Identification and structure activity relationships of quinoline tertiary alcohol modulators of RORγt

Article abstract of DOI:10.1016/j.bmcl.2017.02.044

A high-throughput screen of the ligand binding domain of the nuclear receptor retinoic acid-related orphan receptor gamma t (RORγt) employing a thermal shift assay yielded a quinoline tertiary alcohol hit. Optimization of the 2-, 3- and 4-positions of the quinoline core using structure-activity relationships and structure-based drug design methods led to the discovery of a series of modulators with improved RORγt inhibitory potency and inverse agonism properties.

Full text of DOI:10.1016/j.bmcl.2017.02.044

Accepted Manuscript
Identification and Structure Activity Relationships of Quinoline Tertiary Alco-
hol Modulators of RORγt
David A. Kummer, Maxwell D. Cummings, Marta Abad, Joseph Barbay,
Glenda Castro, Ronald Wolin, Kevin D. Kreutter, Umar Maharoof, Cynthia
Milligan, Rachel Nishimura, Joan Pierce, Celine Schalk-Hihi, John Spurlino,
Maud Urbanski, Hariharan Venkatesan, Aihua Wang, Craig Woods, Xiaohua
Xue, James P. Edwards, Anne M. Fourie, Kristi Leonard
PII:
S0960-894X(17)30163-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.02.044
BMCL 24716
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 January 2017
10 February 2017
14 February 2017
Please cite this article as: Kummer, D.A., Cummings, M.D., Abad, M., Barbay, J., Castro, G., Wolin, R., Kreutter,
K.D., Maharoof, U., Milligan, C., Nishimura, R., Pierce, J., Schalk-Hihi, C., Spurlino, J., Urbanski, M., Venkatesan,
H., Wang, A., Woods, C., Xue, X., Edwards, J.P., Fourie, A.M., Leonard, K., Identification and Structure Activity
Relationships of Quinoline Tertiary Alcohol Modulators of RORγt, Bioorganic & Medicinal Chemistry Letters
(2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.02.044
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Identification and Structure Activity Relationships of Quinoline Tertiary Alcohol
Modulators of RORt
Authors: David A. Kummer*,a, Maxwell D. Cummings*,b, Marta Abadb, Joseph Barbayc,
Glenda Castroa, Ronald Wolina, Kevin D. Kreutterc, Umar Maharoofc, Cynthia Milliganb, Rachel
Nishimuraa, Joan Piercec, Celine Schalk-Hihib, John Spurlinob, Maud Urbanskic, Hariharan
Venkatesana, Aihua Wangc, Craig Woodsa, Xiaohua Xuea, James P. Edwardsa, Anne M. Fouriea,
Kristi Leonard*,c
Corresponding authors: mcummin1@its.jnj.com (M. Cummings), dkummer1@its.jnj.com (D.
Kummer), kleonar1@its.jnj.com (K. Leonard).
Addresses:
aDiscovery Immunology, Janssen Research and Development, 3210 Merryfield Row, San Diego,
CA 92121
bDiscovery Sciences, Janssen Research and Development, Welsh and McKean Roads, Spring
House, PA 19477
cDiscovery Immunology, Janssen Research and Development, Welsh and McKean Roads, Spring
House, PA 19477
Keywords. RORt, retinoic acid-related orphan nuclear receptor gamma t, agonist, neutral
antagonist, inverse agonist, Th17, IL-17
Abstract. A high-throughput screen of the ligand binding domain of the nuclear receptor
retinoic acid-related orphan receptor gamma t (RORt) employing a thermal shift assay yielded a
quinoline tertiary alcohol hit. Optimization of the 2-, 3- and 4-positions of the quinoline core
using structure-activity relationships and structure-based drug design methods led to the

discovery of a series of modulators with improved RORt inhibitory potency and inverse
agonism properties.
The retinoic acid-related orphan receptor (ROR) family of nuclear receptors comprises three
members: ROR, ROR and ROR, with each member generating multiple isoforms. The
ROR isoform is widely expressed in many tissues including kidney, adipose, liver and skeletal
muscle, whereas the RORt isoform is only expressed in a few distinct cell types of the immune
system.1 RORt is the key transcription factor that drives the differentiation of naïve CD4+ T
helper cells to Th17 cells, and induces the transcription of IL-17A and IL-17F.2 There is
abundant evidence that IL-17A and the Th17 pathway play an important role in the pathogenesis
of psoriasis3 and biologics known to inhibit the Th17/IL-17 pathway are clinically validated for
the treatment of psoriasis.4 Due to its effect on Th17 cells, RORt may also play a role in the
development of diseases such as rheumatoid arthritis,5,6 inflammatory bowel disease,7 and
multiple sclerosis.8 Therefore, targeting the inhibition of Th17 differentiation and IL-17
production through modulation of RORt with small molecules has generated much interest in
drug discovery research.9-12
For nuclear receptors such as RORt, transcriptional activity can be modulated by the binding of
small molecule effectors to the ligand binding domain, and several mechanisms could affect this
activity.13 In the first scenario, small molecule effectors can bind to the nuclear receptor and
induce a conformational change that enhances binding of a co-activator peptide to the nuclear
receptor. These ligands would be considered agonists and binding should result in an increase in

RORt-driven gene transcription.14 Secondly, small molecule effectors can also bind to the
nuclear receptor and induce a conformation that yields enhanced binding of a co-repressor
peptide to the nuclear receptor. These ligands would be considered inverse agonists and binding
should lead to decreased gene transcription.15 Finally, ligands may also bind to the nuclear
receptor and induce a conformational change that precludes co-activator or co-repressor peptide
binding. These ligands could have various functional activities including inverse agonism or
neutral antagonism, the latter term referring to compounds that bind to the nuclear receptor with
no functional response.10,13
Early crystal structures of the RORγt ligand binding domain (LBD16) complexed with the agonist
25-hydroxycholesterol17 and with the inverse agonist digoxin17,18 revealed structural details of
the binding interactions for these effector molecules. When bound, the agonist 25-
hydroxycholesterol is completely buried in the center of the LBD. Interestingly, the bound sterol
makes no direct contact with the bound co-activator peptide17 (Figure 1a). Helix 12 of the LBD
is a key structural element of the co-activator peptide binding site, and the bound sterol makes
indirect contact with Tyr502 of helix 12 via a water-bridged hydrogen bond. The bound sterol
also approaches the imidazole of His479 in helix 11, and His479 in turn is involved in close
contacts with the sidechains of both Tyr502 and Phe506 of helix 1217 (see also Figure 1a).
Additionally, and of particular relevance to the present work, a previous small-molecule study
suggested a key mechanistic role for His479 in synthetic effector function.19 Bound digoxin also
occupies the sterol binding site of the LBD, with the trisaccharide moiety extending well beyond
the region occupied by the alcohol-bearing side chain of the bound sterol.20 This binding mode
forces helix 10/11 out of the agonist conformation observed in the sterol complex structure, and

this adjustment in turn likely precludes helix 12 from adopting the observed agonist
conformation. Thus, binding of digoxin causes conformational adjustments that inhibit co-
activator binding (Figure 1b).
Figure 1a. Interactions of His479 in the sterol agonist complex structure.17 A view down the
axis of helix 10/11 of the LBD with the imidazole of His479 projecting toward both the bound
sterol (blue color-by-atom) and helix 12 of the LBD (PDB ID 3L0L). His479 of helix 10/11 and
Tyr502 and Phe506 of helix 12 participate in specific binding contacts related to the positioning
of helix 12 in the co-activator peptide (SRC2) binding site. The oxygen of a bound water
molecule is shown as a red sphere, and several intermolecular contacts are highlighted with
dashed lines and with the intermolecular atom-atom distances shown.

Figure 1b. Bound digoxin clashes with the agonist conformation of helix 10/11. A view of
superimposed digoxin20 (PDB ID 3B0W) and sterol17 (PDB ID 3L0L) LBD complexes. Helix
11 of the LBD (projecting toward the viewer) is disordered in the digoxin complex (LBD:
orange; digoxin: green color-by-atom), in contrast to what is observed in the sterol complex
(LBD: white; sterol: blue color-by-atom).
In this report we describe the structure-activity relationships (SAR) and structure-based
optimization of the 2-, 3- and 4-positions of a series of quinoline tertiary alcohol RORt
modulators that act through binding at the sterol binding site of the LBD. Modification of these
molecules leads to changes in potency and functional activity, with compounds showing
agonism, inverse agonism, and neutral antagonism. The goal of our program was to identify

potent modulators of RORt that would maintain a full inverse agonism profile in our functional
assays.
Quinoline 1 (Figure 2) was identified from a high-throughput, thermal-shift-based assay using
ThermoFluor® (TF) technology.21,22 In order to establish the SAR around this hit, we employed
a TF binding assay using the RORt LBD22 and a 1-hybrid cell-based functional reporter assay.22
Compounds of interest were then tested in a Th17 cell-based assay measuring IL-17A production
to confirm activity in primary cells. Our optimization effort ultimately led to the discovery of
high affinity RORt modulators encompassing a range of functional activities. Herein we
describe SAR data for substitution on the 2-, 3- and 4-positions of the quinolone core; future
reports will disclose our efforts around other positions of this chemotype.
Figure 2. Racemic quinoline tertiary alcohol 1 was identified using a TF binding assay.
Our initial work on the hit 1 focused on exploring the SAR of the C2- and C4-positions of the
quinoline core. Compound 1 contains a stereocenter at the carbon bearing the tertiary alcohol

group and was initially characterized as a mixture of stereoisomers. In Table 1, we depict two
sets of racemic quinoline compounds that contain distinct substituents on the C6-position of the
quinoline; the first contains a 4-chlorophenyl/1-methyl-1H-imidazol-5-yl substituted alcohol
(compounds 1-9) and the second contains a 1-methyl-1H-imidazol-5-yl/pyridin-4-yl substituted
alcohol (compounds 10-16). Compounds 1 and 10 behaved as inverse agonists and exhibited full
suppression of RORt-driven transcriptional activity in the cell-based reporter assay. Similar
results were observed when comparing compounds 4 and 11, both of which contain a 2-methoxy
group in place of the 2-chloro atom on the quinoline core; the compounds maintained full inverse
agonism activity with >90% efficacy. Replacement of the chlorine atom with a hydrogen atom
at either the 2-position (compound 3) or the 4-position (compound 2) reduced both binding
affinity (KD >4 M) and cell potency (IC50 >2 M). However, in several cases we were able to
maintain or slightly improve potency while maintaining full inverse agonism activity by
retaining the 4-chlorine atom and replacing the 2-chlorine atom with electron-withdrawing or
electron-donating groups such as methoxy, cyano, trifluoromethyl, dimethyl- and diethyl amino
groups (compounds 4, 6, 8, 12, 13). Replacement of both the 2- and 4-chlorine atoms with
trifluoromethyl (compound 9) showed a 16-fold increase in TF binding but only a 2-fold increase
in cellular potency in the reporter assay, whereas 2,4-dicyanoquinoline 7 had a slight increase in
TF binding with no effect on cell potency or efficacy. Also, modifications to the 4-position of
compound 10 with dimethylamine (compound 15) did not impact TF binding or cell potency,
whereas methylamine (compound 16) lost significant binding and cell potency.
Table 1. C2- and C4-quinoline SAR.a

TF
binding
to
RORt
LBD
KD
C4-
substit
uent
RORt cell-based
reporter assay IC50 (M)
(% inhc @ 6 M)
C2-
substituent
(R2)
Cmpdb
(R1)
(M)
Cl
H
Cl
Cl
0.16
10
0.26 (101)
>4 (57)
1
2
Cl
H
4.3
2.7 (78)
3
Cl
OMe
OMe
CN
0.1
0.22 (104)
~2 (97)
4
OMe
Cl
0.57
5
0.051
0.055
0.057
0.0095
0.014
0.005
0.002
0.002
0.11 (101)
0.24 (105)
0.12 (110)
0.11 (97)
0.05 (105)
0.025 (92)
0.0075 (105)
0.011 (100)
6
CN
Cl
CN
7
CF3
CF3
Cl
8
CF3
Cl
9
10
11
12
13
Cl
OMe
NMe2
NEt2
Cl
Cl
Cl
15
1.9 (73)
14
NMe2
Cl
Cl
0.023
9.5
0.11 (101)
>6 (17)
15
16
NHM
e
aAll compounds are racemic mixtures. bCmpd = compound. cinh = inhibition.

After our initial efforts to probe the SAR with racemic material showed promise, methods were
investigated to characterize the TF binding and modulation of RORt-driven transcription in our
cell-based reporter assay with individual enantiomers. This chemical series proved consistently
amenable to isomer separation by supercritical fluid chromatography (SFC), and all isomeric
mixtures were subsequently separated this way. The data in Table 2 shows that one enantiomer
(12a) of compound 12 had significantly greater binding affinity in TF (145-fold) and greater
potency in the cell-based reporter assay (56-fold) than the opposite enantiomer (12b).
Interestingly, both enantiomers maintained full inverse agonism in the reporter assay. From this
point forward, all compounds described in this manuscript are single enantiomers (absolute
stereochemistry not determined) unless specifically indicated otherwise. The data described is for
the more potent isomer with regard to binding to the LBD of RORt as measured by the TF
assay.
Table 2. Binding and cell-based activity of enantiomers of compound 12.
TF binding to
RORt LBD KD
(M)
RORt reporter cell assay IC50
Cmpda
(M) (% inhd @ 6 M)
12b
12ac
0.002
0.0075 (105)
0.011 (105)
0.002
12bc
0.29
0.62 (101)

b
c
aCmpd = compound; racemate; single enantiomer, absolute stereochemistry not determined;
dinh = inhibition.
Figure 3. The (S) enantiomer of 12 binds in the sterol binding pocket. The structure of the
(S) enantiomer of 12 as positioned in the LBD complex (LBD: orange ribbon, orange color-by-
atom; 12(S): orange color-by-atom; deposited as PDB ID 5UFR) superimposed with the sterol
agonist complex structure (LBD: white, white color-by-atom; sterol: purple color-by-atom; PDB
ID 3L0L),17 viewed down the axis of helix 10/11. While the bound (S) enantiomer of 12 does not
clash with the (superimposed) agonist conformation of the LBD, His479 is shifted from the
conformation observed in the agonist complex.
Co-crystallization of RORt LBD with racemate 12 yielded a 2.07Å resolution crystal structure
of the S-enantiomer bound at the previously elucidated sterol binding site of the LBD (Figure

3).17 Interestingly, the overlay with the sterol agonist structure indicates that while the 3-phenyl
group of the bound ligand does not cause an obvious steric clash with the agonist conformation
of the LBD, the residues comprising helix 10/11 in the agonist structure are largely non-helical
and His479 is shifted from the orientation observed in the agonist complex. The specific
structural features of the small-molecule and binding interactions that drive this conformational
change, and, in turn, presumably contribute to this compound behaving as a full inverse agonist
in the reporter assay, remain unclear.
We next focused our chemistry effort on substitution at the C3-position of the quinoline core, in
an effort to maximize chemical diversity while seeking to maintain full inverse agonism in our
functional assay. Table 3 shows compounds that contain a 3-pyridyl-4-CF3/1-methyl-1H-
methylimidazol-5-yl tertiary alcohol at the C6-position of the quinoline, with varying
substituents at the C3-position. Two examples containing a C3-phenyl group with potent TF
binding affinity and full inverse agonism in the cell-based reporter assay are shown in
compounds 17 and 18. The cyanopyrimidine 19 demonstrates an example of a heterocyclic
phenyl replacement that maintains full inverse agonism in the cell-based assay. Interestingly, the
4-(methylsulfonyl)phenyl substituted quinoline 20 maintained similar binding affinity (KD =
0.037 M) to that of compound 19, but demonstrated functional agonism in the reporter assay,
with an EC50 of 0.049 M and stimulation of transcriptional activity of 78% at 6 M.

Table 3. Tertiary alcohol quinolines with varying substituents at the C3-position.a
RORt cell-
TF binding
based reporter
to RORt
Cmpdb
R1
R2
R3
assay IC50 (M)
(% inh.c @ 6
M)
LBD KD
(M)
OMe
Cl
CF3
0.015
0.006
0.19 (93)
17
18
azeti
dine
~0.05 (103)
Cl
Cl
OMe
OMe
0.032
0.037
0.31 (96)
19
20
0.049 (78%)d
b
aAll compounds are single enantiomers, absolute stereochemistry not determined; Cmpd =
compound; cinh = inhibition; ddenotes EC50 (% increase in transcription activity).

Figure 4. The quinoline tertiary alcohol 20 maintains a close-to-agonist conformation for
His479. The structure of the LBD-20 complex (LBD: cyan, cyan color-by-atom; 20: cyan color-
by-atom; deposited as PDB ID 5UFO) superimposed with the sterol agonist complex structure
(LBD: white, white color-by-atom; sterol: purple color-by-atom; PDB ID 3L0L),17 viewed along
the helix 10/11 axis. In contrast to the LBD-12 complex (Figure 3), bound 20 stabilizes His479
in a conformation very close to that seen in the sterol agonist complex.
Using x-ray crystallography conditions similar to those that yielded the initial complex structure
with the (S)-enantiomer of 12, we also succeeded in determining the 2.80Å resolution crystal
structure of the functional agonist 20 bound to the LBD, which shows the (S)-enantiomer is
bound in the complex. Relative to the sterol agonist structure, the LBD-20 complex structure
shows His479 shifted slightly toward the bound ligand, with the ligand sulfone oxygen in close

contact with the His479 sidechain (Figure 4). Helix 10/11 is fully resolved and completely
helical, and overall is shifted slightly from its position in the sterol agonist complex. This
contrasts with the inverse agonist complexes disclosed in this report (Figures 3 and 5), in which
the residues comprising helix 10/11 in the agonist structure are resolved but largely or
completely non-helical (see also eg. Ref. 19). The water-bridged hydrogen bond and ring-ring
contacts with helix 12 observed in the sterol agonist complex are not observed in the LBD-20
complex structure (c.f. Figures 1a and 3). We hypothesize that the observed sulfone-imidazole
contact serves to partially stabilize helix 10/11 in a “close to agonistic” conformation that allows
or facilitates co-activator binding, thus contributing to agonistic function. These speculations are
consistent with the observed complex structures and functional activities of these compounds.
While complicating the understanding of emerging SAR for a chemical series, overall it seems
an attractive feature of a chemotype when a range of biological function can be achieved through
relatively minor modifications of the parent structure, as noted here for these quinoline tertiary
alcohols. Interestingly, similar behavior has been reported by other research groups working in
the RORt field.19,23
Medicinal chemistry efforts led us to exchange the 3-aryl group with 3-substituted benzylic
groups. Structural and modeling studies later established that this targeted a slightly different
region of the binding pocket, potentially allowing diverse substituents in close proximity to helix
10/11. Given the established importance of helices 10-12 for nuclear receptor function,24 we
were keenly interested in targeting interactions with this region of the LBD. Additionally,

modeling indicated that sufficient extension of a benzylic substituent could project out of the
buried pocket and into a solvent-exposed region (c.f. digoxin, as in Figure 1b), thus providing an
opportunity to incorporate more general chemical modifications aimed at modulating the
physicochemical properties of these ligands. This design hypothesis was productive, with biaryl
substituents proving particularly effective.
As shown in Table 4, data for compounds 21 and 22 indicates that unsubstituted or 4-fluoro-
benzyl groups are tolerated at the C3-position. Racemic mixture 21 and single enantiomer 22
were found to have binding affinities of 20 nM and 6.6 nM for RORt, respectively. Although
the C3-benzyl compounds 21 and 22 were potent binders in TF, they both showed only slight
functional activity up to 6 M in the cellular reporter assay with partial impact on transcription,
behaving as neutral antagonists at lower concentrations and partial inverse agonists at higher
concentrations. Interestingly, the addition of larger substituents at the 4-benzyl position, such as
trifluoromethyl and pyrazole (compounds 23 and 24) resulted in single digit nanomolar binding
affinities in TF and potency of 0.12 M and 0.089 M in the cellular reporter assay, respectively,
while demonstrating a full inverse agonism profile. We also explored the C3-benzyl SAR with
the 4-chlorophenyl/1-methyl-1H-imidazol-5-yl tertiary alcohol at the C6-position of the
quinoline (Table 4). Similar to compounds 21 and 22, 4-fluorobenzyl compound 25 was a neutral
antagonist at lower concentrations with partial inverse agonism observed at high concentrations,
whereas compounds 26-28, containing a 4-cyano, -trifluoromethyl or –pyrazolyl group were all
potent molecules displaying full inverse agonism.

The data clearly indicates that structural changes at the C3-position of the quinoline can have a
dramatic effect on the transcriptional activity in the 1-hybrid cell-based reporter assay. It is
interesting to observe the full spectrum of functional activities - agonism, neutral antagonism and
inverse agonism, within this one chemical series. These results are consistent with the proximity
of the C3 substituent of our ligands to helix 10/11, a structural element critical to the functional
activity of the LBD.
Table 4. C3-benzyl SAR. All are single enantiomers except compound 21.a
RORt cell-based
TF binding to
reporter assay IC50
Cmpdb
R3
RORt LBD
KD (M)
(M) (% inh.d @ 6
M)
21c
22
0.020
>6 (20)
~6 (42)
0.0066
0.0015
0.005
0.012
0.12 (102)
0.089 (97)
~6 (62)
23
24
25

0.0062
0.0077
0.0092
0.14 (94)
0.041 (103)
0.073 (99)
26
27
28
aAll compounds are single enantiomers unless otherwise denoted, absolute stereochemistry not
determined; bCmpd = compound; cracemate; dinh = inhibition.

Figure 5. The 4-pyrazolylbenzyl quinoline 28 sterically clashes with helix 10/11. The
structure of the LBD-28 complex (LBD: yellow, yellow color-by-atom; 28: yellow color-by-
atom; deposited as PDB ID 5UHI) superimposed with the sterol agonist complex structure
(LBD: white, white color-by-atom; sterol: purple color-by-atom; PDB ID 3L0L),17 viewed along
the helix 10/11 axis. The benzyl-pyrazole moiety of compound 28 precludes the agonist
conformation of His479.
Larger substituents at the 3-position of the quinoline core can disrupt the His479-Phe502-Tyr506
interaction, leading to full inverse agonism. As with the (S)-enantiomers of compounds 12 and
20, the structure of the LBD-28 complex was determined, in this case at 3.2Å resolution. In this
complex, the pyrazol-1-yl substituent on the benzyl group displaces His479 from its position in
the sterol agonist complex structure (Figure 5). It is likely that this obvious steric incompatibility

leads to the complete disruption of helix 10/11 observed in this complex structure, which in turn
disturbs helices 11’ and 12 and ultimately precludes co-activator and co-repressor binding.
Thus, compound 28 and related molecules function as full inverse agonists in our cell-based
assay.
To understand the correlation in functional activity between the transfected cell-based reporter
assay and relevant human primary cells, we tested a selection of compounds in a Th17 cell assay
that measures production of IL-17A under conditions that favor Th17 cell differentiation (Table
5). Compound 20 was found to be an agonist in both the reporter assay and the Th17 cell assay.
Compounds 22 and 25, behaving like neutral antagonists in the reporter assay with less potency
than expected when compared to TF binding, also showed less potency in the Th17 cells with
inhibition curves only starting to develop at high compound concentrations. Compounds 23 and
27, containing a 4-trifluromethylbenzyl substituent, were full inverse agonists in the reporter
assay (>100% inhibition at 6 M compound concentration), and showed 83% and 95% inhibition
in Th17 cells, respectively, a slight reduction in efficacy. This same pattern was observed for
compounds 24, 26 and 28, which displayed full inverse agonism in the cell-based reporter assay
with a reduction in efficacy in the Th17 assay (ranging from 65 to 87% inhibition). All
compounds tested showed similar potency in the reporter assay and Th17 assays, except for
compound 20 which was 5-fold less potent in Th17 cells compared to the reporter assay. These
data support the concept that the cell-based reporter assay is showing a good correlation to the
Th17 cell assay, with minor to moderate differences in efficacy in some cases.

Table 5. Functional activity correlation between the transfected cell-based reporter assay and
relevant human primary cells.
RORt reporter cell Human Th17 assay
IC50 (% inh.b @ 6
Cmpda
assay IC50 (M) (%
inh. @ 6 M)
M)
0.049c (78)
0.24 (85)c, d
2.74 (78)
20
22
23
24
25
26
27
28
~6 (42)
0.053 (83)d
0.057 (78)
0.42 (78)
0.12 (102)
0.089 (97)
~6 (62)
0.045 (65)d
0.062 (95)d
0.14 (94)
0.041 (103)
0.073 (99)
0.085 (87)
b
c
aCmpd = compound; inh = inhibition; denotes EC50 (% increase in transcription activity);
ddenotes % inhibition at 1.2 M instead of 6 M.
Structural information has been valuable in furthering our understanding of the SAR in our
optimization of this series of quinoline tertiary alcohols. Three crystal structures of the RORt
LBD complexed with analogs of this chemotype are generally consistent with the observed
functional activities, and provide mechanistic insights into the range of functional activities we
describe. The structure of the LBD-28 complex is, perhaps, the most straightforward of the three
to understand. Compound 28 functions as a full inverse agonist by preventing co-activator or co-
repressor binding through disruption of the conformation of the helix 10-12 region via a steric
clash with the protein in the region of His479. We report an agonist, 20, that slightly disturbs
His479 from the conformation observed in the sterol agonist complex. We speculate that this

ligand stabilizes a conformation of helix 10/11 that is competent to allow or facilitate binding of
co-activator. The complex with the S-enantiomer of compound 12 is especially interesting.
Comparison of the LBD-12 and LBD-sterol complexes indicates that bound compound 12 has no
obvious steric clash with the sterol agonist conformation of the LBD that would cause the
observed structural changes, yet this compound disrupts agonist conformation and functions as a
full inverse agonist in the cell-based reporter assay. Comparison of functional data generated in
the cell-based reporter assay versus the Th17 assay shows a trend that suggests the potency is
similar between the two assays, however, some subtle differences in efficacy were noted.
Synthetic routes to racemic quinoline tertiary alcohols25-28 utilized a key carbon-carbon bond
forming reaction between two intermediates; diaryl ketones 29‒31 and an organometallic reagent
derived from 6-haloquinolines (32) (Scheme 1). In this section we detail reaction conditions for
the synthesis of these components, their coupling and further elaboration into final product
analogs described herein.
Scheme 1. Retrosynthetic route to racemic quinoline tertiary alcohols.

Synthesis of ketone and quinoline intermediates. The synthesis of ketones 29‒31 is summarized
in Scheme 2. 4-Chlorobenzoyl chloride, 4-picolinic acid, and 6-(trifluoromethyl)nicotinoyl
chloride were transformed into the corresponding N,O-dimethyl hydroxamic acid derivatives
33‒35, respectively. The reaction conditions and precursor material used to introduce the N-
methyl imidazole group varied slightly. For example, Grignard reagents derived from 5-bromo-
1-methyl-1H-imidazole were coupled with amides 33 and 35 to afford ketones 29 and 31,
respectively. The organometallic reagent precursor needed for the synthesis of 4-pyridyl ketone
30 was formed by a deprotonation‒protection‒deprotonation sequence. First, deprotonation of
the C2-position of 1-methyl-1H-imidazole with n-BuLi and trapping with chlorotriethylsilane
followed by a second deprotonation of the C5-position of 1-methyl-2-(triethylsilyl)-1H-
imidazole with n-BuLi provided the requisite C5-lithio imidazole which was coupled with amide
34 to afford ketone 30 (the triethylsilyl protecting group was removed during aqueous workup).
Scheme 2. Synthesis of ketone intermediates. Reagents and reaction conditions:
(a) N,O-dimethylhydroxylamine hydrochloride, pyridine, CH2Cl2; (b) 5-bromo-1-methyl-1H-
imidazole, EtMgBr, THF; (c) N,O-dimethylhydroxylamine hydrochloride, CDI, CH2Cl2; (d) 1-

methyl-1H-imidazole, n-BuLi, TESCl, ‒78 °C; then n-BuLi, 10 °C; then 34, ‒78 °C; (e) N,O-
dimethylhydroxylamine hydrochloride, (i-Pr)2EtN, CH2Cl2;
imidazole, i-PrMgCl•LiCl, THF.
(f) 5-bromo-1-methyl-1H-
The second precursor required for racemic tertiary alcohol formation was a substituted 6-
bromoquinoline compound. Acylation of methyl 2-amino-5-bromobenzoate (36) with phenyl
acetyl chloride, benzyloxy acetyl chloride or 3-phenylpropanoyl chloride provided amides 37, 38
and 39, respectively (Scheme 3). Base-mediated cyclization followed by chlorination with
phosphorus oxychloride afforded 6-bromo-2,4-dichloro-quinolines 40‒42. Synthesis of
monochloro-substituted quinolines 46 and 47 began by coupling of 4-bromoaniline with ethyl 3-
oxo-2-phenylpropanoate or 2-phenylacetyl chloride to afford 44 and 45, respectively. Cyclization
of 46 with polyphosphoric acid and subsequent chlorination provided quinoline 44. Cyclization
of 47 with phosphorus oxychloride in the presence of dimethyl formamide furnished quinoline
45.

Scheme 3. Synthesis of 6-bromoquinoline intermediates. Reagents and reaction conditions: (a)
2-phenylacetyl chloride, benzyloxyacetyl chloride, or 3-phenylpropanoyl chloride; Et3N,
CH2Cl2; (b) LiHMDS, THF for amide 37; (c) KHMDS, THF for amides 38 and 39; (d) POCl3;
o
(e) ethyl 3-oxo-2-phenylpropanoate, EtOH; (f) PPA, 150 C; (g) 2-phenylacetyl chloride, Et3N,
DMAP, DCM; (h) POCl3, DMF.
Two additional methods which were utilized to form 6-bromoquinolines are described in Scheme
4. Condensation/cyclization of 4-bromoaniline with 2,2-dimethyl-1,3-dioxan-4,6-dione followed
by chlorination with phosphorus oxychloride afforded dichloroquinoline 48. Deprotonation of
the C3-position of 48 with LDA and coupling with 4-(bromomethyl)benzonitrile provided 49
after methoxylation of the C2-position of the core. Alternatively, the 1,3-dicarboxylic acids 51-
53 could be synthesized by a condensation/reduction sequence between 4-substituted
benzaldehydes 50 and 2,2-dimethyl-1,3-dioxan-4,6-dione using catalytic proline and the
stoichiometric reducing reagent diethyl 1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate29

followed by base- or acid-mediated hydrolysis. Condensation/cyclization with 4-bromoaniline,
chlorination, and methoxylation afforded quinolines 54‒56.
Scheme 4. Synthesis of 6-bromoquinoline intermediates. Reagents and reaction conditions:
(a) 2,2-dimethyl-1,3-dioxan-4,6-dione; then Eaton’s reagent; (b) POCl3; (c) LDA, 4-
(bromomethyl)benzonitrile, THF; (d) NaOMe; (e) 2,2-dimethyl-1,3-dioxane-4,6-dione, catalytic
L-proline, EtOH; then diethyl 1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate; (f) aqueous
NaOH for 51 and 53; aqueous TFA for 52; (g) 4-bromoaniline, POCl3.
The synthesis of trifluoromethyl-substituted quinolines is described in Scheme 5. Cyclization of
2-amino-5-iodo-benzoic acid with 1,1,1-trifluoro-3-phenylpropan-2-one and Eaton’s reagent
afforded hydroxyl quinoline 57. Chlorination with phosphorus oxychloride followed by
methoxylation furnished iodoquinoline 59. Synthesis of 2,4-bistrifluoromethyl quinoline 60
proceeded by a four step sequence starting with deprotonation of 1-bromo-4-fluorobenzene with
LDA and coupling of the formed aryl lithium reagent with ethyl trifluoroacetate. A two-step,
one-pot azidation‒reduction reaction provided aniline 61. Cyclization with 1,1,1-trifluoro-3-
phenylpropan-2-one in hot DMF in the presence of tributylamine and a subsequent iodination
completed the synthesis of quinoline 60.

Scheme 5. Synthesis of trifluoromethyl-substituted quinoline intermediates. Reagents and
o
reaction conditions: (a) 1,1,1-trifluoro-3-phenylpropan-2-one, Eaton's reagent, 100 C; (b)
POCl3, 110 oC; (c) NaOCH3/CH3OH; (d) LDA, THF, ‒78 oC; then ethyl trifluoroacetate, 23 oC;
o
(e) NaN3, DMSO, 95 C; then SnCl2•dihydrate; (f) 1,1,1-trifluoro-3-phenylpropan-2-one, DMF,
Bu3N, 130 oC; (g) CuI, DMEDA, t-BuOH, NaI, microwave, 150 oC.
Tertiary alcohol formation reaction conditions.
Tertiary alcohol construction relied on a
carbon-carbon bond forming reaction between a diaryl ketone and an organometallic reagent
derived from a 6-halo quinoline. This synthetic step was executed using two strategies as
exemplified in Scheme 6. In the first, the diaryl ketone component was added to a pre-formed
organometallic reagent derived from 6-bromo or -iodo quinolines 40 and 60 using n-butyllithium
or i-propylmagnesium chloride, respectively, at low temperature (‒78 °C). In the second method,
n-butyllithium was added to a ‒78 °C solution of ketone 29 and quinoline 49 in tetrahydrofuran
(in-situ coupling conditions). These methods were applied to the synthesis of other quinoline
tertiary alcohols described herein.

Scheme 6. Tertiary alcohol synthesis.
Transformations of quinoline tertiary alcohols. The chlorine atoms of 2,4-dichloroquinolines 1,
10, and 62 can be replaced with numerous groups as described in Scheme 7. Selective
displacement of the C2-chlorine atom with methoxide proceeds in hot toluene30,31 (compounds 4
and 11) whereas replacement of both chlorine atoms requires methoxide in hot methanol
(compound 5). The introduction of nitrile groups was also achieved in a selective manner; the
substitution of the C2-chlorine atom proceeded within 4 hours and the substitution of both
chlorine atoms in 22 hours under palladium-catalyzed cyanation reaction conditions (compounds
6 and 7). Reaction conditions that installed amino groups gave rise to C2-amino quinoline

products as the major products (compounds 12, 13, 14 and 18) and in two instances the C4-
amino product was isolated (compounds 15 and 16).
Scheme 7. Displacement of chlorine atoms. Reagents and reaction conditions: (a) NaOCH3
(excess), toluene, reflux; (b) NaOMe (excess), methanol, reflux; (c) Pd2dba3, dppf, Zn(CN)2,
zinc nanopowder, DMA, 120 oC, 4 hr (22 hr to displace both chlorine atoms); (d) 2M NH(CH3)2
in MeOH, 80 °C; (e) NHEt2, DMF, 130 °C; (f) 2-(methylamino)ethanol, 80 °C, 16 hr; (g)
33% CH3NH2 in EtOH, TFA, 80 °C, 17 hr; (h) azetidine, DMF, 100 °C; then chiral SFC
separation.
The synthesis of quinoline tertiary alcohols 19 and 20 was accomplished by way of palladium-
catalyzed cross coupling reactions between triflate 63 and (4-(methylsulfonyl)phenyl)boronic
acid or 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine-2-carbonitrile (Scheme 8).

Scheme 8. Synthesis of quinoline tertiary alcohols 19 and 20. Reagents and reaction conditions:
(a) n-BuLi, THF, -78 °C; then 31, -78 ° to 0 °C; (b) NaOMe, MeOH; (c) H2, 10% Pd/C, CH3OH,
1.5 hr; (d) trifluoromethanesulfonic anhydride, pyridine; (e) (4-(methylsulfonyl)phenyl)boronic
acid or 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine-2-carbonitrile, PdCl2(dppf),
K2CO3, 1,4-dioxane, 65 °C, 15 hr.
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