed compounds 10

(2-pyrrole) and 11 (pyrazole), with resolutions of 1.95 and

1.84 Å, respectively (Tables S6 and S7, Figure S6). The co-

crystal structure of RORγt with 10 reveals a clear ligand

electron density in the allosteric site with the ether linker in

the same preferred conformation as for 3 (Figure 3D,E). The

2-pyrrole at the C-5 position, with the nitrogen at a diﬀerent

position, does not establish a direct hydrogen bond interaction

with the protein, as expected, potentially explaining the lower

potency of the compound for RORγt. Interestingly, the NH of

the pyrrole substituent of 10 forms an alternative hydrogen

bond network via a water molecule in the binding pocket at a

distance of 3.2 Å from the NH of the pyrrole (Figure 3F) (this

water molecule appears to be present in all crystal structures

with an allosteric ligand, and thus has a structural role). For

pyrazole 11, the binding mode is highly similar to 3, again

establishing a hydrogen bond interaction between the NH of

the pyrazole and the backbone carbonyls of RORγt (Figure

3G−I).

Figure 4. IL-17a mRNA expression levels in EL4 cells treated with

ligands 1, 2, 3, 6, 10, 11, and 12 (10 μM, 24 h) or DMSO and fold

decrease of the IL-17a expression relative to DMSO. The level of IL-

17a expression was normalized to that of GAPDH expression. All

These results show that small changes at the pyrrole C-5

moiety lead to relevant changes in the potency of the

compounds. The 4.5-fold decrease in potency for the

compound with a changed position of the nitrogen in the

pyrrole ring (10) (compared to 3) is most probably due to the

loss of the characteristic H-bond interaction between the

pyrrole ring and the backbone of the protein. The additional

H-bond interaction with a water molecule explains why 10 is

data are expressed as mean

s.d. (n = 3). The relative gene

expression was calculated by the 2^−ΔΔC_t(Livak) method using DMSO

control as a calibrator. Statistical analysis was performed using a one-

way analysis of variance (ANOVA) compared against the DMSO

control following Dunnett’s post hoc test; ***P < 0.001 and ****P <

0.0001.

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Table 3. EC₅₀Values Observed in the TR-FRET Coactivator Recruitment Assay, IC₅₀Values from the TR-FRET Competition

a

Assay with PPARγ, and ΔT_mValues (°C) from TSA Assays

fold-selective for RORγt over

PPARγ

compound

EC₅₀(μM) PPARγ

IC₅₀(μM) PPARγ (competition rosiglitazone)

ΔT_m(°C) PPARγ

TR-FRET

TSA

1

2

3

4

5

6

7

8

0.34 0.02

8.2 0.3

3.1 0.1

6.3 0.7

2.5 0.2

0.3 0.0

0.6 0.0

0.7 0.0

0.0 0.0

1.1 0.2

0.1 0.0

0.4 0.0

1.2 0.0

0.7 0.0

1.5 0.0

26

30

100

2.7

3.1

8.0

8.1

0.5

81

63

7

8

18

1

>100

64

>100

2.5 0.1

7.5 0.3

3

125

15

1.6

67

7.1

33

0.36

0.27

5.8

7.0

7.5

2.3

3.9

<0.1

>100

67

15

1

9

5.8 0.2

1.7 0.1

3.6 0.1

1.2 0.1

0.79 0.10

6

4

10

11

12

13

29

79 11

20

19

2

3

a

TR-FRET and TSA data are recorded in triplicate; values are representative of ≥3 repeated experiments.

activity. Compounds 10 and 11 induced a 12- and 6.6-fold

decrease in IL-17a mRNA levels, correlating with their slightly

lower biochemical potency compared to 3 and 6. Compound

12 only resulted in a 2.4-fold decrease compared to DMSO, as

expected, given its signiﬁcantly lower potency in the TR-FRET

coactivator recruitment assay.

2.6. The Isoxazole Series Shows an Improved

Selectivity Proﬁle for RORγt. Previous studies have shown

that 1 is selective for RORγt over other NRs, except for

PPARγ on which it shows a signiﬁcant cross-reactivity to the

orthosteric binding site, as was also supported by the co-crystal

structure.^24,44Isoxazole 2 already showed a higher selectivity

for RORγt over PPARγ than 1,²⁸so we were interested to

establish if the novel isoxazole compounds further improved

this promising selectivity proﬁle.

19

3 μM resp.). Compound 10 showed some cross-

reactivity with PPARγ as well, although with an IC₅₀value >

20 μM.

Next, the compounds were evaluated in a PPARγ TSA

(Table 3). Compounds 10, 12, and 13 showed the highest

thermal stabilization eﬀect (ΔT_mof 1.2 and 1.5 °C), in line

with the TR-FRET data, and also 6 appeared to show a

signiﬁcant stabilization eﬀect (ΔT_mof 1.1 °C). Compound 1

again showed the most signiﬁcant activity on PPARγ, with a

ΔT_mof 2.5 °C. The selectivity of the compounds for RORγt

over PPARγ was calculated (Table 3) by comparing the data

for PPARγ and RORγt for both the TR-FRET coactivator

recruitment assay and TSA (see Tables 1−3). Interestingly,

compounds 3 and 6, which are highly potent RORγt inverse

agonists (IC₅₀values < 35 nM), show a 100- and 125-fold

selectivity for RORγt over PPARγ, respectively, based on the

TR-FRET results, which is a signiﬁcant improvement

compared to 2 (30-fold). Contrastingly, 12 and 13 containing

a methyl substituent at the pyrrole moiety show higher cross-

reactivity toward PPARγ, in the same range as for 1. Together,

the cross-reactivity of the isoxazoles on PPARγ can be

modulated by only small changes in the pyrrole substitution

pattern. Gratifyingly, the most potent compounds for RORγt

(3 and 6) also feature the highest selectivity for RORγt over

PPARγ.

Lastly, the isoxazole compounds were tested on the

farnesoid X receptor (FXR) to exclude cross-reactivity because

trisubstituted isoxazole ligands, such as GW4064, have been

reported as potent FXR agonists.^46,47The most potent RORγt

isoxazole ligands and GW4064 were evaluated in a

ﬂuorescence polarization (FP) assay. GW4064 showed a

typical agonistic dose−response curve for FXR, whereas the

novel isoxazole ligands did not elicit an agonistic response on

FXR (Figure S7A). Additionally, the EC₅₀value of GW4064

was not signiﬁcantly aﬀected by the presence of isoxazole 3

(Figure S7B), thus also excluding an antagonistic behavior of 3

on FXR.

In a TR-FRET coactivator recruitment assay (Table 3), 1

showed PPARγ agonism with an EC₅₀value of 0.34

μM, while the EC₅₀value for 2 was >20-fold higher (EC₅₀

0.02

=

8.2 0.3 μM).^24,28All novel isoxazole ligands developed in

this study were at least ﬁve times less active on PPARγ than 1

(EC₅₀values > 1.7 μM), except for compounds 12 and 13

(both bearing a methyl substituent at the pyrrole moiety),

which showed a higher cross-reactivity on PPARγ (EC₅₀values

of 1.2 0.1 and 0.79 0.10 μM resp.). This might be due to

the more bulky substitution pattern around the pyrrole C-5

substituent.

In order to further explore the binding site of the isoxazole

compounds on PPARγ, a TR-FRET assay was performed

whereby the competition between the isoxazole ligands and a

known orthosteric PPARγ agonist, rosiglitazone, was measured

(Table 3).^28,45All compounds showed competition with

rosiglitazone at high concentrations, indicating binding to the

orthosteric site of PPARγ. Interestingly, at least a 3-fold lower

competition with rosiglitazone was observed for the isoxazole

compounds compared to 1 (i.e., at least a 3-fold increase of

the IC₅₀values compared to 1), with most analogues

demonstrating IC₅₀values > 50 μM. Overall, a similar trend

was observed for the IC₅₀values in this competition assay as

for the EC₅₀values in the previously mentioned TR-FRET

coactivator recruitment assay (Table 2). Again, compounds 12

and 13 appeared to be less selective for RORγt than the more

potent RORγt-binding isoxazoles (IC₅₀values of 20 2 and

2.7. Absorption, Distribution, Metabolism, and

Excretion Proﬁle. Previous studies on the absorption,

distribution, metabolism, and excretion (ADME) parameters

of the isoxazoles showed that these were promising but not

optimal yet for compound 2, most probably governed by the

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Table 4. ADME Properties for Compounds 1, 2, 3, 6, 9, 10, and 11

microsomal stability

kinetic solubility

PAMPA

(% ﬂux)

phase 1

(Cl_int, μL/min/mg)

glucuronidation in vitro

plasma stability

(% remaining)

plasma protein binding

(% bound)

compound

(μM)

(% remaining)

1

2

3

6

9

10

11

423

460

436

392

445

421

476

29.7

36.1

40.9

40.3

37.3

50.5

23.0

0.4

31.2

57.8

69.0

77.0

68.0

8.8

37.7

73.0

30.7

58.3

54.6

68.9

39.1

95.5

79.1

74.2

59.5

97.1

89.5

100

99.7

99.9

99.7

99.9

99.8

99.3

98.8

pyrrole moiety and the amine linker, which are both likely to

be prone to oxidation.^28,48The ADME proﬁling of the novel

isoxazoles (Table 4) demonstrates that compounds 3, 6, 9,

and 10 in general show comparable properties to hit

compound 2, with promising values for the kinetic solubility

and (passive) membrane permeability, especially because the

compounds contain a carboxylic acid moiety which generally

leads to lower permeability values. The metabolic stability in

human liver microsomes showed that the clearance values in

phase I metabolism are rather high, similar to 2. Glucur-

onidation assays (as the most important phase II pathway)

showed promising improved values (% remain), especially for

compound 10. The plasma stability (except for 6) was slightly

lower than for 1, and relatively high levels of blood plasma

protein binding were measured. Contrastingly, compound 11,

containing a pyrazole instead of a pyrrole substituent at the C-

5 position, showed a rather diﬀerent ADME proﬁle. It

demonstrated a promising stability in phase I metabolism

and was highly stable in plasma, although the phase II stability

(glucuronidation) and permeability were lower than for the

other isoxazoles.

at the C-5 position to deliver lead compounds for further

optimization. The optimization of the linker at the C-4

position revealed a clear correlation between potency and the

lipophilicity and ﬂexibility of the linker, with an ether or alkene

linker resulting in the highest potency. The co-crystal structure

of RORγt in complex with ether compound 3 revealed the role

of hydrophobic interactions and conformation of the linker at

the C-4 position. The most potent compounds 3 (FM156)

and 6 (FM260) were highly selective for RORγt over PPARγ,

which is a valuable improvement compared to the exemplary

indazole 1.

The focused library of C-5 isoxazoles revealed several routes

for chemical exploration. Changing the position of the

hydrogen bond donor via the repositioning of the pyrrole

ring (10) led only to a 3-fold lower potency, with the

formation of another hydrogen bond interaction network via a

water molecule in the RORγt pocket. Changing the pyrrole to

a pyrazole (with a lower pK_a) in compound 11 (FM257) was

also tolerated and resulted in a promising PK proﬁle, opening

up routes for aﬃnity and ADME optimization.

In conclusion, by probing the eﬀect of the linker at the C-4

position and the importance of the polar character of the C-5

moiety, this study has led to valuable leads as allosteric inverse

agonists for RORγt with improved potency, increased

selectivity against PPARγ and FXR, and promising initial

ADME properties. The results not only provide new insights

into the SAR for this speciﬁc isoxazole class of allosteric

RORγt ligands but can also most probably similarly be

translated to other allosteric RORγt chemotypes. Overall, the

trisubstituted isoxazole class of allosteric RORγt ligands shows

high potential for chemical biology approaches as well as for

future development in drug discovery programs against

autoimmune diseases.

The open-source program OSIRIS Property Explorer⁴⁹was

used to predict the drug likeness and toxicity of the

compounds (Table S3) to have a ﬁrst indication on these

aspects. All isoxazole compounds (except 5) were predicted to

have no side eﬀects (i.e., mutagenic, tumorigenic, irritant, and

reproductive eﬀects), whereas 1 was predicted to have a

potentially high risk of reproductive toxicity. Additionally, all

isoxazole ligands showed higher drug scores than 1, with

pyrazole 11 showing the best properties, again highlighting its

potential for further development.

Combined, the change of an amine to an ether/alkene linker

(2 vs 3 and 6) does not signiﬁcantly aﬀect the ADME(T)

properties (except for the glucuronidation of 3), while the

change of a pyrrole to a pyrazole moiety (11) results in a

diﬀerentiated, promising proﬁle. The C-4/C-5 isoxazole

modiﬁcations thus provide valuable entry points for

optimization of the ADME properties.

4. EXPERIMENTAL SECTION

4.1. In Silico Studies. 4.1.1. Molecular Docking Studies. The

receptor−ligand complex structure (PDB code: 4YPQ) was prepared

using the Protein Preparation Wizard within Maestro (version 12.3

̈

(2020-1), Schrodinger LLC, New York, NY, USA) (default

parameters). A receptor grid centered on the bound ligand was

generated using the Protein Preparation Wizard. All parameters were

kept as the default. The examined ligands were drawn in ChemDraw.

Energy-minimized 3D ligand conformations for each molecule to be

investigated were generated using the Ligand Preparation Wizard

within Maestro (default parameters). Ligands were docked using

Glide in standard precision mode with ﬂexible ligand sampling. The

predicted binding modes of all ligands were ranked according to their

glide score.

4.1.2. In Silico Drug Likeness and Toxicity Predictions. OSIRIS

Property Explorer utilizes the database of traded drugs and

commercially available compounds (Fluka), assumable as nondrug-

like data set, to assess the occurrence frequency of each fragment in

3. CONCLUSIONS

The NR RORγt has an important regulatory role in the

immune system, and inhibition of RORγt via allosteric inverse

agonists could be a promising strategy in the treatment of

autoimmune disorders. Apart from indazoles, with 1 (MRL-

871) as a prominent example, there has been little chemical

diversity of allosteric RORγt modulators. Trisubstituted

isoxazoles constitute a distinct novel chemotype in this matter,

with 2 (FM26) as an exemplary potent hit compound.

Here, we further optimized this isoxazole series, speciﬁcally

focusing on the linker at the C-4 position and the heterocycle

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the individual structure.⁴⁹The program was used to estimate the risks

of side eﬀects, such as mutagenic, tumorigenic, irritant, and

reproductive eﬀects, as well as drug likeness and overall drug score

(by combining the outcome of clog P, log S (solubility), MW, toxicity

risks, and drug likeness). The drug score is a measure of the

compound’s potential to meet the criteria of a possible drug

candidate.

4.2. General Chemistry. All nonaqueous reactions were

performed under an argon atmosphere unless otherwise stated.

Water-sensitive reactions were performed in oven-dried glassware,

cooled under argon before use. All solvents were supplied by Biosolve

or Sigma-Aldrich and used without further puriﬁcation. Dry solvents

were obtained from a MBRAUN solvent puriﬁcation system (MB-

SPS-800). Water was puriﬁed by a Millipore puriﬁcation train.

Deuterated solvents were obtained from Cambridge Isotope

over MgSO₄, ﬁltered, and concentrated in vacuo to aﬀord the title

compound which was puriﬁed as described.

4.2.3. General Procedure for Mesylation and Substitution. Under

an inert atmosphere, triethylamine (3.0 equiv) was added to a

solution of alcohol compound 16 (1.0 equiv) in anhydrous CH₂Cl₂

(0.1 M). Methanesulfonic anhydride (1.5 equiv) was added to the

ﬂask, and the reaction mixture was heated at 40 °C. The reaction was

monitored using NMR analysis. Subsequently, aniline (5.0 equiv) was

added, and the reaction mixture was stirred for 1 h, cooled to room

temperature, and CH₂Cl₂was removed in vacuo. The crude product

was puriﬁed by ﬂash column chromatography using the speciﬁed

eluent.

4.2.4. General Procedure for Ester Hydrolysis and N-Boc

Deprotection. LiOH·H₂O (10.0 equiv) was added to a suspension

of ester (1.0 equiv) in a 4:1 mixture of EtOH/H₂O (0.05 M). The

reaction mixture was heated to 95 °C followed by TLC analysis.

Upon complete consumption of the starting material, EtOH was

removed in vacuo, and the resulting aqueous mixture was acidiﬁed to

pH 3 using 10% v/v aqueous HCl and extracted with a 9:1 mixture of

CH₂Cl₂/MeOH (5×). The combined organic phase was dried

(MgSO₄), ﬁltered, and concentrated in vacuo to furnish the ﬁnal

compound which was puriﬁed as described.

4.2.5. General Procedure for Mitsunobu Coupling. Under an

inert atmosphere, triphenylphosphine (2.0 equiv) and DIAD (2.0

equiv) were dissolved in anhydrous tetrahydrofuran (THF) (0.05 M)

and stirred for 15 min at 0 °C. Subsequently, the alcohol compound

(1.0 equiv), the benzoate (1.1 equiv), and triethylamine (1.0 equiv)

were added, and the reaction mixture was heated at 80 °C for 3 h,

cooled to room temperature, and THF was removed in vacuo. The

crude product was puriﬁed by ﬂash column chromatography using

the speciﬁed eluent.

4.2.6. 4-(((3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-5-(1H-pyrrol-3-

yl)isoxazol-4-yl)methyl) amino) Benzoic Acid (2). According to the

general procedure for ester hydrolysis and N-Boc deprotection, ester

17 (0.035 g, 0.067 mmol) was hydrolyzed with a concomitant loss of

the Boc protecting group. The crude product was puriﬁed by column

chromatography, eluted with 1.5% MeOH in CH₂₁Cl₂, to furnish

carboxylic acid 2 (10.3 mg, 33%) as a white solid. H NMR (400

MHz, DMSO-d₆): δ (ppm) 11.98 (1H, br s, CO₂H), 11.52 (1H, s,

pyrrole-NH), 7.90 (1H, d, J = 8.0 Hz, ArH-3), 7.84 (1H, d, J = 7.8

Hz, ArH-5), 7.72 (1H, app. t, J = 8.0 Hz, ArH-4), 7.53 (2H, d, J = 8.7

Hz, benzoate C-2), 7.39 (1H, m, pyrrole H-2), 6.98 (1H, m, pyrrole

H-5), 6.55 (1H, m, pyrrole H-4), 6.41 (2H, d, J = 8.7 Hz, benzoate

H-3), 6.34 (1H, app. t, J = 4.4 Hz, CH₂NH), 4.07 (1H, dd, J = 14.4,

5.0 Hz, CH_aNH), 4.02 (1H, dd, J = 14.5, 4.5 Hz, CH_bNH); ¹³C

NMR (100 MHz, DMSO-d₆): δ (ppm) 167.9 (CO₂H), 165.7 (C-5),

159.2 (C-3), 152.3 (benzoate C-4), 136.0 (ArC-2), 134.1 (ArC-3),

132.4 (ArC-4), 131.3 (benzoate C-2), 130.9 (q, J = 30.4 Hz, ArC-6),

126.8 (ArC-1), 125.8 (q, J = 5.0 Hz, ArC-5), 122.0 (q, J = 274.3 Hz,

CF₃), 120.5 (pyrrole C-5), 119.2 (pyrrole C-2), 117.8 (benzoate C-

1), 111.2 (benzoate C-3), 110.5 (pyrrole C-3), 109.2 (C-4), 106.7

(pyrrole C-4), 36.2 (CH₂NH). LC−MS (ESI): calcd for

C₂₂H₁₅ClF₃N₃O₃[M + H]⁺, 462.08; observed, 462.17 (R_t= 6.13

min). HRMS (ESI): calcd for C₂₂H₁₅ClF₃N₃O₃[M + H]⁺, 462.0832;

observed, 462.0835.

4.2.7. 4-((3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-5-(1H-pyrrol-3-

yl)isoxazol-4-yl)methoxy) Benzoic Acid (3). According to the general

procedure for ester hydrolysis and N-Boc deprotection, ester 20

(0.020 g, 0.035 mmol) was hydrolyzed with a concomitant loss of the

Boc-protecting group. The crude product was puriﬁed by column

chromatography, eluting with 1.5% MeOH in CH₂₁Cl₂, to furnish

carboxylic acid 3 (12.0 mg, 75%) as a white solid. H NMR (400

MHz, acetone-d₆): δ (ppm) 10.78 (1H, br s, CO₂H), 7.92 (4H, m,

ArH-3, ArH-5 and benzoate C-2), 7.77 (1H, app. t, J = 8.0 Hz, ArH-

4), 7.47 (1H, m, pyrrole H-2), 7.03 (1H, m, pyrrole H-5), 6.94 (2H,

d, J = 8.8 Hz, benzoate H-3), 6.64 (1H, m, pyrrole H-4), 5.04 (2H, s,

CH₂O); ¹³C NMR (100 MHz, acetone-d₆): δ (ppm) 167.6 (C-5),

167.3 (benzoate C-4), 163.1 (CO₂H), 159.8 (C-3), 137.2 (ArC-2),

134.4 (ArC-3), 132.8 (q, J = 30.4 Hz, ArC-6), 132.6 (benzoate C-2),

132.5 (ArC-4), 127.9 (ArC-1), 126.1 (q, J = 5.0 Hz, ArC-5), 124.2

Laboratories. Solvents were removed in vacuo using a Buchi rotary

̈

evaporator and a diaphragm pump. Commercially available starting

materials were obtained from Sigma-Aldrich, TCI Chemicals and

Fluorochem. Proton (¹H) NMR (400 MHz), carbon (¹³C) NMR

(100 MHz), and 2D NMR (400 MHz) were recorded on a Bruker

Avance 400 MHz NMR spectrometer. Proton spectra are referenced

to tetramethyl silane (TMS). Carbon spectra are referenced to TMS

or the solvent peak of the deuterated spectrum. NMR spectra are

reported as follows: chemical shift (δ) in parts per million (ppm),

multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m =

multiplet, dd = doublet of doublet, td = triplet of doublets, app. =

apparent), coupling constant (J) in Hertz (Hz) (if applicable), and

integration (proton spectra only). Peak assignments are based on

additional 2D NMR techniques (COSY, HMBC, and HSQC).

Analytical liquid chromatography coupled with mass spectrometry

(LC−MS) was performed on a C4 Jupiter SuC4300A 150 × 2.0 mm

column using ultrapure water with 0.1% formic acid (FA) and

acetonitrile with 0.1% FA, in general with a gradient of 5−100%

acetonitrile for over 10 min, connected to a Thermo Fisher LCQ ﬂeet

ion-trap mass spectrometer. The purity of the samples was assessed

using a UV detector at 254 nm. Unless otherwise stated all ﬁnal

compounds were >95% pure as judged by HPLC. High-resolution

mass spectra (HRMS) were recorded using a Waters ACQUITY

UPLC I-Class LC system coupled to a Xevo G2 quadrupole time-of-

ﬂight (Q-TOF) mass spectrometer. Preparative HP-LC was

performed on a Gemini S4 110A 150 × 21.20 mm column using

ultrapure water with 0.1% FA and acetonitrile with 0.1% FA with

various gradients (mentioned for each compound speciﬁcally).

Column chromatography was either performed manually using silica

gel (60−200 μm particle size, 60 AÅ) or using an automated Grace

Reveleris X2 chromatograph with pre-packed silica columns supplied

by Buchi/Grace (40 μm particle size). The reaction progress was

monitored by thin-layer liquid chromatography (TLC) using Merck

TLC silica gel 60 F254 plates. The visualization of the plates was

achieved using an ultraviolet lamp (λ_max= 254 nm).

4.2.1. General Procedure for Suzuki Coupling. Under an inert

atmosphere, pinacol boronate (2.0 equiv), Cs₂CO₃(2.0 equiv), and

Pd(dppf)Cl₂(0.1 equiv) were added to a solution of bromide 14 (1.0

equiv) in degassed 1,2-dimethoxyethane (DME) (0.1 M). The

reaction mixture was heated at 85 °C for 8 h, cooled to room

temperature, diluted with H₂O, and extracted with EtOAc (3×). The

combined organic phase was washed with brine, dried over MgSO₄,

ﬁltered, and concentrated in vacuo. The crude product was puriﬁed by

ﬂash column chromatography using the speciﬁed eluent to aﬀord the

desired product.

4.2.2. General Procedure for the Reduction of Esters to Alcohols.

Under an inert atmosphere, DIBAL (1.23 g/mL in cyclohexane, 15.0

equiv) was added dropwise to a solution of ester (1.0 equiv) in

anhydrous CH₂Cl₂(0.1 M) at −78 °C. The reaction mixture was

followed by TLC analysis. Upon complete consumption of the

starting material, the reaction mixture was warmed to room

temperature, quenched by the addition of a saturated Rochelle salt

(KNaC₄H₄O₆·4H₂O) solution and stirred vigorously for 60 min.

Subsequently, the mixture was extracted with CH₂Cl₂(3×) and

separated. The combined organic phase was washed with brine, dried

9249

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(benzoate C-1), 122.7 (q, J = 274.3 Hz, CF₃), 121.0 (pyrrole C-5),

120.0 (pyrrole C-2), 115.1 (benzoate C-3), 111.6 (pyrrole C-3),

108.3 (C-4), 107.4 (pyrrole C-4), 60.8 (CH₂O). LC−MS (ESI):

calcd for C₂₂H₁₄ClF₃N₂O₄[M + H]⁺, 463.06; observed, 463.25 (R_t=

4.62 min). HRMS (ESI): calcd for C₂₂H₁₄ClF₃N₂O₄[M + H]⁺,

463.0672; observed, 463.0650.

4.90 min). HRMS (ESI): calcd for C₂₃H₁₄ClF₃N₂O₃[M + H]⁺,

459.0723; observed, 459.0726.

4.2.11. (Z)-4-(2-(3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-5-(1H-

pyrrol-3-yl)isoxazol-4-yl) vinyl) Benzoic Acid (7). According to the

general procedure for ester hydrolysis and N-Boc deprotection, ester

25 (0.040 g, 0.070 mmol) was hydrolyzed with a concomitant loss of

the Boc-protecting group. The crude product was puriﬁed via

preparative HPLC (gradient of 70−73% acetonitrile in H₂O) to

4.2.8. 4-(((3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-5-(1H-pyrrol-3-

yl)isoxazol-4-yl)methyl)thio) Benzoic Acid (4). According to the

general procedure for ester hydrolysis and N-Boc deprotection, ester

22 (0.020 g, 0.034 mmol) was hydrolyzed with a concomitant loss of

the Boc protecting group. The crude product was puriﬁed by column

chromatography, eluting with 2% MeOH in CH₂Cl₂, to furnish

1

furnish carboxylic acid 7 (26.0 mg, 81%) as a white solid. H NMR

(400 MHz, acetone-d₆): δ (ppm) 10.50 (1H, br s, CO₂H), 7.80 (5H,

m, ArH-3, ArH-5, ArH-4 and benzoate C-2), 7.36 (2H, d, J = 8.2 Hz,

benzoate H-3), 7.21 (1H, m, pyrrole H-2), 6.79 (1H, m, pyrrole H-

5), 6.74 (1H, d, J = 12.2 Hz, benzoate C4-HCCH), 6.49 (1H, m,

pyrrole H-4), 6.29 (1H, d, J = 12.2 Hz, C4-HCCH); ¹³C NMR

(100 MHz, acetone-d₆): δ (ppm) 166.3 (CO₂H), 163.7 (C-5), 158.4

(C-3), 141.1 (benzoate C-4), 136.1 (ArC-2), 133.5 (benzoate C4-

HCCH), 133.5 (ArC-3), 131.6 (ArC-4), 131.3 (q, J = 30.4 Hz,

ArC-6), 129.4 (benzoate C-2), 128.4 (benzoate C-3), 127.2

(benzoate C-1), 125.3 (q, J = 5.0 Hz, ArC-5), 124.5 (ArC-1),

121.8 (q, J = 274.3 Hz, CF₃), 119.3 (pyrrole C-5), 118.8 (pyrrole C-

2), 118.7 (C4-HCCH), 111.2 (pyrrole C-3), 108.6 (C-4), 106.3

(pyrrole C-4). LC−MS (ESI): calcd for C₂₃H₁₄ClF₃N₂O₃[M + H]⁺,

459.06; observed, 459.25 (R_t= 4.87 min). HRMS (ESI): calcd for

C₂₃H₁₄ClF₃N₂O₃[M + H]⁺, 459.0723; observed, 459.0715.

1

carboxylic acid 4 (8.00 mg, 49%) as a white solid. H NMR (400

MHz, acetone-d₆): δ (ppm) 10.77 (1H, br s, CO₂H), 7.89 (4H, m,

ArH-3, ArH-5 and benzoate C-2), 7.79 (1H, app. t, J = 8.0 Hz, ArH-

4), 7.52 (1H, m, pyrrole H-2), 7.31 (2H, d, J = 8.5 Hz, benzoate H-

3), 7.03 (1H, m, pyrrole H-5), 6.68 (1H, m, pyrrole H-4), 4.22 (2H,

s, CH₂S); ¹³C NMR (100 MHz, acetone-d₆): δ (ppm) 167.2

(CO₂H), 166.8 (C-5), 159.8 (C-3), 144.6 (benzoate C-4), 137.2

(ArC-2), 134.5 (ArC-3), 132.7 (ArC-4), 132.5 (q, J = 30.4 Hz, ArC-

6), 131.4 (benzoate C-1), 130.9 (benzoate C-2), 128.3 (ArC-1),

127.3 (benzoate C-3), 126.3 (q, J = 5.0 Hz, ArC-5), 122.7 (q, J =

274.3 Hz, CF₃), 120.9 (pyrrole C-5), 119.7 (pyrrole C-2), 111.6

(pyrrole C-3), 107.3 (C-4), 107.3 (pyrrole C-4), 26.2 (CH₂S). LC−

MS (ESI): calcd for C₂₂H₁₄ClF₃N₂O₃S [M + H]⁺, 479.04; observed,

479.25 (R_t= 4.86 min). HRMS (ESI): calcd for C₂₂H₁₄ClF₃N₂O₃S

[M + H]⁺, 479.0444; observed, 479.0438.

4.2.9. 4-(((3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-5-(1H-pyrrol-3-

yl)isoxazol-4-yl)methyl) (methyl)amino) Benzoic Acid (5). Accord-

ing to the general procedure for ester hydrolysis and N-Boc

deprotection, ester 18 (0.040 g, 0.068 mmol) was hydrolyzed with

a concomitant loss of the Boc protecting group. The crude product

was puriﬁed by column chromatography, eluting with 2% MeOH in

CH₂Cl₂, to furnish carboxylic acid 5 (19.3 mg, 60%) as a white solid.

¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 12.08 (1H, br s, CO₂H),

4.2.12. 4-(((3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-5-(1H-pyrrol-

3-yl)isoxazol-4-yl)methyl) amino)-2-ﬂuorobenzoic Acid (8). Ac-

cording to the general procedure for ester hydrolysis and N-Boc

deprotection, ester 19 (0.008 g, 0.016 mmol) was hydrolyzed. The

crude product was puriﬁed by column chromatography, eluting with

1.5% MeOH in CH₂Cl₂, to furnish carboxylic acid 8 (2.2 mg, 29%) as

1

a white solid. H NMR (400 MHz, DMSO-d₆): δ 12.09 (1H, br s,

CO₂H), 11.5 (1H, s, pyrrole-NH), 7.87 (1H, d, J = 8.0 Hz, ArH-3),

7.82 (1H, d, J = 7.9 Hz, ArH-5), 7.72 (1H, app. t, J = 7.9 Hz, ArH-4),

7.47 (1H, app. t, J = 8.8 Hz, benzoate H-2), 7.38 (1H, m, pyrrole H-

2), 6.97 (1H, m, pyrrole H-5), 6.64 (1H, app. t, J = 4.8 Hz, CH₂NH),

6.55 (1H, m, pyrrole H-4), 6.24 (1H, dd, J = 8.8, 2.1 Hz, benzoate H-

3_a), 6.10 (1H, dd, J = 14.4, 1.8 Hz, benzoate H-3_b), 4.06 (2H, m,

CH₂NH). LC−MS (ESI): calcd for C₂₂H₁₄ClF₄N₃O₃[M + H]⁺,

480.07; observed, 480.25 (R_t= 6.21 min). HRMS (ESI): calcd for

C₂₂H₁₄ClF₄N₃O₃[M + H]⁺, 480.0738; observed, 480.0759.

11.60 (1H, s, pyrrole-NH), 7.80 (1H, d, J = 8.1 Hz, ArH-3), 7.76

(1H, d, J = 7.8 Hz, ArH-5), 7.64 (1H, app. t, J = 8.0 Hz, ArH-4), 7.53

(1H, m, pyrrole H-2), 7.49 (2H, d, J = 8.9 Hz, benzoate C-2), 7.02

(1H, m, pyrrole H-5), 6.63 (1H, m, pyrrole H-4), 6.33 (2H, d, J = 8.9

Hz, benzoate H-3), 4.48 (2H, s, CH₂NCH₃), 2.54 (3H, s,

CH₂NCH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 167.3

(CO₂H), 165.0 (C-5), 158.5 (C-3), 151.7 (benzoate C-4), 135.5

(ArC-2), 133.6 (ArC-3), 131.8 (ArC-4), 130.5 (benzoate C-2), 130.1

(q, J = 30.4 Hz, ArC-6), 126.5 (ArC-1), 125.4 (q, J = 5.0 Hz, ArC-5),

124.2 (q, J = 274.3 Hz, CF₃), 120.2 (pyrrole C-5), 118.8 (pyrrole C-

2), 117.3 (benzoate C-1), 110.5 (benzoate C-3), 109.7 (pyrrole C-3),

109.0 (C-4), 106.3 (pyrrole C-4), 43.9 (CH₂NCH₃), 37.0

(CH₂NCH₃). LC−MS (ESI): calcd for C₂₃H₁₇ClF₃N₃O₃[M +

H]⁺, 476.09; observed, 476.17 (R_t= 6.33 min). HRMS (ESI): calcd

for C₂₃H₁₇ClF₃N₃O₃[M + H]⁺, 476.0989; observed, 476.0971.

4.2.10. (E)-4-(2-(3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-5-(1H-

pyrrol-3-yl)isoxazol-4-yl) vinyl) Benzoic Acid (6). According to the

general procedure for ester hydrolysis and N-Boc deprotection, ester

24 (0.012 g, 0.021 mmol) was hydrolyzed with a concomitant loss of

the Boc protecting group. The crude product was puriﬁed via

preparative HPLC (gradient of 70−73% acetonitrile in H₂O) to

4.2.13. 4-((3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-5-(1H-pyrrol-

3-yl)isoxazol-4-yl)methoxy)-2-ﬂuorobenzoic Acid (9). According to

the general procedure for ester hydrolysis and N-Boc deprotection,

ester 21 (0.017 mg, 0.029 mmol) was hydrolyzed with a concomitant

loss of the Boc protecting group. The crude product was puriﬁed by

column chromatography, eluting with 1.5% MeOH in CH₂Cl₂, to

1

furnish carboxylic acid 9 (8.0 mg, 57%) as a white solid. H NMR

(400 MHz, acetone-d₆): δ (ppm) 10.81 (1H, br s, CO₂H), 7.98 (3H,

m, ArH-3, ArH-5 and benzoate C-2), 7.78 (1H, app. t, J = 8.0 Hz,

ArH-4), 7.48 (1H, m, pyrrole H-2), 7.02 (1H, m, pyrrole H-5), 6.73

(2H, m, benzoate H-3), 6.64 (1H, m, pyrrole H-4), 5.06 (2H, s,

CH₂O); ¹³C NMR (100 MHz, acetone-d₆): δ (ppm) 167.4 (C-5),

165.5 (CO₂H), 164.0 (benzoate C-4), 162.9 (benzoate C−F), 159.7

(C-3), 137.2 (ArC-2), 134.4 (ArC-3), 132.7 (q, J = 30.4 Hz, ArC-6),

132.6 (Ar C-4), 132.4 (benzoate C-2), 127.8 (ArC-1), 126.1 (q, J =

5.0 Hz, ArC-5), 125.4 (benzoate C-1), 122.7 (q, J = 274.3 Hz, CF₃),

121.0 (pyrrole C-5), 120.0 (pyrrole C-2), 111.7 (benzoate C-3_a),

111.5 (pyrrole C-3), 108.0 (C-4), 107.4 (pyrrole C-4), 103.7

(benzoate C-3_b), 61.3 (CH₂O). LC−MS (ESI): calcd for

C₂₂H₁₃ClF₄N₂O₃[M + H]⁺, 481.05; observed, 481.25 (R_t= 4.92

min). HRMS (ESI): calcd for C₂₂H₁₃ClF₄N₂O₃[M + H]⁺, 481.0578;

observed, 481.0572.

4.2.14. 4-((3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-5-(1H-pyrrol-

2-yl)isoxazol-4-yl)methoxy) Benzoic Acid (10). According to the

general procedure for Mitsunobu coupling (without the addition of

triethylamine), alcohol 30 (0.080 g, 0.180 mmol) was reacted with

methyl 4-hydroxybenzoate (0.030 g, 0.200 mmol). The crude product

was puriﬁed by column chromatography, eluting with a gradient of

5−25% EtOAc in n-heptane, to furnish the ether compound (22.0

mg, 21%). The resulting product (0.012 g, 0.021 mmol) was subject

1

furnish carboxylic acid 6 (6.1 mg, 63%) as a white solid. H NMR

(400 MHz, acetone-d₆): δ (ppm) 10.85 (1H, br s, CO₂H), 8.00 (2H,

ArH-3 and ArH-5), 7.92 (3H, m, benzoate C-2 and ArH-4), 7.62

(1H, m, pyrrole H-2), 7.39 (3H, m, benzoate H-3 and C4-HC

CH), 7.08 (1H, m, pyrrole H-5), 6.74 (1H, m, pyrrole H-4), 6.15

(1H, s, benzoate C4-HCCH); ¹³C NMR (100 MHz, acetone-d₆):

δ (ppm) 166.3 (CO₂H), 165.2 (C-5), 156.8 (C-3), 141.5 (benzoate

C-4), 136.1 (ArC-2), 133.8 (ArC-3), 131.9 (ArC-4), 131.4 (q, J =

30.4 Hz, ArC-6), 130.0 (benzoate C-2), 129.4 (benzoate C-1), 128.1

(benzoate C4-HCCH), 128.0 (ArC-1), 126.0 (benzoate C-3),

125.6 (q, J = 5.0 Hz, ArC-5), 121.9 (q, J = 274.3 Hz, CF₃), 120.2

(pyrrole C-5), 119.3 (pyrrole C-2), 118.8 (C4-HCCH), 110.9

(pyrrole C-3), 110.3 (C-4), 106.7 (pyrrole C-4). LC−MS (ESI):

calcd for C₂₃H₁₄ClF₃N₂O₃[M + H]⁺, 459.06; observed, 459.25 (R_t=

9250

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to ester hydrolysis with a concomitant loss of the Boc protecting

group according to the general procedure for ester hydrolysis and N-

Boc deprotection. The crude product was puriﬁed by column

chromatography, eluting with 1.5% MeOH in CH₂₁Cl₂, to furnish

carboxylic acid 10 (4.0 mg, 41%) as a white solid. H NMR (400

MHz, acetone-d₆): δ (ppm) 11.13 (1H, br s, CO₂H), 7.91 (4H, m,

ArH-3, ArH-5 and benzoate C-2), 7.80 (1H, app. t, J = 8.0 Hz, ArH-

4), 7.19 (1H, m, pyrrole H-5), 6.93 (2H, d, J = 8.4 Hz, benzoate H-

3), 6.79 (1H, m, pyrrole H-3), 6.34 (1H, m, pyrrole H-4), 5.09 (2H,

s, CH₂O); ¹³C NMR (100 MHz, acetone-d₆): δ (ppm) 162.4 (C-5),

162.0 (CO₂H), 161.2 (benzoate C-4), 159.1 (C-3), 136.2 (ArC-2),

133.5 (ArC-3), 131.9 (benzoate C-2), 131.6 (ArC-4), 131.5 (q, J =

30.4 Hz, ArC-6), 126.5 (ArC-1), 125.3 (q, J = 5.0 Hz, ArC-5), 123.2

(benzoate C-1), 122.7 (pyrrole C-5), 121.8 (q, J = 274.3 Hz, CF₃),

118.7 (pyrrole C-2), 114.2 (benzoate C-3), 111.6 (pyrrole C-3),

111.3 (pyrrole C-4), 107.6 (C-4), 59.8 (CH₂O). LC−MS (ESI):

calcd for C₂₂H₁₄ClF₃N₂O₄[M + H]⁺, 463.06; observed, 463.17 (R_t=

4.87 min). HRMS (ESI): calcd for C₂₂H₁₄ClF₃N₂O₄[M + H]⁺,

463.0672; observed, 463.0665.

122.7 (q, J = 274.0 Hz, CF₃), 115.1 (benzoate C-3), 111.6 (pyrrole

C-3), 108.2 (C-4), 107.8 (pyrrole C-4), 60.8 (CH₂O), 36.7 (NCH₃).

LC−MS (ESI): calcd for C₂₃H₁₆ClF₃N₂O₄[M + H]⁺, 477.08;

observed, 477.33 (R_t= 5.06 min). HRMS (ESI): calcd for

C₂₃H₁₆ClF₃N₂O₄[M + H]⁺, 477.0829; observed, 477.0813.

4.2.17. 4-((3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-5-(5-methyl-

1H-pyrrol-3-yl)isoxazol-4-yl)methoxy) Benzoic Acid (13). According

to the general procedure for Mitsunobu coupling, alcohol 33 (0.175

g, 0.383 mmol) was reacted with methyl 4-hydroxybenzoate (0.064 g,

0.421 mmol). The crude product was puriﬁed by column

chromatography, eluting with a gradient of 10−40% EtOAc in n-

heptane, to furnish the ether compound (100 mg, 44%). The

resulting product (0.030 g, 0.051 mmol) was subjected to ester

hydrolysis with a concomitant loss of the Boc protecting group

according to the general procedure for ester hydrolysis and N-Boc

deprotection. The crude product was puriﬁed via preparative HPLC

(gradient of 65−70% acetonitrile in H₂O) to furnish carboxylic acid

1

13 (20.0 mg, 83%) as a white solid. H NMR (400 MHz, acetone-

d₆): δ (ppm) 10.47 (1H, br s, CO₂H), 7.90 (4H, m, ArH-3, ArH-5

and benzoate C-2), 7.77 (1H, app. t, J = 8.0 Hz, ArH-4), 7.29 (1H,

m, pyrrole H-2), 6.93 (2H, d, J = 8.5 Hz, benzoate H-3), 6.44 (1H,

m, pyrrole H-4), 5.02 (2H, s, CH₂O), 2.29 (3H, s, NCCH₃); ¹³C

NMR (100 MHz, acetone-d₆): δ (ppm) 167.7 (C-5), 167.3 (benzoate

C-4), 163.1 (CO₂H), 159.7 (C-3), 137.2 (ArC-2), 134.4 (ArC-3),

132.7 (q, J = 30.4 Hz, ArC-6), 132.5 (benzoate C-2), 132.4 (ArC-4),

130.7 (pyrrole C-5), 128.0 (ArC-1), 126.1 (q, J = 5.0 Hz, ArC-5),

124.2 (benzoate C-1), 122.7 (q, J = 274.3 Hz, CF₃), 119.7 (pyrrole

C-2), 115.1 (benzoate C-3), 111.7 (pyrrole C-3), 108.0 (C-4), 105.2

(pyrrole C-4), 60.8 (CH₂O), 12.6 (NCCH₃). LC−MS (ESI): calcd

for C₂₃H₁₆ClF₃N₂O₄[M + H]⁺, 477.08; observed, 477.25 (R_t= 4.88

min). HRMS (ESI): calcd for C₂₃H₁₆ClF₃N₂O₄[M + H]⁺, 477.0829;

observed, 477.0830.

4.2.15. 4-((3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-5-(1H-pyrazol-

4-yl)isoxazol-4-yl) methoxy) Benzoic Acid (11). According to the

general procedure for Mitsunobu coupling, alcohol 31 (0.100 g, 0.234

mmol) was reacted with methyl 4-hydroxybenzoate (0.039 g, 0.257

mmol). The crude product was puriﬁed by column chromatography,

eluting with a gradient of 10−25% EtOAc in n-heptane, to furnish the

ether compound (65.0 mg, 49%). The ether compound (0.045 g,

0.080 mmol), containing a THP-protected pyrazole, was diluted in a

mixture of TFA/CH₂Cl₂(50/50) (0.05 M), and the reaction mixture

was stirred at 40 °C for 2 h. Afterward, it was cooled to room

temperature, and the solvent was removed in vacuo. The crude

product was puriﬁed by column chromatography, eluting with 25−

40% EtOAc in n-heptane, to furnish the pyrazole product (28.0 mg,

88%). The resulting product was subjected to ester hydrolysis

according to the general procedure for ester hydrolysis and N-Boc

deprotection. The crude product was puriﬁed via preparative HPLC

(gradient of 62−67% acetonitrile in H₂O) to furnish carboxylic acid

4.2.18. Methyl 5-Bromo-3-(2-chloro-6-(triﬂuoromethyl)phenyl)-

isoxazole-4-carboxylate (15). According to the general procedure

for Suzuki coupling, bromide 14 (0.900 g, 2.34 mmol) was reacted

with tert-butyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-

pyrrole-1-carboxylate (1.37 g, 4.68 mmol). The crude product was

puriﬁed by ﬂash column chromatography, eluting with a gradient of

5−10% EtOAc in n-heptane, to furnish 15 (0.553 g, 50%) as colorless

1

11 (26.0 mg, 89%) as a white solid. H NMR (400 MHz, acetone-

d₆): δ (ppm) 8.24 (2H, s, pyrazole H-3 and H-5), 7.92 (4H, m, ArH-

3, ArH-5 and benzoate C-2), 7.80 (1H, app. t, J = 7.9 Hz, ArH-4),

6.94 (2H, d, J = 8.1 Hz, benzoate H-3), 5.12 (2H, s, CH₂O); ¹³C

NMR (100 MHz, acetone-d₆): δ (ppm) 167.2 (CO₂H), 164.6 (C-5),

162.9 (benzoate C-4), 159.9 (C-3), 137.1 (ArC-2), 134.4 (ArC-3),

133.6 (pyrazole C-3 and C-5), 133.0 (q, J = 30.4 Hz, ArC-6), 132.8

(ArC-4), 132.4 (benzoate C-2), 127.4 (ArC-1), 126.2 (q, J = 5.0 Hz,

ArC-5), 124.3 (benzoate C-1), 122.7 (q, J = 274.3 Hz, CF₃), 115.1

(benzoate C-3), 110.2 (C-4), 109.6 (pyrrole C-4). LC−MS (ESI):

calcd for C₂₁H₁₃ClF₃N₃O₄[M + H]⁺, 464.05; observed, 464.08 (R_t=

4.45 min). HRMS (ESI): calcd for C₂₁H₁₃ClF₃N₃O₄[M + H]⁺,

464.0625; observed, 464.0610.

1

oil. H NMR (400 MHz, CDCl₃): δ (ppm) 8.44 (1H, app. t, J = 2.0

Hz, pyrrole H-2), 7.76−7.65 (2H, m, ArH-3 and ArH-5), 7.55 (1H,

app. t, J = 8.0 Hz, ArH-4), 7.36 (1H, dd, J = 3.4, 2.2 Hz, pyrrole H-

5), 6.99 (1H, dd, J = 3.4, 1.6 Hz, pyrrole H-4), 3.61 (3H, s,

CO₂CH₃), 1.64 (9H, s, C(CH₃)₃); ¹³C NMR (100 MHz, CDCl₃): δ

(ppm) 168.9 (C-5), 161.5 (CO₂CH₃), 158.9 (C-3), 148.1 (NCO₂),

136.2 (ArC-2), 132.7 (ArC-3), 131.4 (q, J = 31.4 Hz, ArC-6), 130.5

(ArC-4), 127.7 (ArC-1), 124.6 (q, J = 5.0 Hz, ArC-5), 124.1 (pyrrole

C-2), 121.6 (q, J = 274.4 Hz, CF₃), 120.9 (pyrrole C-5), 113.7

(pyrrole C-3), 111.5 (pyrrole C-4), 107.1 (C-4), 85.1 (C(CH₃)₃),

51.6 (CO₂CH₃), 27.9 (C(CH₃)₃). LC−MS (ESI): calcd for

C₂₁H₁₈ClF₃N₂O₅[M + H]⁺, 471.09; observed, 470.92 (R_t= 6.04

min).

4.2.16. 4-((3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-5-(1-methyl-

1H-pyrrol-3-yl)isoxazol-4-yl)methoxy) Benzoic Acid (12). According

to the general procedure for Mitsunobu coupling, alcohol 32 (0.175

g, 0.490 mmol) was reacted with methyl 4-hydroxybenzoate (0.082 g,

0.540 mmol). The crude product was puriﬁed by column

chromatography, eluting with a gradient of 5−20% EtOAc in n-

heptane, to furnish the ether compound (109 mg, 45%). The

resulting product (0.060 g, 0.122 mmol) was subjected to ester

hydrolysis according to the general procedure for ester hydrolysis and

N-Boc deprotection. The crude product was puriﬁed by column

chromatography, eluting with 1.5% MeOH in CH₂Cl₂, to furnish

4.2.19. tert-Butyl 3-(3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-4-

(hydroxymethyl)isoxazol-5-yl)-1H-pyrrole-1-carboxylate (16).

Ester 15 (0.100 g, 0.212 mmol) was treated according to the general

procedure for the reduction of esters to alcohols. The crude product

was puriﬁed by ﬂash column chromatography, eluting with a gradient

of 10−20% EtOAc in n-heptane, to furnish alcohol 16 (0.075 g, 80%)

1

as colorless oil. H NMR (400 MHz, CDCl₃): δ (ppm) 7.88 (1H, s,

pyrrole H-2), 7.78−7.68 (2H, m, ArH-3 and ArH-5), 7.57 (1H, app.

t, J = 8.0 Hz, ArH-4), 7.37−7.33 (1H, m, pyrrole H-5), 6.79−6.73

(1H, m, pyrrole H-4), 4.42 (2H, br s, CH₂OH), 1.63 (9H, s,

C(CH₃)₃); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 164.6 (C-5),

158.7 (C-3), 148.2 (NCO₂), 136.5 (ArC-2), 133.1 (ArC-3), 132.2 (q,

J = 31.2 Hz, ArC-6), 131.0 (ArC-4), 126.7 (ArC-1), 124.9 (q, J = 5.1

Hz, ArC-5), 121.5 (q, J = 274.5 Hz, CF₃), 121.4 (pyrrole C-5), 120.0

(pyrrole C-2), 114.5 (pyrrole C-3), 113.1 (C-4), 110.3 (pyrrole C-4),

84.9 (C(CH₃)₃), 53.8 (CH₂OH), 27.9 (C(CH₃)₃). LC−MS (ESI):

calcd for C₂₀H₁₈ClF₃N₂O₄[M + H]⁺, 443.09; observed, 443.08 (R_t=

7.23 min).

1

carboxylic acid 12 (50.0 mg, 86%) as a white solid. H NMR (400

MHz, acetone-d₆): δ (ppm) 7.92 (4H, m, ArH-3, ArH-5 and

benzoate C-2), 7.76 (1H, app. t, J = 8.0 Hz, ArH-4), 7.38 (1H, m,

pyrrole H-2), 6.92 (2H, d, J = 8.8 Hz, benzoate H-3), 6.88 (1H, m,

pyrrole H-5), 6.56 (1H, m, pyrrole H-4), 5.04 (2H, s, CH₂O), 3.78

(3H, s, NCH₃); ¹³C NMR (100 MHz, acetone-d₆): δ (ppm) 167.3

(C-5), 167.1 (CO₂H), 163.1 (benzoate C-4), 159.7 (C-3), 137.1

(ArC-2), 134.3 (ArC-3), 132.7 (q, J = 30.4 Hz, ArC-6), 132.5 (ArC-

4), 132.4 (benzoate C-2), 127.9 (ArC-1), 126.1 (q, J = 5.1 Hz, ArC-

5), 124.8 (pyrrole C-5), 124.1 (benzoate C-1), 123.3 (pyrrole C-2),

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4.2.20. tert-Butyl 3-(3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-4-

(((4-(methoxycarbonyl) phenyl)amino)methyl)isoxazol-5-yl)-1H-

pyrrole-1-carboxylate (17). According to the general procedure for

mesylation and substitution, alcohol 16 (0.072 g, 0.163 mmol) was

mesylated and reacted with methyl 4-aminobenzoate (0.122 g, 0.805

mmol) in situ. The crude product was puriﬁed by column

chromatography, eluting with a gradient of 5−20% EtOAc in n-

heptane, to furnish 17 (32.2 mg, 36%) as colorless oil. ¹H NMR (400

MHz, CDCl₃): δ 7.83 (2H, d, J = 8.2 Hz, benzoate H-2), 7.69 (3H,

m, ArH-3, ArH-5 and pyrrole H-2), 7.53 (1 H, app. t, J = 8.0 Hz,

ArH-4), 7.34 (1H, m, pyrrole H-5), 6.65 (1H, m, pyrrole H-4), 6.51

(2H, d, J = 8.6 Hz, benzoate H-3), 4.09 (2H, m, CH₂NH), 4.06 (1H,

m, CH₂NH), 3.84 (3H, s, OCH₃), 1.52 (9H, s, (C(CH₃)₃); ¹³C

NMR (100 MHz, CDCl₃): δ (ppm) 167.1 (COOCH₃), 164.3 (C-5),

159.1 (C-3), 150.9 (benzoate C-4), 148.0 (NCO₂), 136.5 (ArC-2),

133.2 (ArC-3), 132.1 (q, J = 31.2 Hz, ArC-6), 131.5 (benzoate C-2),

131.2 (ArC-4), 126.4 (ArC-1), 125.0 (q, J = 5.0 Hz, ArC-5), 121.6

(pyrrole C-5), 121.5 (q, J = 274.5 Hz, CF₃), 119.7 (pyrrole C-2),

119.2 (benzoate C-1), 114.2 (pyrrole C-3), 110.3 (C-4), 111.5

(benzoate C-3), 109.9 (pyrrole C-4), 85.0 (C(CH₃)₃), 51.6 (OCH₃),

36.5 (CH₂NH), 27.8 (C(CH₃)₃). LC−MS (ESI): calcd for

C₂₈H₂₅ClF₃N₃O₅[M + H]⁺, 576.14; observed, 576.08 (R_t= 8.23

min).

(MgSO₄), ﬁltered, and concentrated in vacuo. The crude product was

puriﬁed by ﬂash column chromatography, eluting with a gradient of

30−40% EtOAc in n-heptane, to furnish compound 19 (10.5 mg,

1

17%). H NMR (400 MHz, DMSO-d₆): δ 11.4 (1H, s, pyrrole-NH),

7.89 (1H, d, J = 8.0 Hz, ArH-5), 7.84 (1H, d, J = 7.5 Hz, ArH-3),

7.72 (1H, app. t, J = 8.0 Hz, ArH-4), 7.47 (1H, app. t, J = 8.7 Hz,

benzoate H-2), 7.40 (1H, m, pyrrole H-2), 6.99 (1H, m, pyrrole H-

5), 6.76 (1H, app. t, J = 4.6 Hz, CH₂NH), 6.55 (1H, m, pyrrole H-4),

6.25 (1H, dd, J = 8.9, 1.9 Hz, benzoate H-3_a), 6.10 (1H, d, J = 14.6

Hz, benzoate H-3_b), 4.17 (2H, m, CH₂NH). LC−MS (ESI): calcd for

C₂₃H₁₆ClF₄N₃O₃[M + H]⁺, 494.08; observed, 494.25 (R_t= 7.14

min).

4.2.23. tert-Butyl 3-(3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-4-((4-

(methoxycarbonyl) phenoxy)methyl)isoxazol-5-yl)-1H-pyrrole-1-

carboxylate (20). According to the general procedure for Mitsunobu

coupling, alcohol 16 (0.023 g, 0.053 mmol) was reacted with methyl

4-hydroxybenzoate (0.010 g, 0.058 mmol). The crude product was

puriﬁed by column chromatography, eluting with a gradient of 5−

25% EtOAc in n-heptane, to furnish 20 (16.6 mg, 55%) as a white

solid. ¹H NMR (400 MHz, CDCl₃): δ 7.92 (2H, d, J = 8.9 Hz,

benzoate H-2), 7.71 (3H, m, ArH-3, ArH-5 and pyrrole H-2), 7.56 (1

H, app. t, J = 8.0 Hz, ArH-4), 7.35 (1H, m, pyrrole H-5), 6.82 (2H, d,

J = 8.9 Hz, benzoate H-3), 6.66 (1H, m, pyrrole H-4), 4.83 (2H, s,

CH₂O), 3.87 (3H, s, OCH₃), 1.55 (9H, s, (C(CH₃)₃); ¹³C NMR

(100 MHz, CDCl₃): δ (ppm) 166.7 (COOCH₃), 165.1 (C-5), 161.8

(benzoate C-4), 158.9 (C-3), 148.1 (NCO₂), 136.7 (ArC-2), 133.1

(ArC-3), 132.3 (q, J = 31.2 Hz, ArC-6), 131.6 (benzoate C-2), 131.1

(ArC-4), 126.4 (ArC-1), 124.9 (q, J = 5.0 Hz, ArC-5), 123.2

(benzoate C-1), 121.6 (pyrrole C-5), 121.4 (q, J = 274.5 Hz, CF₃),

120.0 (pyrrole C-2), 114.2 (pyrrole C-3), 114.1 (benzoate C-3),

110.2 (pyrrole C-4), 109.3 (C-4), 85.0 (C(CH₃)₃), 59.4 (CH₂O),

51.9 (OCH₃), 27.8 (C(CH₃)₃). LC−MS (ESI): calcd for

C₂₈H₂₄ClF₃N₂O₆[M + H]⁺, 577.13; observed, 577.17 (R_t= 8.57

min).

4.2.21. tert-Butyl 3-(3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-4-

(((4-(methoxycarbonyl) phenyl) (methyl)amino)methyl)isoxazol-5-

yl)-1H-pyrrole-1-carboxylate (18). According to the general

procedure for mesylation and substitution, alcohol 16 (0.120 g,

0.271 mmol) was mesylated and reacted with methyl 4-

(methylamino)benzoate (0.190 g, 1.15 mmol) in situ. The crude

product was puriﬁed by column chromatography, eluting with a

gradient of 5−20% EtOAc in n-heptane, to furnish 18 (70.3 mg,

1

52%) as colorless oil. H NMR (400 MHz, CDCl₃): δ 7.70 (3H, m,

benzoate H-2 and pyrrole H-2), 7.60 (1H, d, J = 7.8 Hz, ArH-5), 7.50

(1 H, d, J = 8.0 Hz, ArH-3), 7.41 (1H, app. t, J = 8.0 Hz, ArH-4),

7.38 (1H, m, pyrrole H-4), 6.68 (1H, m, pyrrole H-5), 6.39 (2H, d, J

= 8.8 Hz, benzoate H-3), 4.42 (2H, m, CH₂NCH₃), 3.84 (3H, s,

OCH₃), 2.66 (3H, s, CH₂NCH₃), 1.52 (9H, s, (C(CH₃)₃); ¹³C NMR

(100 MHz, CDCl₃): δ (ppm) 167.2 (COOCH₃), 163.3 (C-5), 159.0

(C-3), 152.2 (benzoate C-4), 148.1 (NCO₂), 136.7 (ArC-2), 133.0

(ArC-3), 131.7 (q, J = 31.2 Hz, ArC-6), 131.0 (benzoate C-2), 130.8

(ArC-4), 126.9 (ArC-1), 124.9 (q, J = 5.0 Hz, ArC-5), 121.6 (q, J =

274.5 Hz, CF₃), 121.5 (pyrrole C-5), 119.5 (pyrrole C-2), 117.8

(benzoate C-1), 114.3 (pyrrole C-3), 111.5 (C-4), 110.9 (benzoate

C-3), 110.2 (pyrrole C-4), 85.1 (C(CH₃)₃), 51.5 (OCH₃), 44.2

(CH₂NCH₃), 36.8 (CH₂NCH₃), 27.9 (C(CH₃)₃). LC−MS (ESI):

calcd for C₂₉H₂₇ClF₃N₃O₅[M + H]⁺, 590.16; observed, 590.00 (R_t=

8.64 min).

4.2.22. Methyl 4-(((3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-5-(1H-

pyrrol-3-yl)isoxazol-4-yl) methyl)amino)-2-ﬂuorobenzoate (19).

Alcohol compound 16 (0.256 g, 0.580 mmol) was dissolved in

anhydrous CH₂Cl₂(8 mL). To this was added Dess−Martin

periodinane (0.369 mg, 0.870 mmol), and the reaction mixture was

stirred at room temperature for 3 h. The reaction mixture was

quenched by the addition of 10% aqueous Na₂S₂O₃solution and

extracted with CH₂Cl₂(3×). The combined organic phase was

washed with saturated aqueous NaHCO₃and H₂O, dried over

MgSO₄, ﬁltered, and concentrated in vacuo. The crude product was

puriﬁed by column chromatography, eluting with a gradient of 20−

30% EtOAc in n-heptane, to furnish the aldehyde (112.0 mg, 44%).

Methyl 4-amino-2-ﬂuorobenzoate (0.052 g, 0.310 mmol) was added

to a solution of the aldehyde (0.112 g, 0.250 mmol) and AcOH (1.43

μL, 0.025 mmol) in MeOH (2 mL). The reaction mixture was heated

at the reﬂux for 24 h and then concentrated in vacuo. The

intermediate imine was isolated by ﬂash column chromatography,

eluting with a gradient of 10−30% EtOAc in n-heptane (62.3 mg,

42%), and then dissolved in EtOH (1 mL), cooled to 0 °C (ice), and

treated with NaBH₄(0.020 mg, 0.526 mmol). The reaction mixture

was stirred at 86 °C for 4 h. The solvent was removed in vacuo, and

the mixture was dissolved in CH₂Cl₂and washed with water. The

aqueous phase was further extracted with CH₂Cl₂(3×), dried

4.2.24. tert-Butyl 3-(3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-4-((3-

ﬂuoro-4-(methoxy carbonyl)phenoxy)methyl)isoxazol-5-yl)-1H-

pyrrole-1-carboxylate (21). According to the general procedure for

Mitsunobu coupling, alcohol 16 (0.050 g, 0.113 mmol) was reacted

with methyl 2-ﬂuoro-4-hydroxybenzoate (0.021 g, 0.124 mmol). The

crude product was puriﬁed by column chromatography, eluting with a

gradient of 5−25% EtOAc in n-heptane, to furnish 21 (17.0 mg,

1

25%) as a white solid. H NMR (400 MHz, CDCl₃): δ 7.85 (1H,

app. t, J = 8.6 Hz, benzoate H-2), 7.73 (3H, m, ArH-3, ArH-5 and

pyrrole H-2), 7.57 (1 H, app. t, J = 8.0 Hz, ArH-4), 7.36 (1H, m,

pyrrole H-5), 6.65 (2H, m, pyrrole H-4 and benzoate H-3_a), 6.52

(1H, dd, J = 12.4, 2.3 Hz, benzoate H-3_b), 4.82 (2H, s, CH₂O), 3.89

(3H, s, OCH₃), 1.57 (9H, s, (C(CH₃)₃); ¹³C NMR (100 MHz,

CDCl₃): δ (ppm) 165.2 (C-5), 164.5 (COOCH₃), 162.9 (benzoate

C−F), 161.9 (benzoate C-4), 158.9 (C-3), 148.0 (NCO₂), 136.7

(ArC-2), 133.6 (benzoate C-2), 133.2 (ArC-3), 132.3 (q, J = 31.2 Hz,

ArC-6), 131.2 (ArC-4), 126.2 (ArC-1), 125.0 (q, J = 5.0 Hz, ArC-5),

121.7 (pyrrole C-5), 121.4 (q, J = 274.5 Hz, CF₃), 120.0 (pyrrole C-

2), 114.1 (pyrrole C-3), 111.6 (benzoate C-3_a), 110.4 (benzoate C-

1), 110.2 (pyrrole C-4), 108.8 (C-4), 103.1 (benzoate C-3_b), 85.1

(C(CH₃)₃), 59.9 (CH₂O), 52.0 (OCH₃), 27.8 (C(CH₃)₃). LC−MS

(ESI): calcd for C₂₈H₂₃ClF₄N₂O₆[M + H]⁺, 595.12; observed,

595.00 (R_t= 6.14 min).

4.2.25. tert-Butyl 3-(3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-4-

(((4-(methoxycarbonyl) phenyl)thio)methyl)isoxazol-5-yl)-1H-pyr-

role-1-carboxylate (22). According to the general procedure for

Mitsunobu coupling, alcohol 16 (0.055 g, 0.124 mmol) was reacted

with methyl 4-mercaptobenzoate (0.024 g, 0.137 mmol). The crude

product was puriﬁed by column chromatography, eluting with a

gradient of 0−20% EtOAc in n-heptane, to furnish 21 (20.0 mg,

27%) as a white solid. ¹H NMR (400 MHz, CDCl₃): δ 7.84 (2H, d, J

= 8.5 Hz, benzoate H-2), 7.72 (3H, m, ArH-3, ArH-5 and pyrrole H-

2), 7.57 (1 H, app. t, J = 8.5 Hz, ArH-4), 7.34 (1H, m, pyrrole H-5),

7.18 (2H, d, J = 8.9 Hz, benzoate H-3), 6.68 (1H, m, pyrrole H-4),

4.01 (2H, s, CH₂O), 3.89 (3H, s, OCH₃), 1.58 (9H, s, (C(CH₃)₃).

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LC−MS (ESI): calcd for C₂₈H₂₄ClF₃N₂O₅S [M + H]⁺, 593.10;

observed, 592.92 (R_t= 6.28 min).

(benzoate C-4), 136.6 (ArC-2), 133.9 (C4-HCCH), 133.1 (ArC-

3), 132.0 (q, J = 31.5 Hz, ArC-6), 130.8 (ArC-4), 129.5 (benzoate C-

2), 129.1 (benzoate C-1), 128.2 (benzoate C-3), 126.8 (ArC-1),

124.9 (q, J = 5.0 Hz, ArC-5), 121.5 (q, J = 274.5 Hz, CF₃), 120.6

(pyrrole C-5), 119.7 (pyrrole C-2), 118.2 (benzoate C4-HCCH),

114.6 (pyrrole C-3), 110.8 (C-4), 110.0 (pyrrole C-4), 84.7

(C(CH₃)₃), 52.1 (OCH₃), 27.9 (C(CH₃)₃). LC−MS (ESI): calcd

for C₂₉H₂₄ClF₃IN₂O₅[M + H]⁺, 573.13; observed, 573.00 (R_t= 6.26

min).

4.2.26. tert-Butyl 3-(3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-4-

formylisoxazol-5-yl)-1H-pyrrole-1-carboxylate (23). Alcohol com-

pound 16 (0.150 g, 0.407 mmol) was dissolved in anhydrous CH₂Cl₂

(5 mL). To this was added Dess−Martin periodinane (0.216 mg,

0.610 mmol), and the reaction mixture was stirred at room

temperature for 3 h. The reaction mixture was quenched by the

addition of 10% aqueous Na₂S₂O₃solution and extracted with

CH₂Cl₂(3×). The combined organic phase was washed with

saturated aqueous NaHCO₃and H₂O, dried over MgSO₄, ﬁltered,

and concentrated in vacuo. The crude product was puriﬁed by column

chromatography, eluting with a gradient of 15−30% EtOAc in n-

4.2.28. Methyl 3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-5-(1H-pyr-

rol-2-yl)isoxazole-4-carboxylate (26). According to the general

procedure for Suzuki coupling, bromide 14 (0.500 g, 1.30 mmol)

was coupled to tert-butyl 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-

yl)-1H-pyrrole-1-carboxylate (0.762 g, 2.60 mmol). The crude

product was puriﬁed by ﬂash column chromatography, eluting with

a gradient of 5−10% EtOAc in n-heptane, to furnish 26 (0.298 g,

1

heptane, to furnish 21 (150 mg, 100%) as colorless oil. H NMR

(400 MHz, CDCl₃): δ (ppm) 9.67 (1H, s, CHO), 8.54 (1H, s,

pyrrole H-2), 7.81−7.73 (2H, m, ArH-3 and ArH-5), 7.63 (1H, app.

t, J = 8.0 Hz, ArH-4), 7.43−7.38 (1H, m, pyrrole H-5), 6.97−6.92

(1H, m, pyrrole H-4), 1.66 (9H, s, C(CH₃)₃). LC−MS (ESI): calcd

for C₂₀H₁₆ClF₃N₂O₄[M + H]⁺, 441.08; observed, 440.92 (R_t= 5.87

min).

1

49%) as colorless oil. H NMR (400 MHz, CDCl₃): δ (ppm) 7.77−

7.67 (2H, m, ArH-3 and ArH-5), 7.60−7.55 (2H, m, ArH-4 and

pyrrole H-5), 6.88 (1H, dd, J = 3.6, 1.7 Hz, pyrrole H-3), 6.36 (1H,

app. t, J = 3.4 Hz, pyrrole H-4), 3.57 (3H, s, CO₂CH₃), 1.52 (9H, s,

C(CH₃)₃); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 166.7 (C-5),

161.1 (CO₂CH₃), 158.4 (C-3), 148.3 (NCO₂), 136.3 (ArC-2), 132.8

(ArC-3), 131.5 (q, J = 31.4 Hz, ArC-6), 130.7 (ArC-4), 127.1 (ArC-

1), 126.2 (pyrrole C-5), 124.6 (q, J = 5.0 Hz, ArC-5), 121.6 (q, J =

274.4 Hz, CF₃), 121.2 (pyrrole C-3), 118.5 (pyrrole C-2), 111.1

(pyrrole C-4), 110.2 (C-4), 85.3 (C(CH₃)₃), 51.7 (CO₂CH₃), 27.7

(C(CH₃)₃). LC−MS (ESI): calcd for C₂₁H₁₈ClF₃N₂O₅[M + H]⁺,

471.09; observed, 470.92 (R_t= 5.73 min).

4.2.27. tert-Butyl (E) or (Z)-3-(3-(2-Chloro-6-(triﬂuoromethyl)-

phenyl)-4-(4-(methoxy carbonyl)styryl)isoxazol-5-yl)-1H-pyrrole-1-

carboxylate (24 and 25). Under an inert atmosphere, triphenyl-

phosphine (0.890 g, 3.39 mmol) and methyl 4-(bromomethyl)-

benzoate (0.519 g, 2.27 mmol) were dissolved in acetonitrile (20

mL). The reaction mixture was stirred at 82 °C for 3 h, when TLC

indicated the full conversion of the starting material. The reaction

mixture was cooled to room temperature and precipitated in toluene

(150 mL). The solid was ﬁltered, collected, and dried under reduced

pressure to yield (4-(methoxycarbonyl)benzyl) triphenylphospho-

nium as a crystalline white solid (930 mg, 100%). The resulting

product (0.152 g, 0.369 mmol) was dissolved in anhydrous THF (2

mL) under an inert atmosphere. The solution was cooled to −78 °C,

LiHMDS (0.4 mL, 1 M in hexane) was added and the solution was

stirred at −78 °C for 2 h. Aldehyde 23 (0.125 g, 0.284 mmol),

dissolved in anhydrous THF (2 mL), was added dropwise. The

reaction mixture was slowly warmed to room temperature and stirred

for 24 h. The reaction was quenched with H₂O (10 mL) and

extracted with EtOAc (3×). The combined organic phase was washed

with H₂O and brine, dried over MgSO₄, ﬁltrated, and concentrated in

vacuo. The crude product was puriﬁed by column chromatography,

eluting with a gradient of 5−20% EtOAc in n-heptane, to furnish a

mixture of cis/trans isomers (3:1) (70 mg). The mixture of cis/trans

isomers was separated via preparative HPLC (gradient of 80−100%

acetonitrile in H₂O) to furnish 24 (trans isomer, 12.0 mg, 7%) and

25 (cis isomer, 44.0 mg, 27%), both as white solids. Trans isomer

4.2.29. Methyl 3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-5-(1H-pyr-

azol-4-yl)isoxazole-4-carboxylate (27). According to the general

procedure for Suzuki coupling, bromide 14 (0.500 g, 1.30 mmol) was

reacted with tert-butyl 1-(3,4-dihydro-2H-pyran-6-yl)-4-(4,4,5-tri-

methyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.723 g, 2.60 mmol).

The crude product was puriﬁed by ﬂash column chromatography,

eluting with a gradient of 5−10% EtOAc in n-heptane, to furnish 27

(0.203 g, 34%) as colorless oil. ¹H NMR (400 MHz, CDCl₃): δ

(ppm) 8.78 (1H, s, pyrazole H-5), 8.34 (1H, s, pyrazole H-3), 7.75−

7.67 (2H, app. t, J = 9.2 Hz, ArH-3 and ArH-5), 7.56 (1H, app. t, J =

8.0 Hz, ArH-4), 5.51−5.46 (1H, m, NCHCO), 4.14−4.04 (1H, m,

THP-H), 3.78−3.72 (1H, m, THP-H), 3.60 (3H, s, CO₂CH₃), 2.20−

2.12 (2H, m, THP-H), 1.75−1.62 (4H, m, THP-H); ¹³C NMR (100

MHz, CDCl₃): δ (ppm) 167.8 (C-5), 161.6 (CO₂CH₃), 158.6 (C-3),

139.7 (pyrazole C-3), 136.2 (ArC-2), 132.7 (ArC-3), 131.4 (q, J =

31.3 Hz, ArC-6), 130.6 (ArC-4), 130.6 (pyrazole C-5), 127.5 (ArC-

1), 124.6 (q, J = 4.9 Hz, ArC-5), 121.6 (q, J = 274.4 Hz, CF₃), 109.6

(pyrazole C-4), 106.8 (C-4), 88.0 (NCHCO), 67.8 (THP-C), 51.7

(CO₂CH₃), 30.6 (THP-C), 24.8 (THP-C), 20.1 (THP-C). LC−MS

(ESI): calcd for C₂₀H₁₇ClF₃N₃O₄[M + H]⁺, 456.09; observed,

456.08 (R_t= 5.37 min).

1

(24): H NMR (400 MHz, CDCl₃): δ 7.92 (2H, d, J = 7.9 Hz,

benzoate H-2), 7.80 (3H, m, ArH-3, ArH-5 and pyrrole H-2), 7.63 (1

H, app. t, J = 7.8 Hz, ArH-4), 7.39 (1H, m, pyrrole H-5), 7.24 (2H, d,

J = 7.9 Hz, benzoate H-3), 7.03 (1H, d, J = 16.5 Hz, C4-HCCH),

6.73 (1H, m, pyrrole H-4), 6.17 (1H, d, J = 16.4 Hz, benzoate C4-

HCCH), 3.90 (3H, s, OCH₃), 1.64 (9H, s, (C(CH₃)₃); ¹³C NMR

(100 MHz, CDCl₃): δ (ppm) 166.7 (COOCH₃), 162.7 (C-5), 157.1

(C-3), 148.1 (NCO₂), 141.3 (benzoate C-4), 136.7 (ArC-2), 133.2

(ArC-3), 132.2 (q, J = 31.5 Hz, ArC-6), 131.1 (ArC-4), 129.9

(benzoate C-2), 129.8 (benzoate C4-HCCH), 129.2 (benzoate C-

1), 127.6 (ArC-1), 126.0 (benzoate C-3), 125.1 (q, J = 5.0 Hz, ArC-

5), 121.5 (pyrrole C-5), 121.4 (q, J = 274.5 Hz, CF₃), 119.7 (pyrrole

C-2), 118.0 (C4-HCCH), 114.6 (pyrrole C-3), 112.6 (C-4), 110.4

(pyrrole C-4), 85.1 (C(CH₃)₃), 52.1 (OCH₃), 27.9 (C(CH₃)₃). LC−

MS (ESI): calcd for C₂₉H₂₄ClF₃IN₂O₅[M + H]⁺, 573.13; observed,

573.00 (R_t= 6.44 min).

4.2.30. Methyl 3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-5-(1-

methyl-1H-pyrrol-3-yl)isoxazole-4-carboxylate (28). Under an

inert atmosphere 1-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-

2-yl)-1H-pyrrole (0.404 g, 1.95 mmol), Na₂CO₃(0.276 g, 2.6

mmol)), and Pd(PPh₃)₄(0.150 g, 0.13 mmol) were added to a

solution of bromide 14 (0.500 g, 1.3 mmol) in de-gassed DME/H₂O

(4:1) (15 mL). The reaction mixture was heated at 85 °C for 8 h,

cooled to room temperature, diluted with H₂O, and extracted with

EtOAc (3×). The combined organic phase was dried (MgSO₄),

ﬁltered, and concentrated in vacuo. The crude product was puriﬁed by

column chromatography, eluting with 5−15% EtOH in n-heptane, to

1

furnish 28 (0.275 g, 55%) as a yellow solid. H NMR (400 MHz,

CDCl₃): δ (ppm) 7.97 (1H, s, pyrrole H-2), 7.75−7.64 (2H, m, ArH-

3 and ArH-5), 7.52 (1H, app. t, J = 8.0 Hz, ArH-4), 6.92 (1H, br s,

pyrrole H-5), 6.69 (1H, br s, pyrrole H-4), 3.74 (3H, s, NCH₃), 3.56

(3H, s, CO₂CH₃); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 170.4 (C-

5), 162.1 (CO₂CH₃), 158.6 (C-3), 136.2 (ArC-2), 132.6 (ArC-3),

131.3 (q, J = 31.3 Hz, ArC-6), 130.3 (ArC-4), 128.2 (ArC-1), 126.8

(pyrrole C-2), 124.5 (q, J = 5.0 Hz, ArC-5), 123.1 (pyrrole C-5),

121.6 (q, J = 274.4 Hz, CF₃), 110.6 (pyrrole C-3), 109.4 (pyrrole C-

4), 104.9 (C-4), 51.4 (CO₂CH₃), 36.7 (NCH₃). LC−MS (ESI): calcd

Cis isomer (25): ¹H NMR (400 MHz, CDCl₃): δ 7.77 (2H, d, J =

8.0 Hz, benzoate H-2), 7.68 (2H, m, ArH-3 and ArH-5), 7.53 (1 H,

app. t, J = 8.0 Hz, ArH-4), 7.38 (1H, br s, pyrrole H-2), 7.28 (2H, d, J

= 8.0 Hz, benzoate H-3), 7.08 (1H, br s, pyrrole H-5), 6.62 (1H, d, J

= 12.1 Hz, C4-HCCH), 6.50 (1H, br s, pyrrole H-4), 6.18 (1H, d,

J = 12.1 Hz, benzoate C4-HCCH), 3.87 (3H, s, OCH₃), 1.57 (9H,

s, (C(CH₃)₃); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 166.7

(COOCH₃), 161.3 (C-5), 159.0 (C-3), 148.1 (NCO₂), 140.8

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for C₁₇H₁₂ClF₃N₂O₃[M + H]⁺, 385.05; observed, 385.17 (R_t= 5.27

min).

MS (ESI): calcd for C₁₉H₁₇ClF₃N₃O₃[M + H]⁺, 428.09; observed,

428.08 (R_t= 4.64 min).

4.2.31. Methyl 3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-5-(5-

methyl-1H-pyrrol-3-yl)isoxazole-4-carboxylate (29). Under an

inert atmosphere, a Schlenk tube was charged with tert-butyl 2,4-

dimethyl-1H-pyrrole-1-carboxylate (1.10 g, 6.05 mmol). Two

separate ﬂasks under argon were charged with [Ir(OMe)(COD)]₂

(0.060 g, 0.091 mmol) and dtbpy (0.049 g, 0.182 mmol). HBPin

(1.16 g, 9.08 mmol) was added to the [Ir(OMe)(COD)]₂containing

ﬂask. Anhydrous THF (18 mL) was added to the dtbpy-containing

ﬂask, and the solution was mixed with the [Ir(OMe)(COD)]₂and

HBPin mixture. The resulting solution was transferred to the Schlenk

ﬂask, and the reaction mixture was stirred at 60 °C for 6 h. Afterward,

the reaction mixture was cooled to room temperature and THF was

removed in vacuo. The crude product was puriﬁed by ﬂash column

chromatography, eluting with a gradient of 0−5% EtOAc in n-

heptane, to furnish tert-butyl 2-methyl-4-(4,4,5-trimethyl-1,3,2-

dioxaborolan-2-yl)-1H-pyrrole-1-carboxylate (1.30 g, 70%).⁵⁰

Subsequently, according to the general procedure for Suzuki

coupling, bromide 14 (1.09 g, 2.82 mmol) was reacted with the

synthesized pinacol boronate (1.30 g, 4.23 mmol). The crude product

was puriﬁed by ﬂash column chromatography, eluting with a gradient

of 30−60% CH₂Cl₂in n-heptane, to furnish 29 (0.325 g, 24%) as

4.2.34. (3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-5-(1-methyl-1H-

pyrrol-3-yl)isoxazol-4-yl) Methanol (32). Under an inert atmos-

phere, LiAlH₄(1 M in THF, 1.07 mL, 1.01 mmol) was added

dropwise to a solution of ester 28 (0.275 g, 0.710 mmol) in

anhydrous THF (6 mL) at 0 °C. The reaction mixture was warmed

to room temperature and stirred for 2 h. The reaction mixture was

quenched by the addition of saturated aqueous NH₄Cl solution and

stirred vigorously for 60 min. Subsequently, the mixture was extracted

with EtOAc (3×). The combined organic phase was washed with

brine, dried over MgSO₄, ﬁltered, and concentrated in vacuo. The

crude product was puriﬁed by ﬂash column chromatography, eluting

with a gradient of 10−20% EtOAc in n-heptane, to furnish alcohol 32

(0.192 g, 76%) as colorless oil. ¹H NMR (400 MHz, CDCl₃): δ

(ppm) 7.76−7.68 (2H, m, ArH-3 and ArH-5), 7.56 (1H, app. t, J =

8.3 Hz, ArH-4), 7.30 (1H, app. t, J = 1.9 Hz, pyrrole H-2), 6.70 (1H,

app. t, J = 2.5 Hz, pyrrole H-5), 6.68 (1H, app. t, J = 2.5 Hz, pyrrole

H-4), 4.38 (2H, br s, CH₂OH), 3.73 (3H, s, NCH₃); ¹³C NMR (100

MHz, CDCl₃): δ (ppm) 166.3 (C-5), 158.6 (C-3), 136.6 (ArC-2),

133.1 (ArC-3), 132.2 (q, J = 31.3 Hz, ArC-6), 130.9 (ArC-4), 127.1

(ArC-1), 124.9 (q, J = 5.0 Hz, ArC-5), 123.4 (pyrrole C-5), 122.5

(pyrrole C-2), 121.5 (q, J = 274.4 Hz, CF₃), 111.3 (pyrrole C-3),

110.9 (C-4), 107.5 (pyrrole C-4), 54.2 (CH₂OH), 36.6 (NCH₃).

LC−MS (ESI): calcd for C₁₆H₁₂ClF₃N₂O₂[M + H]⁺, 357.05;

observed, 357.17 (R_t= 4.63 min).

4.2.35. (3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-5-(5-methyl-1H-

pyrrol-3-yl)isoxazol-4-yl) Methanol (33). Ester 29 (0.400 g, 0.825

mmol) was treated according to the general procedure for the

reduction of esters to alcohols. The crude product was puriﬁed by

ﬂash column chromatography, eluting with a gradient of 10−25%

EtOAc in n-heptane, to furnish alcohol 33 (0.220 g, 58%) as colorless

oil. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.83 (1H, s, pyrrole H-2),

7.77−7.58 (2H, m, ArH-3 and ArH-5), 7.58 (1H, app. t, J = 8.0 Hz,

ArH-4), 6.48 (1H, s, pyrrole H-4), 4.42 (2H, br s, CH₂OH), 2.50

(3H, s, NCCH₃), 1.63 (9H, s, C(CH₃)₃); ¹³C NMR (100 MHz,

CDCl₃): δ (ppm) 164.8 (C-5), 158.7 (C-3), 149.1 (NCO₂), 136.5

(ArC-2), 133.4 (pyrrole C-5), 133.1 (ArC-3), 132.2 (q, J = 31.4 Hz,

ArC-6), 131.0 (ArC-4), 126.8 (ArC-1), 124.9 (q, J = 4.9 Hz, ArC-5),

121.5 (q, J = 274.4 Hz, CF₃), 120.7 (pyrrole C-2), 112.9 (C-4), 112.3

(pyrrole C-3), 109.9 (pyrrole C-4), 84.6 (C(CH₃)₃), 53.9 (CH₂OH),

28.0 (C(CH₃)₃), 15.4 (NCCH₃). LC−MS (ESI): calcd for

C₂₁H₂₀ClF₃N₂O₄[M + H]⁺, 457.11; observed, 456.92 (R_t= 5.53

min).

1

colorless oil. H NMR (400 MHz, CDCl₃): δ (ppm) 8.39 (1H, s,

pyrrole H-2), 7.74−7.65 (2H, m, ArH-3 and ArH-5), 7.54 (1H, app.

t, J = 8.0 Hz, ArH-4), 6.70 (1H, s, pyrrole H-4), 3.61 (3H, s,

CO₂CH₃), 2.51 (3H, s, NCCH₃), 1.64 (9H, s, C(CH₃)₃); ¹³C NMR

(100 MHz, CDCl₃): δ (ppm) 169.0 (C-5), 161.6 (CO₂CH₃), 158.9

(C-3), 148.9 (NCO₂), 136.2 (ArC-2), 132.8 (pyrrole C-5), 132.6

(ArC-3), 131.3 (q, J = 31.4 Hz, ArC-6), 130.5 (ArC-4), 127.7 (ArC-

1), 124.9 (pyrrole C-2), 124.5 (q, J = 4.9 Hz, ArC-5), 121.6 (q, J =

274.4 Hz, CF₃), 111.6 (pyrrole C-3), 110.9 (pyrrole C-4), 106.9 (C-

4), 84.7 (C(CH₃)₃), 51.6 (CO₂CH₃), 28.0 (C(CH₃)₃), 15.3

(NCCH₃). LC−MS (ESI): calcd for C₂₂H₂₀ClF₃N₂O₅[M + H]⁺,

485.10; observed, 485.00 (R_t= 6.22 min).

4.2.32. (3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-5-(1H-pyrrol-2-

yl)isoxazol-4-yl)methanol (30). Ester 26 (0.260 g, 0.552 mmol)

was treated according to the general procedure for the reduction of

esters to alcohols. The crude product was puriﬁed by ﬂash column

chromatography, eluting with a gradient of 10−20% EtOAc in n-

1

heptane, to furnish alcohol 30 (169 mg, 69%) as colorless oil. H

NMR (400 MHz, CDCl₃): δ (ppm) 7.77−7.69 (2H, m, ArH-3 and

ArH-5), 7.57 (1H, app. t, J = 8.0 Hz, ArH-4), 7.51 (1H, dd, J = 3.2,

1.7 Hz, pyrrole H-5), 6.70 (1H, dd, J = 3.4, 1.7 Hz, pyrrole H-3), 6.34

(1H, app. t, J = 3.4 Hz, pyrrole H-4), 4.34 (2H, br s, CH₂OH), 1.53

(9H, s, C(CH₃)₃); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 161.9 (C-

5), 158.6 (C-3), 148.7 (NCO₂), 136.5 (ArC-2), 133.2 (ArC-3), 132.1

(q, J = 31.2 Hz, ArC-6), 131.0 (ArC-4), 126.8 (ArC-1), 125.5

(pyrrole C-5), 125.0 (q, J = 5.1 Hz, ArC-5), 121.5 (q, J = 274.5 Hz,

CF₃), 119.9 (pyrrole C-3), 118.7 (pyrrole C-2), 116.9 (C-4), 111.3

(pyrrole C-4), 85.5 (C(CH₃)₃), 54.1 (CH₂OH), 27.8 (C(CH₃)₃).

LC−MS (ESI): calcd for C₂₀H₁₈ClF₃N₂O₄[M + H]⁺, 443.09;

observed, 443.00 (R_t= 5.27 min).

4.3. Biophysical Assays. 4.3.1. RORγt-LBD Expression and

Puriﬁcation. His-tagged RORγt LBD was expressed and puriﬁed as

described in previous studies.²⁸

4.3.2. TR-FRET Coactivator Recruitment Assay. Assays were

conducted using 100 nM N-terminal biotinylated SRC-1 box2

coactivator peptide (Biotin-N-PSSHSSLTARHKILHRLLQEGSPSD-

CONH₂) and 20 nM His₆-RORγt-LBD in buﬀer containing 10 mM

HEPES (pH 7.5), 150 mM NaCl, 5 mM dithiothreitol (DTT), 0.1%

bovine serum albumin (BSA) (w/v), and 0.1 mM CHAPS. A

terbium-labeled anti-His antibody (CisBio Bioassays, 61HISTLA)

and D2-labeled streptavidin (CisBio Bioassays, 610SADLA) were

used at the concentrations recommended by the supplier.

Compounds (dissolved in DMSO) were titrated using a 2× dilution

series in Corning white low volume, low binding, 384-well plates at a

ﬁnal volume of 10 μL. The ﬁnal DMSO concentration was 1% v/v

throughout. The plate was directly measured and centrifuged before

reading (excitation = 340 nm; emission = 665 and 620 nm) on a

Tecan inﬁnite F500 plate reader using the parameters recommended

by CisBio Bioassays. The data were analyzed with GraphPad Prism

7.0 software. The dose−response curve was ﬁtted represented by

4.2.33. (3-(2-Chloro-6-(triﬂuoromethyl)phenyl)-5-(1H-pyrazol-4-

yl)isoxazol-4-yl)methanol (31). Ester 27 (0.200 g, 0.434 mmol) was

treated according to the general procedure for the reduction of esters

to alcohols. The crude product was puriﬁed by ﬂash column

chromatography, eluting with a gradient of 30−50% EtOAc in n-

1

heptane, to furnish alcohol 31 (0.100 g, 71%) as colorless oil. H

NMR (400 MHz, CDCl₃): δ (ppm) 8.25 (1H, s, pyrazole H-5), 8.09

(1H, s, pyrazole H-3), 7.80−7.69 (2H, m, ArH-3 and ArH-5), 7.59

(1H, app. t, J = 8.0 Hz, ArH-4), 5.47 (1H, t, J = 6.1 Hz, NCHCO),

4.41 (1H, br s, CH₂OH), 4.14−3.98 (1H, m, THP-H), 3.76−3.71

(1H, m, THP-H), 2.17−1.98 (3H, m, THP-H), 1.78−1.58 (3H, m,

THP-H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 163.3 (C-5), 158.6

(C-3), 137.9 (pyrazole C-3), 136.5 (ArC-2), 133.2 (ArC-3), 132.2 (q,

J = 31.2 Hz, ArC-6), 131.1 (ArC-4), 127.5 (pyrazole C-5), 126.6

(ArC-1), 125.0 (q, J = 5.1 Hz, ArC-5), 121.5 (q, J = 274.4 Hz, CF₃),

112.8 (C-4), 110.2 (pyrazole C-4), 87.9 (NCHCO), 67.8 (THP-C),

53.9 (CH₂OH), 30.7 (THP-C), 24.9 (THP-C), 22.1 (THP-C). LC−

A₁− A₂

y = A₁+

x^p

1 +

p

x₀

where y is the FRET ratio ((acceptor/donor) × 1000), A₁is the

bottom asymptote, A₂is the top asymptote, p is the Hill slope, x is

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the ligand concentration in μM, and x₀is the IC₅₀value in μM.

Where dose−response curves did not reach a bottom asymptote, this

was ﬁxed at the value of the negative control. The data were recorded

in triplicate in three independent experiments (one representative

data set shown). Error bars represent the SD of the mean.

4.3.3. Competition TR-FRET Coactivator Recruitment Assay.

Competition assays were performed in an analogous fashion to that

described above, only in the presence of ﬁxed concentrations of

cholesterol: 0 μM (DMSO), 0.25 μM, and 1.0 μM such that the ﬁnal

concentration of DMSO remained at 1.2% v/v.

4.3.4. TR-FRET AlexaFluor-MRL-871 Recruitment Assay. Assays

were conducted using 100 nM AlexaFluor647-labeled MRL-871 and

20 nM His₆-RORγt-LBD in buﬀer as described above. A terbium-

labeled anti-His antibody (CisBio Bioassays, 61HISTLA) was used at

the concentrations recommended by the supplier. The assay was

carried out in the same manner as described above.

4.3.5. Thermal Shift Assay. TSA were performed using 40 μL

samples containing 5 μM RORγt-LBD, 10 μM compound, and 2.5x

SYPRO Orange (Sigma) in buﬀer containing 150 mM NaCl, 10 mM

HEPES (pH 7.5), and 1% DMSO. Hard-Shell 96-well PCR plates

(low proﬁle, thin wall, skirted, green/white #hsp9645) were used.

The samples were heated from 25 to 80 °C at a rate of 0.5 °C per 5 s

in a CFX96 touch real-time PCR detection system (Bio-Rad). After

each increment, Sypro Orange intensity was measured using the plate

read option in Bio-Rad CFX Manager 3.1. Melting temperatures were

determined by Bio-Rad CFX Manager 3.1 software in negative mode.

The data were recorded in triplicate in three independent

experiments (one representative data set shown). Error bars represent

the SD of the mean.

4.4. Quantitative IL-17a mRNA RT-PCR Assay. Cell culture,

RT-PCR experiments, and data analysis were performed as described

in previous studies.²⁸Statistical analysis was performed using a one-

way ANOVA, comparing against the DMSO control following the

Dunnett’s post hoc test (GraphPad Prism 7.0 software). A P value <

0.05 was considered statistically signiﬁcant. (Data recorded in

triplicate from two independent experiments. Data shown are

representative of two independent experiments. Error bars represent

the SD of the mean.)

4.5. Protein X-ray Crystallography. 4.5.1. RORγt-LBD Ex-

pression and Puriﬁcation (Used for Crystallography). His-tagged

RORγt LBD containing a C455H mutation was expressed and

puriﬁed as described in previous studies.²⁸

4.5.2. X-ray Crystallography. X-ray crystallography experiments

and data analysis were performed as described in previous studies.²⁸

Diﬀraction data were collected at the P11 beamline of the PETRA III

facility at DESY (Hamburg, Germany), the ID30B beamline of ESRF

(Grenola, France), and the i04 beamline of Diamond Light Source

(Didcot, UK). The structures of RORγtC455H in complex with 3, 9,

10, and 11 were deposited in the Protein Data Bank (PDB) under

codes 7NPC, 7NP5, 7NEC, and 7NP6.

4.6. Selectivity TR-FRET Coactivator Recruitment Assays.

4.6.1. TR-FRET Coactivator Recruitment Assays on PPARγ. TR-

FRET competition assays were performed in an analogous fashion to

that described above, only using 10 nM His₆-PPARγ LBD instead of

20 nM His₆-RORγt LBD and 200 nM N-terminal biotinylated

PGC1a coactivator peptide (Biotin-N-GTPPPQEAEEPSLLKLLLA-

PANTQ-CONH₂) instead of 100 nM SRC-1 box 2 coactivator

peptide.

4.6.2. Competition TR-FRET Coactivator Recruitment Assays on

PPARγ. TR-FRET competition assays were performed in an

analogous fashion to that described above, only using 100 nM

His₆-PPARγ LBD instead of 20 nM His₆-RORγt LBD. The assay was

performed in the presence of 1 μM rosiglitazone, in order to initially

activate PPARγ.

tagged FXR-LBD in buﬀer containing 10 mM HEPES (pH 7.5),

150 mM NaCl, 5 mM DTT, 0.1% BSA (w/v), and 0.1 mM CHAPS.

For the competition assay, the assay was performed in the presence of

0, 1, 10, or 50 μM of ligand 3. Compounds (dissolved in DMSO)

were titrated using a 2× dilution series in Corning black round-

bottom, low-volume, low-binding, 384-well plates at a ﬁnal volume of

10 μL. The ﬁnal DMSO concentration was 1% v/v throughout. The

plate was directly measured (excitation = 485 nm; emission = 535

nm) on a Tecan inﬁnite F500 plate reader. The data were analyzed

with GraphPad Prism 7.0 software. The dose−response curve was

ﬁtted in the same way as described for the TR-FRET data on RORγt.

The data were recorded in triplicate in three independent

experiments (one representative data set shown). Error bars represent

the SD of the mean.

4.7. ADME Experiments. 4.7.1. Kinetic Solubility. Aqueous

solubility of compounds was determined by the spectrophotometrical

measurement of the kinetic solubility of a 500 μM compound

solution in HEPES buﬀer pH 7.4 compared to a solution containing

50% of the organic solvent acetonitrile. To this end, saturated

samples of the compounds in buﬀer were prepared starting from

DMSO stocks, and samples were shaken for 90 min at room

temperature. The precipitated material was removed by ﬁltration and

samples were further diluted with the same volume of acetonitrile.

Absorbance spectra (250−500 nm) were recorded and relative

solubility was calculated in comparison to the acetonitrile−buﬀer

solution of the compounds using absorbance ratios.

4.7.2. Parallel Artiﬁcial Membrane Permeability Assay. Perme-

ability through artiﬁcial membranes [parallel artiﬁcial membrane

permeability assay (PAMPA)] was performed at an initial

concentration of 500 μM of the compound in the donor

compartment. After an incubation period of 20 h, the absorption of

the receiver wells was measured by spectrophotometry and

permeation was calculated by the normalization of the compound

ﬂux across a blank ﬁlter.

4.7.3. Microsomal StabilityPhase I. Metabolic stability under

oxidative conditions was measured using NADPH-supplemented

human liver microsomes. Compound depletion was analyzed by

liquid chromatography with tandem mass spectrometry (LC−MS/

MS) at a concentration of 1 μM over a time up to 50 min at 37 °C.

Based on compound half-life t_1/2, in vitro intrinsic clearance Cl_intwas

V × 0.693

t_1/2× mg

calculated. Cl_int

=

, with V = assay volume and mg = amount

of microsomal protein.

4.7.4. Microsomal StabilityIn Vitro Glucuronidation. Metabolic

stability under conjugative conditions was measured in the

glucuronidation assay by the LC−MS-based determination of %

remaining of selected compounds. Prior to the assay, human liver

microsomes were activated using alamethicin and further supple-

mented with 5 mM of the cofactor UDPGA. 5 μM compound was

added to the reaction mixture, and it was incubated for 1 h at 37 °C.

4.7.5. Plasma Stability. Plasma stability was measured by the LC−

MS-based determination of % remaining of selected compounds at a

concentration of 5 μM after incubation in 50% plasma in phosphate-

buﬀered saline (obtained from humans) for 1 h at 37 °C.

4.7.6. Plasma Protein Binding. Plasma protein binding was

determined by equilibrium dialysis. Plasma containing 5 μM of test

compound was allowed to equilibrate with the buﬀer compartment

for 6 h at 37 °C. Compound concentrations on both sides of the

semipermeable membrane were analyzed by LC−MS/MS and %

bound was calculated.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00475.

■

sı

4.6.3. TSA on PPARγ. TSA were performed in an analogous fashion

to that described above, only using PPARγ LBD instead of RORγt

LBD.

4.6.4. FP Assays on FXR. Assays were conducted using 100 nM N-

terminal FITC-labeled SRC-1 box2 coactivator peptide (FITC-N-

PSSHSSLTARHKILHRLLQEGSPSD-CONH₂) and 1 μM GST-

Chemical structure of AlexaFluor-MRL probe; dose−

response curves; schematic representation of TR-FRET

assays; docking poses of 4 and 12 in overlay with 3;

protein−ligand interaction plot for 3; electron density

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J. Med. Chem. 2021, 64, 9238−9258

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

around compounds 3, 9, 10, and 11; ﬂuorescence

polarization assay; synthesis route for isoxazole scaﬀold

14; original synthesis route for isoxazole compound

FM26; library of compounds used in docking studies;

toxicity risks, drug likeness, and drug score for all

compounds; data collection and reﬁnement statistics

(molecular replacement) of 3, 9, 10, and 11; synthesis,

biochemical assays, and docking experiments; NMR and

LC−UV spectra for assayed compounds; and details on

crystallography experiments (PDF)

Eindhoven, 5612 AZ Eindhoven, The Netherlands;

orcid.org/0000-0001-7315-0315

Peter J. Cossar − Laboratory of Chemical Biology,

Department of Biomedical Engineering and Institute for

Complex Molecular Systems, Technische Universiteit

Eindhoven, 5612 AZ Eindhoven, The Netherlands

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00475

Author Contributions

Molecular formula strings (CSV)

The manuscript was written through contributions of all the

authors. F.A.M. and A.O.W.M.S. performed synthesis; F.A.M.

performed biochemical studies; G.J.M.O., R.M.J.M.d.V., and

B.A.S. performed crystallography studies; G.J.M.O. performed

the expression of the FXR protein; AU performed ADME

measurements; F.A.M., R.G.D., B.K., C.O., P.J.C., and L.B.

designed the studies. All the authors have given approval to

the ﬁnal version of the manuscript.

Accession Codes

Coordinates and structure factors for the RORγt bound to

compounds 3, 9, 10, and 11 have been deposited in the

Protein Data Bank under accession codes 7NPC, 7NP5,

7NEC, and 7NP6. The authors will release the atomic

coordinates and experimental data upon article publication.

AUTHOR INFORMATION

Corresponding Author

Funding

■

This work was supported by the Netherlands Organization for

Scientiﬁc Research through Gravity program 024.001.035, Vici

grant 016.150.366, ECHO grants 711.018.003 and

711.017.014, the European Union through a Marie Skłodow-

ska-Curie Actions (MSCA) Individual Fellowship (R.G.D.,

H2020-MSCA-IEF-2016, grant no. 705188) and Eurotech

Postdoctoral Fellow program (grant no. 754462).

Luc Brunsveld − Laboratory of Chemical Biology,

Department of Biomedical Engineering and Institute for

Complex Molecular Systems, Technische Universiteit

Eindhoven, 5612 AZ Eindhoven, The Netherlands;

orcid.org/0000-0001-5675-511X; Email: l.brunsveld@

tue.nl

Notes

Authors

The authors declare the following competing ﬁnancial

interest(s): F.A.M., R.G.D., C.O. and L.B. are coinventors of

patent WO2020149740: Substituted heterocyclic compounds

and their use as retinoid-related orphan receptor (ROR)

gamma-t inhibitors.

Femke A. Meijer − Laboratory of Chemical Biology,

Department of Biomedical Engineering and Institute for

Complex Molecular Systems, Technische Universiteit

Eindhoven, 5612 AZ Eindhoven, The Netherlands;

orcid.org/0000-0003-4412-9968

Annet O.W.M. Saris − Laboratory of Chemical Biology,

Department of Biomedical Engineering and Institute for

Complex Molecular Systems, Technische Universiteit

Eindhoven, 5612 AZ Eindhoven, The Netherlands

Richard G. Doveston − Laboratory of Chemical Biology,

Department of Biomedical Engineering and Institute for

Complex Molecular Systems, Technische Universiteit

Eindhoven, 5612 AZ Eindhoven, The Netherlands; Leicester

Institute of Structural and Chemical Biology and School of

Chemistry, University of Leicester, LE1 7RH Leicester, U.K.

Guido J.M. Oerlemans − Laboratory of Chemical Biology,

Department of Biomedical Engineering and Institute for

Complex Molecular Systems, Technische Universiteit

Eindhoven, 5612 AZ Eindhoven, The Netherlands;

orcid.org/0000-0001-7562-2654

Rens M.J.M. de Vries − Laboratory of Chemical Biology,

Department of Biomedical Engineering and Institute for

Complex Molecular Systems, Technische Universiteit

Eindhoven, 5612 AZ Eindhoven, The Netherlands

Bente A. Somsen − Laboratory of Chemical Biology,

Department of Biomedical Engineering and Institute for

Complex Molecular Systems, Technische Universiteit

Eindhoven, 5612 AZ Eindhoven, The Netherlands

Anke Unger − Lead Discovery Center GmbH, 44227

Dortmund, Germany

ACKNOWLEDGMENTS

■

We thank Iris A. Leijten-van de Gevel for expression of the

RORγt protein used for biophysical assays, Auke A. Koops for

the expression of the PPARγ protein, and Joost L.J. van

Dongen for performing HRMS measurements. Additionally,

we thank Saskia W.C. van Mil for providing the plasmid for

FXR expression and for providing GW4064 (FXR agonist).

We also thank the DLS-CCP4 Data Collection and Structure

Solution Workshop 2021 at Diamond Light Source (Oxford-

shire, UK).

ABBREVIATIONS

■

ADME, absorption, distribution, metabolism, and excretion;

FP, ﬂuorescence polarization; FXR, farnesoid X receptor; H12,

helix 12; HPLC−UV, high-performance liquid chromatog-

raphy/ultraviolet; IPTG, isopropyl-β-^D-thiogalactoside; LBD,

ligand-binding domain; LC−MS, liquid chromatography−

mass spectrometry; NR, nuclear receptor; PAMPA, parallel

artiﬁcial membrane permeability assay; PK, pharmacokinetic;

PPARγ, peroxisome proliferated-activated receptor γ; Q-TOF,

quadrupole time-of-ﬂight; RORγt, retinoic-acid-receptor-re-

lated orphan receptor γt; RT-PCR, reverse transcriptase PCR;

SAR, structure−activity relationship; Th17, T helper 17; T_m,

melting temperature; TR-FRET, time-resolved FRET; TSA,

thermal shift assay

Bert Klebl − Lead Discovery Center GmbH, 44227

Dortmund, Germany

Christian Ottmann − Laboratory of Chemical Biology,

Department of Biomedical Engineering and Institute for

Complex Molecular Systems, Technische Universiteit

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Article Doi

Structure-Activity Relationship Studies of Trisubstituted Isoxazoles as Selective Allosteric Ligands for the Retinoic-Acid-Receptor-Related Orphan Receptor γt

DOI: 10.1021/acs.jmedchem.1c00475

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Article abstract of DOI:10.1021/acs.jmedchem.1c00475

Full text of DOI:10.1021/acs.jmedchem.1c00475

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