Discovery of a Series of Pyrazinone RORγAntagonists and Identification of the Clinical Candidate BI 730357

Identi ﬁ cation of the Clinical Candidate BI 730357

Letter

Discovery of a Series of Pyrazinone RORγ Antagonists and

Christian Harcken,* Johanna Csengery, Michael Turner, Lifen Wu, Shuang Liang, Robert Sibley,

Steven Brunette, Mark Labadia, Kathleen Hoyt, Anita Wayne, Thomas Wieckowski, Gregg Davis,

Mark Panzenbeck, Donald Souza, Stanley Kugler, Donna Terenzio, Delphine Collin, Dustin Smith,

Ryan M. Fryer, Yin-Chao Tseng, Jorg P. Hehn, Kim Fletcher, and Robert O. Hughes

Cite This: ACS Med. Chem. Lett. 2021, 12, 143 −154

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sı Supporting Information

ABSTRACT: The interleukin (IL)-23/T helper (Th)17 axis plays a critical role in autoimmune diseases, and there is an increasing

number of biologic therapies that target IL-23 and IL-17. The transcription factor retinoic acid receptor-related orphan nuclear

receptor γt (RORγt) is important for the activation and diﬀerentiation of Th17 cells and thus is an attractive pharmacologic target

for the treatment of Th17-mediated diseases. A novel series of pyrazinone RORγ antagonists was discovered through hybridization

of two distinct screening hits and scaﬀold hopping. The series oﬀers attractive potency and selectivity in combination with favorable

druglike properties, such as metabolic stability and aqueous solubility. Lead optimization identiﬁed a clinical candidate, compound

(S)-11 (BI 730357), for the treatment of autoimmune diseases.

KEYWORDS: RORγ modulators, IL-17 axis, anti-inﬂammatory agents, autoimmune diseases, pyrazinone, scaﬀold hopping

he interleukin (IL)-23/T helper 17 (Th17) axis plays a

protected against the development of experimentally induced

autoimmune encephalitis.

Unlike biologic therapies that target individual cytokines,

such as IL-17 or IL-23 inhibitors, the inhibitory action of

RORγ has the potential to have a broad eﬀect in Th17-

mediated disease due to its central role in the IL-23/Th17 axis.

critical role in the pathogenesis of autoimmune diseases,

such as plaque psoriasis. Recent successes in clinical

development have led to the approval of several biologic

therapies that target IL-17 or IL-23 for the treatment of plaque

psoriasis and other autoimmune diseases; however, there is a

strong unmet clinical need for an oral therapeutic agent that

Thus, there is increasing interest in the identiﬁcation of RORγ

5,8−14

targets the IL-23/Th17 axis.

antagonists for clinical development.

It is not known

Retinoic acid receptor-related orphan nuclear receptor γ

RORγ), more precisely the splice variant RORγt, is a

whether RORγ antagonists function as true antagonists or

inverse agonists. The ﬁrst ligands for ROR were identiﬁed in

2011, with compound 1 (SR1001) as a synthetic ligand for

(

transcription factor that plays an important role in the

activation and diﬀerentiation of Th17 cells and is expressed

in distinct subsets of lymphoid cells, such as Th17 cells, γδ T

15,16

both RORα and RORγ (Figure 1).

Subsequently, a

diphenylpropanamide series of selective modulators of RORγ

were reported, and the ﬁrst inverse agonist with dual selectivity

−5

cells, natural killer T cells, and innate lymphoid cells.

Upon

17,18

for RORβ and RORγ was identiﬁed.

Since the discovery of

cell activation, RORγ regulates the transcription of IL-17A, IL-

7F, IL-22, and the IL-23 receptor (IL-23R). The

involvement of RORγt in regulating immune homeostasis

was ﬁrst suggested when proinﬂammatory Th17 cells were

found to be largely absent in RORγt-knockout mice, with later

studies reporting synergistic activity for RORγ and RORα

Received: November 11, 2020

Accepted: December 11, 2020

Published: January 5, 2021

coexpression. Mice deﬁcient in both RORγ and RORα

exhibited impaired production of Th17 cells and were fully

2021 American Chemical Society

ACS Medicinal Chemistry Letters

ACS Med. Chem. Lett. 2021, 12, 143−154

Letter

Figure 1. Representative structures of RORγ antagonist classes.

compound 2 (T0901317; Tularik/Amgen), further acyclic

sulfonamides have been developed, as exempliﬁed by

compound 3 (GSK2981278; GSK), a topical compound that

was investigated in a Phase I/II trial in patients with plaque

psoriasis; compound 4 (ABBV-157; AbbVie), a cyclic

sulfonamide, is also under investigation for the treatment of

(desmosterol) was identiﬁed as an endogenous RORγ

27,28

agonist;

to date, compound 10 (RTA-1701; Reata) is

the only steroidal compound to have entered the clinical

development pipeline. The structures of key compounds are

summarized in Figure 1.

Herein, we describe the extensive exploration of the

structure−activity relationship (SAR) and optimization of the

pyrazinone series of RORγ antagonists, which led to the

identiﬁcation of the clinical compound (S)-11 (BI 730357), an

RORγ antagonist developed by Boehringer Ingelheim currently

in Phase II clinical development for the treatment of plaque

0,19,20

plaque psoriasis.

Other classes of compounds that have

entered clinical development include carboxamides, as

exempliﬁed by compound 5 (JTE-151; Japan Tobacco), the

development of which was terminated in 2016; aryl sulfones as

exempliﬁed by compound 6 (VTP-43742/vimirogant; Abb-

Vie/Vitae), which reached Phase IIa development for the

treatment of psoriasis before development was discontinued in

30,31

psoriasis (NCT03635099; NCT03835481).

All compounds described in this paper have been

018; and compound 7 (AZD-0284; AstraZeneca), the

,10,13,21−25

32,33

development of which was suspended in 2019.

Currently, the most advanced compound in clinical develop-

ment that targets RORγ is compound 8 (JTE-451; Japan

Tobacco), an RORγ antagonist for which a Phase II clinical

trial in plaque psoriasis (IMPACT-PS) was completed in

February 2020; the structure of compound 8 has not been

substituted aminopyrimidine 12 was subjected to regioselective

nucleophilic substitution with the appropriate amine contain-

ing the alkyl group R (in the following called “northern

group”), to create the aminopyrimidine 13. For some

reactions, the S Ar reaction was performed with a nitro-

published to date.

Recently, the steroidal ligand 9

pyrimidine, and the substitution was followed by reduction of

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Letter

Scheme 1. General Synthesis of Pyrazinone RORγ

Antagonists

To identify attractive starting points as initial targets for

optimization, an extensive lead identiﬁcation campaign using

multiple parallel approaches was initiated. An initial reporter

gene assay (RGA) screen identiﬁed three singleton hits. While

these were successfully used as tools to establish the

mechanism of RORγ antagonism, including the disturbance

of helix 12 of the ligand-binding domain (LBD), SAR

expansion of these hits did not lead to a series with suﬃcient

optimization potential. One of these hits, the azaindazole 17,

with moderate inhibition of RORγ activity (RGA 50%

inhibitory concentration [IC ] 1600 nM), was used in a

scaﬀold-hopping approach that led to the indoline compound

8 (RGA IC₅₀410 nM) (Figure 2). Compound 18 showed

weak inhibitory activity (Th17 IL-17 IC₅₀200 nM); however,

the compound was very lipophilic (predicted log P 5.15),

which led to challenging druglike properties (human liver

microsome [HLM] stability 58% hepatic blood ﬂow [Qh];

kinetic high-throughput solubility [HTSol] < 0.1 μg/mL; and

cytochrome P450 3A [CYP3A] inhibition IC₅₀0.9 μM).

Through multiparameter optimization, the indoline series

progressed to the identiﬁcation of potential candidate

compounds, such as compound 19 (predicted logP 4.25;

the nitro group. The aminopyrimidine 13 was cyclized with

oxochloroacetate to give the pyrazinedione 14. Treatment of

the pyrazinedione 14 with oxalyl chloride, and the subsequent

regioselective introduction of the right-hand side (RHS) group

through S Ar reaction, led to the intermediate compound 15

Th17 IL-17 IC 200 nM; HLM < 24% Qh; HTSol 78 μg/mL;

containing the RHS group R . The left-hand side (LHS)

moiety containing R was introduced last, through Suzuki

CYP3A4 inhibition IC > 30 μM; hERG IC > 25 μM; rat in

vivo pharmacokinetic [PK] intravenous [IV] clearance 34%

Qh; and peroral [PO; by mouth] bioavailability 90% [3 mg/kg

coupling, to give the ﬁnal compound 16. In rare cases, the

order of the penultimate and ultimate steps was reversed. Most

of the synthetic eﬀort was spent on generating the required

135 PO, 1 mg/kg IV]). Advanced indolines were used as in

substituted R -containing amines and substituted R -contain-

ing pyrimidine boronic acid derivatives. Synthetic schemes

outlining the synthesis of each individual compound are given

in the Supporting Information. All compounds discussed have

been synthesized and tested as indicated enantiomers where

applicable.

vivo pharmacology tools, and they have been described

,34−36

elsewhere.

The advanced indolines were used for

scaﬀold hopping with the pteridin-7(8H)-one core (here,

referred to as a substituted pyrazinone) in compound 20,

which was identiﬁed in a high-concentration fragment screen.

Compound 20 was one of 44 hits identiﬁed using an RORγ

Figure 2. Lead identiﬁcation.

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Letter

ﬂuorescence polarization (FP) binding assay (IC₅₀780 nM)

out of 50 000 fragment-like compounds tested at 200 μM.

Compound 20 showed only very weak activity in the RORγ

RGAs (IC ₅ ₀ 13 000 nM). In Figure 3 a, the ROR γ LBD X-ray

and PO bioavailability 70%). However, further proﬁling

revealed that compound 22 needed further improvement

with regard to, for example, functional potency (human whole

blood IL-17 IC₅₀240 nM), in vivo half-life (projected human

half-life 8 h), thermodynamic crystalline equilibrium solubility

(

3.0 μg/mL), and safety pharmacology (acute heart rate

increase in rat at a concentration ≥11-fold the anticipated

human C ).

max

Following the promising initial proﬁle of the pyrazinone lead

2, an extensive SAR exploration was performed. In addition

to RORγ RGAs and PBMC IL-17 inhibition, key druglike

properties, such as HLM stability and kinetic solubility, were

screened for in most compounds. Table 1 shows data for

several core ring system modiﬁcations. As the crystal structure

of compound 20 had revealed key interactions of the

pyrazinone ring system with the RORγ LBD, the focus was

on modiﬁcations of the pyrimidine core ring. Removing both

nitrogen atoms from the pyrimidine core ring in compound 23

led to a predictable substantial reduction in aqueous solubility

(

0.2 μg/mL); the analogue with an additional nitrogen atom in

-position, compound 24, also showed very low solubility (0.1

μg/mL). Interestingly, the analogue with an additional

nitrogen atom in 6-position, compound 25, maintained

acceptable solubility (40 μg/mL), but was much less potent

(

PBMC IC 300 nM). The introduction of a nitrogen atom in

the 5-position was not tolerated, as compound 26 lost potency

in the RORγ RGA (IC₅₀4100 nM); the related pyrazine 27

lost potency in the functional assay (PBMC IC₅₀330 nM).

The pyridazine isomer (R)-28 was also 30-fold less active than

compound 22. Therefore, it was concluded from this limited,

synthetically challenging core ring system exploration that the

original 6,8-diazapyrazinone core was the most promising.

Further SAR exploration was performed on the LHS using

the 6,8-diazapyrazinone core and the ethylsulfonylpyridine

RHS that had originated from the indoline series (Table 2).

Removal of both substituents from the pyrimidine LHS ring, as

in compound 29, led to the loss of all RORγ activity (RGA

IC₅₀> 30 000 nM). While the corresponding analogue with

either of the two LHS substituents removed was not examined

in this series, the monosubstituted isopropylpyrimidine 30

exempliﬁes that signiﬁcant potency is lost by removing one of

the two LHS substituents (Th17 IC₅₀200 nM). The

corresponding cyclopropyl compound was only prepared in

combination with the other RHS and northern groups, but lost

potency to a similar degree to the corresponding isopropyl

compound. The replacement of the methoxy group in

compound 22 with a methyl group led to compound 31,

which showed slightly reduced RORγ activity (PBMC IC50 110

nM), with increased solubility (HTSol 60 μg/mL). This LHS

group was examined in further SAR studies and combination

molecules. The ethyl cyclopropyl pyrimidine 32, a closer

isostere to compound 22, was less potent (PBMC IC50 360

nM); however, by increasing the size of the second substituent,

as in the bis(cyclopropyl) compound 33, potency was regained

(PBMC IC 13 nM), while solubility decreased (HTSol 5 μg/

Figure 3. (a) Overlay of the X-ray cocrystal structures of the RORγ

LBD, with 20 (green), with indoline 21 (magenta), and key binding

interactions. (b) Overlay of the X-ray cocrystal structures of the

RORγ LBD, with the optimized pyrazinone series antagonist (R)-43

(blue), with indoline 21 (magenta), and key binding interactions.

cocrystal structure and binding interactions of the pyrazinone

fragment 20 is overlaid with the X-ray crystal structure of the

second-generation indoline 21. While compound 20 does not

extend far enough into the binding pocket to reach the Arg367

anchor, it does appear to ﬁll the lower portion of the binding

pocket well; this partial occupation may explain the weak

RORγ RGA activity of compound 20 that has been previously

observed. The core ring system of compound 20 does not

overlay well with compound 21; however, it functions as a

scaﬀold to position key peripheral groups in the correct

trajectories.

Preferred RHS and LHS fragments from the indoline series

(

such as the aryl sulfone RHS fragments that are well

established in the literature) were hybridized onto the

identiﬁed pyrazinone core, leading to compound 22, which

showed an attractive proﬁle, with substantially improved

druglike properties (predicted logP 0.37) compared with the

indolines. This contributed to an improved overall proﬁle

mL). The introduction of two polar groups into the

bis(methoxy) analogue 34 also led to reduced RORγ activity

(PBMC IC₅₀450 nM) and the removal of the methyl group

from the methoxy group, thus introducing a very polar group

that led to the loss of all activity, as observed with compound

35 (RGA IC₅₀> 30 000 nM). The introduction of a third

substituent in the 2-position of the pyrimidine LHS ring (e.g.,

(

peripheral blood mononuclear cell [PBMC] IC₅₀48 nM;

HLM < 24% Qh; HTSol 17 μg/mL; no CYP inhibition <30

μM; hERG IC₅₀12 μM; rat in vivo PK IV clearance 32% Qh;

aminopyrimidine 36) led to reduced potency (PBMC IC 120

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Table 1. Core Modiﬁcations

NT, not tested. Diﬀerent group elsewhere in the molecule, uses 1-cyclopropylethyl northern group.

nM), and the introduction of larger alkoxy groups (e.g.,

isopropoxy analogue 37) led to reduced metabolic stability

ﬂexibility for further optimization. Both enantiomers of the

secondary butyl-substituted compounds (R)-43 and (S)-44

retained potency (PBMC IC₅₀28 nM and 19 nM,

respectively), with reduced solubility (HTSol 4.0 μg/mL and

3.4 μg/mL, respectively). Figure 3b shows the X-ray cocrystal

structure of compound(R)-43 as an example of the binding

mode of these optimized compounds as overlaid with the

indazole lead compound 21. Compound 45, using the

isomeric, albeit primary alkyl, isobutyl group, showed a similar

proﬁle of retained activity but reduced solubility (HTSol 3.6

μg/mL). The 2-pentyl group in compound(R)-46, with

increased lipophilicity, led to a reduced solubility (HTSol 0.3

μg/mL) and decreased metabolic stability (HLM 50% Qh).

Cycloalkyl substitution, as in the cyclobutyl analogue 47, also

maintained potency but did not improve solubility (HTSol 3.2

μg/mL). The N-cyclopropyl was only explored in combination

with other RHS and LHS fragments, but it was approximately

4-fold less potent than the corresponding isopropyl compound.

The introduction of a neopentyl substitution and/or the

introduction of a very polar substituent in compound 48 led to

(

HLM 79% Qh). Other related heteroaromatic ring systems

were explored, here exempliﬁed by the quinoline 38, which lost

metabolic stability (HLM 50% Qh), and the pyrazine 39,

which lost RORγ activity (RGA IC₅₀2000 nM). The removal

of one or both nitrogen atoms from the pyrimidine LHS ring

was not extensively explored in this series, but the, albeit

diﬀerently substituted, phenyl compound 40 exempliﬁes that

this change also led to substantial loss of RORγ activity (RGA

IC₅₀2800 nM).

The SAR of the N-substituent in 1-position of the core

pyrazinone ring system in the following “northern group” was

also investigated, while maintaining the LHS, RHS, and core

ring system of compound 22 for direct comparison (Table 3).

Generally, the northern group proved to be very sensitive to

changes. Reducing the size of the isopropyl group in

compound 22 by one methylene group created the N-ethyl

pyrazinone 41 (this compound was synthesized using the

similarly active methylcyclopropylpyrimidine LHS) with a 10-

fold reduction in RORγ potency (RGA IC₅₀3000 nM).

Adding a methyl group to give the tert-butyl analogue 42 also

led to a reduction in potency (PBMC IC₅₀220 nM). Keeping

the substituent as a secondary alkyl group allowed more

a marked reduction in potency (RGA IC 17 000 nM) due to

its position in the lipophilic region of the binding pocket

(Table 3). Based on the attractive proﬁle of the sec-Bu

compounds (R)-43 and (S)-44, close variations of these

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Table 2. Left-Hand Side Modiﬁcations

NT, not tested. Determined from Th17 cell IL-17 production assay.

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Table 3. Northern Group Modiﬁcations

NT, not tested. Diﬀerent group elsewhere in the molecule, uses 4-cyclopropyl-6-methylpyrimidin-5-yl LHS.

groups were explored. The 3-methyl-2-butyl enantiomers (S)-

reduced binding, as demonstrated by the aniline derivative 56

(RGA IC50 3800 nM).

9 and (R)-50 both retained potency (PBMC IC 13 nM and

6 nM, respectively), but solubility decreased (HTSol 0.5 μg/

Lastly, the SAR on the RHS pyridine ring system was

investigated (Table 4). This SAR is shown in combination with

the preferred moieties identiﬁed on the LHS and the northern

group. The methylsulfone (S)-11 (BI 730357) showed a

similar proﬁle to the comparator compound, the ethylsulfone

mL and 0.8 μg/mL, respectively). However, the closely related

-cyclopropylethyl groups in the two enantiomeric compounds

S)-51 and (R)-52 regained some solubility (HTSol 8 μg/

(

mL). The (S)-isomer 51 was one of the most potent

compounds identiﬁed in this series (PBMC IC₅₀8.4 nM),

and this group was used as one of the preferred groups in

further SAR studies and combination molecules. The

triﬂuoromethyl analogue 53 showed reduced solubility

(

S)-57; both compounds were included in advanced proﬁling.

The propylsulfone (S)-58 and the cyclopropylsulfone (S)-59

maintained potency (PBMC 15 nM and 22 nM, respectively),

but increasing the size of the alkylsulfonyl group led to a

reduction in solubility (HTSol 1.6 μg/mL and 1.4 μg/mL,

respectively). The isomeric 3-pyridyl methylsulfone (S)-60 and

ethylsulfone (S)-61 both maintained the desired initial proﬁle,

while removing the pyridine nitrogen entirely, as in compound

(S)-62, reduced solubility (HTSol 7 μg/mL). Other

heteroaromatic rings were also explored; the thiazole (S)-63

showed low solubility (HTSol 0.3 μg/mL), while the pyrazine

(S)-64 showed reduced potency (PBMC IC₅₀80 nM).

(

HTSol 0.6 μg/mL), but when the cyclopropyl moiety of

the northern group in compound (S)-51 was replaced by the

isosteric triﬂuoromethyl group, the resulting compound (S)-54

^a^l^s^o^s^h^o^w^e^d^a^f^a^v^o^r^a^b^l^e^p^r^o^ﬁ^l^e⁽^P^B^M^C^I^C⁵0 9.9 nM; HTSol

2 μg/mL); this group was also pursued further in

combination molecules. The introduction of an ether oxygen

atom, as in compound (S)-55, was not as potent (PBMC IC₅₀

8 nM) compared with the isosteric pentyl compound (R)-46

The pyrimidine (S)-65 maintained an attractive proﬁle, but

the introduction of the ninth nitrogen atom increased the polar

(

PBMC IC₅₀15 nM). Aromatic groups as N-1-substituents

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Table 4. Right-Hand Side Modiﬁcations

surface area to 158 Å ; therefore, this compound required

compound (S)-67, decreased metabolic stability (HLM 42%

and 34% Qh, respectively) and did not provide a substantial

solubility beneﬁt. The sulfonamide substitution in compound

(S)-68 maintained potency (PBMC IC₅₀26 nM), while the

additional proﬁling for permeability and absorption. The

replacement of the pyridine with a saturated ring system, as in

the piperidine in compound (S)-66 or the spiro azetidine in

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Table 5. Advanced Proﬁling

Equilibrium

solubility pH 7

[μg/mL]

hERG patch-

clamp IC

Rat CV assessment

(anesthetized or

telemetry)

Compound Th17 IL-17 Human Human hepatocyte

Rat PK IV clearance [% Qh];

PO bioavailability [%]

IC [nM] PPB [%] clearance [% Qh]

[μM]

(

S)-11

2.0

16; 70

32; 70

16; 71

42; 20

56; NT

44; NT

10; NT

23; NT

19; 61

29; NT

4.8

3.1

5.6

4.7

8.8

no eﬀect up to 10 μM

HR ↑ ≥ 10 μM

HR ↓ ≥ 40 μM

no eﬀect up to 15 μM

8.0

3.0

(

R)-43

S)-51

R)-52

S)-54

S)-57

S)-61

S)-65

S)-70

8.2

HR ↓ ≥ 4.6 μM

HR ↓ ≥ 10 μM

HR ↓ ≥ 12 μM

1.2

57; NT

CV, cardiovascular; HR, heart rate; NT, not tested; PPB, plasma protein binding. Most stable polymorph.

methylsulfoximide (S)-69 was less potent (most potent

diastereomer shown; PBMC IC₅₀120 nM). The N-

methylsulfonamide (S)-70 provided another initially promising

proﬁle (PBMC IC₅₀21 nM). The non-sulfur-containing

hydrogen bond acceptor substitution in the carboxamide (S)-

moderate human hepatocyte and rat PK clearance (28% and

29%, respectively). Compound (S)-70 showed reduced

metabolic stability (43% Qh).

The methylsulfone (S)-11 (BI 730357) was selected as a

potential development candidate. Additional preclinical

pharmacology data showed potent IL-17 inhibition in the

human whole blood setting (IC 140 nM); in human PBMCs,

1 led to reduced activity (PBMC IC₅₀160 nM).

Based on the previously described SAR exploration, selected

compounds were proﬁled further. Key advanced proﬁling

parameters that emerged in this pyrazinone series included

additional cell-speciﬁc functional assessments, including

inhibition of IL-17 from diﬀerentiated Th17 cells, human

plasma protein binding (PPB), human hepatocyte clearance,

thermodynamic crystalline equilibrium solubility, rat PK

clearance and bioavailability, patch-clamp hERG ion channel

inhibition, and a rat hemodynamic assessment (see the

Supporting Information for method descriptions). Other

parameters were proﬁled but did not present challenges in

this series, as most compounds showed an acceptable proﬁle;

these parameters included RORα and RORβ selectivity,

nuclear receptor and general receptor selectivity, mechanistic

in vivo mouse pharmacology studies, Caco-2 cell permeability,

CYP inhibition and induction, and a dog electrophysiology

assessment. Compounds showed typically good cross-reactivity

to mouse and rat RORγ, which allowed the relevant proﬁling in

rodent pharmacology and safety studies.

human RORγ-mediated IL-22 production was also inhibited

(IC₅₀43 nM). Cross-reactivity to other nuclear receptors was

minimal, as the only activity identiﬁed was liver X receptor-α

antagonism with IC₅₀10 μM. No oﬀ-target activity was

detected in the typical screening panels of receptors and

kinases.

To assess the eﬀect of RORγ antagonism on skin

inﬂammation, synovitis/enthesitis, and aberrant bone forma-

tion, an in vivo murine IL-23 minicircle model was used.

This model is characterized by a pronounced skin, gut, and

rheumatic phenotype, and IL-23 minicircle DNA is introduced

into B10.RIII mice via hydrodynamic injection. Minicircle

DNA was injected on Day 0, and compound (S)-11 (BI

730357) was dosed orally (0.3, 3, and 30 mg/kg) twice daily,

starting on Day 1, for 28 days. In vivo activity was assessed

using tissue biomarker end points and arthritic score (severity

was assessed using a visual scoring system, where 0 = normal;

1 = erythema and edema in 1−2 digits; 2 = erythema and

edema in >2 digits or mild erythema and edema, usually in the

ankle joint; 3 = moderate erythema and edema encompassing

the tarsal joint; and 4 = severe erythema and edema

encompassing the tarsal and metatarsal joint), and mRNA

expression of disease-relevant tissue biomarkers (e.g., IL-17A,

IL-22, S100a8, S100a9, and β-defensin) was measured in the

skin, entheses, and ileum. Signiﬁcant inhibition of IL-17A

mRNA (the target engagement biomarker) was observed in

entheses (up to 101%, p < 0.05), skin (up to 109%, p < 0.05),

and ileum (up to 101%, p < 0.05) at mid-range and high doses

of compound (S)-11 (BI 730357). In addition, signiﬁcant

inhibition was observed for the disease-relevant biomarkers

S100a8 (up to 95%, p < 0.05), S100a9 (up to 95%, p < 0.05),

and β-defensin (up to 95%, p < 0.05) in the skin, with ED <

Table 5 shows that none of the proﬁled advanced compound

examples are free of challenges. Compound 31 showed

reduced potency in the functional Th17 IL-17 assay (IC₅₀71

nM). Compound (R)-43 was discontinued due to high PPB

(

98%) and decreased metabolic stability in human hepatocytes

(

34% Qh) that also translated into increased clearance in rat

PK (42%); these both contributed to a high human dose

estimation. Compounds (S)-51 and (R)-52 also showed

increased human hepatocyte clearance (44% and 42% Qh,

respectively) and rat PK clearance (56% and 44%, respectively)

and single-digit micromolar hERG inhibition (IC 3.1 μM and

.6 μM, respectively). Compound (S)-54 showed high PPB

(

97%) and hERG inhibition (IC₅₀4.7 μM), while compound

S)-57 showed potent eﬀects on hemodynamic parameters in

rats (e.g., marked acute bradycardia). Compound (S)-11 (BI

0.3 mg/kg. These eﬀects translated into dose-dependent

reductions in arthritic scores and reduced histological changes.

Regarding the additional proﬁling of druglike properties,

compound (S)-11 (BI 730357) showed excellent Caco-2 cell

permeability (110 × 10 cm s ), with an eﬄux ratio near 1.0.

Good in vitro−in vivo correlation in relation to PK parameters

30357) showed low thermodynamic solubility for the most

stable polymorph (2 μg/mL) but otherwise had the most

attractive proﬁle. Compound (S)-61 also showed bradycardia

in rats. Compound (S)-65 suﬀered from low solubility (1.2

μg/mL), increased hERG inhibition (IC₅₀8.8 μM), and

−

−1

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ACS Medicinal Chemistry Letters

The Supporting Information is available free of charge at

Letter

was observed; human PK parameters were projected using

allometric scaling to support once-daily dosing, with a

predicted mean residence time of 34 h. The drug−drug

interaction potential was estimated as moderate based on the

absence of in vitro CYP inhibition (all IC₅₀> 23 μM) and

moderate CYP3A4 induction potential (2.4-fold at 3 μM). The

compound’s in vitro and acute general pharmacology safety

proﬁle supported advancement into preclinical development. A

highly crystalline form with excellent physicochemical proper-

ties was identiﬁed as the most stable of only two polymorphs.

Based on this promising and attractive preclinical proﬁle, the

compound (S)-11 (BI 730357) was selected as the clinical

development compound and advanced into clinical trials, and

we believe this to be one of the most attractive RORγ

antagonist candidates known. Results from Phase II trials

assessing the eﬃcacy and safety of compound (S)-11 (BI

Kathleen Hoyt − Department of Immunology and Respiratory

Disease Research, Boehringer Ingelheim Pharmaceuticals, Inc.,

Ridgeﬁeld, Connecticut 06877, United States

Anita Wayne − Department of Immunology and Respiratory

Disease Research, Boehringer Ingelheim Pharmaceuticals, Inc.,

Ridgeﬁeld, Connecticut 06877, United States

Thomas Wieckowski − Department of Immunology and

Respiratory Disease Research, Boehringer Ingelheim

Pharmaceuticals, Inc., Ridgeﬁeld, Connecticut 06877, United

States

Gregg Davis − Department of Immunology and Respiratory

Disease Research, Boehringer Ingelheim Pharmaceuticals, Inc.,

Ridgeﬁeld, Connecticut 06877, United States

Mark Panzenbeck − Department of Immunology and

Respiratory Disease Research, Boehringer Ingelheim

Pharmaceuticals, Inc., Ridgeﬁeld, Connecticut 06877, United

States

Donald Souza − Department of Immunology and Respiratory

Disease Research, Boehringer Ingelheim Pharmaceuticals, Inc.,

Ridgeﬁeld, Connecticut 06877, United States

Stanley Kugler − Department of Small Molecule Discovery

Research, Boehringer Ingelheim Pharmaceuticals, Inc.,

Ridgeﬁeld, Connecticut 06877, United States

Donna Terenzio − Department of Small Molecule Discovery

Research, Boehringer Ingelheim Pharmaceuticals, Inc.,

Ridgeﬁeld, Connecticut 06877, United States

30357) in patients with plaque psoriasis will be reported in

30,31

the near future (NCT03635099; NCT03835481).

ASSOCIATED CONTENT

sı Supporting Information

■

https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00575.

Methodology described for the RORγ reporter gene

assay, PBMC IL-17 production, human liver microsomal

stability, high-throughput solubility, Th17 cell IL-17

production, human plasma protein binding, human

hepatocyte clearance, equilibrium solubility measure-

ment, rat pharmacokinetics, hERG inhibition, rat

cardiovascular assessment; synthesis and character-

ization for all target compounds are described (PDF)

Delphine Collin − Department of Small Molecule Discovery

Research, Boehringer Ingelheim Pharmaceuticals, Inc.,

Ridgeﬁeld, Connecticut 06877, United States

Dustin Smith − Department of Small Molecule Discovery

Research, Boehringer Ingelheim Pharmaceuticals, Inc.,

Ridgeﬁeld, Connecticut 06877, United States

Ryan M. Fryer − Department of Cardiometabolic Diseases

Research, Boehringer Ingelheim Pharmaceuticals, Inc.,

Ridgeﬁeld, Connecticut 06877, United States

Yin-Chao Tseng − Department of Small Molecule Discovery

Research, Boehringer Ingelheim Pharmaceuticals, Inc.,

Ridgeﬁeld, Connecticut 06877, United States

AUTHOR INFORMATION

Corresponding Author

Christian Harcken − Department of R&D Project

Management and Development Strategies, Boehringer

Ingelheim Pharmaceuticals, Inc., Ridge ﬁ eld, Connecticut

■

6877, United States ; Email: christian.harcken@

Boehringer Ingelheim Pharma GmbH & Co. KG, 88397

Biberach an der Riß, Germany

boehringer-ingelheim.com

Kim Fletcher − Department of Small Molecule Discovery

Research, Boehringer Ingelheim Pharmaceuticals, Inc.,

Ridgeﬁeld, Connecticut 06877, United States

Robert O. Hughes − Department of Small Molecule Discovery

Research, Boehringer Ingelheim Pharmaceuticals, Inc.,

Ridgeﬁeld, Connecticut 06877, United States

Authors

Johanna Csengery − Department of Small Molecule Discovery

Research, Boehringer Ingelheim Pharmaceuticals, Inc.,

Ridgeﬁeld, Connecticut 06877, United States

Michael Turner − Department of Small Molecule Discovery

Research, Boehringer Ingelheim Pharmaceuticals, Inc.,

Ridgeﬁeld, Connecticut 06877, United States

Complete contact information is available at:

Lifen Wu − Department of Small Molecule Discovery Research,

Boehringer Ingelheim Pharmaceuticals, Inc., Ridgeﬁeld,

Connecticut 06877, United States

https://pubs.acs.org/10.1021/acsmedchemlett.0c00575

Author Contributions

Shuang Liang − Department of Small Molecule Discovery

Research, Boehringer Ingelheim Pharmaceuticals, Inc.,

Ridgeﬁeld, Connecticut 06877, United States

Robert Sibley − Department of Small Molecule Discovery

Research, Boehringer Ingelheim Pharmaceuticals, Inc.,

Ridgeﬁeld, Connecticut 06877, United States

Steven Brunette − Department of Small Molecule Discovery

Research, Boehringer Ingelheim Pharmaceuticals, Inc.,

Ridgeﬁeld, Connecticut 06877, United States

Mark Labadia − Department of Immunology and Respiratory

Disease Research, Boehringer Ingelheim Pharmaceuticals, Inc.,

Ridgeﬁeld, Connecticut 06877, United States

All authors contributed to the design and/or synthesis of the

described compounds, or to the generation of the discussed

data for the compounds. All authors participated in the

conceptualization, writing, review, and editing of the manu-

script, and provided approval of the ﬁnal submitted version.

Funding

This study is funded by Boehringer Ingelheim.

Notes

The authors declare no competing ﬁnancial interest.

ACS Med. Chem. Lett. 2021, 12, 143−154

ACS Medicinal Chemistry Letters

Letter

12) Cyr, P.; Bronner, S. M.; Crawford, J. J. Recent progress on

nuclear receptor RORgamma modulators. Bioorg. Med. Chem. Lett.

016, 26 (18), 4387−93.

13) Bronner, S. M.; Zbieg, J. R.; Crawford, J. J. RORgamma

ACKNOWLEDGMENTS

■

The authors would like to thank Stefan Ho

rer for his help in

(14) Cherney, R. J.; Cornelius, L. A. M.; Srivastava, A.; Weigelt, C.

generating views of X-ray crystal structure data. This study is

funded by Boehringer Ingelheim. Editorial assistance in the

preparation of this manuscript was provided by Tina Borg,

PhD, of OPEN Health Medical Communications (London,

UK) and funded by Boehringer Ingelheim.

antagonists and inverse agonists: a patent review. Expert Opin. Ther.

Pat. 2017, 27 (1), 101−12.

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15) Kumar, N.; Kojetin, D. J.; Solt, L. A.; Kumar, K. G.; et al.

221−7.

ABBREVIATIONS

■

CV, cardiovascular; CYP, cytochrome P450; FP, ﬂuorescence

polarization; HLM, human liver microsome; HTSol, high-

throughput solubility; IC , 50% inhibitory concentration; IL,

Identification of SR3335 (ML-176): a synthetic RORalpha selective

inverse agonist. ACS Chem. Biol. 2011, 6 (3), 218−22.

(16) Solt, L. A.; Kumar, N.; Nuhant, P.; Wang, Y.; et al. Suppression

of TH17 differentiation and autoimmunity by a synthetic ROR ligand.

Nature 2011, 472 (7344), 491−4.

interleukin; IV, intravenous; LBD, ligand-binding domain;

LHS, left-hand side; NT, not tested; PBMC, peripheral blood

mononuclear cell; PK, pharmacokinetic/s; PO, peroral; PPB,

plasma protein binding; Qh, hepatic blood ﬂow; RGA, reporter

gene assay; RHS, right-hand side; ROR, retinoic acid receptor-

related orphan receptor; RORγ, retinoic acid receptor-related

orphan nuclear receptor γ; SAR, structure−activity relation-

ship; Th, T helper

(17) Khan, P. M.; El-Gendy, B. E.-D. M.; Kumar, N.; Garcia-

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