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Design, Synthesis, and Biological Evaluation of Retinoic-Acid-Related Orphan Receptor #t (ROR#t) Agonist -Structure-based functionality switching approach from in house ROR#t inverse agonist to ROR#t agonistTomoya Yukawa, Yoshi Nara, Mitsunori Kono, Ayumu Sato, Tsuneo Oda, Terufumi Takagi, Takayuki Sato, Yoshihiro Banno, Naohiro Taya, Takashi Imada, Zenyu Shiokawa, Nobuyuki Negoro, Tetsuji Kawamoto, Ryokichi Koyama, Noriko Uchiyama, Robert Skene, Isaac D. Hoffman, Chien-Hung Chen, Biching Sang, Gyorgy P. Snell, Ryosuke Katsuyama, Satoshi Yamamoto, and Junya Shirai J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01181 • Publication Date (Web): 17 Jan 2019 Downloaded from http://pubs.acs.org on February 3, 2019

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

Design, Synthesis, and Biological Evaluation of Retinoic-Acid-Related Orphan Receptor γt (RORγt) Agonist -Structure-based functionality switching approach from in house RORγt inverse agonist to RORγt agonistTomoya Yukawa,*† Yoshi Nara,† Mitsunori Kono,† Ayumu Sato,† Tsuneo Oda,† Terufumi Takagi,† Takayuki Sato,† Yoshihiro Banno,† Naohiro Taya,† Takashi Imada,† Zenyu Shiokawa,† Nobuyuki Negoro,† Tetsuji Kawamoto,† Ryokichi Koyama,† Noriko Uchiyama,† Robert Skene,‡ Isaac Hoffman,‡ Chien-Hung Chen,‡ BiChing Sang,‡ Gyorgy Snell,‡ Ryosuke Katsuyama,† Satoshi Yamamoto,*† Junya Shirai† †Pharmaceutical

Research Division, Takeda Pharmaceutical Company Limited, 26-1 Muraoka-Higashi

2-chome, Fujisawa, Kanagawa 251-8555, Japan ‡Takeda

California, 10410 Science Center Drive, San Diego, CA 92121, USA

ABSTRACT RORγt agonists are expected to provide a novel class of immune-activating anticancer drugs via activation of Th17 cells and Tc17 cells. Herein, we describe a novel structure-based functionality switching approach from in house well-optimized RORγt inverse agonists to potent RORγt agonists. We succeeded in identification of potent RORγt agonist 5 without major chemical structure change. The biochemical response was validated by molecular dynamics (MD) simulation studies that showed a helix 12 stabilization effect of RORγt agonists. These results indicate that targeting helix-12 is an attractive and novel medicinal chemistry strategy for switching existing RORγt inverse agonists to agonists. Introduction 1 Environment ACS Paragon Plus

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Th17 cells and inflammatory cytokines (such as IL-17A and IL-17F) produced by them are thought to be involved in the pathology of various cancers by immunologically modifying cancer cells and surrounding tissues. Recently, it has become clear that Retinoid-related Orphan Receptor (ROR) γt, an orphan nuclear receptors, plays an important role in differentiation of Th17 cells and IL-17A / IL-17F production. RORγt, which is mainly expressed in T cell, functions as a transcription factor of IL-17A and IL-17F and is a master regulator of Th17 cell differentiation. RORγt promotes the activation and differentiation of CD4+ and CD8+ T cells into Th17 cells and IL-17-secreting CD8+ T cells (Tc17).1 Since these cells induce adaptive immune and pro-inflammatory responses, an agent that enhances the activity of RORγt is expected to exert a therapeutic effect in various cancers by promoting the differentiation and activation of Th17 and Tc17 cells. Recently, a Scripps research group reported the potential utility of synthetic RORγt agonists in oncology.2 These synthetic agonists activated Th17 and Tc17 cells and induced IL-17 production. Lycera also reported that orally-available RORγt agonists show anti-tumor efficacy in syngeneic tumor models. They have initiated a Phase 1/2A study of RORγt agonist LYC55716 for the treatment of solid tumors and have reported some clinical trial results.3 However, despite the therapeutic potential, reports about RORγt agonists are limited.4 RORγt inverse agonists have been well-investigated with compelling preclinical biology data observed. We previously reported phenylglycine, tetrahydroqunoline and tetrahydronaphthyridine derivatives as potent RORγt inverse agonists.5 Through our research, we succeeded in the identification of clinical compound TAK-828F (Figure 1). In order to explore novel RORγt agonists quickly, a medicinal chemistry strategy of ‘functionality switching’ from well-investigated RORγt inverse agonist to RORγt agonist was deployed. This approach would allow us to leverage the following existing assets. 1) Potent binders with favorable ADMET properties are available as promising lead compounds. 2) Co-crystal structures and structure activity relationship (SAR) information that would enable us to develop clinical candidates quickly via rational drug design. The ligand-induced conformational change of ligand-binding domain (LBD) between nuclear receptor agonists and antagonists/inverse agonists has been well-investigated.6 In general, coactivators and corepressors can interact with a nuclear receptor LBD to regulate gene transcription.7 A coactivator complex is thought to involve the movement and stabilization of helix 12, indicating this helix plays a key role in controlling function. NMR experiment of peroxisome proliferator-activated receptor (PPAR) γ indicate agonists afford helix 12 stabilization compared with apo form.6b Through direct stabilization of the activation function 2 (AF-2) coactivator binding site, full agonists would promote co-activator recruitment. The stabilization effect of helix 12 in response to farnesoid X receptor (FXR) agonist binding has also been reported by nanosecond time-scale molecular dynamics (MD).8 Similar conformational changes of helix 12 have been confirmed in RORγt nuclear receptors by the comparison of agonist with 2 Environment ACS Paragon Plus

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inverse agonist crystal structures9 and by hydrogen/deuterium exchange (HDX) mass spectrometry.2 These data suggest the interaction between His479 in helix 11 and Tyr502 in helix 12 (agonist lock residues) is required to stabilize the secondary structure of helix 12 in the agonist conformation, and synthetic agonists which engage the agonist lock residues act by stabilizing this helix 12 conformation. As part of our TAK-828 inverse agonist program, the X-ray crystal structure of an inverse agonist 1 with RORγt was analyzed. This structure showed that the left-side terminal indane ring, while directed toward helix 12 was unable to reach the agonist lock residue and confer the stabilization effect to helix 12. This observation led us to hypothesize that introducing a R-group onto compound 1 designed to promote helix 12 stabilization could lead to ‘functionality switching’ from inverse agonist to agonist without major structural changes (Figure 1). In this paper, we describe the design, synthesis, and biological evaluation of a novel tetrahydronaphthyridine series of RORγt agonists. Utilizing rational compound design, we performed a structure-based ‘functionality switching’ approach starting from our inverse agonist 1. Consistency between the observed biochemical agonistic/inverse agonist functions and synthetic agonists with helix 12 stabilization was further validated by molecular dynamics (MD) simulation studies.

Inverse agonist O

O

H N

F

N O

O

O

Helix 12

N

N F

Agonist

H N

N O

O

N

R

O

Stabilize OH

H N

N O

O

O OH

CO2H

TAK-828F

1 (TAK-828 related compound)

Design Strcture

Figure 1. Concept of functionality switching from inverse agonist to agonist. Result and Discussion Lead compound generation The synthesized compounds were initially evaluated using an RORγt binding test, cofactor (steroid receptor co-activator-1: SRC-1) recruitment test and RORγt reporter gene assay.5c From our set of HTS hit compounds, compound 2 was identified as an RORγt agonist with potent binding activity, SRC-1 recruitment activity and agonistic activity in reporter gene assay. The docking model indicated that the terminal methoxyphenyl moiety of 2 projected toward helix 11 and 12, occupying the agonist lock region (His479 of helix 11 and Tyr502 of helix 12) (Figure 2a). At the opposite end of the pocket from helix 11 and 12, the sulfonamide moiety interacts with Arg367 in helix 5. In this binding pose, the terminal methoxyphenyl moiety appears to stabilize helix 12 by occupying the agonist lock region, leading to the agonistic properties of compound 2. Interestingly, when analyzing the superposition 3 Environment ACS Paragon Plus


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of compounds 1 and 2, close alignment was observed between ring A of compound 2 and ring B of inverse agonist 1 (Figure 3, Figure 2b). The information suggested that the terminal methoxyphenyl moiety of compound 2 could also work to stabilize the agonist lock residues if combined with inverse agonist 1. This hypothesis led to the design and synthesis of the para-substituted compound 3a as a novel series of RORγt agonists (Figure 3). Regarding the carboxylic part III, we proceeded design without the parts based on our experience to keep the molecule weight lower. As we expected, compound 3a showed potent binding affinity with agonist activity in the biochemical coactivator recruitment and reporter gene assay. We successfully discovered a unique RORγt agonist starting from an inverse agonist compound, and subsequently set out on the optimization study of the compound 3a.

Figure 2. (a) Docking model of compound 2 with RORγt agonist structure (b) Superposition of compounds 1 (inverse agonist, green) and 2 (agonist, blue). O

II

O NH2 S

II O

O N

I

O

O

A

N H

O

2 (Hit agonist) Binding IC50: 14 nM SRC-1 recruitment EC50: 31 nM, % activation (1 M): +1184% Reporter (3 M): +120%

III

H N

F B

N

I

N O

O

H N

O

O OH

1 (Inverse agonist) Binding IC50: 3.3 nM SRC-1 recruitment IC50: 31 nM, % activation (1 M): -81%

Figure 3. Design strategy of novel RORγt agonist. 4 Environment ACS Paragon Plus

C

D O

N O

O

3a (agonist) Binding IC50: 44 nM SRC-1 recruitment EC50: 53 nM, % activation (1 M): +1323% Reporter (3 uM): +114%, EC50: 8100 nM

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We succeeded in acquiring the crystal structure of RORγt protein in complex with the agonist 3a. This structure showed that 3a is bound within the ligand binding pocket of RORγt, and that the central paraaminooxy phenyl ring of compound 3a and the phenyl ring of terminal indane parts of compound 1 overlap as we expected (Figure 4). Additionally, the structure revealed that the terminal methoxyphenyl ring of compound 3a does occupy the agonist lock region, and play a key role in switching functionality. The terminal phenyl ring is observed making van der Waals interactions, packing against Trp317, His479 and Tyr502, with the methoxy group directed toward Trp317, forming CH- interaction with distance of 3.65 Å. This stabilize helix 12, with the H-bond between His479 and Tyr502 remaining intact, with an observed distance of 2.80 Å.

Figure 4. The crystal structure of the agonist ligand 3a within the LBD of RORγt. (left: superimposition with compound 1, right: interaction picture of compound 3a). Initial SAR study and molecular dynamics study To better understand the agonistic response of 3a and the helix 12 stabilization effect of the terminal methoxyphenyl ring, we initially investigated the ring C which occupy the agonist lock region (Table 1). However, the (S)-isomer 3b showed no binding affinity and functional effect of RORγt. In addition, the non-substituted benzyl derivative 3c showed weak binding affinity with no agonistic properties. The chloro derivative 3d showed moderate binding affinity, with no agonistic effects confirmed. These results indicated that occupation of the pocket toward Trp317 was important to binding affinity (3a, 3d vs 3c) and that an electron-donating effect was essential for the agonistic function (3a vs 3d in SRC-1 recruiting and reporter gene assay). Also, the ability of ring C to make van der Waals interactions in the agonist lock region would be important for stabilizing agonist conformation.

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Table 1. In vitro RORγt binding, SRC-1 recruitment and reporter activities of acyclic derivatives. Bindinga,c compound R

Stereo

O N H N

R C

D

* O

O

N

IC50 (nM)

EC50 (nM)

Reporter geneb,c % of control (3 M) EC50 (nM)

3a

OMe

R

44 [35-55]

53 [39-72]

114

8100 [2600-25000]

3b

OMe

S

>10000

>10000

96

>30000

3c

H

racemate

1200 [1100-1400]

>10000

N.D.

>30000

3d

Cl

racemate

290 [260-330]

>10000

80

>30000

O

3

SRC-1 recruitingc

a

Displacement assay of BODIPY-labeled ligand with apo RORt Jurkat RE, cell-based assay c 95% confidence intervals (CI) conducted in duplicate (n = 2) b

We conducted 500 ns MD simulation of RORγt with agonist compound 3a and inverse agonist compound 1 using a structure of RORγt in the agonist conformation to better understand the helix 12 stabilization effect of the terminal ring. Conditions of the MD simulation are summarized in the experimental section and the results are shown in Figure 5a. With agonist 3a, helix 12 remained in the active conformation along all the 500 ns simulations, while a major conformational change was observed with inverse agonist compound 1 (Figure 5a, left, 3a: magenta line, 1: green line). As we mentioned above, the interaction between His479 in helix 11 and Tyr502 in helix 12 is considered to stabilize the position of helix 12 in the active form. Therefore, we observed the distance between the nitrogen atom of His479 with the oxygen atom of Tyr502 in 500 ns scale simulation. For agonist 3a, no dynamic change of the distance was observed with the average rmsd in 500 ns of 2.99 Å, while the interaction was clearly disrupted with inverse agonist 1 (Figure 5a, right, 3a: magenta line, 1: green line). This observation is consistent with our hypothesis, which the hydrogen bond between His479 and Tyr502 (agonist lock residues) has a key role to stabilize helix 12 conformation. In addition to the validation of agonistic/inverse agonistic response by MD, we also conducted MD simulation of neutral ligand (R)-3d, which has similar structure with agonist 3a (methoxy vs chloro-group). Interestingly, neutral binder (R)-3d gave different behavior of helix 12/hydrogen bond between Tyr502 and His479 compared with 3a and 1 within 500 ns trajectory (Figure 5a, blue line). Figure 5b shows the ratio of MD studies, which gave helix 12 movement (rmsd >2 Å) and hydrogen bond between His479Try502 disruption (>4 Å) are not linked (n = 4). This result highlights the difference of helix 12/His479Tyr502 hydrogen bond behavior between agonist 3a/inverse agonist 1 and neutral ligand (R)-3d. 38.3% of MD study of (R)-3d demonstrates unlink between helix 12 movement and His479-Tyr502 hydrogen bond disruption. Our investigation clearly suggested that not only hydrogen bond between His479-Tyr502, but also other interactions have key roles of stabilizing helix 12 as an active form. Just recently, Sun et al. 6 Environment ACS Paragon Plus

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independently reported a critical role of Trp317 conformation, in addition to hydrogen bond between His479-Tyr502, for agonistic response of RORγt by MD simulation study.10 They showed Trp317 was stayed as “gauche” conformation when a synthetic agonist bound, and that helped to form the hydrogen bond between His479 and Trp502, and a large hydrophobic network among helix 11 and helix 12, while an inverse agonist led Trp317 to “trans” conformation. In fact, methoxy group of 3a has CH- interaction with Trp317 in co-crystal structure (Figure 4), while 3d is supposed not to have the interaction due to the lacking of hydrogen atoms or size difference of chloro atom with methoxy group. Consistent with our SAR study, interaction with Trp317 would also play a key role for agonistic responses of our molecules. We used the active state of RORγt structures bound to each RORγt ligand as the initial structure of our MD simulation and identified three different behaviors of helix 12/hydrogen bond between Tyr502 and His479, while Sun et al. utilized active conformation for their agonist and inactive conformation for their inverse agonist, respectively. In our MD simulation study which side chain of Trp317 was initially “gauche” conformation (Figure 5c, white), conformational change to “trans” conformation of Trp317 was observed with inverse agonist 1 (Figure 5c, right, green), but not with agonist 3a (Figure 5c, left, magenta). Our MD study is undoubtedly consistent with the conclusion by Sun et al. which was supported by some crystal structure information, and it clearly suggests an important role of Trp317 for the stabilization of helix 12. More detail analysis to clarify both activation and inactivation mechanism by agonist and inverse agonist will be reported in the future. Figure 5d shows the comparison of before and after conformations of RORγt with agonist 3a, neutral ligand (R)-3d or inverse agonist 1. These MD experiments, with 1, 3a and (R)-3d bound to RORγt, provide additional validation of the helix 12 stabilization strategy designed to affect an agonist biochemical response.

Figure 5a. Variation of the rmsd (Å) of helix 12 (Phe498-Ser507) during the 500 ns trajectory.



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Figure 5b. The ratio which rmsd of helix 12 (>2 Å) and hydrogen bond disruption between His479Try502 (>4 Å) are NOT linked in MD studies (n = 4).

Figure 5c. Graphical representation of the conformational state of Trp317 observed in MD simulation. (white: crystal structure, left: with agonist 3a (magenta), right: with inverse agonist 1 (green)).

Figure 5d. Graphical representation of the helix 12. (left: with agonist 3a, center: with neutal ligand (R)-3d, right: with inverse agonist 1, white: crystal structure, green, blue and magenta: structure at the end of MD simulations.) 8 Environment ACS Paragon Plus

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Medicinal chemistry lead optimization With agonist ligand 3a and the insight about agonistic response in hand, we investigated modification around the D ring in order to identify potent and drug-like RORγt agonists. The ortho-substituent on ring C was fixed to an alkoxy group. Previously, in our RORγt inverse agonist program, we noticed that ring B modification, such as conversion from monocyclic to bicyclic ring, gave favorable SAR. In addition, ring B-tert-carbon (Figure 6, blue of compound 1) and the ring D-oxygen (Figure 6, blue of compound 3a) overlay closely when superimposed (Figure 4). Keeping this in mind, we designed 5,6-membered bicyclic indoline derivative 4. H N

F

O

B C Compound 1

C

H N

H N D O

Compound 3a

RO

N

Design Structure 4

Figure 6. Design strategy of indoline derivative. The compound 4a showed marked enhancement of binding and agonistic activities compared with acyclic derivative 3a (Table 2). Replacement of the methoxy group with an ethoxy group or trifluoromethoxy group maintained the binding activity with potent reporter gene activity. We proceed 4b for further investigation, however, 4b shows poor PK profiles in mice. Next, we introduced a carboxylic acid tether to compound 4b to improve PK profiles with maintaining affinity based on our experience. As expected, compound 5 maintained the agonistic properties with drastically improvement of PK profiles in mice (Cmax = 226.7 ng/mL, AUC0-8h: 1722 ng*h/mL, MRT = 4.1 h, F = 19%) like compound 1.11 Compound 5 also doesn‘t show CYP3A4 inhibition at 10 μM and cytotoxicity at 100 μM in HepG2 cell. Finally, we progressed indoline derivatives 4b and 5 into a mouse spleen Th17 differentiation assay12 and observed dose dependent IL-17 productions of synthetic agonists 4b and 5, while an inverse agonist 1 showed inhibition of IL-17 production (Figure 7). In agreement with their agonistic mechanism of action, compounds 4b and 5 showed Th17 cell differentiation activities. Table 2. In vitro RORγt binding, SRC-1 recruitment, reporter activities and Th17 differentiation of indoline derivatives.



Bindingb,c compound

R1

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R2 IC50 (nM)

O

EC50 (nM)

Reporter genec,d % of control EC50 (nM) (3 M)

N H N R 1O

N

4aa

Me

Me

14 [7.5-25]

9.5 [8.0-11]

134

270 [68-1100]

4b

Et

Me

7.2 [5.1-10]

4.1 [2.9-57]

157

140 [91-220]

4ca

CF3

20 [18-22] 6.0 (CH2)3COOH [5.6-6.5]

11 [8.8-15] 2.1 [1.5-2.8]

N O

O

R2

4a-4c, 5

5

Et

Me

139 147

2000 [850-4700] 68 [36-130]

a

The compounds are racemic. Displacement assay of BODIPY-labeled ligand with apo RORt c 95% confidence intervals (CI) conducted in duplicate (n = 2) d Jurkat RE, cell-based assay

b

Figure 7. In vitro IL-17A production assay of compounds 1 (left, inverse agonist), 4b (center, agonist) and 5 (right, agonist). Results were expressed as mean ± SE in each group (N=3). *p< 0.025 as compared with vehicle (Williams test). Chemistry The synthesis of the tetrahydronaphthyridine derivative is shown in Scheme 1. Key intermediate rac-6 and (R)-6 were prepared by reported method.5c Benzyl protection of the carboxylic acid rac-6 and removal of Boc group gave amine derivative 7. After acetylation and following benzyl group deprotection, conjugation with anilines afforded compounds 3a-3d and 4a. Boc-protected amino indoline was similarly conjugated with carboxylic acid 8 and then Boc group of indoline ring was removed. Alkylation with benzyl halide gave indoline derivatives 4c. Optically pure indoline derivatives were prepared using intermediate (R)-6, which was prepared by optical resolution of rac-6 by diastereomeric salt formation. 2-Ethoxy benzyl indoline was introduced to (R)-6 by using DMT-MM as a condensation reagent to avoid epimerization, and then de-protection and following condensation with corresponding acid chloride or carboxylic anhydride afforded ethoxy indoline derivatives (4b, 5). [Scheme 1] 10 Environment ACS Paragon Plus

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Journal of Medicinal Chemistry O

HO

a, b

O

O

O

N

c, d

N

N Boc

HO

N H

R N

e, (f) H N

N O

7

rac-6

O

O

O N

MeO

O

N

O N

N e, b

g H N

H N

N O

HN

F3CO

O

N O

N

O

4c

9 O

O N

rac-6

h 6 (R)

4a

8

O

8

R = OMe (R: 3a, S: 3b) H (3c) Cl (3d)

N O

O

O

e, b

c or i

H N O

O

N

N

N H

H N O

N

O

N R

R = Ac (4b) (CH2)3COOH (5)

10

a

Reagents and conditions: (a) BnBr, K2CO3, DMF, rt, quant.; (b) TFA, neat or toluene, rt, 74%–quant.;

(c) AcCl, Et3N, THF, rt, 73%–84%; (d) Pd/C, H2, MeOH, rt, 84%; (e) corresponding aniline, HATU, DIPEA, DMF, rt or DMT-MM, EtOH, rt, 16%–84%; (f) optical resolution by SFC; (g) 2(trifluoromethoxy)benzyl

bromide,

K2CO3,

DMF,

rt,

60%;

(h)

((1R,2S)-2-

(benzylamino)cyclohexyl)methanol, EtOH, CH3CN, 50 °C, 28%, >99.9% d.e. and then 5% citric acid aq., EtOAc, rt, 100% (i) Succinic anhydride, Et3N, THF, rt, 84%.

Conclusion In conclusion, we demonstrated a structure-based ‘functionality switching’ strategy to convert existing RORγt inverse agonists to RORγt agonists can successfully deliver a unique and drug-like compound. We utilized MD simulation to characterize the stabilization effect of helix 12 by synthetic RORγt agonists and found that the helix 12 stabilization effect were properly simulated even in similar chemical series with varied biochemical response. Structure-based drug design was used to discover the potent indoline derivative RORγt agonists 4b. By introducing carboxylic motif based on our experience, we succeed to identify orally-available RORγt agonist 5. We identified that appropriate substituent introductions can stabilize the helix 12 conformation with his479-Tyr502 hydrogen bond of RORγt. Although several group reported medicinal chemistry efforts of RORγt agonist with an investigation of helix 12 conformation difference between inverse agonist and agonist, to the best of my knowledge, this is a first strategy to 11 Environment ACS Paragon Plus


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obtain potent and orally-available RORγt agonists by structure-based drug design of existing RORγt inverse agonists. These results indicate that helix 12-targeting ‘functionality switching’ approach from existing RORγt inverse agonists is novel and attractive medicinal chemistry strategy of switching a chemotype functionality, and MD simulation can support drug design within this approach. We also believe that the ‘functionality switching’ approach will be applicable for other targets. By carefully selecting drug-like lead compounds (ex, favorable ADMET profiles) with accessibility to ‘hot spot’ based on crystal structure and SAR information, drug discovery process would be shortened dramatically.

Experimental Section Chemistry Melting points were determined with a Yanagimoto melting point apparatus or a Büchi melting point apparatus B-545 and are uncorrected. 1H NMR spectra were obtained at 300 MHz on a Varian Ultra-300, or a Bruker DPX-300 spectrometer. Chemical shifts are given in δ values (ppm) using tetramethylsilane as the internal standard. Peak multiplicities are expressed as follows: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublet; ddd, doublet of doublet of doublets; dt, double triplet; td, triple doublet; quin., quintet; brs, broad singlet; m, multiplet. Elemental analyses were carried out by Takeda Analytical Laboratories Ltd. Reactions were followed by TLC on Silica gel 60 F 254 precoated TLC plates (E. Merck) or NH TLC plates (Fuji Silysia Chemical Ltd.). Chromatographic separations were carried out on silica gel 60 (0.063–0.200 or 0.040–0.063 mm, E. Merck) or basic silica gel (Chromatorex® NH, 100– 200 mesh, Fuji Silysia Chemical Ltd.) using the indicated eluents. Yields are unoptimized. The HPLC analyses were performed using a Shimadzu UFLC instrument. Elution was done with a gradient of 5– 90% solvent B in solvent A (solvent A was 0.1% TFA in water, and solvent B was 0.1% TFA in MeCN) through a L-column 2 ODS (3.0 × 50 mm, 2 μm) column at 1.2 mL min-1. Area % purity was measured at 254 nm. The purity of all key test compounds was assessed by HPLC (>95%. If there is no note.). Abbreviations for chemicals are the following: MeOH, methanol; EtOH, ethanol; EtOAc, ethyl acetate; THF, tetrahydrofuran; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; MeCN, acetonitrile; CDCl3, chroloform-d; Et3N, triethylamine; Boc2O, di-tert-butyl dicarbonate; HATU, 2-(3H[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate (V); DMT-MM, 4-(4,6-dimethoxy[1.3.5]triazin-2-yl)-4-methylmorpholinium chloride; TFA, trifluoroacetic acid; DIPEA, N,N-diisopropylethylamine (R)-6-Acetyl-2-methoxy-N-(4-((2-methoxybenzyl)oxy)phenyl)-5,6,7,8-tetrahydro-1,6naphthyridine-5-carboxamide (3a), (S)-6-Acetyl-2-methoxy-N-(4-((2-methoxybenzyl)oxy)phenyl)5,6,7,8-tetrahydro-1,6-naphthyridine-5-carboxamide (3b). A mixture of 8 (237 mg, 0.95 mmol), 412 Environment ACS Paragon Plus

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((2-methoxybenzyl)oxy)aniline (217 mg, 0.95 mmol), HATU (432 mg, 1.14 mmol) and DIPEA (0.25 mL, 1.42 mmol) in DMF (10 mL) was stirred at rt for 3 h. The mixture was quneched with brine and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4 and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexane/EtOAc) and following purification by preparative SFC gave 3a (32.0 mg, 7%) as tR1 fraction (shorter retention time) and 3b (52.0 mg, 12%) as tR3 fraction (longer retention time). 3a: 1H NMR (300 MHz, CDCl3) δ 2.27 (3H, s), 2.96–3.06 (2H, m), 3.64–3.79 (1H, m), 3.85 (3H, s), 3.90–3.99 (4H, m), 5.07 (2H, s), 6.03 (1H, s), 6.65 (1H, d, J = 8.3 Hz), 6.83–7.02 (4H, m), 7.25–7.33 (1H, m), 7.38–7.47 (4H, m), 8.87 (1H, s). MS m/z: 462.2 [M+H]+. [α]25D +94.8 (c 0.25, MeOH), HPLC purity 99.8%. 3b: 1H NMR (300 MHz, DMSO-d6) δ 2.13–2.26 (3H, m), 2.84–3.11 (2H, m), 3.77–3.85 (6H, m), 3.88 (1H, brs), 3.97–4.15 (1H, m), 5.01 (2H, s), 5.78 (1H, s), 6.72 (1H, d, J = 8.7 Hz), 6.83–6.99 (3H, m), 7.04 (1H, d, J = 8.7 Hz), 7.24–7.40 (2H, m), 7.47 (2H, d, J = 8.7 Hz), 7.79 (1H, d, J = 8.7 Hz), 9.97–10.37 (1H, m). MS m/z: 462.2 [M+H]+. [α]25D 91.8 (c 0.25, MeOH), HPLC purity 100%. 6-Acetyl-N-(4-(benzyloxy)phenyl)-2-methoxy-5,6,7,8-tetrahydro-1,6-naphthyridine-5-carboxamide (3c). To a mixture of 4-(benzyloxy)aniline (119 mg, 0.60 mmol), 8 (100 mg, 0.40 mmol) and DIPEA (140 μl, 0.80 mmol) in DMF (1.3 ml) was added HATU (182 mg, 0.48 mmol). After being stirred at 80 °C overnight the mixture was poured into water and extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexane/EtOAc) to give 3c (127 mg, 73%) as a brown powder. 1H NMR (300 MHz, CDCl3) δ 2.27 (3H, s), 2.80 (3H, s), 2.94–3.03 (2H, m), 3.65–3.77 (1H, m), 3.87–3.99 (4H, m), 5.04 (2H, s), 6.03 (1H, s), 6.65 (1H, d, J = 8.7 Hz), 6.83–6.98 (2H, m), 7.29–7.49 (8H, m), 8.80 (1H, s). MS m/z: 432.2 [M+H]+, HPLC purity 92.9%. 6-Acetyl-N-(4-((2-chlorobenzyl)oxy)phenyl)-2-methoxy-5,6,7,8-tetrahydro-1,6-naphthyridine-5carboxamide (3d). To a mixture of 4-((2-chlorobenzyl)oxy)aniline (44.8 mg, 0.19 mmol), 8 (40 mg, 0.16 mmol) and DIPEA (55.8 μl, 0.32 mmol) in DMF (530 μl) was added HATU (91 mg, 0.24 mmol). After being stirred at rt overnight, the mixture was poured into water and extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexane/EtOAc) to give 3d (66.6 mg, 89%) as a colorless foam. 1H NMR (300 MHz, CDCl3) δ 2.27 (3H, s), 2.99 (2H, dd, J = 7.6, 4.5 Hz), 3.65–3.78 (1H, m), 3.88–3.99 (4H, m), 5.14 (2H, s), 6.03 (1H, s), 6.66 (1H, d, J = 8.7 Hz), 6.88–6.98 (2H, m), 7.21– 7.31 (1H, m), 7.35–7.57 (5H, m), 8.02 (1H, s), 8.82 (1H, s). MS m/z: 466.2 [M+H]+, HPLC purity 94.0%. 6-Acetyl-2-methoxy-N-(1-(2-methoxybenzyl)indolin-5-yl)-5,6,7,8-tetrahydro-1,6-naphthyridine-5carboxamide (4a). To a mixture of 1-(2-methoxybenzyl)indolin-5-amine (80 mg, 0.31 mmol), 8 (79 13 Environment ACS Paragon Plus


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mg, 0.31 mmol) and DIPEA (104 μl, 0.63 mmol) in DMF (4 ml) was added HATU (144 mg, 0.38 mmol). After being stirred at rt overnight the mixture was poured into water and extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over MgSO4 and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexane/EtOAc) to give 4a (24 mg, 16%) as a colorless solid. 1H NMR (300 MHz, CDCl3) δ 2.26 (3H, s), 2.83–3.10 (4H, m), 3.38 (2H, t, J = 8.3 Hz), 3.78–4.07 (8H, m), 4.23 (2H, s), 5.91–6.09 (1H, m), 6.40 (1H, d, J = 8.3 Hz), 6.65 (1H, d, J = 8.7 Hz), 6.77–7.01 (2H, m), 7.10 (1H, d, J = 8.3 Hz), 7.36 (4H, dd, J = 18.3, 9.3 Hz), 8.56 (1H, s). MS m/z: 487.3 [M+H]+, HPLC purity 91.4%. (R)-6-Acetyl-N-(1-(2-ethoxybenzyl)indolin-5-yl)-2-methoxy-5,6,7,8-tetrahydro-1,6-naphthyridine5-carboxamide (4b). To a mixture of 10 (2.40 g, 5.25 mmol) and TEA (1.46 mL, 10.5 mmol) in THF (26 mL) was added acetyl chloride (560 μL, 7.87 mmol) at 0 °C. The mixture was stirred at rt under Ar for 2 h. The mixture was poured into water and extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexane/EtOAc) and tritulated with iPr2O to give 4b (2.20 g, 84%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 1.41 (3H, t, J = 7.0 Hz), 2.26 (3H, s), 2.86–3.04 (4H, m), 3.39 (2H, t, J = 8.3 Hz), 3.64–3.82 (1H, m), 3.85–3.99 (4H, m), 4.05 (2H, q, J = 6.8 Hz), 4.24 (2H, s), 6.02 (1H, s), 6.41 (1H, d, J = 8.3 Hz), 6.64 (1H, d, J = 8.3 Hz), 6.84–6.92 (2H, m), 7.10 (1H, dd, J = 8.5, 2.1 Hz), 7.17–7.35 (3H, m), 7.40 (1H, d, J = 8.3 Hz), 8.62 (1H, brs). MS m/z: 501.2 [M+H]+. [α]25D +92.1 (c 0.20, MeOH), HPLC purity 97.7%. 6-Acetyl-2-methoxy-N-(1-(2-(trifluoromethoxy)benzyl)indolin-5-yl)-5,6,7,8-tetrahydro-1,6naphthyridine-5-carboxamide (4c). A mixture of 9 (50 mg, 0.14 mmol), 2-(trifluoromethoxy)benzyl bromide (33 μL, 0.20 mmol) and K2CO3 (56.6 mg, 0.41 mmol) in DMF (2 mL) was stirred at rt for 3 h. The mixture was quneched with brine and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4 and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexane/EtOAc) to give 4c (44.1 mg, 60%) as a pale yellow amorphous solid. 1H

NMR (300 MHz, CDCl3) δ 2.10–2.18 (3H, m), 2.82–3.06 (4H, m), 3.19–3.29 (2H, m), 3.83 (3H, s),

3.84–3.92 (1H, m), 3.98–4.13 (1H, m), 4.27 (2H, s), 5.77 (1H, s), 6.40–6.50 (1H, m), 6.66–6.75 (1H, m), 7.09–7.19 (1H, m), 7.26–7.33 (1H, m), 7.34–7.46 (3H, m), 7.48–7.55 (1H, m), 7.72–7.81 (1H, m), 10.06 (1H, s). MS m/z: 541.2 [M+H]+, HPLC purity 98.8%. (R)-4-(5-((1-(2-Ethoxybenzyl)indolin-5-yl)carbamoyl)-2-methoxy-7,8-dihydro-1,6-naphthyridin6(5H)-yl)-4-oxobutanoic acid (5). Succinic anhydride (709 mg, 7.09 mmol) was added to a mixture of 10 (2.50 g, 5.45 mmol) and Et3N (1.14 mL, 8.18 mmol) in THF (55 mL) at rt under Ar. After being stirred at rt for 4 h, the reaction mixture was poured into water, acidified with 2 N HCl and extracted with EtOAc14 Environment ACS Paragon Plus

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THF. The organic layer was washed with water and brine, dried over MgSO4 and concentrated in vacuo. The residue was purified by column chromatography (Diol-silica gel, hexane/EtOAc) to give 5 (2.56 g, 84%) as an off-white amorphous solid 1H NMR (300 MHz, CDCl3) δ 1.40 (3H, t, J = 7.0 Hz), 2.56–3.06 (8H, m), 3.34 (2H, brs), 3.72–3.83 (1H, m), 3.90 (3H, s), 3.92–3.99 (1H, m), 4.04 (2H, q, J = 7.2 Hz), 4.22 (1H, brs), 5.97 (1H, brs), 6.37 (1H, brs), 6.61 (1H, d, J = 8.7 Hz), 6.82–6.91 (2H, m), 6.97–7.33 (5H, m), 7.46 (1H, d, J = 8.7 Hz), 8.53 (1H, brs). MS m/z: 559.3 [M+H]+, HPLC purity 100%. (R)-6-(tert-Butoxycarbonyl)-2-methoxy-5,6,7,8-tetrahydro-1,6-naphthyridine-5-carboxylic acid ((R)-6). The solution of ((1R,2S)-2-(benzylamino)cyclohexyl)methanol (2.19 g, 10.0 mmol) in EtOH (18 mL) was added dropwise to a solution of rac-6 (3.08 g, 10 mmol) in CH3CN (36 mL) at 50 °C. The mixture was stirred at 50 °C for 30 min then at rt overnight. The resulting precipitate was collected by filtration, washed with CH3CN/EtOH (2/1) and dried in vacuo to give a white crystal (2.2 g). The crystals (1.5 g) were re-crystallized MeOH (10 mL)-EtOAt/heptane (1/5) (120 mL) to give ((1R,2S)-2(benzylamino)cyclohexyl)methanol

(R)-6-(tert-butoxycarbonyl)-2-methoxy-5,6,7,8-tetrahydro-1,6-

naphthyridine-5-carboxylate (11, 1.0 g, 28%) as a white crystal. 1H NMR (300 MHz, DMSO-d6) δ 1.13– 1.74 (16H, m), 1.86 (1H, brs), 2.69–2.83 (2H, m), 2.83–2.96 (1H, m), 3.36–3.69 (3H, m), 3.76–3.89 (5H, m), 3.90–4.09 (1H, m), 5.11–5.35 (1H, m), 6.66 (1H, d, J = 8.7 Hz), 7.20–7.42 (5H, m), 7.82 (1H, dd, J = 8.7, 3.4 Hz). MS m/z: 309.2 [M+H]+. The (R) absolute configuration was determine by single-crystal X-ray analysis (see Figure 7 in experimental section). >99.9% d.e. was observed by HPLC (column: SUMICHIRAL OA2500 4.6 mm ID×250 mmL, mobile phase: Methanol/TEA/AA=1000/5/5). The crystal (422 mg, 0.8 mmol) was dissolved in EtOAc (30 mL) and 5% citric acid aq. (100 mL), and the mixture was poured into water and extracted with EtOAc. The organic layer was separated, washed with 5% citric acid aq. and brine, dried over MgSO4 and concentrated in vacuo to give (R)-6 (247 mg, 100 %) as an amorphous solid.1H NMR (300 MHz, CDCl3) δ 1.49 (9H, d, J = 7.9 Hz), 2.92 (2H, brs), 3.52–3.71 (1H, m), 3.86–3.94 (3H, m), 4.06 (1H, d, J = 15.5 Hz), 5.32–5.65 (1H, m), 6.62 (1H, d, J = 8.3 Hz), 7.68 (1H, d, J = 8.7 Hz). MS m/z: 309.2 [M+H]+. Benzyl 2-methoxy-5,6,7,8-tetrahydro-1,6-naphthyridine-5-carboxylate (7). To a mixture of benzyl bromide (7.72 mL, 64.9 mmol) and rac-6 (10 g, 32.4 mmol) in DMF (80 mL) was added K2CO3 (13.4 g, 97.3 mmol). The mixture was stirred at rt overnight. The mixture was poured into water and extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexane/EtOAc) to give 5-benzyl 6-tert-butyl 2-methoxy-7,8-dihydro-1,6-naphthyridine-5,6(5H)-dicarboxylate (14.4 g, quant) as a colorless oil. MS m/z: 399.3 [M+H]+. To the oil was added TFA (40 mL, 519 mmol). The mixture was stirred at rt for 10 min, and then added to ice-NaHCO3 aq.-EtOAc solution. The neutralized 15 Environment ACS Paragon Plus


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mixture was separated, washed with water and brine, dried over Na2SO4 and concentrated in vacuo to give 7 (12.2 g, quant.). MS m/z: 299.3 [M+H]+. 6-Acetyl-2-methoxy-5,6,7,8-tetrahydro-1,6-naphthyridine-5-carboxylic acid (8). To a mixture of 7 (12.2 g, 40.7 mmol) and acetyl chloride (7.24 mL, 102 mmol) in THF (200 mL) was added Et3N (22.7 mL, 163 mmol). The mixture was stirred at rt for 1 h and then concentrated in vacuo to 1/3 volume. The residue was poured into water and extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexane/EtOAc) to give benzyl 6-acetyl-2-methoxy-5,6,7,8-tetrahydro-1,6naphthyridine-5-carboxylate (10.1 g, 73%) as a colorless oil. MS m/z: 341.2 [M+H]+. To a solution of the oil in MeOH (59.4 mL) was added Pd-C (1.0 g, 9.40 mmol) at rt under N2. The mixture was stirred under H2 for 3 h then filtered through Celite and concentrated in vacuo. The residue was crystallized from EtOAc-hexane to give 8 (6.25 g, 84%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 2.25 (3H, s), 2.92– 3.04 (2H, m), 3.78–3.94 (5H, m), 5.85 (1H, s), 6.48–6.73 (1H, m), 7.70 (1H, d, J = 8.7 Hz). MS m/z: 251.3 [M+H]+. 6-Acetyl-N-(indolin-5-yl)-2-methoxy-5,6,7,8-tetrahydro-1,6-naphthyridine-5-carboxamide (9). To a mixture of 8 (470 mg, 1.88 mmol), tert-butyl 5-aminoindoline-1-carboxylate (400 mg, 1.71 mmol) and DIPEA (0.58 mL, 3.41 mmol) in DMF (8.0 mL) was added HATU (779 mg, 2.05 mmol) at rt. The mixture was stirred for 1 h. The mixture was quenched with water and extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over MgSO4 and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexane/EtOAc) to give tert-butyl 5-(6-acetyl-2methoxy-5,6,7,8-tetrahydro-1,6-naphthyridine-5-carboxamido)indoline-1-carboxylate (668 mg, 84%) as a pale yellow amorphous solid. MS m/z: 467.3 [M+H]+. The mixture of the solid and TFA (6.6 mL, 85.7 mmol) was stirred at rt for 5 min. The mixture was poured into sat. NaHCO3 aq. and extracted with EtOAc. The organic layer was separated and dried over MgSO4 and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexane/EtOAc) to give 9 (420 mg, 80%) as a yellow solid. MS m/z: 367.2 [M+H]+. (R)-N-(1-(2-Ethoxybenzyl)indolin-5-yl)-2-methoxy-5,6,7,8-tetrahydro-1,6-naphthyridine-5carboxamide (10). DMT-MM (18.2 g, 65.7 mmol) was added to a solution of (R)-6 (14.2 g, 46 mmol) and 1-(2-ethoxybenzyl)indolin-5-amine (11.8 g, 43.8 mmol) in EtOH (300 mL) at rt under Ar. After being stirred at rt for 2 h, the reaction mixture was poured into water and extracted with EtOAc. The organic layer was washed with water and brine, dried over MgSO4 and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexane/EtOAc) to give (R)-tert-butyl 5-((1-(2ethoxybenzyl)indolin-5-yl)carbamoyl)-2-methoxy-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate 16 Environment ACS Paragon Plus

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(12.1 g, 50%) as a gray amorphous solid. MS m/z: 559.3 [M+H]+. TFA (100 mL) was added to the solid in toluene (100 mL) at rt. After being stirred at rt for 20 min, the reaction mixture was poured into ice sat. NaHCO3 aq. (500 mL), basified (pH 8) by the addition of 8 N NaOH and K2CO3 with stirring, and extracted with EtOAc. The organic layer was washed with water and brine, dried over MgSO4 and concentrated in vacuo to give crystals, which were collected with EtOAc. The filtrate was concentrated in vacuo, and the residue was purified by column chromatography (silica gel, hexane/EtOAc) to give crystals, which were collected by filtration, combined with the above crystals and washed with iPr2Ohexane to give 10 (7.40 g, 74%) as a off-white powder. 1H NMR (300 MHz, DMSO-d6) δ 1.34 (3H, t, J = 7.0 Hz), 2.62–2.81 (2H, m), 2.86 (2H, t, J = 8.3 Hz), 2.95–3.06 (1H, m), 3.14–3.23 (1H, m), 3.29 (2H, t), 3.69 (1H, brs), 3.81 (3H, s), 4.05 (2H, q, J = 6.8 Hz), 4.18 (2H, s), 4.51 (1H, s), 6.46 (1H, d, J = 8.3 Hz), 6.62 (1H, d, J = 8.3 Hz), 6.84–6.92 (1H, m), 6.98 (1H, d, J = 7.6 Hz), 7.16–7.29 (3H, m), 7.34 (1H, d, J = 1.5 Hz), 7.62 (1H, d, J = 8.7 Hz), 9.77 (1H, s). MS m/z: 459.2 [M+H]+. Materials and Methods Cloning, Expression and Purification of RORγt For structure determination, two constructs of the RORγt ligand binding domain (residues 261-494, and 261-508) were amplified from cDNA by PCR and cloned into the pSX70 vector. The human RORγt LBD was over-expressed in fusion with an N-terminal 6x poly-histidine tag and TEV cleavage site. Large scale production of recombinant protein was carried out in E. coli BL21 cells utilizing 5 L shake flasks. For both constructs, RORγt purification was carried out from cell pellets re-suspended in lysis buffer consisting of 50 mM Tris-HCl (pH 7.6), 200 mM NaCl, 20mM imidazole, 0.25 mM TCEP, 3 Roche Complete tablets, and further lysed via polytron for 2-4 minutes. The lysate was centrifuged at 4200xg for 60 minutes and clarified supernatant was batch bound with 5 ml of Probond Ni resin (Invitrogen). The resin slurry was washed then eluted with buffer containing and additional 200 mM imidazole. Subsequent cleavage of the 6x poly-histidine tag was initiated by the addition of 1 mg of TEV followed by dialysed into buffer containing only 20 mM imidazole. TEV cleavage was confirmed by mass spectroscopy and the sample was incubated with an additional 5 ml of Probond Ni resin. The resin slurry was removed by centrifugation and the protein sample was further purified by size-exclusion chromatography utilizing a Superdex 200 column equilibrated in 25 mM Tris-HCl (pH 7.9), 200 mM NaCl, 5% Glycerol, 0.5 mM TCEP. Fractions containing the protein of interest were pooled and concentrated to 11 mg/ml utilizing YM10 centricon (Millipore) and flash-frozen in liquid nitrogen for storage at -80 °C. Crystallization, Data Collection and Structure Solution of RORγt 17 Environment ACS Paragon Plus


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Crystals of RORγt (residues 261-494) in complex with inverse agonist compound 1 were prepared by incubation of 11 mg/ml protein with 1mM compound from 50 mM DMSO stock solutions and left on ice for 1 hour. Initial crystal trials were conducted utilizing Takeda San Diego’s automated nanovolume crystallization technology platform. Large crystals suitable for data collection were obtained from reservoir solution containing 1 - 1.6M Sodium Formate, 3% MPD, and 100 mM HEPES (pH 7.5). Crystals of RORγt (residues 261-508) in complex with agonist compound 3a were prepared by incubation of 11 mg/ml protein with 0.5 mM SRC-1 peptide, 1 mM compound from 50 mM DMSO stock solutions and left on ice for 1 hour. Large crystals suitable for data collection were obtained from reservoir solution containing 20% PEG3350, 150 mM ammonium nitrate, and 100 mM citrate (pH 5.5). Crystals selected for data collection were flash frozen in mother liquor with liquid nitrogen in an ALS compatible crystal mounting cassette. Diffraction data were collected from cryo-cooled crystals at the Advanced Light Source (ALS) beamline 5.0.3, and data reduction were performed using the HLK2000 software package.13 The structures were determined by molecular replacement using the programs MOLREP14 and PHASER15 from the CCP4 program suite. Subsequent structure refinement and model re-building were conducted utilizing REFMAC16 and XtalView17 software packages. Data collection and refinement statistics are included in Supplementary Table 4, and figures were generated using PyMol (http://www.pymol.org). The coordinates and structure factors have been deposited in Protein Data Bank with accession code 6E3E for 1 and 6E3G for 3a.

Table 4. Data reduction and refinement statistics for the X-ray structures of the RORγt compound 1 and 3a complex. Data Collection Compound

1

3a

Wavelength (Å)

0.98

0.98

Space group

P61

P41

Unit cell dimensions a=99.7,b=99.7, c=125.3 (Å)

Resolution (Å)

α=90°,β=90°, γ=120° 2.47

a=62.0,b=62.0, c=155.6 α=90°,β=90°, γ=90° 2.10


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Unique reflections

25326

34235

Redundancy

9.4

5.0

Completeness (%)

99.6 (99.4)

100.0 (100.0)

I/σ(I)

34.2 (2.5)

10.9 (1.4)

Rsyma

0.056 (0.95)

0.13 (0.859)

2

2

Reflections used

23909

32497

RMS Bonds (Å)

0.011

0.012

RMS Angles (°)

1.454

1.611

Refinement Molecules in asymmetric unit

aRsym

Average B value (Å2) 76.6

31.7

R-valueb

0.179

0.185

R freeb

0.215

0.230

= ΣhΣj | - I(h)j | / ΣhΣj , where is the mean intensity of symmetry-related reflections.

bR-value

= Σ | |Fobs| - |Fcalc| | / Σ |Fobs|. Rfree for 5% of reflections excluded from refinement. Values in

parentheses are for the highest resolution shell.

Docking study We used the X-ray cocrystal structure of RORγt in the Protein Data Bank (PDB ID: 6E3G). The protein structure for the docking study was prepared with Protein Preparation Workflow on Maestro version 10.3 (Schrödinger software, http://www.schrodinger. com), as described below. (1) All crystallographic waters were removed and chain A was kept. (2) Hydrogens were added. (3) The orientation of hydroxyl groups of Asn and Gln, and the protonation states of His were optimized. (4) Constrained energy minimization



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was performed with the OPLS3 force field, setting the max root mean square deviation (RMSD) to 0.30 for the heavy atoms. Molecular docking was performed by Glide SP using the default setting. MD preparation Structures for MD simulation are prepared by docking studies described in Docking Study section. Since one of our main purposes was evaluation of the stability of Helix12 of RORγt in complex with an agonist or an inverse agonist, the cofactor SRC-1 was removed from the models for MD simulation. The MD simulation was performed by the AMBER 14 or the AMBER 16 (PMEMD)16 with GPU acceleration. The complex was solvated in a TIP3P cuboid water box with at least 8 Å away from any protein atoms. In addition, the ff14SB force field was employed for the protein and the RESP charge generated by Gaussian 0917 calculation at HF/6-31G(d) basis set was used for the ligand. The structure preparation was followed by two rounds of minimization, each comprising 500 steps of steepest descent followed by 500 steps of conjugate gradient minimization. In the first round of minimization, a weak constraint (1.0 kcal/(mol∙Å2)) was assigned to all heavy atoms. In the second round, no restrains were used to allow the minimization of the entire system. After minimization, the system was gradually heated from 50 K to 300 K within 0.2 ns while the protein was restrained with a constant force of 1.0 kcal/(mol∙Å2). Then the system was further equilibrated for 0.5 ns at constant pressure (1.0 bar) without any restraints. In the final production phase, 501 ns simulation in NPT ensemble was performed without any constraint. The first 1 ns was discarded for the following analysis. Single-crystal X-ray structure analysis Crystal data for ((1R,2S)-2-(benzylamino)cyclohexyl)methanol (R)-6-(tert-butoxycarbonyl)-2-methoxy5,6,7,8-tetrahydro-1,6-naphthyridine-5-carboxylate (11): C15H19N2O5-∙C14H22NO+, MW = 527.66; crystal size, 0.26 x 0.14 x 0.10 mm; colorless, block; orthorhombic, space group P212121, a = 6.2762(1) Å, b = 15.8157(3) Å, c = 27.9559(5) Å, α = β = γ = 90°, V = 2774.97(9) Å3, Z = 4, Dx = 1.263 g/cm3, T = 100 K, μ = 0.719 mm-1, λ = 1.54187 Å, R1 = 0.0299, wR2 = 0.0724, S = 1.130, Flack Parameter18 = -0.04(17). All measurements were made on a Rigaku R-AXIS RAPID-191R diffractometer using multi-layer mirror monochromated Cu-Kα radiation. The structure was solved by direct methods with SIR200819 and was refined using full-matrix least-squares on F2 with SHELXL-97.20 All non-H atoms were refined with anisotropic displacement parameters.


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CCDC 1878847 for compound 11 (((1R,2S)-2-(benzylamino)cyclohexyl)methanol (R)-6-(tertbutoxycarbonyl)-2-methoxy-5,6,7,8-tetrahydro-1,6-naphthyridine-5-carboxylate

)

contains

the

supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/structures.

Figure 8. ORTEP of 11 (((1R,2S)-2-(benzylamino)cyclohexyl)methanol (R)-6-(tert-butoxycarbonyl)-2methoxy-5,6,7,8-tetrahydro-1,6-naphthyridine-5-carboxylate), thermal ellipsoids are drawn at 30% probability. Screening RORγt binding test using fluorescent-labeled synthetic ligand. The binding activity of the test compound to RORγt was measured by a time resolved fluorescence resonance energy transfer method (TR-FRET) utilizing histidine-tagged RORγt, the fluorescent-labeled synthetic ligand and terbiumlabeled anti-histidine tag antibody (Invitrogen). First, 4 μL of a test compound diluted with an assay buffer (20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, 0.1% BSA) was added to a 384 well plate. Then, 4 μL of the mixture of RORγt and the terbium-labeled anti-histidine tag antibody diluted with the assay buffer were added so that their final concentrations were 2 nM and 1 nM, respectively. Finally, 4 μL of the fluorescent-labeled synthetic ligand diluted with the assay buffer was added to make the final concentration of 120 nM. The mixture was incubated at room temperature for 120 min, and fluorescence intensity (excitation wavelength 320 nm, fluorescence wavelength 520 nm) was measured by Envision 21 Environment ACS Paragon Plus


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(PerkinElmer). The fluorescent-labeled synthetic ligand: 5-((2Z)-2-((1-(Difluoroboryl)-3,5-dimethyl-1Hpyrrol-2-yl)methylene)-2H-pyrrol-5-yl)-N-(2-((3,5-difluoro-4-(trimethylsilyl)phenyl)amino)-1-(4(methoxymethyl)phenyl)-2-oxoethyl)pentanamide. Cofactor recruitment test. Cofactor recruitment test was performed by a time resolved fluorescence resonance energy transfer method (TR-FRET) utilizing histidine-tagged RORγt, europium-labeled antihistidine tag antibody, biotinylated SRC-1 peptide and streptavidin XL665. First, a test compound was diluted with an assay buffer (25 mM HEPES(pH 7.5), 100 mM NaCl, 1 mM DTT, 0.01% BSA, 0.01% Tween20) and added to a 384 well plate by 5 μL. Then, the mixture of streptavidin XL665 and the europium-labeled anti-histidine tag antibody were added by 2 μL so that their final concentrations were 1.56 μg/mL and 0.5 nM, respectively. After that, RORγt diluted with the assay buffer was added by 2 μL to

make

the

final

concentration

10

nM,

and

biotinylated

SRC-1

peptide

(biotin-

CLTARHKILHRLLQEGSPSD) diluted with the assay buffer was added by 2 μL each to make the final concentration 0.1 μM. The mixture was incubated in a dark place for 1 hr at room temperature, and the fluorescence intensity (excitation wavelength 320 nm, fluorescence wavelength 665/615 nm) was measured by Envision (PerkinElmer). For calculation of the % activation, the wells containing DMSO were defined as 0% control and the wells without the RORγt protein were defined as - 100% control. Jurkat reporter test. A human IL-17 ROR response element was inserted into the upstream of luciferase of pGL 4.28 reporter vector (Promega)and RORγt sequence was inserted into the downstream of CMV promoter. Jurkat cells (Clontech Laboratories, Inc) used for the reporter test were generated by stably introducing reporter vector and RORγt expression vector. The cells were cultured in a culture medium (RPMI-1640 (Invitrogen), 10% FCS (AusGeneX), 100 U/mL penicillin, 100 μg/mL streptomycin). On the day of the test, frozen cells were collected by a centrifugal operation (1000 rpm, 5 min) and suspended in PBS (phosphate buffered saline) (Invitrogen). The cells were suspended in a cholesterol-depleted reaction medium (RPMI-1640, 10% Lipid reduced FCS (HyClone), 10 mM HEPES (pH 7.5), 100 U/mL penicillin, 100 μg/mL streptomycin, 5 μM lovastatin) or a normal medium (RPMI1640, 10% FCS (AusGeneX), 10 mM HEPES (pH 7.5), 100 U/mL penicillin, 100 μg/mL streptomycin). After doxycycline (final concentration, 1.25 μg/mL) was added to cell suspension of 1X106 cells/mL, cells were seeded into a 384 well plate by 20 μL (final densitity, 20,000 cells/well). After that, a test compound diluted with the reaction medium was added by 5 μL, and the cells were cultured overnight in an incubator. Bright-Glo (Promega) was added by 25 μL, and the mixture was stirred at room temperature for 10 min, and the luminescence level was measured by Envision (PerkinElmer). The cells treated with 1.25 μg/ml Doxycyclin were used as high control and the cells in the absence of Doxycyclin were used as low control. EC50 is calculated as the molar concentration of a substance that produces 50% of that test 22 Environment ACS Paragon Plus

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substance's maximum stimulation. EC50 of the sigmoidal fits were analyzed using Prism 5, and a fourparameter logistic fit equation. Y=Bottom + (Top-Bottom)/(1+10^((LogEC50-X)*hill slope). “X” is the log of compound concentration, “Y” is the response, increasing as X increase. “Y” starts at “Bottom” and goes to “Top” with sigmoid shape. Mouse Th17 cell differentiation test. Mice were sacrificed for splenectomy via cervical dislocation at 8 weeks of age. Spleen was crushed on a cell strainer (Fisher Scientific) by the head of a syringe’s piston. Splenocyte suspensions were collected and were centrifuged at 1500 rpm for 3 min. The supernatants were removed, and blood red cells were hemolyzed by 0.165 M NH4Cl solution. RPMI 1640 medium (Gibco) supplemented with 10% FBS (Gibco), 10 units/ml penicillin and 10 μg/ml streptomycin (Wako), was added to the cell suspensions, and they were centrifuged at 200 x g for 3 min. Collected splenocytes were resuspended in the RPMI1640 medium after the complete destruction of red blood cells. Naive CD4+ T cells were then purified using a CD4+ CD62L+ T Cell Isolation Kit (Miltenyi Biotec) according to the manufacture’s manual. Naive CD4+ T cells (2 x 105 cells/200 μL/well) were cultured in Th17-skewing condition with or without compound on 10 μg/mL anti-mouse CD3 Ab (BD Biosciences) pre-coated 96 well U-bottomed plate (Nunc) for 4 days. Th17-skewing conditions were as follows. Anti-mouse CD28 Ab 5 μg/mL, anti-mouse IL-2 Ab 10 μg/mL (BD Biosciences), anti-mouse IL-4 Ab 10 μg/mL (BD Biosciences), anti-mouse IFN-γ Ab 10 μg/mL (BD Biosciences), recombinant human IL-6 20 ng/mL (BioLegend), recombinant human TGF-β 1 ng/mL (BioLegend), and recombinant mouse IL-23 50 ng/mL (BioLegend). To measure the concentrations of IL-17A in the culture supernatant, ELISA was performed according to the manufacture’s manual (eBioscience). The results (% of control of a test compound) evaluated by the above-mentioned method are shown in Figure 7. Pharmacokinetic study in mice The test compound was administered intravenously (0.1 mg/kg) or orally (1 mg/kg) to 8 week old male ICR mouse (cassette dosing, n=3 each). The blood samples were collected 5, 10, 15, 30 min, 1, 2, 4 and 8 h after intravenous administration and 15, 30 min, 1, 2, 4 and 8 h after oral administration. The blood samples were centrifuged to prepare the plasma samples. The plasma samples were pretreated with protein precipitation method. The concentrations of compounds were determined by LC/MS/MS. Corresponding Author * E-mail: 23 Environment ACS Paragon Plus


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[email protected]. Phone: +1-617-551-8958. [email protected]. Phone: +81-466-32-1219. FAX: +81-466-29-4454. Acknowledgment. We thank Dr. Masato Yoshida for helpful discussions, Mr. Naoki Ishii, Mr. Akito Shibuya, Dr. Yasuo Nakagawa for project supports and Mr. Mitsuyoshi Nishitani and Mr. Motoo iida for the support for X-ray crystallographic analysis and Mr. Kenichi Nagatome for statics discussion. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Present Addresses Tomoya Yukawa, Noriko Uchiyama: Takeda Pharmaceutical Company Limited, 40 Landsdowne street, Cambridge, MA, 02139, USA. Yoshi Nara, Ayumu Sato, Yoshihiro Banno, Naohiro Taya, Tetsuji Kawamoto, Ryosuke Katsuyama: Axcelead Drug Discovery Partners, Inc., 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 2510012, Japan. Ryokichi Koyama: Scohia Pharmaceuticals Inc. c/o Shonan Research Center, Takeda Pharmaceutical Company Ltd., 26-1, MuraokaHigashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan. Zenyu Shiokawa, Junya Shirai: Cardurion Pharmaceuticals K.K., c/o Shonan Research Center, Takeda Pharmaceutical Company Ltd., 26-1, MuraokaHigashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan. ABBREVIATIONS RORγt, retinoic acid receptor-related orphan receptor γt; BODIPY, boron-dipyrromethene; TR-FRET, time-resolved fluorescence resonance energy transfer; SAR, structure-activity relationship; SBDD, structure-based drug design; PK, pharmacokinetics; AUC, area under the plasma concentration-time curve. ASSOCIATED CONTENT Supporting Information. Molecular formula strings is provided in supporting information. All other information is written in experimental section. This material is available free of charge via the Internet at http://pubs.acs.org.”


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Accession Codes. The coordinates and structure factors have been deposited in Protein Data Bank with the accession codes 6E3E (1) and 6E3G (3a). Authors will release the atomic coordinates and experimental data upon article publication. AUTHOR INFORMATION

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(RORÎ³t) Agonist - ACS Publications

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