Discovery of biaryl carboxylamides as potent RORγ inverse agonists

Discovery of biaryl carboxylamides as potent RORγ inverse agonists

Accepted Manuscript Discovery of biaryl carboxylamides as potent RORγ inverse agonists Jianhua Chao, Istvan Enyedy, Kurt Van Vloten, Douglas Marcotte,...

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Accepted Manuscript Discovery of biaryl carboxylamides as potent RORγ inverse agonists Jianhua Chao, Istvan Enyedy, Kurt Van Vloten, Douglas Marcotte, Kevin Guertin, Richard Hutchings, Noel Powell, Howard Jones, Tonika Bohnert, ChiChi Peng, Laura Silvian, Victor Sukbong Hong, Kevin Little, Daliya Banerjee, Liaomin Peng, Arthur Taveras, Joanne L. Viney, Jason Fontenot PII: DOI: Reference:

S0960-894X(15)00476-X http://dx.doi.org/10.1016/j.bmcl.2015.05.026 BMCL 22716

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

26 March 2015 10 May 2015 12 May 2015

Please cite this article as: Chao, J., Enyedy, I., Van Vloten, K., Marcotte, D., Guertin, K., Hutchings, R., Powell, N., Jones, H., Bohnert, T., Peng, C-C., Silvian, L., Hong, V.S., Little, K., Banerjee, D., Peng, L., Taveras, A., Viney, J.L., Fontenot, J., Discovery of biaryl carboxylamides as potent RORγ inverse agonists, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.05.026

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Graphical Abstract

Discovery of biaryl carboxylamides as potent RORγγ inverse agonists

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Jianhua Chao, Istvan Enyedy, Kurt Van Vloten, Douglas Marcotte, Kevin Guertin, Richard Hutchings, Noel Powell, Howard Jones, Tonika Bohnert, Chi-Chi Peng, Laura Silvian, Victor Sukbong Hong, Kevin Little, Daliya Banerjee, Liaomin Peng, Arthur Taveras, Joanne L.Viney, and Jason Fontenot

Bioorganic & Medicinal Chemistry Letters j o ur n al h om e p a g e : w w w . e l s e v i er . c o m

Discovery of biaryl carboxylamides as potent RORγγ inverse agonists Jianhua Chaoa,∗, Istvan Enyedya, Kurt Van Vlotena, Douglas Marcottea, Kevin Guertina, Richard Hutchingsa, Noel Powella, Howard Jonesa, Tonika Bohnerta, Chi-Chi Penga, Laura Silviana, Victor Sukbong Honga, Kevin Littlea, Daliya Banerjeeb, Liaomin Pengb, Arthur Taverasa, Joanne L. Vineyb and Jason Fontenotb a b

Chemical and Molecular Therapeutics, Biogen Idec, 12 Cambridge Center, Cambridge, MA 02142 Immunology Research, Biogen Idec, 12 Cambridge Center, Cambridge, MA 02142

A R T IC LE IN F O

A B S TR A C T

Article history: Received Revised Accepted Available online

RORγt is a pivotal regulator of a pro-inflammatory gene expression program implicated in the pathology of several major human immune-mediated diseases. Evidence from mouse models demonstrates that genetic or pharmacological inhibition of RORγ activity can block the production of pathogenic cytokines, including IL-17, and convey therapeutic benefit. We have identified and developed a biaryl-carboxylamide series of RORγ inverse agonists via a structure based design approach. Co-crystal structures of compounds 16 and 48 supported the design approach and confirmed the key interactions with RORγ protein; the hydrogen bonding with His479 was key to the significant improvement in inverse agonist effect . The results have shown this is a class of potent and selective RORγ inverse agonists, with demonstrated oral bioavailability in rodents.

Keywords: Retinoid-related orphan receptor gamma t RORγt Th-17 IL-17 Biaryl carboxylamides

Retinoid-related orphan receptor gamma (RORγ) is a member of the nuclear receptor superfamily. It belongs to the ROR subfamily of the nuclear receptors which includes two closely related members RORα and RORβ.1 The three RORs share a high degree of sequence similarity, but exhibit distinct tissue distribution patterns and distinct functional roles in the regulation of the many physiological processes including development, immunity, circadian rhythm, and cellular metabolism. Two isoforms of RORγ, RORγ1 and RORγt, are generated by alternative initiation and splicing of the same gene. The two isoforms differ only in their amino terminal domains and notably share the identical ligand binding domains (LBD). RORγ1is expressed in many tissues including liver, skeletal muscle, adipose tissue while RORγt expression is limited to cells of the immune system. Nuclear hormone receptors, including RORγ, are ligand regulated transcription factors whose function is highly amenable to pharmacologic manipulation. RORγt has been demonstrated to control a pro-inflammatory gene expression program implicated in the pathology of several major autoimmune diseases. Thus RORγ has garnered significant attention as a small molecule therapeutic target.2,3,4 RORγt is essential in the development of secondary lymphoid tissues and plays a pivotal role in the development and function of multiple pro-inflammatory lymphocyte lineages including

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2009 Elsevier Ltd. All rights reserved.

TH17cells, LTi cells, γδT cells and iNKT cells. 2,3,4 These cells and the cytokines they produce, including IL-17 and IL-22, have been implicated in many chronic immune-mediated diseases. Increased numbers of RORγt-expressing cells and RORγt regulated cytokines, including IL-17 and IL-22, have been observed in the tissues and circulation of patients suffering from multiple immune-mediated pathologies including rheumatoid arthritis, inflammatory bowel diseases, multiple sclerosis, and psoriasis.5 Biologic therapeutics targeting cytokines in the RORγt pathway including IL-17 and IL-23 have demonstrated encouraging efficacy in clinical trials.6,7,8 Most recently, the first anti-IL-17A mAb CosentyxTM was approved by FDA for the treatment of psoriasis.9 Small molecule inhibition of RORγt activity has been demonstrated to inhibit the production of multiple pathogenic cytokines in this pathway and can reduce or eliminate disease in mouse models. Such small molecule therapeutics may provide broader and more potent therapeutic efficacy when compared to biologics targeting single cytokines in the RORγt pathway.10 Identification of small molecule inhibitors of RORγt activity is of considerable interest. Several RORγ biased inverse agonist series were disclosed in literature in the last two years including the tertiary sulfonamides (1),11 the phenanthridinones (2),12 the aryl-amides (3a, 3b),13 and the diphenylpropanamides (4),14

∗ Jianhua Chao. Tel.: +1-617-679-2166; fax: +1-888-689-6832; e-mail: [email protected]

scaffolds shown in Figure 1. There are a few reviews which summarized a broader chemical landscape including patent literature.3,10,15 JTE-151 (an advanced compound 5, Figure 1) from Japan Tobacco is reported to be the first RORγ inverse agonist entering phase I clinical trial.16 The field is entering an important phase in understanding the therapeutic potential and safety consequences of inhibiting RORγ activity with various classes of inverse agonists. O

O N

N

S

O

F 3C

R

HO CF 3 R Phenanthridinones (2)

Tertiary sulfonamides (1) O

O

Figure 2. A. A depiction of a co-crystal structure of an early undisclosed compound bound in the LBD, grey surface representation of the ligand, green area for the lipophilic region of the LBD; red and blue for the hydrophilic region. B. Conceptual model of a compound in the LBD which reaches out to the solvent at the pocket’s entrance.

R1

S

Y O

X

N H

S

N

OH

O

Z O X

R R2

Aryl amides (3-a) O

Diphenylpropanamides (4)

O

Figure 3. Initial hits identified from two designed libraries

S O N H Aryl amides (3-b)

N R

N N

H N

O

N N O 5

Figure 1. Representative chemical series of RORγ inverse agonists from recent publications

The ligand binding pocket of RORγ is fairly lipophilic, thus highly lipophilic molecules tend to have better binding activity which is at odds with achieving good drug-like properties.17 In our early efforts, we identified a class of biochemically potent RORγ inverse agonists, which were able to perturb H12/H11 by binding in the lipophilic region (green) of the LBD as illustrated in Figure 2, A. Only hydrophobic interactions were observed between these molecules and RORγ protein based on co-crystal structures. Due to the high lipophilicity, this class of compounds had low solubility, high plasma protein binding and microsomal instability. Seeking improvement, we applied a structure-based approach, designing molecules to reach further to the hydrophilic pocket entrance. Our intention was to incorporate polar groups into the molecule and strategically place them at the pocket entrance to allow formation of hydrogen bonding interaction(s) and at the same time to modulate the drug-like properties. The concept is highlighted in Figure 2 picture B.18 Two libraries were prepared. Compound 6 from a 20-mer library and 7 from a 88mer library emerged as good hits, with biochemical IC50 of 0.22 uM and 0.71 uM respectively. 19 Importantly, a recurring benzene-carboxylamide motif was shared by these two hits (Figure 3), which could be utilized as a polar handle for property modulation. Docking suggested the hits adopted a conformation similar to B in Figure 2.

Structure-activity-relationship investigation was prioritized on hit 6 based on the overall library results, potency and its smaller molecular size. The structure template was divided into three areas for probing, defined as head (the benzamide group), core, and tail (see general structure in Table 1). The activity of a compound was first assessed in the RORγ Fret assay for its ability of inhibiting co-activator peptide binding; and those with IC50 < 0.5 uM were tested in the RORγ Gal4 cellular assay.19,20 In the tail region, various substituents to the phenyl ring system were evaluated with respect to the chemical identity of the group and the pattern of substitution (mono, di, and trisubstitution). 2-Cl-5-F-phenyl group as in 6 was found to be one of the best and therefore used as a standard tail group in the investigation of the head and core regions. The head benzamide region was explored using different phenyl or amide substitutions. On the phenyl ring halogen, alkyl and alkoxy groups were evaluated, Table 1 (Entries 8 to 15). 4Substitution on the phenyl is required and 4-Cl appears to be optimal in terms of size and electronics when comparing 6 to the unsubstituted (8), 4-F (11), 4-Me (9), and 4-OMe (10) analogs; there is a size restriction in this area. While 5-substitution is unfavorable (12), 6-subsitution and particularly 6 and 4 disubstitution are tolerated (13 and 15). The primary amide of 6 was further derivatized with various substituents on the N (16 to 23). No significant potency improvement seen with these amide modifications, however, examples 20 and 21 demonstrated that potency can be maintained even when polar groups like hydroxyl were incorporated. The results support the hypothesis that this part of the molecule interacts with the solvent exposed region and can be utilized to modulate polarity. The methyl amide analog 16 was one of the best compounds with good biochemical (IC50 = 0.1 uM) and cellular inhibition (EC50 = 0.2 uM).

23

Table 1. Initial SAR of the benzamide head group

2.12

Assay results are reported as the mean of at least two separate runs a The biochemical assay was run using assay condition A.19 b Cellular evaluation was performed on compounds with biochemical IC50 < ~ 0.5 uM.

hRORγ Fret IC50 (uM)a

hRORγ Gal4 EC50 (uM)b

6

0.22

0.20

8

6.2

9

0.3

10

1.0

11

10.9

12

13

13

9.3

14

7.1

15

0.55

0.14

16

0.1

0.20

17

2.1

18

0.33

0.16

19

0.37

0.33

20

0.19

0.21

21

0.75

22

1.34

Entry Head group R

0.45

Co-crystallization of methyl amide 16 with RORγ protein was successful when H12 was removed.21 The structure confirms that the amide group at the head region forms a direct H-bond interaction with the backbone residue Glu379 at the pocket entrance, the 4-Cl substitution enforces a twist between the head group and the core phenyl group with a 122 degree dihedral angle, the small volume of the cavity where 4-Cl resides is consistent with the SAR results (View 1, Figure 4). The bicyclic central core serves as an anchor to deliver the 2-Chloro-5-Fluoro phenyl tail group toward H12 (even though in the co-crystal structure H12 was removed); the interactions between the core/tail groups with the protein seem to be hydrophobic only.

Figure 4. Co-crystal structure of 16 with RORγ protein (code 4ZJW, 2.5A), views from two different angles. 21

Modification of the core region initially was centered on the bicyclic motif seeking further improvement in potency by adapting it to the shape of the binding pocket. There was also a need to address the metabolic instability issue of the core template. In the initial in vitro ADME screenings, compounds in Table 1 were found to be permeable and with minor to none CYP450 inhibitions. However, they have shown a high clearance rate. When 16 was incubated with liver microsomes, it was quickly metabolized at a Clhep rate (mL/min/Kg) of 88.4 (98.2%Qh) in mouse, 53.2 (96.8% Qh) in rat, and 18.7 (93.7% Qh) in human. The three methylene groups in the tetrahydroquinone core (THQ, as highlighted in Table 2) were identified as major metabolic sites, and oxidative metabolism contributed to the high clearance rates. Various attempts were made to block these metabolic soft sites. Summarized in Table 2 are examples of the bicyclic core modifications and their effects on biochemical inhibition. Reducing the core to a [6,5]-system (24) led to a significant loss of inverse agonist effect; increasing the size to a [6,7]-system (25) was tolerated. Blocking one of the three metabolic sites on the THQ system with methyl had different impact on inhibitory activities: 1-methyl substitution (26) being detrimental, 2,2-dimethyl substitution (27) reduced activity slightly, and 3,3-dimehtyl analog (28) with unchanged activity. When two fluorines were used to block site 3 of the THQ core (29), a great loss of inhibition was observed. It was not obvious why the activity of the morpholino analog 30 was significantly reduced while the piperazinyl substitution 31 maintained activity. Overall, the modifications of the bicyclic core did not yield progressive potency improvement. While

modest reduction of Cl hep rate was achieved with 29 (human/rat/mouse %Qh: 74%, 86%, 83%) and 30 (human/rat/mouse %Qh: 76%, 85%, 82%), these were done at a significant loss of inhibitory activity.

Table 2. SAR of the bicyclic core (THQ-core) F

F O

O N

O N H

3 Cl 16

Cl 1 2

N

O

Cl R

H2N Cl

core

major metabolic sites

hRORγ Fret IC50 (uM)a

hRORγ Gal4 EC50 (uM)b

16

0.22

0.20

24

4.8

25

0.36

26

10.0

27

0.64

Entry Bicyclic core

0.53

0.45

improve activity: for example, if R2 = H, the compound was weakly active (32), and trifluoromethyl (39) and methyoxy (40) analogs were better than the halogens. Compounds 39 and 40 achieved comparable biochemical activity as the best bicyclic core compounds 16 and 31. The R1 group was evaluated as well but found to be limited for potency improvement. In the absence of R2 substitution, ethyl R1 was more potent than methyl (33 vs 32). However, this trend was not applicable to analogs where R2 was larger than H (e.g. 41 and 42). Utilizing the alkoxyl R2 as a means of probing the intended cavity, compounds 43 to 47 were prepared and improvement in potency was seen. Compounds 45 (OPr) and 47 (OCH2CF3 ) were among the best with biochemical IC50 of 11 nM and cellular potency below 100 nM. The deuterated methyl compound 48 has comparable activity to its protio analog 47. Nitrogen insertion to the phenyl core is accommodated only at the ortho position of the internal amide group. The resulting pyridyl analogs (X = N) exhibited reduced cLogP and increased solubility, however, they tend to be less active in cellular inhibition than their phenyl analogs (X = CH), for example 49 vs 47, and 50 vs 48. With R2 modifications, the potency of the pyridyl core compounds was improved, compounds 51 and 52 featuring a cyclobutylmethyleneoxy R2 group were potent biochemically (IC50 = 10 nM) and cellularly (EC50 = 60 nM). Nitrogen insertion to the R2 group i.e. replacing the alkoxy group with the dialkylamino group provided favorable potency enhancement, achieving single digit nM cellular potency as examplified by 54 and 55, while the monoalklamino group (53) was less effective.

Table 3. SAR of the monocyclic core 28

0.2

29

3.6

30

1.9

0.30

Entry X 31

0.25

R1

R2

hRORγ Fret IC50 (uM)a,

hRORγ Gal4 EC50 (uM)b

0.21

Assay results are reported as the mean of at least two separate runs a The biochemical assay was run using assay condition A.19 b Cellular evaluation was performed on compounds with biochemical IC50 < ~ 0.5 uM.

In order to address the metabolic instability issue associated with the bicyclic core and improve potency at the same time, we decided to break open the tetrahydroquinone (THQ) ring system in 16, and explore the monocyclic core motif with an emphasis on the R1 and R2 group modifications as depicted in the general structure in Table 3. A small pocket exists near the aliphatic portion of the THQ ring, as shown in view 2 (Figure 4) of the cocrystal structure of 16 with the RORγ protein. Access of this pocket via R2 may help to achieve potency improvement. Representative examples of R1 and R2 modifications and results were summarized in Table 3. The R2 group was found to be critical to potency. When R1 was maintained as a methyl group, R2 was varied from H to halogen (32, 34-36), small alkyls, and small alkoxyl groups (3740). The results have shown that increasing the size of R2 could

32

CH

CH3

H

8.7

33

CH

C2H5

H

1.29

34

CH

CH3

F

1.93

35

CH

CH3

Cl

2.09

36

CH

CH3

Br

0.82

37

CH

CH3

CH3

4.22

38

CH

CH3

C2 H5

0.73

0.350

39

CH

CH3

CF3

0.23

0.484

40

CH

CH3

OCH3

0.37

0.984

41

CH

C2H5

OCH3

1.5

42

CH

C2H5

CF3

1.6

43

CH

CH3

OC2 H5

0.05

44

CH

CH3

O-iPr

0.054

0.182

45

CH

CH3

O-nPr

0.011

0.058

46

CH

CH3

O-nBu

0.022

0.101

0.772

0.181

47

CH

CH3

OCH2CF3

0.011

0.093

EC50 (uM)

0.097

48

CH

CD3

OCH2CF3

0.029

0.097

Targets24

49

N

CH3

OCH2CF3

0.020

0.246

ERα, ERβ, FXR, GR, LXRα, LXRβ, MR, PPARα, PPARδ, PPARγ, PR, PXR, RARα, RARβ, RARγ, RXRα, RXRβ, RXRγ, TRα, TRβ, VDR, AR

50

N

CD3

OCH2CF3

0.089

0.245

51

N

CH3

OCH2-cBu

0.010

0.058

% inh @ 10 uMa

All < 50% except: ERα (61%), LXRα (53%), LXRβ (55%), MR (59%) and PR (61%)

52

N

CD3

OCH2-cBu

0.010

0.060

% act @ 10 uM

All < 50%

53

CH

CH3

NHC2 H5

0.5

0.717

54

CH

CH3

N(C2 H5 )2

0.003

0.012

55

CH

CH3

piperidyl

0.003

0.008

Assay results are reported as the mean of at least two separate runs a The biochemical assay was run using method A for entries 32 to 41, and method B for entries 42 to 52; details see reference 19. b Cellular evaluation was performed on compounds with biochemical IC50 < ~ 0.5 uM; details see reference 20.

Reaching out to an untapped small pocket in the LBD (View 2, Figure 4) with an alkoxy or dialkyl amino R2 group may have contributed to the potency improvement seen with compounds 43 to 55. The co-crystal structure of 48 and the RORγ protein, however, revealed additional intriguing feature as illustrated in Figure 5. The H-bond interaction between the head amide group and the backbone Glu379 was maintained as in the previous structure (Figure 3); but the OCH2CF3 (R2) group filled up the nearby small pocket, additionally this alkoxy group forces a twist of the internal amide group and anchors the C=O towards His479 on H11, thus forming a second H-bond interaction. This second H-bond interaction disrupts the agonist lock between His479 (on H11) and Tyr502 (on H12), and is believed to be a key factor to the improved inverse agonist effect (not merely filling the small pocket). Compounds carrying an alkoxy or dialkylamino R2 group like those described in Table 3 share this mode of action with compound 48, they generally exhibit potent inverse agonist effects both in biochemical and cellular assays.

a

Antagonist mode, %inhibition was measured; b agonist mode %activation was measured;

The monocyclic compounds described in Table 3 are highly permeable (e.g. 47:Papp(A→B)= 32.2 x 10-6cm/s and Papp(B→A)= 35.3 x 10-6cm/s; 49:Papp(A→B)= 35.9 x 10-6cm/s and Papp(B→A)= 54.5 x 10-6 cm/s) in Caco-2 cells and have no CYP inhibition or minor level; but their clearance rates are still high in general, and R1 and R2 groups remain to be the major metabolic soft spots. Of all the R2 groups screened, the OCH2 CF3 group emerged as the most stable substituent. When CD3 was applied at R1, reduced N-demethylation was observed with phenyl analogs but not the pyridyls (X = N). The deuterated analog 48 has slightly reduced clearance in microsomes (Clhep in hu / rat as %Qh: 51% / 65%) compared to its protio analog 47 (Clhep in hu / rat as %Qh: 88% / 83%). The pair of compounds was profiled in rat, and their pharmacokinetic results were summarized in Table 5. The deuterated 48 exhibited a slightly lower in vivo clearance rate and an improved oral bioavailability over the protio analog 47. The application of deuterium incorporation, i.e. CD3 as the R1 group, and its impact on metabolism needs to be explored in future.

Table 5. Summary of pharmacokinetic results of 47 and 48 in rat

a

Assessment of general nuclear receptor selectivity was performed with representative compounds of this series. Gal4 luciferase reporter assays were used to evaluate compounds’ activity: first against RORγ, RORα and RORβ in a dose responsive manner, and then in a 22-NR panel screening at a single concentration.23,24 The results of 48 were summarized in Table 4 as an example, good selectivity was observed over the tested targets.

Table 4. Selectivity of compound 48 towards other nuclear receptors Targets20,23

hRORγ

hRORα

hRORβ

1.93

b

Entry Rout ea

Figure 5. Co-crystal structure of 48 with RORγ protein (4ZJR, 2.7A).22

8.36

Dose mg/Kg

T1/2 h

AUCinf ng*hr/mL

Cmax ng/mL

CL %Qh

1.3

1127

2354

54

1553

1014

1536

2735

3134

1210

47

iv

2.0

47

po

10

48

iv

2.0

48

po

10

9.6

F%

28 40 41

Vehicles used: SPEW for iv dosing and 0.1% CMC-0.2%Tween for oral.

The biaryl-carboxylamides can be prepared efficiently from commercially available starting materials.25 Preparation of 48 was illustrated in Scheme 1. 5-Bromo-2-nitrophenol (56) was alkylated using electrophile 2,2,2-trifluoroethyltriflate to give 57, and subsequent reduction of the nitro group in 57 using iron and acetic acid provided aniline 58. The aniline was acylated with 59 to form a secondary amide 60. Upon treatment of sodium hydride, 60 reacted with deuterated methyl iodide to furnish key intermediate 61. Under Suzuki coupling conditions, 61 and boronic acid 62 would react to afford the desired product 48. Yields are generally good in this 5-step process.

14.

15. 16. 17. Scheme 1. Reagents and conditions: (a) CF3CH2 OTf (1.5 equiv.), Cs2 CO3 (2 equiv.), DMF, rt, 16h, 98%; (b) Fe (5 equiv.), HOAc, EtOH, reflux, 8h, 74%; (c) 59 (1.2 equiv.), Et3N, rt, 93%; (d) NaH (2 equiv.), CD3 I (1.2 equiv.), DMF, rt, 2 h, 90%; (e) 62, Pd(dppf)Cl2 (0.1 equiv.), Cs2CO3 (2.0 equiv.), DMF – H2O, 1000C, 3h, 50%.

Applying a structure-based drug design approach we have obtained initial hits that can form a H-bonding interaction with the RORγ protein; iterative optimization guided by structural information led to a biaryl-carboxylamide class of RORγ inverse agonists that forges a key contact with His479 of helix 11 and demonstrated low nM cellular potency. Good NR selectivity profiles can be achieved based on preliminary screening results. Representative compounds are shown to be orally available in rodents. However, improvements in potency and pharmacokinetic properties are needed in order to achieve good inhibitors to interrogate RORγ biology in vivo. Further optimization of this class of compounds and the in vitro and in vivo pharmacological evaluation of the lead compounds will be reported in the near future.

18. 19.

20.

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21.

22.

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24.

25.

Zhang, J.; Xiang, J.-N.; Leung, S.; Qiu, Y.; Zhong, Z.; Elliott, J. D.; Lin, X. ACS Med. Chem. Lett. 2014, 5, 65; c) Zhang, W.; Zhang, J.; Fang, L.; Zhou, L.; Wang, S.; Xiang, Z.; Li, Y.; Wisely, B.; Zhang, G.; An, G.; Wang, Y.; Leung, S.; Zhong, Z. Mol. Pharmacol. 2012, 82, 583. Khan, P. M.; El-Gendy, B. E. M.; Kumar, N.; Garcia-Ordonez, R.; Lin, L.; Ruiz, C. H.; Cameron, M. D.; Griffin, P. R.; Kamenecka, T. M. Bioorg. Med. Chem. Lett. 2013, 23, 532. Murali Dhar, T. G.; Zhao, Q.; Markby, D. W. Annual Rep. Med. Chem. 2013, 48, 169. Shiozaki, M. Presented at the 249th National Meeting of the American Chemical Society, Denver, CO, March 2015; paper 192. a) Parker, M. A.; Kurrasch, D. M.; Nichols, D. E. Bioorg. Med. Chem. 2008, 16, 4661; b) Pirovano, A.; Huijbregts, M. A. J.; Ragas, A. M. J.; Hendriks, A. J. Environ. Sci. Technol. 2012, 46, 5168. The details associated with the design of the two libraries will be furnished in a separate manuscript in the near future. hRORγ biochemical activity assay: a FRET-based (fluorescence resonance energy transfer) assay which measures interaction of co-activator TRAP220 peptide and the recombinant human RORγ ligand binding domain (LBD). In this assay, the ligand’s ability to enhance (agonists) or inhibit (inverse agonists) the interaction of RORγ and a TRAP220 co-activator peptide is measured in the EnVision plate reader. Assay condition A: 20 uL of compound + 5 uL of detection mix = 25 uL total assay volume; 20 mM TrisHCl pH7.0, 60 mM NaCl, 5 mM MaCl2, 1 mM DTT, 0.1% BSA; 40 nM Biotin –TRAP220, 10 nM GST-RORγ (LBD), 50 nM SAAPC, 1.5 nM Eu-Anti GST IgG, 1.0% DMSO. Assay condition B: 2.5 nM of GST-hRORγ (LBD) was used instead of 10 nM (above) to improve assay sensitivity as inhibitory activity improved. hRORγ Gal4 cell based assay: it is a functional Gal4:RORγ reporter assay that measures the ability of compounds to either activate (agonist) or suppress (inverse agonist) Gal4: RORγ mediated luciferase reporter transcriptional activity in the human embryonal kidney cell line, 293T. In the presence of RORγ modulators, Luc activation or suppression is observed for agonists or inverse agonists respectively, as measured by the LumiStar Optima plate reader. The performance of the assay takes three days: day1 cells are seeded in 96-well plates in plating medium; day 2 transfection of the cells after removal of plating medium, and treatment of cells with assay medium and then compounds 46h after transfection; day 3 cells are lysed and luciferase buffers are added, and then luminescence is measured in a dual-flash procedure. Details of the assay performance can be found in reference 25. Co-crystal structure of 16 with RORγ protein: the PDB code is 4ZJW; human RORγ ligand binding domain was used with AF2 removed. Co-crystal structure of 48 with RORγ protein: the PDB code is 4ZJR; human RORγ ligand binding domain was used with AF2 removed. hRORα and hRORβ Gal4 cell based assays: assay principle and format are the same as hRORγ Gal4 assay (ref 20). Gal4DBD:RORαLBD or Gal4DBD:RORβLBD are used respectively. NR panel screening: the screening was performed by assay group at ShangPharma (ChemPartner). Assay format: cell-based transient co-transfection reporter assay with Gal4DBD-NRLBD and UAS-Luc. The human NR LBD and HEK293 cells are used. Assay results are validated by commercial reference compounds provided by ChemPartner. There are two testing modes, agonist and antagonist. In each, test compounds and reference compounds are profiled in the same assay. Chao, J.; Enyedy, I. J.; Guertin, K.; Hutchings, R. H.; Jones, J. H.; Powell, N.; Vanvloten, K. D. PCT Int. Appl. (2014), WO2014008214 A1.