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Discovery of 3-Cyano-N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide: A Potent, Selective, and Orally Bioavailable Retinoic Acid Receptor-Related Orphan Receptor C2 Inverse Agonist

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Cite this: J. Med. Chem. 2018, 61, 23, 10415–10439
Publication Date (Web):August 21, 2018
https://doi.org/10.1021/acs.jmedchem.8b00392

Copyright © 2018 American Chemical Society. This publication is licensed under these Terms of Use.

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Abstract

The nuclear hormone receptor retinoic acid receptor-related orphan C2 (RORC2, also known as RORγt) is a promising target for the treatment of autoimmune diseases. A small molecule, inverse agonist of the receptor is anticipated to reduce production of IL-17, a key proinflammatory cytokine. Through a high-throughput screening approach, we identified a molecule displaying promising binding affinity for RORC2, inhibition of IL-17 production in Th17 cells, and selectivity against the related RORA and RORB receptor isoforms. Lead optimization to improve the potency and metabolic stability of this hit focused on two key design strategies, namely, iterative optimization driven by increasing lipophilic efficiency and structure-guided conformational restriction to achieve optimal ground state energetics and maximize receptor residence time. This approach successfully identified 3-cyano-N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide as a potent and selective RORC2 inverse agonist, demonstrating good metabolic stability, oral bioavailability, and the ability to reduce IL-17 levels and skin inflammation in a preclinical in vivo animal model upon oral administration.

 Note

After this paper was published ASAP September 9, 2018, a correction was made to the compound number in ref 43. The corrected version was reposted September 24, 2018.

Introduction

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Interleukin-17 (IL-17) is a proinflammatory cytokine that plays a dominant role in inflammation, autoimmunity, and host defense. (1,2) IL-17 drives an amplification mechanism in inflammatory disease. At a site of inflammation, IL-17 stimulates resident cells to secrete chemokines and other proinflammatory mediators that recruit additional inflammatory cells to the tissue. (3) Monoclonal antibodies directed toward the IL-17 cytokine itself or the IL-17 receptor have demonstrated clinical efficacy in the treatment of psoriasis, (4−6) psoriatic arthritis, (7−9) and ankylosing spondylitis. (10) The IL-17 pathway has also been implicated in other diseases such as rheumatoid arthritis (1,11) and multiple sclerosis. (12,13) IL-17 is produced by a specific subset of T cells and other immune cells, of which T helper 17 (Th17) cells are believed to play a dominant role in disease pathogenesis. (2,14) In these cells, the nuclear hormone receptor RORC2, a member of the retinoic acid receptor-related orphan receptor family and the immune cell specific isoform of the RORC gene, is required for the expression of IL-17 and the differentiation of Th17 cells from naïve CD4+ T cells. (15) Human subjects displaying a homozygous loss-of-function mutation in the RORC gene lack IL-17 producing cells in their peripheral blood, suggesting a critical role for RORC2 in IL-17 production. (16) Although the endogenous ligand of RORC2 remains to be firmly established, evidence suggests that one or more intermediates along the cholesterol biosynthesis pathway may play this role. (17−19) Two other members of the same nuclear receptor family, RORA and RORB, are believed to contribute to the regulation of circadian and motor functions. (20)
In addition to the large molecule IL-17 (or IL-17R) monoclonal antibody approach to modulate the cytokine pathway, targeting RORC2 with a small molecule approach is also an attractive therapeutic strategy. An inverse agonist of the receptor is anticipated to attenuate production of IL-17 not only in the presence of agonist but also in the case where the receptor has a constitutive level of transcriptional activity. (21) Indeed, a diverse range of chemotypes have been reported in the literature by industry and academia that display pharmacology consistent with inverse agonism of the receptor in vitro and in some cases in vivo. (22−30) Most of the RORC2 ligands reported to date have been found to bind to a large, hydrophobic pocket within the ligand binding domain (LBD), presumably the same binding site of the endogenous ligand. The nature of this pocket to favor lipophilic ligands has presented a specific challenge toward the identification of pharmaceutical agents suitable for oral delivery as favorable ligands tend to have poor metabolic stability and modest oral bioavailability. In spite of the anticipated challenges in candidate optimization, we initiated a small molecule program targeting RORC2 based on its strong linkage to inflammatory diseases. Herein, we describe our approach to lead optimization of RORC2 inverse agonists originating from the identification of a high-throughput screening (HTS) hit. The combined approach of structure-guided ligand conformational restriction and lipophilic efficiency focused optimization has led to the identification of a potent, selective, and orally bioavailable inverse agonist of RORC2.

Chemistry

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The synthesis of 5-amino-3-piperidine substituted indoles has been previously described in the literature, (31) and intermediate 1 served as the precursor for the preparation of the initial HTS hit 3 and related analogues, Scheme 1. Differentiation of the two amine functionalities was realized through benzamide formation with the 5-amino substituent of the indole ring to afford compound 2. Subsequent piperidine N-deprotection followed by acylation with cyclopentane carbonyl chloride provided compound 3. Access to the corresponding 1-methylindole analogue was possible by alkylation of the indole nitrogen to provide compound 4, Scheme 1. However, to enable broader structure–activity relationship (SAR) studies, installation of the methyl group prior to amide formation was more advantageous, Scheme 2. 1-Methylindole 5 allowed for the sequential amide bond formation steps suitable for a parallel medicinal chemistry approach to yield analogues such as 7, 8a, and 8b. The representative 1-ethylindole analogue 11 was also prepared from intermediate 1 by alkylation of the indole nitrogen prior to piperidine amide formation, Scheme 3.

Scheme 1

Scheme 1. Synthesis of Screening Hit 3 and 1-Methylindole Analogue 4a

aReagents and conditions: (a) DIPEA, TBTU, 3-cyanobenzoic acid, DMF; (b) HCl, dioxane; (c) DIPEA, cyclopentane carbonyl chloride, DMF; (d) NaH, THF; CH3I.

Scheme 2

Scheme 2. Representative Approach to 1-Methylindole Analogues, Including 7, 8a, and 8ba

aReagents and conditions: (a) benzoyl chloride or 4-cyanopicolinoyl chloride, triethylamine, CH2Cl2; (b) HCl, dioxane, MeOH; (c) cyclopentanecarbonyl chloride, triethylamine, CH2Cl2; or DIPEA, TBTU, DMF, cyclohexanecarboxylic acid, or cyclopentanecarboxylic acid, 40 °C.

Scheme 3

Scheme 3. Synthesis of 1-Ethylindole Analogue 11a

aReagents and conditions: (a) 4-cyanopicolinoyl chloride, triethylamine, CH2Cl2; (b) NaOH, EtI, Aliquat 336, CH2Cl2; (c) HCl, dioxane; (d) cyclopentanecarbonyl chloride, triethylamine, CH2Cl2.

Synthesis efforts next focused on the preparation of pyrrolopyridine and pyrrolopyrimidine scaffolds, Schemes 4 and 5. In these cases, installation of the piperidine substituent was made possible through a Suzuki cross-coupling reaction between vinyl boronate 12 and the corresponding 3-iodo-pyrrolopyridine or 3-iodo-pyrrolopyrimidine scaffold intermediate. The pyrrolo[2,3-b]pyridine iodide was prepared from 5-nitro-1H-pyrrolo[2,3-b]pyridine (13) by a one-pot N-methylation/iodination reaction to provide compound 14, Scheme 4. Subsequent cross-coupling with 12 afforded compound 15. Reduction of the olefin and nitro group concurrently under transfer hydrogenation conditions afforded amine 16, which was then coupled with the appropriate acid chlorides to provide analogues 17 and 18. Pyrrolo[3,2-b]- and pyrrolo[2,3-c]pyridine scaffolds were provided through cross-coupling of 12 with 21a and 21b, respectively, Scheme 5. Each was prepared through iodination of either 19a or 19b (32) followed by N-methylation of the iodinated heterocycle. The cross-coupling reaction occurred with advantageous deprotection of the formamide protecting group during workup to provide compounds 22a and 22b. Subsequent reduction of the olefin by transfer hydrogenation and amide formation afforded analogues 23 and 24. The required coupling partner 28 for the pyrrolo[3,2-d]pyrimidine scaffold arose from initial regioselective reductive dehalogenation of 2,4-dichloro-5H-pyrrolo[3,2-d]pyrimidine (25) followed by iodination and N-methylation, Scheme 5. The cross-coupling of iodoheterocyle 28 with boronate 12 provided compound 29. Installation of the 5-amino functionality was achieved through a catalytic amination reaction between 29 and benzophenone imine followed by transfer hydrogenation. (33) The resulting amine was then reacted with 4-cyanopicolinoyl chloride to provide analogue 31.

Scheme 4

Scheme 4. Synthesis of Pyrrolo[2,3-b]pyridine Analogues 17 and 18a

aReagents and conditions: (a) KOH, I2, DMF; K2CO3, CH3I; (b) 12, PdCl2(PPh3)2, K2CO3, DME, EtOH, H2O, 120 °C; (c) NH4HCO2, Pd, THF:NMP (9:1), 150 °C; (d) pyridine, CH2Cl2; 3-cyanobenzoyl chloride or 4-cyanopicolinoyl chloride.

Scheme 5

Scheme 5. Synthesis of Representative Pyrrolo[3,2-b]pyridine, Pyrrolo[2,3-c]pyridine, and Pyrrolo[3,2-d]pyrimidine Scaffold Analoguesa

aReagents and conditions: (a) N-iodosuccinimide, DMF; (b) Bu4NBr, NaOH, CH3I, CH2Cl2; (c) 12, (PPh3)4Pd, K2CO3, DMF:H2O (8:1), 100 °C; (d) NH4HCO2, Pd/C, DMF:NMP (5:1), 150 °C; (e) triethylamine, 4-cyanopicolinoyl chloride, CH2Cl2; (f) NaHCO3, Pd/C, H2, EtOH; (g) 12, PdCl2(PPh3)2, K2CO3, DMF:EtOH:H2O (4:1:1), 120 °C; (h) Pd(OAc)2, BINAP, dioxane; Ph2CNH, NaOt-Bu, 140 °C; (i) NH4HCO2, Pd/C, THF:NMP (5:1), 150 °C.

The synthesis of analogues bearing substitution at the 4-position of either the indole or pyrrolo[2,3-b]pyridine scaffold was undertaken as follows. The 4-methylindole analogue 36 was prepared through cross-coupling of 12 with 3-iodo-4-methylindole 33, which originated from 4-methyl-5-nitroindole (32) through a one-pot iodination/N-methylation, Scheme 6. Variation of the 4-position substituent to the pyrrolo[2,3-b]pyridine scaffold was challenging to introduce through parallel synthetic methods. Therefore, the corresponding substituted 3-iodo-pyrrolo[2,3-b]pyridines bearing either methyl, isopropyl, trifluoromethyl, or methoxy substitution each required unique synthetic approaches depending on the substituent, Schemes 710. The 4-methyl substituent was introduced through a Kumada coupling (34) between 4-chloro-1H-pyrrolo[2,3-b]pyridine (37) and methylmagnesium bromide to provide precursor 38, Scheme 7. To achieve clean nitration at the 5-position of the ring, it was necessary to convert 38 to the corresponding benzenesulfonamide derivative 39. In the absence of N-sulfonylation, competitive nitration at the C3-position led to complex mixtures of products. Subsequent nitration of 39 with tetramethylammonium nitrate selectively afforded 5-nitro-pyrrolo[2,3-b]pyridine 40. Removal of the phenylsulfonyl protecting group was accomplished by heating 40 with morpholine under basic conditions to afford compound 41. Subsequent N-methylation and C3-iodination provided the desired 4-methyl-3-iodo-pyrrolo[2,3-b]pyridine coupling partner 43. For the corresponding 4-isopropyl intermediate 49, a similar approach was adopted, Scheme 8. Suzuki cross-coupling with isopropenyl boronic acid pinacol ester proceeded cleanly with benzenesulfonamide 44 (35) to afford the isoprenyl intermediate 45. Hydrogenation of the exocyclic olefin then provided the corresponding isopropylpyrrolo[2,3-b]pyridine 46. Following the same sequence of reactions (nitration, iodination and N-methylation) as with the methyl analogue, the isopropyl substituted coupling partner 49 was prepared. Preparation of 4-methoxy-3-iodopyrrolo[2,3-b]pyridine 53 relied on a different approach to introduce the 4-position substituent, Scheme 9. Compound 50, which was obtained by nitration of chloroheterocycle 44, (35) was found to be sufficiently electron deficient to facilitate clean introduction of nucleophiles at the 4-position. Therefore, condensation of 50 with sodium methoxide afforded ether 51 which proceeded with concomitant desulfonylation. Subsequent N-methylation and iodination afforded coupling partner 53.

Scheme 6

Scheme 6. Synthesis of 1,4-Dimethylindole Analogue 36a

aReagents and conditions: (a) KOH, I2, DMF; K2CO3, CH3I; (b) 12, Pd EnCat TPP30, K2CO3, DME:EtOH:H2O (4:1:1), 70 °C; (c) NH4HCO2, Pd/C, EtOH, 85 °C; d) triethylamine, 3-cyanobenzoyl chloride, CH2Cl2.

Scheme 7

Scheme 7. Synthesis of 5-Methylpyrrolo[2,3-b]pyridine Scaffold Intermediate 43a

aReagents and conditions: (a) CH3MgBr, Pd(dppf)Cl2, toluene, 80 °C; (b) Bu4NBr, KOH, CH2Cl2; PhSO2Cl; (c) tetramethylammonium nitrate, (CF3CO)2O, CH2Cl2; (d) K2CO3, morpholine, MeOH, 65 °C; (e) K2CO3, CH3I, DMF; (f) N-iodosuccinimide, DMF.

Scheme 8

Scheme 8. Synthesis of 5-Isopropylpyrrolo[2,3-b]pyridine Scaffold Intermediate 49a

aReagents and conditions: (a) 4,4,5,5-tetramethyl-2-(prop-1-en-2-yl)-1,3,2-dioxaborolane, Pd(PPh3)4, K2CO3, DMF:H2O (10/1), 120 °C; (b) Pd/C, H2, EtOH; c) tetrabutylammonium nitrate, (CF3CO)2O, CH2Cl2, 0 °C; (d) NaOH, THF; (e) KOH, I2, DMF; K2CO3, CH3I.

Scheme 9

Scheme 9. Synthesis of 5-Methoxypyrrolo[2,3-b]pyridine Scaffold Intermediate 53a

aReagents and conditions: (a) tetramethylammonium nitrate, (CF3CO)2O, CH2Cl2; (b) NaOMe, MeOH, reflux; (c) NaH, CH3I, DMF; (d) N-iodosuccinimide, DMF.

Scheme 10

Scheme 10. Synthesis of 5-Trifluoromethylpyrrolo[2,3-b]pyridine Scaffold Intermediate 58a

aReagents and conditions: (a) CH3C(O)Cl, NaI, CH3CN, 100 °C; NaOH, MeOH; (b) (Bu4N)2SO4, NaOH, CH2Cl2, 0 °C; PhSO2Cl; (c) tetramethylammonium nitrate, (CF3CO)2O, CH2Cl2; (d) methyl 2,2-difluoro-2-(fluorosulfonyl)acetate, CuI, DMF, 100 °C; (e) KOH, I2, 2-MeTHF:EtOH (2:1); K2CO3, CH3I.

The desired 4-trifluoromethyl substituted intermediate for the coupling reaction was particularly challenging to prepare, Scheme 10. Starting from 4-chloro-1H-pyrrolo[2,3-b]pyridine (37), in situ protection of the N1 nitrogen atom as the acetyl derivative was required prior to a Finkelstein reaction, which yielded iodide 54. Next, N-phenylsulfonylation followed by ring nitration with trimethylammonium nitrate afforded the 4-iodo-5-nitropyrrolo[2,3-b]pyridine 56. Copper-mediated trifluoromethylation of 56 with methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (36) provided compound 57. Although it was found that the corresponding bromo- and chloro-analogues of 56 could facilitate introduction of the trifluoromethyl group, the iodide was found to be the most efficient. Furthermore, it was also found that the nitro and phenylsulfonyl groups were both critical to the success of the reaction, as all attempts to introduce a trifluoromethyl group onto substrates lacking either one of these functionalities failed to yield synthetically useful product. Compound 57 was then subjected to a one-pot iodination/desulfonylation and pyrrolopyridine N-methylation to afford coupling partner 58.
Iodides 43, 49, 53, and 58 were subjected to Suzuki cross-coupling conditions with N-Boc-tetrahydropyridine-4-boronic acid pinacol ester to provide compounds 59ad, respectively, Scheme 11. Hydrogenation afforded piperidinyl intermediates 60ad, which were then converted to the corresponding 3-cyanobenzamides 61ad. Subsequent deprotection provided piperidine amines 62ad. In the case of 4-methyl substituted intermediate 62a, a variety of amides 63al were prepared to explore the SAR of this substituent. On the other hand, only isobutyrate amides were prepared from 62bd, resulting in analogues 64 (4-iPr), 65 (4-OMe), and 66 (4-CF3), respectively. As shown in Scheme 12, compound 59a was also transformed to amine 68 to allow for diversification of the benzamide substituent as in analogues 69ad.

Scheme 11

Scheme 11. Synthesis of 5-Substituted Pyrrolo[2,3-b]pyridine Analogues 63al and 6466a

aReagents and conditions: (a) tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate, K2CO3, Pd(PPh3)4, DME:EtOH:H2O, 80 °C; (b) (X = Me) triethylamine, Pd/C, H2, MeOH; (X = iPr) NH4HCO2, Pd/C, EtOH; or (X = OCH3, CF3) Pd(OH)2, H2, MeOH; (c) 3-cyanobenzoyl chloride or 3-cyanobenzoic acid, various conditions; (d) CF3CO2H, CH2Cl2; or HCl, dioxane; (e) RCO2H or RC(O)Cl, various conditions.

Scheme 12

Scheme 12. Synthesis of 5-Methylpyrrolo[2,3-b]pyridine Analogues 69ada

aReagents and conditions: (a) HCl, dioxane, MeOH; (b) triethylamine, isobutyryl chloride, CH2Cl2; (c) triethylamine, Pd/C, H2, MeOH; (d) substituted 3-cyanobenzoic acid, DIPEA, HATU, CH2Cl2.

Results and Discussion

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Screening Strategy

A cell-based GAL4 luciferase reporter assay is uniquely suited as a screening approach to identify small molecule modulators of nuclear hormone receptors because the intrinsic transcriptional activity of the receptor is the functional readout. As opposed to a radioligand binding assay based on an endogenous ligand of the receptor, the reporter assay approach allows for the identification of pharmacologically diverse hits that are either competitive with the endogenous ligand or function by an allosteric binding site mechanism. A notable limitation of the reporter assay, however, is the requirement that the modulator is cell permeable. Nonetheless, as an initial screening strategy for a new target, an approach that quickly delivers a cell permeable hit can greatly facilitate validation of a chemotype or mechanism through functional activity in a disease-relevant pharmacological system.
Consequently, a subset of the Pfizer screening collection (ca. 150 000 compounds) was screened at 5 μM compound concentration using a GAL4-RORC2 luciferase reporter assay. HEK293 cells were transiently transfected with a mammalian expression vector containing GAL4-RORC2 LBD and a GAL4-responsive reporter gene containing firefly luciferase. An RORC2 inverse agonist would be expected to suppress the constitutively active, transcriptionally derived luminescence. Compounds demonstrating inhibition of greater than 50% were then counter-screened in a modification of the reporter assay, employing a GAL4-P65 expression vector instead of RORC2. Undesired activity under these assay conditions would suggest nonspecificity for RORC2, transcriptional inhibition through a general cytotoxicity mechanism, or compound specific interference with the luminescence readout. A dose–response curve for compounds demonstrating satisfactory differentiation was next acquired using comparable luciferase reporter assays in murine Neuro2A cells transfected by either GAL4-RORC2 LBD, GAL4-RORA LBD, or GAL4-RORB LBD to characterize isoform selectivity. As a result of this screening cascade, indole benzamide 3 was identified as a moderately potent RORC2 inverse agonist (IC50 = 2.7 μM), which lacked significant potency against RORA or RORB (IC50 > 30 μM), Table 1.
Table 1. Pharmacology Profile of Screening Hit 3 and 1-Alkylindole Analogues 4, 7, 8a, and 8ba
    TR-FRET IC50 (μM)luciferase IC50 (μM) 
cmpdRnKi (μM) RORC2 SPAbRORC2RORARORBRORC2RORARORBIL-17 IC50 (μM)c
3H 1.20.054>30>302.7>30>302.9
4Me 0.050.007>30>300.17>30>300.067
7  ND0.056>30>30NDNDND0.88
8aMe10.020.005>30>300.13>30>300.13
8bMe20.020.005>30>300.12>30>300.042
11Et1190.295>30>30NDNDND>10
a

All values are the mean of two or more independent assays. ND, not determined.

b

Radioligand [3H]-25-hydroxycholesterol displacement assay.

c

Inhibition of IL-17A production by human Th17 cells.

To further confirm that the functional changes in gene transcription observed with compound 3 resulted from specific ligand interactions with RORC2, we characterized compound 3 in a radioligand SPA binding assay and a time-resolved-fluorescence resonance energy transfer (TR-FRET) assay, Table 1. Compound 3 displaced [3H]-25-hydroxycholesterol, a putative in vitro agonist of RORC2, (37) from purified human RORC2 LBD with a Ki of 1.2 μM, comparable to the functional response in the reporter assay. The competitive behavior with 25-hydroxycholesterol was the first suggestion that this new chemotype might bind within the endogenous ligand binding site of the receptor. The TR-FRET assay monitors the ability of the histidine-tagged RORC2-LBD to bind a biotinylated coactivator peptide (SRC1-2) by fluorescence transfer mediated through association of a europium (Eu)-labeled anti-His antibody. An RORC2 inverse agonist that displaces the endogenous ligand would be expected to decrease the affinity of the coactivator peptide toward the RORC2 LBD and thus decrease the resulting fluorescence intensity. Indeed, compound 3 demonstrated potent suppression of SRC1-2 binding in the RORC2 TR-FRET assay (IC50 = 54 nM, 96% maximum suppression) while again not showing significant inverse agonism toward RORA or RORB (IC50 > 30 μM) when profiled in a similar assay format. Most importantly, compound 3 inhibited the production of IL-17 in human primary Th17 cells (IC50 = 2.9 μM). Unfortunately, compound 3 demonstrated poor metabolic stability (HLM CL = 142 μL/min/mg), precluding its utility in in vivo experiments. Nonetheless, encouraged by the evidence that compound 3 could functionally inhibit production of IL-17 in a disease relevant human cell, demonstrate competent linkage of functional activity to binding of the RORC2 receptor, and display promising isoform selectivity, we undertook efforts to further optimize the chemotype for both potency and pharmacokinetic properties.

Lead Optimization

The topology of indole benzamide 3 provides three clear vectors for structure–activity exploration, namely the benzamide, the piperidine amide, and the indole nitrogen substituent. Diversification of the core heterocycle was also considered in our optimization strategy. Our first venture to explore the vector derived from the indole nitrogen was immediately productive. N-Methyl analogue 4 was found to be 16-fold more potent than compound 3 in the GAL4-RORC2 reporter assay (IC50 = 0.17 μM), Table 1. A similar significant improvement in potency was observed in the RORC2 TR-FRET assay (IC50 = 7 nM) and in binding affinity based on the SPA assay (Ki = 50 nM). High isoform selectivity was maintained as evident from no significant inverse agonism of RORA or RORB in either the TR-FRET or reporter assays (IC50 > 30 μM). Compound 4 was also a significantly more potent inhibitor of IL-17 production in Th17 cells (IC50 = 67 nM, 90% maximum inhibition). Although the lipophilicity of 4 is slightly higher than compound 3 (log D = 4.4 and 4.2, respectively), the addition of a single methyl resulted in a net positive improvement to lipophilic efficiency (ΔLIPE = 1.4).
From compound 4, exploration of potency and ADME space for the benzamide substituent and the piperidine amide was well-suited for application of parallel synthetic chemistry technology. Although this effort did not identify a compound with significantly improved properties, it did provide insight into the dominant interactions with the receptor and led to a compound suitable for cocrystallization with the receptor. The region of the receptor occupied by the piperidine amide appeared to preferentially tolerate large hydrophobic residues; however, this did not lead to favorable lipophilic efficiency improvements. On the other hand, the cyano group present in the benzamide substituent was found to play a dominant role in functional potency for the series. Compound 7, where the cyano was replaced by hydrogen, demonstrated a 10-fold reduction in RORC2 TR-FRET and IL-17 inhibition potency compared to lead compound 4, Table 1. Replacement of phenyl by pyridine as in 4-cyanopicolinamides 8a or 8b was one of the few modifications of the benzamide moiety that was tolerated. Although compound 8b neither demonstrated improved potency (IL-17 IC50 = 42 nM) nor metabolic stability (HLM CL = 173 μL/min/mg), it was a successful candidate for cocrystallization with the RORC2-LBD.
The recently reported crystal structure of apo-RORC2 receptor LBD (38) and earlier structures of the receptor in complex with hydroxyl cholesterols (37) have revealed that the receptor has a large endogenous ligand binding pocket. Both of these structures display the receptor in an active conformation with helix-12 (H12) closely associated with the remainder of the receptor and forming a cleft allowing for coactivator peptide binding. A key structural element that stabilizes the positioning of H12 and enabling the active receptor conformation is a triplet residue latch consisting of His479–Tyr502–Phe506, Figure 1. The H12 residue Tyr502 forms a key hydrogen bond with His479 which resides on H11, thus bridging the two helixes. In addition, Phe506 forms a favorable edge to face aromatic stacking arrangement with both His479 and Tyr502. This arrangement of residues allows the charged His479 residue to have a strongly favorable cation-π interaction and hold H12 in the active conformation. (39)

Figure 1

Figure 1. RORC2 active conformation His479–Tyr502–Phe506 triplet latch (3KYT). (37)

The cocrystal structure of compound 8b with the RORC2 LBD confirmed that the ligand occupies the endogenous ligand binding pocket, Figure 2. Most notably from the structure, H12 is highly disordered along with partial unwinding of H11′. As a result, H12 no longer forms the coactivator binding cleft or the hydrogen bond between His479 and Tyr502. In place of the key latch interaction, His479 is now engaged in a hydrogen bond with the piperidine amide carbonyl of ligand 8b, Figure 3a. The cyclohexyl amide substituent occupies a hydrophobic pocket formed by H11 and the junction of H6 and H7. Compared to the active conformation of the receptor, this pocket significantly expanded in size due to a 110° torsional change in Trp317 with the vacancy only partially replaced by movement of Phe486. The rest of the binding site pocket is bordered by H3, H5, and H7 and a β-sheeting underlying the indole core. The important indole N-methyl substituent was found to buttress against H7, making hydrophobic contacts with Ile400, Phe401, and Tyr369 within a small pocket, Figure 3b. Indeed, a detrimental impact on inhibition of coactivator binding and functional IL-17 production was encountered by only increasing the substituent size to ethyl as in compound 11 (IL-17 IC50 > 10 μM), Table 1. The indole ring itself is situated in a hydrophobic region of the receptor positioned between Met365 on top and Val376 from the β-sheet below, Figure 3c. The benzamide substituent occupies a noticeably more hydrophilic region of the pocket, making two key hydrogen bond contacts. First, the cyano group of the ligand makes a bifurcated hydrogen bond to Arg367 and the backbone amide NH of Leu287. The second contact is between the backbone carbonyl of Phe377 and the benzamide NH of the ligand. To accommodate this hydrogen bond, the amide bond torsion (C4–C5–N–C) must deviate from planarity with the indole by 113°.

Figure 2

Figure 2. RORC2 LDB cocrystal structure with compound 8b (6CN5). Representation of ligand binding site with respect to receptor structure (H12 disordered and not shown).

Figure 3

Figure 3. RORC2 LDB cocrystal structure with compound 8b (6CN5). (a) Representation of piperidine amide portion of ligand binding site: residues in gray depict cocrystal structure with compound 8b, and residues in yellow depict RORC2 active conformation (3KYT). (b) Representation of methylindole binding pocket with receptor surface displayed. (c) Representation of benzamide indole portion of ligand binding site.

An analysis of available crystallographic data for similar aryl amides found in the Cambridge Crystallographic Database suggests a strong preference for either of the two coplanar rotamers about the carbon–nitrogen bond. (40) Therefore, to adopt the bound state dihedral angle, a noteworthy energetic penalty must be paid with respect to the ground state conformation. Consequently, structural changes to the ligand that would increase the predominance of the bound conformation in solution would be expected to improve potency. Filla and coworkers (32) have reported a strategy to influence the amide rotamer population in a closely related series of N-acylimidazole 5-HT receptor agonists through replacement of the indole ring carbon atom by nitrogen adjacent to the amide substituent. Their analysis of crystallographic data suggested a preference for an anticonformation between the ring nitrogen and the amide carbonyl, although a planar conformation was still highly favored. To probe this effect in our series, we prepared pyrrolo[3,2-b]pyridine 23 and pyrrolo[2,3-c]pyridine 24, Table 2. Introduction of nitrogen into the indole core was also attractive for the potential to lower lipophilicity and potentially improve metabolic stability. To this effect, we also prepared pyrrolo[2,3-b]pyridine 17 and pyrrolo[3,2-d]pyrimidine 31. Unfortunately, compounds 24 and 31 demonstrated significantly reduced potency in the TR-FRET assay compared to the indole isostere 8a. Although compounds 17 and 23 were potent in the TR-FRET assay, they demonstrated a modest reduction in potency for suppression of IL-17 production in Th17 cells. The most interesting compound of the series was pyrrolo[2,3-b]pyridine 17. Although its cellular potency was diminished, it afforded a 0.5 unit reduction in lipophilicity and maintained comparable lipophilic efficiency compared to the indole isostere 8a. Unfortunately, this reduction in lipophilicity was not sufficient to improve metabolic stability (HLM CL = 144 μL/mg/min).
Table 2. Impact of Azaindole Heterocycles on Pharmacology and Lipophilicitya
    IC50 (nM)  
compoundXYZRORC2bIL-17clog DdLIPEe
8aCHCHCH4.91144.32.6
17CHCHN4.84083.72.7
23NCHCH19.93574.22.2
24CHNCH783NDNDND
31NNCH638NDNDND
a

All values are the mean of two or more independent assays. ND, not determined.

b

TR-FRET cofactor recruitment.

c

Inhibition of IL-17A production by human Th17 cells.

d

Log D pH7.4 measured by reverse phase HPLC (Elog D).

e

Calculated based on IC50 in IL-17 suppression assay and measured log D.

An alternative approach considered to alter the amide rotamer population was to substitute the indole ring adjacent to the amide substituent. For the two possible positions, the cocrystal structure of 8b suggested that only substitution at the 4-position of the indole ring would be tolerated in the binding pocket. To test this hypothesis we pursued 4-methylindole 36, Table 3. As shown in the calculated torsional energy profile about the C4–C5–N–C dihedral angle of a representative model system (Figure 4), (41) substitution at the 4-position of the indole ring with methyl significantly disfavors the proximal-orientation of the carbonyl and methyl (θ = 0°). At the dihedral angle of the observed bound conformation, the calculation suggests that the unsubstituted analogue (R = H) suffers a higher energetic penalty (3.4 kcal/mol) compared to the methyl analogue (2.2 kcal/mol) with respect to the lowest energy conformation (θ = 180° or 0°). This was indeed reflected in the potency of 4-methylindole 36, which was 23-fold more potent in the IL-17 suppression assay (IC50 = 3.3 nM) compared to the unsubstituted analogue 4 (IC50 = 75 nM), Table 3. A cocrystal structure of compound 36 bound to the RORC2 LBD confirmed the same binding orientation in the pocket to that observed with compound 8b, Figure 5. The dihedral angle of the C–N bond (C4–C5–N–C) was maintained at 113°, and the methyl substituent now occupies a previously vacant cavity in the binding pocket bordered by His323 and Phe378. A similar phenomenon was observed for the pyrrolo[2,3-b]pyridine ring (Figure S1, see Supporting Information) where the 4-methyl derivative 63h was 13-fold more potent (IC50 = 21 nM) compared to the unsubstituted analogue 18 (IC50 = 274 nM). In both cases, the addition of the methyl substituent led to a reduction in lipophilicity, Δlog D = −0.4 and −0.9 respectively for compound 36 and 63h. As a result, through increased potency and reduced lipophilicity, the combination of the pyrrolo[2,3-b]pyridine and the 4-methyl substituent into compound 63h led to a significant improvement in the lipophilic efficiency compared to compound 4 (ΔLIPE = 2.1).

Figure 4

Figure 4. Calculated torsional energy profile (B3LYP density functional, 6-31G* basis set) (41) for model systems representing rotation of (a) the 5-benzamide substituent (C4–C5–N–C dihedral) and (b) the 3-piperidine substituent (C2–C3–C–H dihedral) to the indole ring. R substituent is hydrogen (blue), methyl (green), trifluoromethyl (red), methoxy (orange), or isopropyl (brown). Dihedral angle observed in bound conformation represented by dashed line.

Figure 5

Figure 5. Overlay of bound conformation for compound 8b (orange) with cocrystal structure of RORC2 LBD with compound 36 (green with gray residues) (6CN6).

Table 3. Impact of C4-Substitution on Pharmacology and Lipophilicitya
   RORC2b   
compoundXRIC50 (nM)dissociation T1/2 (min)IL-17 IC50 (nM)clog DdLIPEe
4CHH5.5106754.42.7
36CHCH35.013093.34.04.5
18NH7.9502743.82.8
63hNCH312.8631212.94.8
a

All values are the mean of two or more independent assays. ND, not determined.

b

TR-FRET cofactor recruitment.

c

Inhibition of IL-17A production by human Th17 cells.

d

Log D pH7.4 measured by reverse phase HPLC (Elog D).

e

Calculated based on IC50 in IL-17 suppression assay and measured log D.

The methyl substituent at the 4-position of the indole ring may also have a profound effect on the torsional dynamics between the piperidine and indole rings. In cocrystal structures of both 8b and 36, the piperidine ring adopts a dihedral angle of 133° (C2–C3–C–H). The torsional energy profile of this dihedral was also calculated using a simplified model system, Figure 4. To adopt the observed bound conformation, the calculations suggest that the methyl analogue suffers a minor energetic penalty (0.4 kcal/mol) compared to the unsubstituted analogue. However, the methyl group imparts a significantly higher rotational barrier (9.8 kcal/mol) compared to the unsubstituted analogue (3.6 kcal/mol). Likewise, the methyl analogue displays a greater energetic preference (Δ −2.4 kcal/mol) for the bound conformation than the next higher local minimum (θ = 0°) as compared to the hydrogen analogue (Δ −0.3 kcal/mol). Again, a similar profile was observed for the pyrrolo[2,3-b]pyridine ring (Figure S1, see Supporting Information).
It should be noted that a similar potency enhancement as observed for inhibition of IL-17 production was not observed in the recombinant RORC2 TR-FRET assay for compounds 36 and 63h, Table 3. To further understand this apparent disconnect, we explored the dissociation kinetics of our inverse agonists from the RORC2 receptor using an adaptation of the TR-FRET assay. The corresponding inverse agonist was incubated with the receptor for 3 h at the respective concentration to achieve 75% inhibition of response. A high concentration of agonist was then added, and the return of TR-FRET signal as a result of SRC1-2 binding was monitored over time. Using this method, we estimated the effective dissociation half-life of compounds 36 and 63h from the recombinant receptor to be 21.8 and 10.5 h, respectively. In both cases, the dissociation rate decreased by more than an order of magnitude by installing the 4-methyl substituent, Table 3. The half-life of the receptor in human Th17 cells as determined by pulse-chase experiments was found to be 5.0 and 5.1 h either in the presence or absence of ligand, respectively (Figure S2, see Supporting Information), and thus supports the potential for a sustained pharmacological effect dependent on the compound dissociation rate in the case of these compounds. Swinney (42) attributed one characteristic of antagonists that possess high biochemical efficiency (BE) as the capacity to induce a nonequilibrium system such as having a residence time on the target longer than the degradation rate of the receptor. The effective pseudoirreversible behavior of these compounds contributes to their high efficiency in the cellular assay. A subsequent screen of a range of inverse agonists demonstrated a strong correlation of effective dissociation half-life with IL-17 inhibitory potency (Figure S3, see Supporting Information). As a consequence, the measurement of IL-17 inhibition in Th17 cells was viewed as the most appropriate assay to rank order compound potency given the presence of the endogenous ligand in physiological concentrations and the long assay window (6 days) to achieve equilibrium.
Unfortunately, even though a significant reduction in lipophilicity had been achieved with compound 63h, metabolic clearance remained high (HLM CL = 125 μL/min/mg). To further address metabolic stability, we revisited optimization of the piperidine amide substituent in light of the advancements made elsewhere to the molecule. A range of acyclic, cyclic, and aromatic amides was prepared spanning size and hydrophobicity, Table 4. The ability of the compound to inhibit IL-17 production was highly dependent on the size and hydrophobicity of the substituent. For example, potency increased exponentially as the size of the acyclic group increased from methyl (63a, IC50 = 5270 nM) to ethyl (63b, IC50 = 625 nM) and eventually isopropyl (63d, IC50 = 69 nM). Similarly, for cycloalkyl substituents, potency increased with ring size from cyclopropyl (63c, IC50 = 242 nM) to cyclohexyl (63i, IC50 = 13 nM). An example of an aromatic substituent, 63j, also showed excellent potency. Although isosteric with the potent analogues 63h and 63j, the polar analogues based on tetrahydrofuran (63k) and 3,5-dimethylisoxazole (63l) were significantly less potent. On the other hand, metabolic stability was inversely proportional to the size of the hydrophobic substituent in 63aj. Overall, the isopropyl analogue 63d demonstrated the best combination of potency, metabolic stability, and lipophilic efficiency (IC50 = 69 nM, HLM CL = 8 μL/min/mg).
Table 4. Impact of Piperidine Amide Substituent on Pharmacology and Metabolic Stabilitya
  IC50 (nM)   
compoundRRORC2bIL-17cHLM CL (μL/min/mg)log DdLIPEe
63aMe2775270<81.63.7
63bEt67625<82.04.2
63ccyclopropyl16242<82.34.3
63diPr206982.05.2
63ecyclopropylmethyl1397102.44.6
63fiBu10251092.55.1
63gcyclobutyl1156392.64.7
63hcyclopentyl13211252.94.8
63icyclohexyl10132363.94.0
63j2-fluoro-6-toluenyl1814903.14.7
63ktetrahydrofuran-3-yl743800<81.63.8
63l3,5-dimethylisoxazol-4-yl491310<82.03.9
a

All values are the mean of two or more independent assays. ND, not determined.

b

TR-FRET cofactor recruitment.

c

Inhibition of IL-17A production by human Th17 cells.

d

Log D pH7.4 measured by reverse phase HPLC (Elog D).

e

Calculated based on IC50 in IL-17 suppression assay and measured log D.

Now with a better understanding of the chemical space required to achieve good metabolic stability, we pursued further optimization of potency. First, we considered additional substitution to the cyanobenzamide. Based on the cocrystal structures of 8b and 36, the 4-position of the phenyl ring appeared to provide the greatest opportunity for additional hydrophobic or hydrogen bond contacts, Table 5. For the analogues prepared (69ad), all of them showed good metabolic stability except for the 4-chloro analogue 69c, presumably due to the significant increase in lipophilicity. Unfortunately, only the 4-methoxy analogue 69a was more potent, although only modestly (2-fold), for inhibition of IL-17 production.
Table 5. Impact of Phenyl Amide Substitution on Pharmacology and Metabolic Stabilitya
  IC50 (nM)  
compoundRRORC2bIL-17cHLM CL (μL/min/mg)log Dd
63dH206982.0
69aOCH32030<82.4
69bF2857<82.3
69cCl7.661203.6
69dCH31579112.3
a

All values are the mean of two or more independent assays. ND, not determined.

b

TR-FRET cofactor recruitment.

c

Inhibition of IL-17A production by human Th17 cells.

d

Log D pH7.4 measured by reverse phase HPLC (Elog D).

Finally, we investigated the scope of the pyrrolo[2,3-b]pyridine 4-position substituent beyond the initially identified methyl. We surveyed examples in which the substituent was sterically larger or had a different electronic character compared to methyl (6466), Table 6. Replacing the methyl by the sterically larger isopropyl group (64) was predicted through torsional energy calculations to yield the smallest energetic penalty for the anticipated bound state, Figure 4. Nonetheless, compound 64 imparted comparable potency and metabolic stability as compound 63d, possibly due to counterbalancing steric interactions with His323 and Phe378. Compound 65 bearing an electron donating methoxy group, however, demonstrated reduced potency and higher metabolic clearance. This would be anticipated from the torsional energy profile with methoxy yielding the largest energetic penalty presumably due to a favorable intramolecular interaction between the amide hydrogen and the oxygen lone pair when the C4–C5–N–C dihedral angle is 180°. On the other hand, an electron withdrawing trifluoromethyl substituent (66) provided improved potency compared to methyl. Compound 66 (43) achieved optimal inhibition of IL-17 production in Th17 cells (IC50 = 9.5 nM, 90% maximum inhibition) while affording excellent metabolic stability. Favorable metabolic stability was observed for compound 66 in spite of a significant increase in lipophilicity induced by the trifluoromethyl group (Δlog D = 1.7) which is consistent with the trend observed in reported matched molecular pair analyses. (44) Several factors likely contribute to the enhanced potency of the trifluoromethyl analogue 66. Calculations suggest the trifluoromethyl group may further skew the C4–C5–N–C rotamer population toward the bound conformation through an unfavorable dipole interaction with the amide carbonyl as well as favor the bound conformation of the piperidine ring, Figure 4. Second, the electron withdrawing nature of the trifluoromethyl group now reduces the polarization of the pyrrolopyridine nitrogen, which would be better accommodated in the hydrophobic binding pocket. This is supported by the reduced negative character for the calculated electrostatic potential (ESP) of the ring nitrogen compared to methyl analogue 63d, Table 6. Lastly, the lipophilicity of compound 66 is now of the same magnitude of earlier leads with single digit nanomolar potency (e.g., 36, log D = 4.0), and therefore, the hydrophobic nature of the binding pocket may be a strong driver of potency.
Table 6. Electronic and Steric Influence of the C4-Substituent on Pharmacology and Metabolic Stabilitya
   IC50 (nM)  
compoundRESP-N7bRORC2cIL-17dHLM CL (μL/min/mg)log De
63dCH3–32.7206982.0
64iPr–32.9376392.6
65OCH3–33.764381262.0
66CF3–25.64.19.5<83.7
a

All values are the mean of two or more independent assays. ND, not determined.

b

Electrostatic potential calculated using Jaguar, Version 8.1, Schrodinger, LLC, New York, NY, 2013 (B3LYP/6-31G**).

c

TR-FRET cofactor recruitment.

d

Inhibition of IL-17A production by human Th17 cells.

e

Log D pH7.4 measured by reverse phase HPLC (Elog D).

Encouraged by the initial in vitro profile of compound 66, we carried out additional pharmacological characterization. The very slow dissociation rate from the RORC2 recombinant receptor seen previously with 4-substituted derivatives was also evident (T1/2 = 6 h) in compound 66. To demonstrate that compound effect was at the level of gene transcription, we showed that compound 66 inhibited mRNA production of IL-17A and other genes that have previously been described as being controlled by RORC2 such as IL-17F, IL-22, IL-26, and IL-23R (Table S1, see Supporting Information). (15,45) As expected, RORC expression was not inhibited by compound 66. In addition, Th1 and Th2 lymphocyte differentiation as well as cellular viability of lymphocytes were not affected by compound 66 at the highest concentration tested (10 μM) suggesting specificity of the observed effects. High isoform selectivity was demonstrated with no significant inverse agonism toward RORA or RORB in the TR-FRET assay (IC50 > 25 μM). To more broadly explore the receptor selectivity of compound 66, we profiled the compound using the trans-FACTORIAL platform (Attagene Inc.), Figure 6. This multiplexed technology allows for the measurement of reporter RNA levels upon transfection of ligand binding domain chimeric constructs from 48 nuclear receptors with GAL4 DNA. Of the 48 receptors, only RORC2 showed a fold reduction (antagonist effect) of greater than 20% of control at a dose of 1 μM compound. Only the estrogen-related receptor alpha (ERRα) (1.28-fold, p = 0.03) and the liver X receptor alpha (LXRα) (1.30-fold, p = 0.165) showed a modest fold increase (agonist effect) greater than 20% of control at the same dose. These results clearly demonstrate the high selectivity of compound 66 for RORC2 compared to all other nuclear receptors.

Figure 6

Figure 6. Effect of compound 66 (n = 3 replicates) against 48 nuclear receptors in HepG2 cells at 1 μM concentration and 24 h time point. HepG2 cells were transiently transfected with optimized trans-FACTORIAL library. Post-transfection (24 h), cells were washed and supplied with fresh low serum (1% FBS, charcoal stripped) culture medium and treated with inducer for 24 h. Profile of the trans-FACTORIAL activities was determined as fold of induction values versus vehicle-treated (DMSO) control cells. Graph shows average fold-induction data plotted in logarithmic scale.

In addition to low in vitro human microsomal and hepatocyte clearance, compound 66 also demonstrated high passive permeability (Papp = 13.2 × 10–6 cm/sec, RRCK cell monolayer). Our primary concern with regard to predicted human pharmacokinetics was the relatively low thermodynamic solubility of compound 66 (1.0 μM, pH 6.5). In a rat pharmacokinetic study, compound 66 demonstrated low in vivo clearance (CL = 3.4 mL/min/kg) and a moderate half-life (6 h) consistent with the low human (8 μL/min/mL) and rat (<14 μL/min/mL) microsomal clearance, Table 7. Clearance was also very low in dog and mouse (0.46 and 1.2 mL/min/kg, respectively) with the elimination half-life in the dog being exceptionally long (39 h). Upon oral dosing of crystalline solid (3 mg/kg) in rats, oral bioavailability was moderate (21%), consistent with a highly permeable but low solubility compound. Unfortunately, due to the decreased basicity of the pyrrolopyridine nitrogen, salt formation was not a viable approach for solubility enhancement. In hopes of improving the compound dissolution rate and ultimately the oral bioavailability, we formulated the compound as a spray-dried dispersion (SDD) (25%, HPMCAS-H polymer). (46) This approach was highly effective resulting in an increased oral bioavailability in rat of 83%. Oral bioavailability in dog remained high with the SDD formulation (73%, 3 mg/kg). Oral bioavailability in mouse was also acceptable at higher doses using the SDD formulation (30%, 30 mg/kg). Overall, compound 66 demonstrated excellent in vivo pharmacokinetics consistent with in vitro parameters, and the SDD formulation approach was very effective in overcoming the low aqueous solubility of the molecule.
Table 7. Pharmacokinetic Profile of 66 in Rat, Mouse, and Doga
speciesCL (mL/min/kg)T1/2 (h)Vdss (L/kg)F (%)dFu(plasma)
ratb3.461.483 (21)0.016
dogb0.46391.573e0.065
mousec1.2100.8300.021
a

For formulation conditions, see Experimental Section.

b

Dosed IV at 1 mg/kg (n = 2) and PO at 3 mg/kg (n = 2).

c

Dosed IV at 1 mg/kg (n = 3) and PO at 30 mg/kg (n = 3).

d

Oral bioavailability following dosing of compound 66 as a 25% spray-dried dispersion or (crystalline solid).

e

Calculated based on AUC0–24h.

Compound 66 also showed potent inhibition of IL-17 production in mouse derived Th17 cells (IC50 = 32 nM, 92% maximum inhibition) which supported assessment of in vivo efficacy in the mouse imiquimod-induced skin inflammation model. (47) Topical administration of imiquimod can induce a psoriasis-like skin inflammation resulting in epidermal thickening. This clinical outcome is at least in part mechanistically dependent on the IL-23/IL-17 pathway. Mice were challenged with topical imiquimod cream (5%) applied to their back and ear for 3 days (days 1–3). Compound 66 was dosed orally at three dose levels (10, 30, and 100 mg/kg) once a day over 5 days (days 1–5). Compound administration was well tolerated without any compound-related adverse events. Compound plasma AUC and Cmax increased with dose although not proportionally, Figure 7. An anti-IL-17 monoclonal antibody and an anti-p40 (IL-23 and IL-12 subunit) antibody were administered by intraperitoneal injection as controls. At the conclusion of the dosing, ear thickness was measured and IL-17A protein levels in the ear were assessed, Figure 8. Compound 66 demonstrated a dose dependent inhibition of ear swelling in the model. A maximum inhibition of 46% (p < 0.0001) was achieved at a dose of 100 mg/kg which was comparable to the reduction observed with the anti-IL-17A Ab (45%, p = 0.0005) compared to the isotype control. The degree of inhibition correlated with the time period in which compound concentration exceeded the IC80 throughout the dosing interval, Figure 7. Consistent with the clinical outcome and the role RORC2 plays, a significant reduction in the level of IL-17A protein in the ear was also observed at all doses of compound 66 compared to the vehicle control, Figure 8. Similar reductions of IL-17A protein in the ear were seen at both the 30 and 100 mg/kg doses (70 and 72%, respectively) (p < 0.0001).

Figure 7

Figure 7. Unbound plasma concentrations of compound 66 following last dosing in the mouse imiquimod-induced skin inflammation model. Compound dosed at 10 mg/kg (blue), 30 mg/kg (green), and 100 mg/kg (red) once a day by oral gavage. For reference, the respective mouse IL-17 IC50 and IC80 (4 × IC50) adjusted for fraction unbound in the in vitro assay media (Fu = 0.62) are depicted (IC50 = 20 nM, IC80 = 80 nM).

Figure 8

Figure 8. End of study magnitude of ear swelling (Δ μm) (a) and protein levels of IL-17A (pg/mL) (b) in the ear following mouse imiquimod-induced skin inflammation model when treated with compound 66, anti-IL-17 Ab (20 mg/kg), or anti-p40 Ab (20 mg/kg) (mean ±95% CI).

Conclusion

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Through a combination of de novo and structure-guided design, we optimized a novel high-throughput screening hit against RORC2 with modest potency and poor metabolic stability into a highly potent and orally bioavailable lead. To align the two properties of potency and metabolic stability into a single molecule, it was important to exploit two key design strategies, namely, iterative optimization through lipophilic efficiency and conformational restriction to achieve optimal ground state energetics and maximize receptor residence time. The introduction of two “magic methyl” substituents (48,49) profoundly impacted the potency characteristics of the chemotype. The first example was transformation of compound 3 to 1-methylindole 4 resulting in a 43-fold improvement in potency. As revealed by the subsequent cocrystal structure of compound 8b, this methyl group occupies a favorable hydrophobic pocket in the receptor, yielding a beneficial lipophilic efficiency (ΔLIPE = 1.4). The second example was the transformation of compound 4 to 4-methylindole 36, resulting in an additional 20-fold potency improvement and further lipophilic efficiency improvement (ΔLIPE = 1.7). In this case, the methyl substituent is believed to restrict the conformational dynamics of the ligand, leading to a lower solution to bound state energetic penalty as well as imparting a profound resonance time enhancement to the ligand in the binding pocket. Although largely neutral toward lipophilic efficiency, the next two prominent changes to the molecule provided an important lowering of the intrinsic lipophilicity. These changes included the introduction of the pyrrolo[2,3-b]pyridine ring system (63h, Δlog D = −1.1) and replacement of the cyclopentylamide by isopropylamide (63d, Δlog D = −0.9). Effectively, this series of transformations achieved a comparable level of potency to the early lead compound 4, but in a substantially lower lipophilic space (Δlog D = −2.4) and now with excellent metabolic stability. The reduction in lipophilicity was crucial to allow for the final potency enhancement through introduction of the metabolically stable but lipophilic 4-trifluoromethyl group found in compound 66. Indeed, compound 66 was found to be a potent and selective RORC2 inverse agonist with excellent pharmacokinetic properties in preclinical species. The ability of compound 66 to reduce levels of IL-17A in vivo after oral dosing in mice, and corresponding reduction in skin inflammation further supports the potential of small molecule RORC2 modulation as a therapeutic target for the treatment of inflammatory diseases.

Experimental Section

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Chemistry

All reagents and solvents were used as purchased without further purification. The purity of the final compounds was characterized by high-performance liquid chromatography (HPLC) using a gradient elution program (e.g., C18, acetonitrile:water, 0.1% formic acid, 5:95–95:5) and UV-detection (220 nM). The purity of all final compounds was 95% or greater. Proton (1H) NMR chemical shifts are referenced to a residual solvent peak.

tert-Butyl 4-(5-(3-Cyanobenzamido)-1H-indol-3-yl)piperidine-1-carboxylate (2)

DIPEA (0.45 mL, 2.6 mmol) was added to a solution of 3-cyanobenzoic acid (0.367 g, 2.5 mmol) and TBTU (0.881 g, 2.6 mmol) in DMF (10 mL). After stirring at room temperature for 30 min, a solution of tert-butyl 4-(5-amino-1H-indol-3-yl)piperidine-1-carboxylate (31) (1, 0.787 g, 2.5 mmol) in DMF (5 mL) was added, and the mixture was stirred at room temperature overnight. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (MeOH/CH2Cl2, 2/98) to afford 0.8 g (72%) of 2. 1H NMR (300 MHz, CDCl3) δ 9.45 (br s, 1H), 9.10 (br s, 1H), 8.25 (s, 1H), 8.17 (d, J = 8.2 Hz, 1H), 7.95 (s, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.45 (t, J = 7.8, 1H), 7.28 (d, J = 8.2 Hz, 1H), 7.20 (d, J = 8.2 Hz, 1H), 6.80 (s, 1H), 4.20–4.05 (m, 2H), 2.80–2.60 (m, 3H), 1.95–1.80 (m, 2H), 1.55–1.28 (s, 11H).

3-Cyano-N-(3-(1-(cyclopentanecarbonyl)piperidin-4-yl)-1H-indol-5-yl)benzamide (3)

A solution of 4 N HCl in dioxane (0.9 mL) was added to a solution of 2 (201 mg, 0.45 mmol) in methanol (2 mL). The mixture was stirred at room temperature for 2.5 h and then concentrated under reduced pressure at 30 °C. The residue was suspended in toluene and concentrated. The resulting crude amine was dissolved in DMF (1 mL), and DIPEA (0.37 mL, 2.24 mmol) was added. The solution was stirred at room temperature for 30 min, and then a solution of cyclopentanecarbonyl chloride (86 mg, 0.65 mmol) in dichloroethane (1.5 mL) was added. The mixture was stirred at room temperature overnight and then quenched with saturated aqueous NaHCO3 solution. The aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried (Na2SO4), filtered and then concentrated. The residue was purified by silica gel column chromatography (MeOH/CH2Cl2, 0/100–10/90). The crude product was dissolved in EtOAc, washed with water, and concentrated to afford 180 mg (91%) of 3 as a tan solid. MS (ES+) m/z 441 (M+H). 1H NMR (300 MHz, CDCl3) δ 8.40 (s, 1H), 8.35 (s, 1H), 8.22 (s, 1H), 8.18 (d, J = 8.0, 1H), 8.00 (s, 1H), 7.80 (d, J = 8.5, 1H), 7.60 (t, J = 8.3, 1H), 7.30–7.20 (m, 2H), 6.95 (s, 1H), 4.75–4.65 (m, 2H), 4.10–4.00 (m, 2H), 3.20–2.85 (m, 3H), 2.70–2.60 (m, 2H), 2.15–1.50 (m, 9H).

3-Cyano-N-(3-(1-(cyclopentanecarbonyl)piperidin-4-yl)-1-methyl-1H-indol-5-yl)benzamide (4)

NaH (11 mg, 0.27 mmol) was added to a cold (0 °C) solution of 3 (100 mg, 0.23 mmol) in THF (10 mL). The mixture was stirred at room temperature for 30 min, and then iodomethane (38 mg, 0.27 mmol) was added dropwise while maintaining 0 °C. The reaction mixture was stirred at room temperature for 3 h and then was poured into saturated aqueous NH4Cl solution (3 mL). The organic layer was concentrated, and the crude product was purified by preparative HPLC to afford 22 mg (21%) of 4 as a light yellow solid. MS (ES+) m/z 455 (M+H). 1H NMR (400 MHz, CD3OD) δ 7.69 (s, 1H), 7.48–7.46 (m, 2H), 7.30–7.25 (m, 3H), 7.04 (s, 1H), 7.00–6.99 (m, 1H), 4.68–4.60 (m, 1H), 4.20–4.13 (m, 1H), 3.53 (s, 3H), 3.26–3.20 (m, 1H), 3.15–3.05 (m, 1H), 3.05–2.95 (m, 1H), 2.80–2.75 (m, 1H), 1.89–1.80 (m, 4H), 1.76–1.60 (m, 6H), 1.55–1.40 (m, 2H).

tert-Butyl 4-(5-Amino-1-methyl-1H-indol-3-yl)piperidine-1-carboxylate (5)

NaH (2.1 g, 52.48 mmol, 60% w/w in mineral oil) was added to a cold (0 °C) solution of tert-butyl 4-(5-nitro-1H-indol-3-yl)-5,6-dihydropyridine-1(2H)-carboxylate (31) (4.5 g, 13.12 mmol) in THF (80 mL). The mixture was stirred at room temperature for 1 h, and then iodomethane (3.3 mL, 52.48 mmol) was added dropwise at 0 °C. The reaction mixture was then allowed to stir at room temperature overnight. The mixture was quenched by addition of ice/water and then extracted by with ethyl acetate (2 × 100 mL). The combined organic layers were dried (Na2SO4), filtered, and concentrated. Methanol (50 mL) and Pd/C (0.1 g) were added to the residue, and the mixture was heated at 40 °C under a hydrogen atmosphere (Balloon pressure) for 7 h. The reaction mixture was filtered through Celite and washed with methanol. The combined filtrate was concentrated to obtain 3.4 g (82%) of 5 as a light brown solid. 1H NMR (400 MHz, DMSO-d6) δ 7.04 (d, J = 8.4 Hz, 1H), 6.88 (s, 1H), 6.69 (s, 1H), 6.51 (dd, J = 8.4, 1.6 Hz, 1H), 4.47 (s, 2H), 4.06–4.03 (m, 2H), 3.60 (s, 3H), 3.09–2.75 (m, 5H), 1.87 (d, J = 12.4 Hz, 2H), 1.41 (s, 9H).

tert-Butyl 4-(5-Benzamido-1-methyl-1H-indol-3-yl)piperidine-1-carboxylate (6a)

Benzoyl chloride (0.41 mL, 3.52 mmol), triethylamine (0.13 mL, 0.91 mmol), and a catalytic amount of DMAP were added to a solution of indole 5 (301 mg, 0.91 mmol) in CH2Cl2 (2 mL). The mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (heptane/EtOAc, 100/0–0/100) to afford 100 mg (25%) of 6a as an off-white solid.

tert-Butyl 4-(5-(4-Cyanopyridine-2-carboxamido)-1-methyl-1H-indol-3-yl)piperidine-1-carboxylate (6b)

To a cold (0 °C) solution of 5 (3.3 g, 10.0 mmol) in CH2Cl2 (20 mL) was added triethylamine (4.2 mL, 30.1 mmol) followed by a solution of 4-cyanopicolinoyl chloride (2.1 g, 12.53 mmol) in CH2Cl2 (10 mL). After 1 h, the reaction mixture was poured into water and extracted with CH2Cl2. The organic layers were dried (Na2SO4), filtered, and concentrated. The crude compound was purified by silica gel column chromatography to afford 3.7 g (80%) of 6b as a light brown solid. 1H NMR (400 MHz, CDCl3) δ 9.88 (s, 1H), 8.82 (d, J = 4.8 Hz, 1H), 8.13 (s, 1H), 7.71 (dd, J = 4.8, 1.2 Hz, 1H), 7.47 (dd, J = 8.8, 2.0 Hz, 1H), 7.28 (d, J = 8.8 Hz, 1H), 6.84 (s, 1H), 4.23–4.18 (m, 2H), 3.76 (s, 3H), 3.15–2.88 (m, 3H), 2.03 (d, J = 12.8 Hz, 2H), 1.65–1.54 (m, 2H), 1.49 (s, 9H).

N-(3-(1-(Cyclopentanecarbonyl)piperidin-4-yl)-1-methyl-1H-indol-5-yl)benzamide (7)

A solution of 4 M HCl in dioxane (3 mL) was added to 6a (100 mg, 0.23 mmol). The reaction mixture was stirred at room temperature for 1 h and then concentrated. Triethylamine (160 μL, 1.15 mmol) and cyclopentanecarbonyl chloride (28 μL, 0.23 mmol) were added to a solution of the residue in CH2Cl2 (1 mL), and the mixture was stirred at room temperature for 1.5 h. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (heptane/EtOAc, 100/0–0/100) to afford 39 mg (39%) of 7 as a white solid. MS (ES+) m/z 430 (M+H). 1H NMR (400 MHz, CDCl3) δ 8.08 (s, 1H), 7.96–7.82 (m, 2H), 7.64–7.44 (m, 2H), 7.37–7.26 (m, 4H), 6.83 (s, 1H), 4.80 (d, J = 13.3 Hz, 1H), 4.09 (d, J = 13.7 Hz, 1H), 3.76 (s, 2H), 3.21 (t, J = 12.1 Hz, 1H), 3.11 (t, J = 12.1 Hz, 1H), 2.96 (quint, J = 8.1 Hz, 1H), 2.75 (t, J = 13.1 Hz, 1H), 2.27–1.98 (m, 2H), 1.92–1.45 (m, 9H), 1.28 (br. s., 1H), 0.89 (t, J = 6.6 Hz, 1H).

General Method for the Synthesis of 8a and b

A solution of 4 M HCl in dioxane (30 mL) was added to a cold (0 °C) solution of 6b (3.7 g, 8.04 mmol) in methanol (15 mL). The mixture was allowed to warm to room temperature and stir overnight. The reaction mixture was concentrated, added to a saturated aqueous NaHCO3 solution, and extracted with 10% methanol in CH2Cl2. The combined extracts were dried (Na2SO4), filtered, and concentrated to afford 2.2 g (78%) of the amine. The corresponding carboxylic acid (1.11 mmol) and TBTU (430 mg, 1.34 mmol) were dissolved in DMF (15 mL) and DIPEA (720 mg, 5.57 mmol) was added. After stirring at 40 °C for 1 h, amine (400 mg, 1.11 mmol) was added, and the mixture was stirred overnight at 40 °C. The crude product was purified by preparative HPLC.

4-Cyano-N-(3-(1-(cyclopentanecarbonyl)piperidin-4-yl)-1-methyl-1H-indol-5-yl)picolinamide (8a)

Cyclopentanecarboxylic acid; 21 mg (4%), yellow solid. MS (ES+) m/z 456 (M+H). 1H NMR (400 MHz, DMSO-d6) δ 10.65 (s, 1H), 9.01 (d, J = 4.8 Hz, 1H), 8.51 (s, 1H), 8.23 (d, J = 1.6 Hz, 1H), 8.18 (dd, J = 4.8, 1.2 Hz, 1H), 7.65 (dd, J = 8.8, 1.6 Hz, 1H), 7.40 (d, J = 8.8 Hz, 1H), 7.15 (s, 1H), 4.60–4.52 (m, 1H), 4.18–4.09 (m, 1H), 3.75 (s, 3H), 3.28–3.16 (m, 1H), 3.10–2.98 (m, 1H), 2.78–2.68 (m, 2H), 2.10–1.95 (m, 2H), 1.76–1.40 (m, 10H).

4-Cyano-N-(3-(1-(cyclohexanecarbonyl)piperidin-4-yl)-1-methyl-1H-indol-5-yl)picolinamide (8b)

Cyclohexanecarboxylic acid; 40 mg (8%), yellow solid. MS (ES+) m/z 470 (M+H). 1H NMR (400 MHz, DMSO-d6) δ 10.62 (s, 1H), 8.99 (d, J = 4.8 Hz, 1H), 8.49 (s, 1H), 8.21 (d, J = 1.6 Hz, 1H), 8.16 (dd, J = 4.8, 1.6 Hz, 1H), 7.63 (dd, J = 8.8, 1.6 Hz, 1H), 7.38 (d, J = 8.8 Hz, 1H), 7.14 (s, 1H), 5.77 (s, 1H), 4.58–4.52 (m, 1H), 4.10–4.04 (m, 1H), 3.87 (s, 3H), 3.24–3.15 (m, 1H), 3.06–2.95 (m, 1H), 2.72–2.58 (m, 2H), 2.08–1.95 (m, 2H), 1.74–1.10 (m, 12H).

tert-Butyl 4-(5-(4-Cyanopicolinamido)-1H-indol-3-yl)piperidine-1-carboxylate (9)

Triethylamine (14.6 mL, 105 mmol) was added to a suspension of tert-butyl 4-(5-amino-1H-indol-3-yl)piperidine-1-carboxylate (31) (1, 8.25 g, 26.2 mmol) in CH2Cl2 (120 mL), and the mixture was cooled to 0 °C. A solution of 4-cyanopicolinoyl chloride (4.49 g, 26.9 mmol) in CH2Cl2 (60 mL) was slowly added to the mixture. The reaction mixture was stirred at 0 °C for 1 h and then at room temperature for 20 h. Saturated aqueous sodium chloride and the mixture was extracted with CH2Cl2 and EtOAc. The combined organic layers were concentrated. The residue was triturated with ethanol (150 mL) and filtered. The resulting solid was washed with ethanol (50 mL) and dissolved in a mixture of acetone and CH2Cl2. The solution was filtered and concentrated to afford 12.16 g (95%) of 9 as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 10.82 (s, 1H), 10.55 (s, 1H), 8.95 (d, J = 5.2 Hz, 1H), 8.45 (s, 1H), 8.15–8.10 (m, 2H), 7.54 (dd, J = 9.1, 1.0 Hz, 1H), 7.3 (d, J = 9.1 Hz, 1H), 7.12 (d, J = 1.0 Hz, 1H), 4.10–4.00 (m, 2H), 2.95–2.85 (m, 3H), 1.90–1.80 (m, 2H), 1.60–1.46 (m, 2H), 1.40 (s, 9H).

tert-Butyl 4-(5-(4-Cyanopicolinamido)-1-ethyl-1H-indol-3-yl)piperidine-1-carboxylate (10)

A solution of sodium hydroxide (2.5 M, 6.28 mL, 15.7 mmol), ethyl iodide (1.08 mL, 13.5 mmol), and a catalytic amount of Aliquat 336 was added to a solution of 9 (200 mg, 0.45 mmol) in CH2Cl2 (6 mL). The reaction mixture was stirred at room temperature for 48 h and then partitioned with water. The aqueous layer was extracted with CH2Cl2. The combined organic layers were concentrated and the residue was purified by preparative HPLC to afford 110 mg (52%) of 10 as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 9.89 (s, 1H), 8.82 (d, J = 4.5 Hz, 1H), 8.54 (s, 1H), 8.13 (s, 1H), 7.70 (dd, J = 4.8, 1.0 Hz, 1H), 7.46 (dd, J = 7.2, 1.0 Hz, 1H), 7.31 (d, J = 7.2 Hz, 1H), 6.91 (s, 1H), 4.30–4.15 (m, 2H), 4.12 (q, J = 5.9 Hz, 2H), 3.00 (t, J = 10.5 Hz, 1H), 2.95–2.85 (m, 2H), 2.08–2.01 (m, 2H), 1.70–1.60 (m, 2H), 1.49 (s, 9H), 1.45 (t, J = 5.4 Hz, 3H).

4-Cyano-N-(3-(1-(cyclopentanecarbonyl)piperidin-4-yl)-1-ethyl-1H-indol-5-yl)picolinamide (11)

A solution of 4 M HCl in dioxane (0.9 mL) was added to 10 (110 mg, 0.23 mmol) and the mixture was stirred at room temperature for 12 h. The reaction mixture was partitioned between saturated aqueous NaHCO3 and CH2Cl2, and the organic layer was concentrated. Triethylamine (32 μL, 0.23 mmol) and cyclopentanecarbonyl chloride (28 μL, 0.23 mmol) were added to a solution of the residue in CH2Cl2 (5 mL), and the mixture was stirred at room temperature for 1 h. The reaction mixture was partitioned between 1 M HCl (20 mL) and CH2Cl2 (50 mL). The organic layer was concentrated, and the residue was purified by preparative HPLC to afford 60 mg (64%) of 11 as a white solid. MS (ES+) m/z 470 (M+H). 1H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 9.00 (d, J = 4.7 Hz, 1H), 8.50 (s, 1H), 8.20 (d, J = 1.6 Hz, 1H), 8.16 (dd, J = 1.4, 4.9 Hz, 1H), 7.62 (dd, J = 1.6, 9.0 Hz, 1H), 7.43 (d, J = 8.6 Hz, 1H), 7.21 (s, 1H), 4.57 (d, J = 12.5 Hz, 1H), 4.19–4.08 (m, 3H), 3.22 (t, J = 12.5 Hz, 1H), 3.09–3.00 (m, 2H), 2.73 (t, J = 12.3 Hz, 1H), 2.09–1.97 (m, 2H), 1.86–1.44 (m, 10H), 1.36 (t, J = 7.2 Hz, 3H).

Cyclopentyl(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridin-1(2H)-yl)methanone (12)

Trifluoroacetic acid (472 g, 4.14 mol) was added dropwise to a cold (0 °C) solution of tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (160 g, 517 mmol) in CH2Cl2 (640 mL). The mixture was allowed warm to 20 °C and was stirred for 1 h. The reaction mixture was concentrated under reduced pressure and then azeotroped with CH2Cl2 (4 × 200 mL). The residue was divided into two equal portions and each dissolved into CH2Cl2 (1.5 L). Triethylamine (209.6 mL) and then cyclopentanecarbonyl chloride (99.7 g, 751.8 mmol) were added dropwise into the reaction mixture while keeping the internal temperature at 0–5 °C. The mixture was allowed warm to 20 °C and was stirred overnight. The reaction mixture was washed with aqueous NaHCO3 (2 × 750 mL). The organic layer was washed with brine, dried (Na2SO4) and concentrated. The crude product was purified by silica gel column chromatograph (PE/EtOAc, 50/1–1/1) to afford 107.98 g (68%) of 12 as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 6.48 (m, 1H), 4.10 (m, 2H), 3.65 (m, 1H), 3.52 (m, 1H), 2.95–2.80 (m, 1H), 2.30–2.20 (m, 2H), 1.90–1.65 (m, 6H), 1.60–1.50 (m, 2H), 1.26 (s, 12H).

3-Iodo-1-methyl-5-nitro-1H-pyrrolo[2,3-b]pyridine (14)

Potassium hydroxide (241 mg, 4.29 mmol, pellets) was added to a suspension of 5-nitro-1H-pyrrolo[2,3-b]pyridine (500 mg, 3.06 mmol) in DMF (15 mL), and the mixture was stirred at room temperature for 10 min. Iodine (856 mg, 3.37 mmol) was then added, and stirring was continued for 1.5 h. K2CO3 (974 mg, 7.05 mmol) followed by iodomethane (1.14 mL, 18.4 mmol) were added and stirring was continued at room temperature for 2.5 h. The mixture was diluted with water (50 mL), treated with NaHSO3 until yellow, and was then stirred for 30 min. The precipitate was collected by filtration, washed with water, and dried in vacuo to provide 845 mg (91%) of 14 as a yellow solid. MS (ES+) m/z 304 (M+H). 1H NMR (500 MHz, DMSO-d6) δ 9.16 (d, J = 2.5 Hz, 1H), 8.43 (d, J = 2.6 Hz, 1H), 8.08 (s, 1H), 3.90 (s, 3H).

Cyclopentyl(4-(1-methyl-5-nitro-1H-pyrrolo[2,3-b]pyridin-3-yl)-3,6-dihydropyridin-1(2H)-yl)methanone (15)

A mixture of 14 (300 mg, 1.0 mmol), 12 (287 mg, 1.29 mmol), PdCl2(PPh3)2 (35 mg, 0.05 mmol), and K2CO3 (274 mg, 2.0 mmol) in a mixture of DME:ethanol:water (4:1:1, 4 mL) was degassed and flushed with nitrogen gas. The reaction mixture was heated under microwave irradiation at 120 °C for 20 min. The reaction mixture was partitioned with CHCl3. The organic phase was concentrated, and the residue was purified by silica gel column chromatography to provide 95 mg (27%) of 15. MS (ES+) m/z 355 (M+H). 1H NMR (500 MHz, CDCl3) δ 9.25 (s, 1H), 8.95 (d, J = 10.4 Hz, 1H), 7.30 (d, J = 18.0 Hz, 1H), 6.20 (m, 1H), 4.35–4.25 (m, 2H), 3.95 (s, 3H), 3.90–3.85 (m, 1H), 3.80–3.75 (m, 1H), 3.00–2.90 (m, 1H), 2.65–2.50 (m, 2H), 1.90–1.50 (m, 8H).

(4-(5-Amino-1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)piperidin-1-yl)(cyclopentyl)methanone (16)

A mixture of 15 (95 mg, 0.27 mmol), ammonium formate (676 mg, 10.8 mmol), and palladium black (2 mg) in THF:NMP (9:1, 10 mL) was heated with microwave irradiation at 150 °C for 1.5 h. The reaction mixture was filtered through Celite and concentrated. The residue was purified by preparative HPLC to afford the corresponding formamide which was dissolved in methanol (2 mL) and treated with an aqueous solution of HCl (2 M, 0.5 mL). The mixture was allowed to stir at room temperature overnight. The reaction mixture was made basic with a solution of aqueous NaOH (2 M) and extracted with CH2Cl2. The organic layer was concentrated to afford 17 mg (19%) of 16. 1H NMR (500 MHz, CDCl3) δ 7.90 (d, J = 3.0 Hz, 1H), 7.22 (d, J = 3.0 Hz, 1H), 6.84 (s, 1H), 4.80–4.75 (m, 1H), 4.10–4.05 (m, 1H), 3.75 (s, 3H), 2.50 (br.s., 2H), 3.20–3.10 (m, 1H), 2.98–2.90 (m, 2H), 2.75–2.65 (m, 1H), 2.10–1.95 (m, 2H), 1.90–1.50 (m, 10H).

4-Cyano-N-(3-(1-(cyclopentanecarbonyl)piperidin-4-yl)-1-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)picolinamide (17)

Pyridine (5 μL, 0.06 mmol) was added to a cold (0 °C) solution of 16 (17 mg, 0.05 mmol) in CH2Cl2 (0.5 mL). A solution of 4-cyanopicolinoyl chloride (4.3 mg, 0.03 mmol) in CH2Cl2 (0.5 mL) was added, and the mixture was allowed to stir at room temperature overnight. The reaction mixture was concentrated, and the residue was purified by preparative HPLC to afford 6 mg (47%) of 17. MS (ES+) m/z 457 (M+H). 1H NMR (500 MHz, CDCl3) δ 9.92 (s, 1H), 8.85 (d, J = 7.0 Hz, 1H), 8.65 (s, 1H), 8.54 (s, 1H), 8.40 (s, 1H), 7.75 (d, J = 7.0 Hz, 1H), 6.98 (s, 1H), 4.85–4.75 (m, 1H), 4.15–4.05 (m, 1H), 3.85 (s, 3H), 3.25–3.15 (m, 1H), 3.15–3.05 (m, 1H), 3.00–2.90 (m, 1H), 2.80 (m, 1H), 2.20–2.00 (m, 2H), 1.90–1.50 (m, 10H).

3-Cyano-N-(3-(1-(cyclopentanecarbonyl)piperidin-4-yl)-1-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (18)

Pyridine (5 μL, 0.06 mmol) was added to a cold (0 °C) solution of 16 (17 mg, 0.05 mmol) in CH2Cl2 (0.5 mL). A solution of 3-cyanobenzoyl chloride (4.3 mg, 0.03 mmol) in CH2Cl2 (0.5 mL) was added, and the mixture was allowed to stir at room temperature overnight. The reaction mixture was concentrated, and the residue was purified by preparative HPLC to afford 2 mg (18%) of 18. MS (ES+) m/z 456 (M+H). 1H NMR (500 MHz, CDCl3) δ 8.47 (d, J = 3.0 Hz, 1H), 8.32 (d, J = 3.0 Hz, 1H), 8.26 (s, 1H), 8.19 (d, J = 7.6 Hz, 1H), 8.11 (s, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.67 (t, J = 7.9 Hz, 1H), 6.98 (s, 1H), 4.85–4.75 (m, 1H), 4.15–4.05 (m, 1H), 3.85 (s, 3H), 3.25–3.15 (m, 1H), 3.10–3.00 (m, 1H), 3.00–2.90 (m, 1H), 2.75–2.65 (m, 1H), 2.15–2.00 (m, 2H), 1.90–1.50 (m, 10H).

(E)-N′-(3-Iodo-1H-pyrrolo[3,2-b]pyridin-5-yl)-N,N-dimethylformimidamide (20a)

N-Iodosuccinimide (590 mg, 2.62 mmol) was added to a stirred solution of (E)-N,N-dimethyl-N′-(1H-pyrrolo[3,2-b]pyridin-5-yl)formimidamide (32) (19a) (470 mg, 2.50 mmol) in DMF (2 mL) at 0 °C, and the mixture was stirred for 18 h at room temperature. The reaction mixture was concentrated, and the residue was purified by neutral alumina chromatography to provide 785 mg (100%) of 20a. MS (ES+) m/z 315 (M+H). 1H NMR (500 MHz, CD3OD) δ 8.32 (s, 1H), 7.65 (d, J = 4.0 Hz, 1H), 7.50 (s, 1H), 6.85 (d, J = 4.0 Hz, 1H), 3.20 (s, 3H), 3.10 (s, 3H).

(E)-N′-(3-Iodo-1-methyl-1H-pyrrolo[3,2-b]pyridin-5-yl)-N,N-dimethylformimidamide (21a)

Tetrabutylammonium bromide (80 mg, 0.25 mmol), NaOH (4 mL, 2M), and iodomethane (200 μL, 3.25 mmol) were added to a stirred solution of 20a (785 mg, 2.5 mmol) in CH2Cl2 (22 mL), and the mixture was stirred for 16 h at room temperature. The reaction mixture was partitioned between water and CH2Cl2/EtOAc. The organic layer was concentrated, and the residue was purified by neutral alumina chromatography to provide 686 mg (84%) of 21a. MS (ES+) m/z 329 (M+H). 1H NMR (500 MHz, acetone-d6) δ 8.58 (s, 1H), 7.62 (d, J = 4.0 Hz, 1H), 7.48 (s, 1H), 6.80 (d, J = 4.0 Hz, 1H), 3.85 (s, 3H), 3.12 (s, 3H), 3.02 (s, 3H).

(4-(5-Amino-1-methyl-1H-pyrrolo[3,2-b]pyridin-3-yl)-5,6-dihydropyridin-1(2H)-yl)(cyclopentyl)methanone (22a)

A mixture of 21a (40 mg, 0.12 mmol), 12 (89 mg, 0.29 mmol), Pd(PPh3)4 (14 mg, 0.01 mmol), and K2CO3 (50 mg, 0.37 mmol) in DMF/water (8:1, 1.5 mL) was degassed and flushed with nitrogen gas. The mixture was stirred at 100 °C for 16 h. The reaction mixture was partitioned between water and CH2Cl2. The organic layer was concentrated, and the residue was purified by preparative HPLC to provide 15 mg (35%) of 22a. MS (ES+) m/z 325 (M+H).

4-Cyano-N-(3-(1-(cyclopentanecarbonyl)piperidin-4-yl)-1-methyl-1H-pyrrolo[3,2-b]pyridin-5-yl)picolinamide (23)

A mixture of DMF/NMP (5:1, 1 mL) was added to a vessel containing 22a (12 mg, 0.04 mmol), ammonium formate (47 mg, 0.74 mmol), and palladium black (2 mg, 0.02 mmol). The mixture was heated under microwave irradiation at 150 °C for 1 h. The reaction mixture was filtered through Celite, and the filtrate was concentrated. Triethylamine (30 μL, 0.22 mmol) and 4-cyanopicolinoyl chloride (11 mg, 0.07 mmol) were added to a solution of the residue in CH2Cl2 (0.5 mL). The mixture was stirred at room temperature for 30 min, whereupon the solvents were evaporated. The residue was purified by preparative HPLC to provide 10 mg (59%, 2 steps) of 23. MS (ES+) m/z 457 (M+H). 1H NMR (500 MHz, CD3OD) δ 8.96 (dd, J = 5.0, 1.0, 1H) 8.54 (s, 1H), 8.25 (d, J = 9.0, 1H), 7.98 (dd, J = 5.0, 1.56, 1H), 7.89 (d, J = 9.0, 1H), 7.31 (s, 1H), 4.75–4.68 (m, 1H), 4.28–4.23 (m, 1H), 3.82 (s, 3H), 3.20–3.13 (m, 1H), 2.90–2.82 (m, 1H), 2.25–2.11 (m, 2H), 1.94–1.60 (m, 12H).

4-Cyano-N-(3-(1-(cyclopentanecarbonyl)piperidin-4-yl)-1-methyl-1H-pyrrolo[2,3-c]pyridin-5-yl)picolinamide (24)

Following procedures analogous to compound 23, (E)-N,N-dimethyl-N′-(1H-pyrrolo[2,3-c]pyridin-5-yl)formimidamide (32) was converted into compound 24. MS (ES+) m/z 457 (M+H). 1H NMR (500 MHz, acetone-d6) δ 9.02 (d, J = 5.0, 1H), 8.62 (s, 1H), 8.57 (d, J = 10.0, 2H), 8.10 (dd, J = 5.0, 1.70, 1H), 7.34 (s, 1H), 4.75–4.60 (m, 1H), 4.25–4.20 (m, 1H), 3.93 (s, 3H), 3.35–3.28 (m, 1H), 3.25–3.15 (m, 1H), 3.12–3.05 (m, 1H), 2.18–2.10 (m, 1H), 1.94–1.52 (m, 12H).

2-Chloro-5H-pyrrolo[3,2-d]pyrimidine (26)

A mixture of 2,4-dichloro-5H-pyrrolo[3,2-d]pyrimidine (134 mg, 0.71 mmol), NaHCO3 (66 mg, 0.78 mmol) and Pd/C (1.52 mg, 10%) in EtOH (4 mL) was stirred at room temperature for 2.5 h under a hydrogen atmosphere (3 psi) The mixture was filtered passed through Celite, and the filtrate was concentrated. The residue was purified by silica gel column chromatography (CH2Cl2/MeOH, 100/0–80/20) to afford 90 mg (88%) of 26. MS (ES+) m/z 154 (M+H).

2-Chloro-7-iodo-5H-pyrrolo[3,2-d]pyrimidine (27)

N-Iodosuccinimide (138 mg, 0.62 mmol) was added to a stirred solution of 26 (90 mg, 0.59 mmol) in DMF (1 mL) at 0 °C. The mixture was then stirred at room temperature for 17 h. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (CH2Cl2/Et2O/MeOH, 1/0/0–9/1/0–9/0/1)to provide 110 mg (67%) of 27. MS (ES+) m/z 280 (M+H).

2-Chloro-7-iodo-5-methyl-5H-pyrrolo[3,2-d]pyrimidine (28)

Tetrabutylammonium bromide (19 mg, 0.06 mmol), NaOH (1 mL, 2M), and iodomethane (47 μL, 0.47 mmol) were added to a stirred solution of 27 (110 mg, 0.39 mmol) in CH2Cl2 (5 mL), and the mixture was stirred for 16 h at room temperature. The reaction mixture was partitioned between water and CH2Cl2/EtOAc. The organic layer was concentrated, and the residue was triturated with Et2O to afford 110 mg (94%) of 28. MS (ES+) m/z 294 (M+H). 1H NMR (500 MHz, acetone-d6) δ 8.93 (s, 1H), 8.04 (s, 1H), 4.05 (s, 3H).

(4-(2-Chloro-5-methyl-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-5,6-dihydropyridin-1(2H)-yl)(cyclopentyl)methanone (29)

A mixture of 28 (25 mg, 0.09 mmol), 12 (34 mg, 0.11 mmol), PdCl2(PPh3)2 (6 mg, 0.01 mmol), and K2CO3 (26 mg, 0.19 mmol) in DME:EtOH:H2O (4:1:1, 1 mL) was purged with nitrogen. The mixture was heated at 120 °C for 20 min in a microwave reactor. The reaction mixture was partitioned between water and CH2Cl2/EtOAc. The organic layer was concentrated, and the crude product was purified by preparative HPLC to afford 10 mg (34%) of 29. MS (ES+) m/z 345 (M+H).

Cyclopentyl(4-(2-(diphenylmethyleneamino)-5-methyl-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-5,6-dihydropyridin-1(2H)-yl)methanone (30)

Pd(OAc)2 (1 mg, 0.15 mmol) and BINAP (4 mg, 0.22 mmol) were dissolved in degassed dioxane (0.5 mL) and stirred for 5 min. This solution was added to a mixture of 29 (10 mg, 0.03 mmol), diphenylmethanimine (16 mg, 0.09 mmol), and sodium tert-butoxide (6 mg, 0.06 mmol) in degassed dioxane (0.5 mL). The mixture was heated under microwave irradiation for 30 min at 140 °C. CH2Cl2/EtOAc and a small amount of water were added, and the phases were separated. The organic layer was concentrated, and the crude product was purified by preparative HPLC to afford 10 mg (71%) of 30. MS (ES+) m/z 490 (M+H).

4-Cyano-N-(7-(1-(cyclopentanecarbonyl)piperidin-4-yl)-5-methyl-5H-pyrrolo[3,2-d]pyrimidin-2-yl)picolinamide (31)

A mixture of THF:NMP (5:1, 0.5 mL), 30 (10 mg, 0.02 mmol), ammonium formate (52 mg, 0.82 mmol), and Pd black (1 mg, 0.01 mmol) was heated under microwave irradiation at 150 °C for 1 h. The mixture was filtered through Celite, and the filtrate was concentrated. Triethylamine (30 μL), CH2Cl2 (0.5 mL) and 4-cyanopicolinoyl chloride (12 mg) were added to the residue. The reaction mixture was stirred at room temperature for 30 min, whereupon the solvents were evaporated. The residue was purified by preparative HPLC to provide 0.4 mg (4%, 2 steps) of 31. MS (ES+) m/z 458 (M+H). 1H NMR (500 MHz, acetone-d6) δ 9.02 (d, J = 5.0, 1H), 8.86 (s, 1H), 8.54 (s, 1H), 8.11 (dd, J = 5.0, 1.8, 1H), 7.58 (s, 1H), 4.72–4.65 (m, 1H), 4.22–4.15 (m, 1H), 3.96 (s, 3H), 3.30–3.18 (m, 2H), 3.10–3.05 (m, 1H), 2.30–2.20 (m, 1H), 1.90–1.50 (m, 12H).

3-Iodo-1,4-dimethyl-5-nitro-1H-indole (33)

Potassium hydroxide pellets (0.78 g, 14 mmol) were added to a cooled solution (ice-water bath) of 4-methyl-5-nitroindole (1.76 g, 10 mmol) in DMF (30 mL). The cooling bath was removed, and the mixture was stirred at room temperature for 10 min. Iodine (2.79 g, 11 mmol) was added, and the stirring was continued for 5 h at room temperature. Potassium carbonate (3.17 g, 23 mmol) and methyl iodide (3.1 mL, 50 mmol) were added, and stirring was continued at room temperature for 16 h. The mixture was diluted with water (150 mL) and treated with solid NaHSO3 with stirring until all excess iodine was quenched. The crude product was collected by filtration, washed with water, and dried. The solid was suspended in 96% EtOH. After the solution was stirred for 15 min, the precipitate was collected and washed with two small portions of EtOH to provide 2.78 g (88%) of 33 as a golden-brownish solid. MS (ES+) m/z 316 (M+H). 1H NMR (500 MHz, DMSO-d6) δ 7.79 (s, 1H), 7.75 (d, J = 9.1 Hz, 1H), 7.53 (d, J = 9.2 Hz, 1H), 4.381 (s, 3H), 2.92 (s, 3H).

Cyclopentyl(4-(1,4-dimethyl-5-nitro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)methanone (34)

Compound 33 (1.24 g, 3.9 mmol), 12 (1.56 g, 5.1 mmol), Pd EnCat TPP30 (palladium acetate/PPh3, encapsulated, Aldrich 644706, 200 mg), and K2CO3 (1.08 g, 7.9 mmol) were suspended in DME:EtOH:H2O (4:1:1, 24 mL). The mixture was degassed with nitrogen and then heated at 70 °C for 18 h. The reaction mixture was partitioned between CH2Cl2 and water. The aqueous layer was extracted with CH2Cl2. The combined organic layers were concentrated, and the residue was purified by silica gel column chromatography (heptane/EtOAc, 100/0–80/20) to afford 1.14 g (79%) of 34 as a yellow solid. MS (ES+) m/z 368 (M+H). 1H NMR (500 MHz, acetone-d6) δ 7.85 (d, J = 9.0 Hz, 1H), 7.42 (d, J = 11.3 Hz, 1H), 7.3 (s, 1H), 5.8 (s, 1H), 4.30–4.20 (m, 2H), 3.90 (s, 3H), 3.90–3.80 (s, 2H), 3.20–3.05 (m, 1H), 2.75 (s, 3H), 2.60–2.40 (m, 2H), 1.90–1.55 (m, 8H).

(4-(5-Amino-1,4-dimethyl-1H-indol-3-yl)piperidin-1-yl)(cyclopentyl)methanone (35)

A mixture of 34 (1.37 g, 3.69 mmol), ammonium formate (2.79 g, 44.3 mmol), 5% Pd/C (270 mg), and 96% EtOH (50 mL) was heated at 85 °C for 90 min under an atmosphere of nitrogen. The mixture was filtered through Celite, and the filtrate was concentrated. The residue was diluted with water and extracted with CHCl3. The combined organic layers were dried (Na2SO4) and concentrated to provide 1.15 g (92%) of 35 as an off-white foam. MS (ES+) m/z 340 (M+H). 1H NMR (500 MHz, acetone-d6) δ 8.04 (s, 1H), 6.95 (d, J = 9.4 Hz, 1H), 6.90 (s, 1H), 6.70 (d, J = 9.4 Hz, 1H), 4.75–4.60 (m, 1H), 4.22–4.15 (m, 1H), 3.90 (br.s., 1H), 3.65 (s, 3H), 3.40–3.30 (m, 1H), 3.30–3.20 (m, 1H), 3.10–3.00 (m, 1H), 2.75–2.65 (m, 1H), 2.45 (s, 3H), 2.15–2.05 (m, 2H), 1.90–1.40 (m, 10H).

3-Cyano-N-(3-(1-(cyclopentanecarbonyl)piperidin-4-yl)-1,4-dimethyl-1H-indol-5-yl)benzamide (36)

Triethylamine (66 μL, 0.47 mmol) was added to a cooled (ice-water bath) solution of 35 (44 mg, 0.12 mmol) in CH2Cl2 (2 mL) and then a solution of 3-cyanobenzoyl chloride (22 mg, 0.13 mmol) in CH2Cl2 (2 mL) was added slowly. The mixture was allowed to warm to room temperature and was then stirred for 16 h. Brine was added, and the mixture was then extracted with CH2Cl2. The combined organic layers were concentrated and the crude product was purified by preparative HPLC to provide 44 mg (80%) of 36. MS (ES+) m/z 469 (M+H). 1H NMR (500 MHz, acetone-d6) δ 9.40 (s, 1H), 8.42 (s, 1H), 8.38 (d, J = 8.3 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.78 (t, J = 7.7 Hz, 1H), 7.20 (d, J = 8.5 Hz, 1H), 7.16 (d, J = 8.5 Hz, 1H), 7.09 (s, 1H), 4.75–4.68 (m, 1H), 4.25–4.12 (m, 1H), 3.77 (s, 3H), 3.42–3.35 (m, 1H), 3.25–3.18 (m, 1H), 3.08–3.00 (m, 1H), 2.70–2.63 (m, 1H), 2.62 (s, 3H), 2.25–2.10 (m, 1H), 2.05–2.00 (m, 1H), 1.85–1.45 (m, 10H).

4-Methyl-1H-pyrrolo[2,3-b]pyridine (38)

A solution of methylmagnesium bromide (3.28 L, 9.83 mol, 3 M in ether) was added dropwise maintaining 30 °C to a suspension of 4-chloro-1H-pyrrolo[2,3-b]pyridine (300 g, 1.97 mol) and Pd(dppf)Cl2 (28.77 g, 39.32 mmol) in toluene (6 L) under nitrogen. After addition, the reaction mixture was heated at 80 °C for 3 h. The reaction mixture was then cooled to 30 °C and poured into ice-water slowly. The mixture was allowed to stand overnight and then was poured into saturated aqueous NH4Cl (10 L). The mixture was extracted with EtOAc (3 × 20 L). The combined organic layers were washed with brine (10 L), dried (Na2SO4), and filtered. The filtrate was concentrated, and the residue was triturated with petroleum ether (4 L) at 30 °C for 16 h to afford 230 g (89%) of 38 as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 10.67 (br. s., 1H), 8.21 (d, J = 5.2 Hz, 1H), 7.33 (d, J = 2.8 Hz, 1H), 6.91 (d, J = 4.4 Hz, 1H), 6.53 (d, J = 2.8 Hz, 1H), 2.59 (s, 3H).

4-Methyl-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (39)

To a stirred solution of 38 (260 g, 1.97 mol) in CH2Cl2 (6 L) was added n-Bu4NBr (31.71 g, 98.36 mmol) and a solution of KOH (220.75 g, 3.93 mol) in water (450 mL) at 28 °C. The mixture was stirred for 10 min, and then benzenesulfonyl chloride (556 g, 3.15 mol) was added dropwise maintaining a temperature of 30 °C. The mixture was stirred at 30 °C for 4 h. The reaction mixture was poured into water (5 L), and extracted with CH2Cl2. The organic layer was washed with brine (5 L), dried (Na2SO4), and filtered. The filtrate was concentrated, and the crude product was purified by silica gel column chromatography (CH2Cl2/PE, 10/90–100/0) to afford 348 g (65%) of 39 as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.30 (d, J = 5.2 Hz, 1H), 8.18 (d, J = 7.6 Hz, 2H), 7.70 (d, J = 4.0 Hz, 1H), 7.56 (t, J = 7.6 Hz, 1H), 7.47 (t, J = 7.4 Hz, 2H), 6.98 (d, J = 4.8, 1H), 6.63 (t, J = 4 Hz, 1H), 2.48 (s, 3H).

4-Methyl-5-nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (40)

Tetramethylammonium nitrate (314.97 g, 2.31 mol) was added to a cold (−10 °C) solution of 39 (420 g, 1.54 mol) in CH2Cl2 (7 L). Trifluoroacetic anhydride (485.89 g, 2.31 mol) was then added dropwise to the mixture maintaining a temperature of −5 °C. The mixture was then stirred at 30 °C for 16 h and then was partitioned into water (5 L). The organic layer was dried (Na2SO4) and filtered. The filtrate was concentrated, and the crude product was triturated with MTBE (3 L) to afford 316 g (65%) of 40 as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 9.06 (s, 1H), 8.21 (d, J = 7.6 Hz, 2H), 7.88 (d, J = 4.0 Hz, 1H), 7.63 (t, J = 7.4 Hz, 1H), 7.53 (t, J = 7.8, 2H), 6.80 (d, J = 4.0, 1H), 2.79 (s, 3H).

4-Methyl-5-nitro-1H-pyrrolo[2,3-b]pyridine (41)

To a stirred suspension of 40 (300 g, 0.95 mol) in MeOH (11 L) was sequentially added K2CO3 (261 g, 1.9 mol) and morpholine (824 g, 9.5 mol) at 25 °C. The mixture was heated at 65 °C for 20 min. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in CH2Cl2 (6 L), and to this mixture was added saturated aqueous NH4Cl (6 L) and water (3 L). The mixture was stirred at room temperature overnight and then filtered. The solids were rinsed with water followed by CH2Cl2, and then triturated with acetone (300 mL) to give 153 g (91%) of 41 as an off-yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.34 (br. s., 1H), 8.90 (s, 1H), 7.68 (d, J = 3.6 Hz, 1H), 6.84 (d, J = 3.6 Hz,1H), 2.81 (s, 3H).

1,4-Dimethyl-5-nitro-1H-pyrrolo[2,3-b]pyridine (42)

K2CO3 (255.1 g, 1.85 mol) was added to a stirred suspension of 41 (109 g, 0.615 mol) in DMF (1.9 L). Iodomethane (489 g, 3.5 mol) was added dropwise over 1 h. The reaction mixture was stirred at room temperature for 2 h and then was poured into water (4 L). The resulting mixture was stirred for 20 min and then filtered. The solids were washed with EtOAc followed by water and then dried under vacuum. The filtrate was extracted with EtOAc (1 L). The organic layer was washed with water (3 × 0.5 L) followed by brine, dried (Na2SO4), filtered, and concentrated. The residue was combined with the solids and purified by silica gel column chromatography (CH2Cl2) to afford 94 g (80%) of 42 as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 9.05 (s, 1H), 7.29 (d, J = 3.6 Hz, 1H), 6.67 (d, J = 3.6 Hz, 1H), 3.92 (s, 3H), 2.86 (s, 3H).

3-Iodo-1,4-dimethyl-5-nitro-1H-pyrrolo[2,3-b]pyridine (43)

N-Iodosuccinimide (141 g, 627 mmol) was added to a stirred suspension of 42 (100 g, 523 mmol) in DMF (1.14 L) at room temperature. The mixture was stirred for 2 h. The reaction mixture was then poured into water (3 L), and the resulting mixture was stirred at room temperature for 20 min. The mixture was filtered and the collected solids were dissolved in CH2Cl2 (20 L). The mixture was washed with water (2 × 5 L) followed by brine (3.5 L), dried (Na2SO4), and filtered. The filtrate was concentrated, and the crude product was triturated with a mixture of CH2Cl2 and MTBE (3 L, 1:3) to afford 143 g (86%) of 43 as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.88 (s, 1H), 8.00 (s, 1H), 3.85 (s, 3H), 3.00 (s, 3H).

1-(Phenylsulfonyl)-4-(prop-1-en-2-yl)-1H-pyrrolo[2,3-b]pyridine (45)

A mixture of DMF/H2O (10/1, 11 mL) was added to a vial containing 4-chloro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (35) (500 mg, 1.7 mmol), 4,4,5,5-tetramethyl-2-(prop-1-en-2-yl)-1,3,2-dioxaborolane (373 mg, 2.2 mmol), Pd(PPh3)4 (197 mg, 0.2 mmol), and K2CO3 (472 mg, 3.4 mmol). The vial was purged with nitrogen and heated at 120 °C by microwave irradiation for 1.5 h. After cooling, the reaction mixture was poured into water and extracted with CH2Cl2. The combined organic layers were concentrated and the crude product was purified by preparative HPLC to afford 448 mg (88%) of 45 as a colorless glass. MS (ES+) m/z 299 (M+H). 1H NMR (400 MHz, CDCl3) δ 8.40 (d, J = 3.8 Hz, 1H), 8.23 (d, J = 7.3 Hz, 2H), 7.75 (d, J = 3.8 Hz, 1H), 7.65–7.60 (m, 1H), 7.55–7.48 (m, 2H), 7.10 (d, J = 3.8, 1H), 6.78 (d, J = 3.8 Hz, 1H), 5.42 (s, 2H), 2.20 (s, 3H).

4-Isopropyl-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (46)

A mixture of 45 (445 mg, 1.5 mmol) and 5% Pd/C (45 mg) in EtOH (5 mL) was stirred at room temperature under a hydrogen atmosphere (1 bar) for 18 h. The mixture was filtered through Celite, and the filtrate was concentrated to afford 436 mg (97%) of 46 as a colorless glass. MS (ES+) m/z 301 (M+H). 1H NMR (400 MHz, CDCl3) δ 8.48 (m, 1H), 8.23 (d, J = 7.6 Hz, 2H), 7.72 (d, J = 3.8 Hz, 1H), 7.58 (t, J = 6.9 Hz, 1H), 7.50 (t, J = 6.9 Hz, 2H), 7.06 (m, 1H), 6.70 (d, J = 4.4 Hz, 1H), 3.30–3.20 (m, 1H), 1.32 (d, J = 5.7 Hz, 6H).

4-Isopropyl-5-nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (47)

Tetrabutylammonium nitrate (795 mg, 2.61 mmol) followed by trifluoroacetic anhydride (0.37 mL, 2.61 mmol) were added to a cold (0 °C) solution of 46 (436 mg, 1.45 mmol) in CH2Cl2 (25 mL), and the mixture was then stirred at 0 °C for 30 min. The reaction mixture was poured into water and extracted with CH2Cl2. The combined organic layers were concentrated, and the crude product was purified by preparative HPLC to afford 417 mg (83%) of 47 as a yellow solid. MS (ES+) m/z 346 (M+H). 1H NMR (400 MHz, CDCl3) δ 8.73 (s, 1H), 8.25 (d, J = 6.3 Hz, 2H), 7.9 (s, 1H), 7.68 (t, J = 6.6 Hz, 1H), 7.58 (t, J = 6.6 Hz, 2H), 6.92 (d, J = 4.5 Hz, 1H), 3.65–3.55 (m, 1H), 1.48 (d, J = 6.3 Hz, 6H).

4-Isopropyl-5-nitro-1H-pyrrolo[2,3-b]pyridine (48)

An aqueous 2N NaOH solution (10 mL) was added to a solution of 47 (417 mg, 1.21 mmol) in THF (20 mL). The mixture was then stirred at room temperature for 16 h. The reaction mixture was neutralized with acetic acid, and the THF solvent was removed under reduced pressure. The aqueous mixture was extracted with CH2Cl2. The combined organic layers were concentrated to afford 245 mg (99%) of 48 as a yellow solid. MS (ES+) m/z 206 (M+H). 1H NMR (400 MHz, CDCl3) δ 10.25 (br. s., 1H), 8.75 (s, 1H), 7.48 (d, J = 6.3 Hz, 1H), 6.98 (d, J = 6.3 Hz, 1H), 3.82–3.73 (m, 1H), 1.56 (d, J = 6.4 Hz, 6H).

3-Iodo-4-isopropyl-1-methyl-5-nitro-1H-pyrrolo[2,3-b]pyridine (49)

KOH pellets (94 mg, 1.67 mmol) were added to a cold (0 °C) solution of 48 (245 mg, 1.19 mmol) in DMF (10 mL). After the solution was stirred for 10 min, iodine (333 mg, 1.31 mmol) was added, and then the mixture was stirred at room temperature for 2 h. K2CO3 (379 mg, 2.74 mmol) and iodomethane (0.59 mL, 9.55 mmol) were added and the mixture was stirred an additional 2 h at room temperature. The reaction mixture was poured into ice-water (50 mL) and then extracted with CH2Cl2. The combined organic layers were concentrated, and the crude product was purified by preparative HPLC to afford 239 mg (59%) of 49 as a yellow solid. MS (ES+) m/z 346 (M+H). 1H NMR (400 MHz, CDCl3) δ 8.58 (s, 1H), 7.49 (s, 1H), 4.62–4.52 (m, 1H), 3.61 (s, 3H), 1.51 (d, J = 6.0 Hz, 6H).

4-Chloro-5-nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (50)

Tetramethylammonium nitrate (420 g, 3.08 mol) was added to a solution of 4-chloro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (35) (451 g, 1.54 mol) in CH2Cl2 (6 L). Trifluoroacetic anhydride (647 g, 3.08 mol) was then added dropwise over 30 min while keeping the temperature between 0 and 5 °C. The resulting mixture was stirred at 0 °C for 30 min and then for 20 h at room temperature. The reaction mixture was diluted with water (1 L). The organic layer was washed with water (2 × 2 L) and brine (4 L), dried (Na2SO4), and filtered. The filtrate was concentrated to give 348 g (66%) of 50 as a yellow solid. MS (ES+) m/z 338 (M+H). 1H NMR (400 MHz, DMSO-d6) δ 9.09 (s, 1H), 8.28 (d, J = 4.0 Hz, 1H), 8.17 (d, J = 8.4 Hz, 2H), 7.79 (t, J = 7.6 Hz, 1H), 7.68 (t, J = 7.8 Hz, 2H), 7.11 (d, J = 4.0 Hz, 1H).

4-Methoxy-1-methyl-5-nitro-1H-pyrrolo[2,3-b]pyridine (52)

Sodium methoxide (320 g, 5.9 mol) was carefully added to a suspension of 50 (397 g, 1.18 mol) in MeOH (8 L) at room temperature. The mixture was heated to reflux for 18 h. After cooling, the reaction mixture was filtered and concentrated. The resulting solid was washed with cold water and chilled MeOH several times and then dried to afford a mixture of 51 and 52. The resulting solid was dissolved in dry DMF (2 L), and the solution was cooled to 0 °C. Sodium hydride in dispersion oil (60%, 51 g, 1.26 mol) was added portionwise carefully over 30 min while keeping the temperature at 0 °C. Iodomethane (400 g, 2.9 mol) was added dropwise over 15 min. The reaction mixture was then warmed gradually to 25 °C and stirred for 18 h. The reaction mixture was carefully poured into water (8 L), stirred for 10 min, and then was filtered. The resulting solid was washed with water (6 × 500 mL) and dried in a vacuum oven to give 149 g (76%) of 52 as a yellow solid. MS (ES+) m/z 208 (M+H). 1H NMR (400 MHz, CDCl3) δ 8.85 (s, 1H), 7.19 (d, J = 4.0 Hz, 1H), 6.84 (d, J = 4.0 Hz, 1H), 4.44 (s, 3H), 3.90 (s, 3H).

3-Iodo-4-methoxy-1-methyl-5-nitro-1H-pyrrolo[2,3-b]pyridine (53)

N-Iodosuccinimide (195 g, 864 mmol) was added to a solution of 52 (149 g, 720 mmol) in dry DMF (150 mL) at 25 °C. The mixture was stirred at 25 °C for 24 h, and then water (800 mL) was added. The mixture was stirred for 5 min, and then the resulting precipitate was filtered. The solids were washed with water (3 × 500 mL) and dried under reduced pressure. The crude product was triturated with EtOAc (600 mL) overnight, filtered, and dried under reduced pressure to provide 151 g (63%) of 53 as a yellow solid. MS (ES+) m/z 334 (M+H). 1H NMR (400 MHz, CDCl3) δ 8.94 (s, 1H), 7.35 (s, 1H), 4.17 (s, 3H), 3.91 (s, 3H).

4-Iodo-1H-pyrrolo[2,3-b]pyridine (54)

Acetyl chloride (140 mL, 1.96 mol) was added slowly to a mixture of 4-chloro-1H-pyrrolo[2,3-b]pyridine (75 g, 0.49 mol) and NaI (442 g, 2.95 mol) in acetonitrile (2.1 L). The mixture was heated at 100 °C for 24 h. After cooling, the reaction mixture was poured into crushed ice with vigorous stirring. Saturated aqueous solutions of NaHCO3 and NaHSO3 were added sequentially to the biphasic solution, which was stirred vigorously for 30 min. The reaction mixture was then extracted with EtOAc. The combined organic layers were concentrated, and the resulting residue was dissolved in MeOH (2 L). Aqueous 2N NaOH (1 L) was added, and the mixture was stirred at room temperature overnight. The methanol was removed under reduced pressure, and the residue was partitioned between water and CH2Cl2. The organic layer was dried (Na2SO4), filtered, and concentrated to afford 100 g (84%) of 54 as an off-white solid. 1H NMR (400 MHz, CDCl3) δ 11.21 (br. s., 1H), 7.95 (d, J = 4.8 Hz, 1H), 7.52 (d, J = 4.8 Hz, 1H), 7.43 (d, J = 4.0 Hz, 1H), 6.42 (d, J = 4.0 Hz, 1H).

4-Iodo-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (55)

Iodide 54 (700 g, 2.87 mol) was added portionwise over the course of 25 min to a mixture of tetrabutylammonium sulfate (48.7 g, 143.4 mmol) and NaOH (344 g, 8.61 mol) in CH2Cl2 (4 L) while maintaining an internal reaction temperature of 0 °C. The mixture was stirred for 0.5 h at 0 °C, and then phenylsulfonyl chloride (760 g, 4.30 mol) was added dropwise to the mixture over the course of 20 min while maintaining an internal reaction temperature of 0 °C. The reaction mixture was stirred at room temperature overnight and then was partitioned between CH2Cl2 and water. The organic layer was washed with water (2 × 1 L) followed by brine (10 L), dried (Na2SO4), and filtered. The filtrate was concentrated, and the crude product was triturated with MeOH (3 L) to afford 983 g (89%) of 55 as yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.18 (d, J = 1.0 Hz, 1H), 8.16 (d, J = 1.0 Hz, 1H), 8.03 (d, J = 4.8 Hz, 1H), 7.79 (d, J = 4.0 Hz, 1H), 7.60–7.57 (m, 2H), 7.51–7.46 (m, 2H), 6.51 (d, J = 4.0 Hz, 1H). MS (ES+) m/z 385 (M+H).

4-Iodo-5-nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (56)

A solution of trifluoroacetic anhydride (31.2 g, 148 mmol) in CH2Cl2 (100 mL) was added dropwise to a stirred, room temperature solution of tetramethylammonium nitrate (19.1 g, 141 mmol) in CH2Cl2 (200 mL). The resulting slurry was stirred at room temperature for 1.5 h and then cooled in a dry ice/acetone bath. A solution of 55 (30.02 g, 78.1 mmol) in CH2Cl2 (100 mL) was added dropwise to the mixture while maintaining the temperature at −65 °C. The reaction mixture was allowed to slowly warm to room temperature and stir for 16 h. The stirred reaction was quenched with saturated aqueous NaHCO3. The organic layer was washed with water (5 × 200 mL). The combined aqueous layers were extracted with CH2Cl2 (3 × 200 mL). The combined organic layers were dried (Na2SO4) and filtered. The filtrate was concentrated, and the crude product was triturated with EtOAc (150 mL, 6 h) to afford 22.0 g (66%) of 56 as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.89 (s, 1H), 8.25–8.20 (m, 2H), 7.99 (d, J = 4.1 Hz, 1H), 7.70–7.63 (m, 1H), 7.59–7.52 (m, 2H), 6.73 (d, J = 4.1 Hz, 1H).

5-Nitro-1-(phenylsulfonyl)-4-(trifluoromethyl)-1H-pyrrolo[2,3-b]pyridine (57)

Methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (43 g, 224 mmol) and CuI (35.5 g, 186 mmol) were added to a solution of 56 (80 g, 190 mmol) in DMF (480 mL). The mixture was degassed with nitrogen and then heated to 100 °C for 2 h. After cooling, the reaction mixture was filtered through Celite, and the filter cake was washed with MTBE (2 × 200 mL). The filtrate was washed with water and brine. The organic layer was dried (Na2SO4) and filtered. The filtrate was concentrated, and the crude product was triturated with CH2Cl2:PE (1:5, 300 mL) to afford 42 g (61%) of 57 as a yellow solid. 1H NMR (CDCl3) δ 8.88 (s, 1H), 8.23 (d, J = 7.6 Hz, 2H), 8.10 (d, J = 4.0 Hz, 1H), 7.70–7.66 (m, 1H), 7.59–7.55 (m, 2H), 6.96 (d, J = 2.0 Hz, 1H).

3-Iodo-1-methyl-5-nitro-4-(trifluoromethyl)-1H-pyrrolo[2,3-b]pyridine (58)

KOH (31.7 g, 566 mmol) was added to a cold (0 °C), stirred solution of 57 (42 g, 110 mmol) in 2-methyltetrahydrofuran:EtOH (2:1, 600 mL). The reaction mixture was allowed to warm to room temperature and stir for 1 h. Iodine (89.3 g, 352 mmol) was added, and the mixture was stirred for 1 h. K2CO3 (76 g, 550 mmol) and iodomethane (46.8 g, 330 mmol) were added, and the mixture was stirred for 2 h. The reaction mixture was concentrated, and the residue was dissolved in CH2Cl2 (2 L). The solution was washed with 10% of aqueous NaHSO3 (2 × 200 mL) and brine. The organic layer was dried (Na2SO4) and filtered. The filtrate was concentrated, and the crude product was triturated with MTBE (200 mL, 12 h) followed by water (200 mL, 12 h) to afford 27 g (66%) of 58 as a yellow solid. 1H NMR (CDCl3) δ 8.64 (s, 1H), 7.75 (s, 1H), 3.98 (s, 3H).

tert-Butyl 4-(1,4-Dimethyl-5-nitro-1H-pyrrolo[2,3-b]pyridin-3-yl)-3,6-dihydropyridine-1(2H)-carboxylate (59a)

tert-Butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (6.6 g, 21 mmol), K2CO3 (7.9 g, 57 mmol), and water (16 mL) were added to a stirred solution of 43 (4.5 g, 14 mmol) in DME/EtOH (4:1, 160 mL). The mixture was sparged with argon for 5 min, and Pd(PPh3)4 (0.82 g, 0.71 mmol) was added. The mixture was heated at 70 °C for 18 h under argon. After cooling to room temperature the reaction mixture was concentrated under reduced pressure, and the residue was partitioned between water (100 mL) and CH2Cl2 (100 mL). The organic layer was washed with water followed by brine, dried (Na2SO4) ,and filtered. The filtrate was concentrated, and the residue was purified by silica gel column chromatography (EtOAc/PE, 15/85) to afford 2.1 g (40%) of 59a as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.98 (s, 1H), 7.10 (s, 1H), 5.74 (br. s., 1H), 4.08–4.06 (m, 2H), 3.88 (s, 3H), 3.68–3.65 (m, 2H), 2.81 (s, 3H), 2.40 (m, 2H), 1.51 (s, 9H). Compounds 59bd were prepared in a similar fashion from 49, 53, and 58, respectively (see Supporting Information).

tert-Butyl 4-(5-Amino-1,4-dimethyl-1H-pyrrolo[2,3-b]pyridin-3-yl)piperidine-1-carboxylate (60a)

A Parr vessel was charged with 59a (1.08 g, 2.9 mmol), triethylamine (0.61 mL, 4.4 mmol), 10% Pd/C (309 mg, 0.29 mmol), and MeOH (9.7 mL). The mixture was shaken under a hydrogen atmosphere (100 psi) at room temperature for 5.5 h. The reaction mixture was filtered through Celite and rinsed with MeOH (2 mL). The filtrate was concentrated under reduced pressure to afford 999 mg (100%) of 60a as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.90 (s, 1H), 6.86 (s, 1H), 4.30 (br. s., 2H), 3.79 (s, 3H), 3.11 (tt, J = 11.7, 3.9 Hz, 1H), 2.87 (t, J = 12.5 Hz, 2H), 2.50 (s, 3H), 2.02 (d, J = 13.7 Hz, 2H), 1.67–1.47 (m, 4H), 1.48 (s, 9H). Compounds 60bd were prepared in a similar fashion from 59bd, respectively (see Supporting Information).

tert-Butyl 4-(5-(3-Cyanobenzamido)-1,4-dimethyl-1H-pyrrolo[2,3-b]pyridin-3-yl)piperidine-1-carboxylate (61a)

Triethylamine (1.6 g, 16.0 mmol) and DMAP (20 mg, 0.16 mmol) were added to a solution of 60a (1.1 g, 3.2 mmol) in THF (50 mL). The mixture was stirred at room temperature for 5 min, and then 3-cyanobenzoic acid (470 mg, 3.2 mmol) followed by HOBT (440 mg, 3.2 mmol) were sequentially added. The mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (MeOH/CH2Cl2, 2/98) to afford 980 mg (82%) of 61a as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.40 (s, 1H), 8.33 (d, J = 8.0 Hz, 1H), 8.14 (s, 1H), 8.00 (d, J = 7.8 Hz, 1H), 7.77 (t, J = 7.6 Hz, 1H), 7.25 (s, 1H), 4.24 (d, J = 13.6 Hz, 2H), 3.83 (s, 3H), 3.31–3.22 (m, 1H), 2.93 (br. s., 2H), 2.65 (s, 3H), 2.06 (d, J = 12.8 Hz, 2H), 1.65–1.52 (m, 2H), 1.50 (s, 9H). MS (ES+) m/z 474 (M+H). Compounds 61bd were prepared in a similar fashion from 60bd, respectively (see Supporting Information).

3-Cyano-N-(1,4-dimethyl-3-(piperidin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide hydrochloride (62a)

A solution of HCl in dioxane (4N, 50 mL) was added to a flask containing 61a (980 mg, 2.07 mmol) and the resulting mixture was stirred at room temperature for 30 min. The reaction mixture was then concentrated under reduced pressure to afford 850 mg (99%) of 62a as a light yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.48 (s, 1H), 8.40 (s, 1H), 8.34 (d, J = 7.6 Hz, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.78 (t, J = 7.8 Hz, 1H), 7.58 (s, 1H), 3.97 (s, 3H), 3.58–3.50 (m, 3H), 3.26–3.23 (m, 2H), 2.82 (s, 3H), 2.35–2.30 (m, 2H), 1.99–1.90 (m, 2H). MS (ES+) m/z 374 (M+H). Compounds 62bd were prepared in a similar fashion from 61bd, respectively (see Supporting Information).

General Methods for the Preparation of 63al and 6466

Method A: The corresponding acid chloride (1 equiv) was added to a solution of amine (1 equiv) in pyridine (0.1 M), and the mixture was stirred at room temperature for 18 h. The reaction mixture was then concentrated, and the crude residue was purified by preparative HPLC. Method B: A mixture of amine (1 equiv), DIPEA (10 equiv), HATU (1.5 equiv), and the corresponding carboxylic acid (1.5 equiv) in DMF (0.1 M) was stirred at room temperature for 18 h. The reaction mixture was diluted with CH2Cl2 and washed with saturated aqueous NaHCO3. The organic layer was dried (Na2SO4), filtered, and concentrated. The reaction mixture was then concentrated, and the crude residue was purified by preparative HPLC. Method C: A mixture of triethylamine (4 equiv) and amine (1 equiv) in CH2Cl2 (0.1M) was cooled to 0 °C and the corresponding acid chloride (1.2 equiv) was added dropwise. The mixture was stirred at room temperature for 18 h. The reaction mixture was diluted with CH2Cl2 and was washed with a saturated aqueous NaHCO3 solution. The organic layer was washed with brine, dried (Na2SO4), and concentrated. The reaction mixture was then concentrated, and the crude residue was purified by preparative HPLC. Method D: A mixture of DIPEA (20 equiv) and the corresponding acid chloride (1 equiv) were added to a solution of amine (1 equiv) in CH2Cl2 (0.1 M), and the mixture was stirred at room temperature for 18 h. The reaction mixture was diluted with CH2Cl2 and was washed with a saturated aqueous NH4Cl solution. The organic layer was dried (MgSO4) and filtered. The filtrate was concentrated, and the residue was purified by preparative HPLC.

N-(3-(1-Acetylpiperidin-4-yl)-1,4-dimethyl-1H-pyrrolo[2,3-b]pyridin-5-yl)-3-cyanobenzamide (63a)

Method B. 62a, acetic acid, 45 mg (40%), white solid. MS (ES+) m/z 416 (M+H). 1H NMR (400 MHz, DMSO-d6) δ 10.26 (s, 1H), 8.46 (t, J = 1.4 Hz, 1H), 8.32 (dt, J = 8.0 Hz, J = 1.4 Hz, 1H), 8.10–8.07 (m, 2H), 7.77 (t, J = 7.81 Hz, 1H), 7.32 (s, 1H), 4.52 (d, J = 13.2 Hz, 1H), 3.92 (d, J = 13.8 Hz, 1H), 3.75 (s, 3H), 3.33–3.16 (m, 2H), 2.71–2.62 (m, 1H), 2.52 (s, 3H), 2.03 (s, 3H), 2.02–1.93 (m, 2H), 1.60–1.48 (m, 1H), 1.48–1.35 (m, 1H).

3-Cyano-N-(1,4-dimethyl-3-(1-propionylpiperidin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (63b)

Method C. 62a, propionyl chloride, 42 mg (57%), white solid. MS (ES+) m/z 430 (M+H). 1H NMR (400 MHz, CDCl3) δ 8.29 (s, 1H), 8.24–8.20 (m, 2H), 7.98 (s, 1H), 7.85 (d, J = 8.1 Hz, 1H), 7.65 (t, J = 8.1 Hz, 1H), 6.94 (s, 1H), 4.77 (d, J = 12.8 Hz, 1H), 3.98 (d, J = 12.8 Hz, 1H), 3.82 (s, 3H), 3.26–3.10 (m, 2H), 2.70–2.62 (m, 1H), 2.60 (s, 3H), 2.39 (q, J = 7.6 Hz, 2H), 2.13–2.08 (m, 2H), 1.62–1.48 (m, 2H), 1.77 (t, J = 7.6 Hz, 3H).

3-Cyano-N-(3-(1-(cyclopropanecarbonyl)piperidin-4-yl)-1,4-dimethyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (63c)

Method C. 62a, cyclopropanecarbonyl chloride, 68 mg (84%), white solid. MS (ES+) m/z 442 (M+H). 1H NMR (400 MHz, CDCl3) δ 8.29 (s, 1H), 8.26–8.21 (m, 2H), 8.13 (s, 1H), 7.85 (d, J = 8.1 Hz, 1H), 7.65 (t, J = 8.1 Hz, 1H), 6.93 (s, 1H), 4.78–4.58 (m, 1H), 4.42–4.32 (s, 1H), 3.82 (s, 3H), 3.29–3.20 (m, 2H), 2.78–2.62 (s, 1H), 2.60 (s, 3H), 2.16–1.95 (m, 2H), 1.84–1.72 (m, 1H), 1.65–1.35 (m, 2H), 1.04–0.98 (m, 2H), 0.82–0.74 (m, 2H).

3-Cyano-N-(3-(1-isobutyrylpiperidin-4-yl)-1,4-dimethyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (63d)

Method A. 62a, isobutyryl chloride, 7 mg (30%). MS (ES+) m/z 444 (M+H). 1H NMR (500 MHz, CDCl3) δ 8.37–8.35 (m, 2H), 8.28 (d, J = 7.9 Hz, 1H), 8.25 (s, 1H), 7.87 (d, J = 8.5 Hz, 1H), 7.67 (t, J = 7.3 Hz, 1H), 6.95 (s, 1H), 4.80–4.75 (m, 1H), 4.15–4.05 (m, 1H), 3.85 (s, 3H), 3.25–3.15 (m, 2H), 2.90–2.85 (m, 1H), 2.70–2.60 (m, 1H), 2.60 (s, 3H), 2.19–2.10 (m, 1H), 2.05–1.99 (m, 1H), 1.60–1.30 (m, 2H), 1.20–1.15 (m, 6H).

3-Cyano-N-(3-(1-(2-cyclopropylacetyl)piperidin-4-yl)-1,4-dimethyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (63e)

Method B. 62a, 2-cyclopropylacetic acid, 40 mg (33%), white solid. MS (ES+) m/z 456 (M+H). 1H NMR (400 MHz, DMSO-d6) δ 10.27 (s, 1H), 8.46 (s, 1H), 8.32 (d, J = 8.0 Hz, 1H), 8.11–8.06 (m, 2H), 7.77 (t, J = 7.9 Hz, 1H), 7.33 (s, 1H), 4.55 (d, J = 12.8 Hz, 1H), 3.95 (d, J = 13.6 Hz, 1H), 3.75 (s, 3H), 3.12–3.33 (m, 2H), 2.67 (t, J = 12.1 Hz, 1H), 2.51 (s, 3H), 2.28 (d, J = 6.8 Hz, 2H), 1.97 (t, J = 10.4 Hz, 2H), 1.56–1.37 (m, 2H), 1.03–0.91 (m, 1H), 0.49–0.42 (m, 2H), 0.10–0.05 (m, 2H).

3-Cyano-N-(1,4-dimethyl-3-(1-(3-methylbutanoyl)piperidin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (63f)

Method C. 62a, 3-methylbutanoyl chloride, 115 mg (36%), white solid. MS (ES+) m/z 458 (M+H). 1H NMR (400 MHz, CDCl3) δ 8.30 (s, 1H), 8.25–8.20 (m, 2H), 7.90–7.85 (m, 2H), 7.67 (t, J = 7.8 Hz, 1H), 6.95 (s, 1H), 4.35–4.22 (m, 1H), 4.05–4.00 (m, 1H), 3.83 (s, 3H), 3.25–3.10 (m, 2H), 2.70–2.62 (m, 1H), 2.61 (s, 3H), 2.26 (d, J = 7.0 Hz, 2H), 2.15–2.00 (m, 3H), 1.55–1.45 (m, 2H), 0.99 (d, J = 6.8 Hz, 6H).

3-Cyano-N-(3-(1-(cyclobutanecarbonyl)piperidin-4-yl)-1,4-dimethyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (63g)

Method B. 62a, cyclobutanecarboxylic acid, 75 mg (61%), white solid. MS (ES+) m/z 456 (M+H). 1H NMR (400 MHz, DMSO-d6) δ 10.26 (s, 1H), 8.45 (s, 1H), 8.32 (d, J = 8.0 Hz, 1H), 8.10–8.06 (m, 2H), 7.77 (t, J = 7.4 Hz, 1H), 7.32 (s, 1H), 4.51 (d, J = 12.5 Hz, 1H), 3.79 (d, J = 13.8 Hz, 1H), 3.75 (s, 3H), 3.24 (t, J = 11.9 Hz, 2H), 3.10 (t, J = 11.9 Hz, 1H), 2.72–2.63 (m, 1H), 2.52 (s, 3H), 2.23–2.06 (m, 4H), 2.00–1.85 (m, 3H), 1.79–1.69 (m, 1H), 1.50–1.35 (m, 2H).

3-Cyano-N-(3-(1-(cyclopentanecarbonyl)piperidin-4-yl)-1,4-dimethyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (63h)

Method A. 62a, cyclopentanecarbonyl chloride, 43 mg (50%), white solid. 1H NMR (500 MHz, acetone-d6) δ 9.58 (s, 1H), 8.45 (s, 1H), 8.39 (d, J = 7.7 Hz, 1H), 8.20 (s, 1H), 8.01 (d, J = 7.8 Hz, 1H), 7.78 (t, J = 7.8 Hz, 1H), 7.24 (s, 1H), 4.71 (d, J = 12.6 Hz, 1H), 4.20 (d, J = 13.1 Hz, 1H), 3.80 (s, 3H), 3.40–3.30 (m, 1H), 3.28–3.20 (m, 1H), 3.10–3.00 (m, 1H), 2.72–2.65 (m, 1H), 2.64 (s, 3H), 2.12 (d, J = 12.7 Hz, 1H), 2.03 (d, J = 13.8 Hz, 1H), 1.84–1.44 (m, 10H). MS (ES+) m/z 470 (M+H).

3-Cyano-N-(3-(1-(cyclohexanecarbonyl)piperidin-4-yl)-1,4-dimethyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (63i)

Method B. 62a, cyclohexanecarboxylic acid, 45 mg (43%), white solid. MS (ES+) m/z 484 (M+H). 1H NMR (400 MHz, CDCl3) δ 8.34 (s, 1H), 8.30–8.25 (m, 2H), 8.23 (s, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.65 (t, J = 7.8 Hz, 1H), 6.91 (s, 1H), 4.76–4.72 (m, 1H), 4.10–4.00 (m, 1H), 3.83 (s, 3H), 3.25–3.10 (m, 2H), 2.70–2.48 (m, 5H), 2.15–1.90 (m, 2H), 1.85–1.65 (m, 5H), 1.60–1.45 (m, 3H), 1.40–1.15 (m, 4H).

3-Cyano-N-(3-(1-(2-fluoro-6-methylbenzoyl)piperidin-4-yl)-1,4-dimethyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (63j)

Method B. 62a, 2-fluoro-6-methylbenzoic acid, 220 mg (46%), white solid. MS (ES+) m/z 510 (M+H). 1H NMR (400 MHz, CDCl3) δ 8.30–8.15 (m, 3H), 8.00 (d, J = 18.4 Hz, 1H), 7.83 (d, J = 7.2, 1H), 7.68 (t, J = 7.8 Hz, 1H), 7.30–7.15 (m, 1H), 7.05–6.85 (m, 3H), 5.00–4.90 (m, 1H), 3.83 (s, 3H), 3.65–3.55 (m, 1H), 3.30–3.10 (m, 2H), 3.00–2.85 (m, 1H), 2.57 (s, 3H), 2.36 (s, 1.5H), 2.28 (s, 1.5H), 2.22–2.10 (m, 1H), 2.05–1.95 (m, 1H), 1.75–1.40 (m, 2H).

3-Cyano-N-(1,4-dimethyl-3-(1-(tetrahydrofuran-3-carbonyl)piperidin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (63k)

Method B. 62a, tetrahydrofuran-3-carboxylic acid, 75 mg (59%), white solid. MS (ES+) m/z 472 (M+H). 1H NMR (400 MHz, DMSO-d6) δ 10.26 (s, 1H), 8.46 (t, J = 1.4 Hz, 1H), 8.32 (dt, J = 8.0, 1.4 Hz, 1H), 8.11–8.06 (m, 2H), 7.78 (t, J = 7.8 Hz, 1H), 7.35 (s, 1H), 4.55 (d, J = 13.1 Hz, 1H), 4.08 (d, J = 13.9 Hz, 1H), 3.89 (dt, J = 13.3, 8.1 Hz, 1H), 3.75 (s, 3H), 3.74–3.67 (m, 3H), 3.44–3.14 (m, 3H), 2.76–2.65 (m, 1H), 2.53 (s, 3H), 2.08–1.94 (m, 4H), 1.57–1.37 (m, 2H).

3-Cyano-N-(3-(1-(3,5-dimethylisoxazole-4-carbonyl)piperidin-4-yl)-1,4-dimethyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (63l)

Method B. 62a, 3,5-dimethylisoxazole-4-carboxylic acid, 48 mg (45%), white solid. MS (ES+) m/z 497 (M+H). 1H NMR (400 MHz, CD3OD) δ 8.39 (s, 1H), 8.32 (d, J = 8.0 Hz, 1H), 8.14 (s, 1H), 7.99 (d, J = 7.3 Hz, 1H), 7.76 (t, J = 7.9 Hz, 1H), 7.29 (s, 1H), 4.66–4.56 (s, 2H), 3.83 (s, 4H), 3.50–3.35 (m, 2H), 3.18–2.95 (m, 1H), 2.66 (s, 3H), 2.46 (s, 3H), 2.30 (s, 3H), 2.24–2.05 (m, 2H), 1.67 (m, 2H).

3-Cyano-N-(3-(1-isobutyrylpiperidin-4-yl)-4-isopropyl-1-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (64)

Method A. 62b, isobutyryl chloride, 14.6 mg (97%), white solid. MS (ES+) m/z 472 (M+H). 1H NMR (400 MHz, acetone-d6) δ 9.38 (br. s., 1H), 8.48 (s, 1H), 8.42 (d, J = 6.3 Hz, 1H), 8.10 (s, 1H), 8.05 (d, J = 6.3 Hz, 1H), 7.84 (t, J = 6.3 Hz, 1H), 7.30 (s, 1H), 4.80–4.70 (m, 1H), 4.25–4.15 (m, 1H), 3.95–3.90 (m, 1H), 3.82 (s, 3H), 3.35–3.25 (m, 2H), 3.00–2.90 (m, 1H), 2.85–2.80 (m, 1H), 2.74–2.65 (m, 1H), 2.20–2.10 (m, 1H), 1.74–1.50 (m, 2H), 1.49 (d, J = 6.5 Hz, 6H), 1.15–1.05 (m, 6H).

3-Cyano-N-(3-(1-isobutyrylpiperidin-4-yl)-4-methoxy-1-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (65)

Method C. 62c, Isobutyryl chloride, 50 mg (40%), white solid. MS (ES+) m/z 460 (M+H). 1H NMR (400 MHz, DMSO-d6) δ 10.28 (s, 1H), 8.46 (s, 1H), 8.32 (d, J = 8.0 Hz, 1H), 8.13–8.09 (m, 2H), 7.77 (t, J = 7.8 Hz, 1H), 7.23 (s, 1H), 4.56–4.53 (m, 1H), 4.07–4.04 (m, 1H), 3.92 (s, 3H), 3.73 (s, 3H), 3.09–3.05 (m, 2H), 2.95–2.85 (m, 1H), 2.70–2.55 (m, 1H), 2.05–1.90 (m, 2H), 1.55–1.35 (m, 2H), 1.03–0.99 (m, 6H).

3-Cyano-N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (66). (43)

Method D. 62d, isobutyryl chloride, 6 mg (29%), white solid. MS (ES+) m/z 498 (M+H). 1H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 8.42 (s, 1H), 8.35–8.29 (m, 2H), 8.12 (d, J = 7.6 Hz, 1H), 7.88 (s, 1H), 7.80 (t, J = 7.6 Hz, 1H), 4.65–4.55 (m, 1H), 4.15–4.05 (m, 1H), 3.86 (s, 3H), 3.18–3.05 (m, 2H), 2.98–2.88 (m, 1H), 2.60–2.50 (m, 1H), 2.00–1.82 (m, 2H), 1.60–1.40 (m, 2H), 1.10–1.00 (m, 6H).

1-(4-(1,4-Dimethyl-5-nitro-1H-pyrrolo[2,3-b]pyridin-3-yl)-5,6-dihydropyridin-1(2H)-yl)-2-methylpropan-1-one (67)

A solution of HCl (13.5 mL, 4 M in dioxane) was added to a cold (0 °C) solution of 59a (5 g, 13.4 mmol) in MeOH (10 mL) The mixture was warmed to room temperature and stirred for 3 h. Solvents were removed under reduced pressure below 30 °C. The residue was neutralized with saturated aqueous NaHCO3 solution and was extracted with a mixture of CH2Cl2/MeOH (97/3). The combined organic layers were washed with brine, dried (Na2SO4), and filtered. The filtrate was concentrated. Triethylamine (5.3 mL, 38.5 mmol) and CH2Cl2 (50 mL) was added to the residue followed by isobutyryl chloride (1.6 mL, 15.4 mmol) at 0 °C. The mixture was allowed to stir at room temperature for 1 h. The reaction mixture was diluted with CH2Cl2 and washed with a saturated aqueous NaHCO3 solution. The organic layer was washed with brine, dried (Na2SO4), filtered, and concentrated. The crude product was triturated with diethyl ether to afford 4.0 g (91%) of 67. 1H NMR (400 MHz, CDCl3) δ 8.98 (s, 1H), 7.11 (s, 1H), 5.85–5.71 (m, 1H), 4.25–4.20 (m, 2H), 3.89 (s, 3H), 3.76 (t, J = 5.0 Hz, 2H), 2.49–2.43 (m, 2H), 2.93–2.85 (m, 1H), 2.81 (s, 3H), 1.19 (d, J = 6.5 Hz, 6H). MS (ES+) m/z 343 (M+H).

1-(4-(5-Amino-1,4-dimethyl-1H-pyrrolo[2,3-b]pyridin-3-yl)piperidin-1-yl)-2-methylpropan-1-one (68)

Triethylamine (0.17 mL, 1.19 mmol) and 10% Pd/C (1.3 g) were added to a solution of 67 (3.7 g, 10.7 mmol) in MeOH (110 mL). The mixture was stirred under a hydrogen atmosphere (50 psi) at room temperature for 5 h. The reaction mixture was filtered through Celite, and the filtrate was concentrated to afford 3.0 g (88%) of 68. 1H NMR (400 MHz, CDCl3) δ 7.89 (s, 1H), 6.84 (s, 1H), 4.82 (d, J = 12.7 Hz, 1H), 4.07 (d, J = 12.7 Hz, 1H), 3.75 (s, 3H), 3.25–3.16 (m, 2H), 2.88–2.81 (m, 1H), 2.72–2.65 (m, 1H), 2.49 (s, 3H), 2.16–2.05 (m, 2H), 1.58–1.52 (m, 2H), 1.21–1.16 (m, 6H). MS (ES+) m/z 315 (M+H).

General Method for the Preparation of 69ad

To a solution of the corresponding acid (1.2 equiv) in CH2Cl2 (5 mL) was added DIPEA (3 equiv) followed by HATU (2 equiv), and the mixture was allowed to stir at room temperature for 1 h. Amine 68 (100 mg, 0.32 mmol, 1 equiv) was then added, and the reaction mixture was further stirred for 16 h at room temperature. The reaction mixture was diluted with water and extracted using CH2Cl2. The combined organic layers were dried (Na2SO4) and filtered. The filtrate was concentrated, and the residue was purified by preparative HPLC.

3-Cyano-N-(3-(1-isobutyrylpiperidin-4-yl)-1,4-dimethyl-1H-pyrrolo[2,3-b]pyridin-5-yl)-4-methoxybenzamide (69a)

3-Cyano-4-methoxybenzoic acid, 52 mg (35%). 1H NMR (400 MHz, MeOH-d4) δ 8.32–8.29 (m, 2H), 8.09 (s, 1H), 7.34 (d, J = 8.8 Hz, 1H), 7.23 (s, 1H), 4.69 (d, J = 12.8 Hz, 1H), 4.18 (d, J = 12.8 Hz, 1H), 4.06 (s, 3H), 3.81 (s, 3H), 3.39–3.25 (m, 2H), 3.03–2.99 (m, 1H), 2.81–2.75 (m, 1H), 2.63 (s, 3H), 2.19–2.09 (m, 2H), 1.68–1.52 (m, 2H), 1.14–1.11 (m, 6H). MS (ES+) m/z 474 (M+H).

3-Cyano-4-fluoro-N-(3-(1-isobutyrylpiperidin-4-yl)-1,4-dimethyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (69b)

3-Cyano-4-fluorobenzoic acid, 22 mg (15%). 1H NMR (400 MHz, MeOH-d4) δ 8.45 (dd, J = 6.4, 2.4 Hz, 1H), 8.39–8.35 (m, 1H), 8.12 (s, 1H), 7.56 (t, J = 8.8 Hz, 1H), 7.24 (s, 1H), 4.39 (d, J = 13.2 Hz, 1H), 4.18 (d, J = 13.2 Hz, 1H), 3.81 (s, 3H), 3.40–3.26 (m, 2H), 3.05–2.95 (m, 1H), 2.81–2.75 (m, 1H), 2.64 (s, 3H), 2.19–2.03 (m, 2H), 1.68–1.51 (m, 2H), 1.15 (d, J = 6.8 Hz, 3H), 1.11 (d, J = 7.2 Hz, 3H). MS (ES+) m/z 462 (M+H).

4-Chloro-3-cyano-N-(3-(1-isobutyrylpiperidin-4-yl)-1,4-dimethyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (69c)

4-Chloro-3-cyanobenzoic acid, 62 mg (41%). 1H NMR (400 MHz, CDCl3) δ 8.36 (d, J = 11.0 Hz, 2H), 8.24–8.16 (m, 2H), 7.65 (d, J = 8.2 Hz, 1H), 6.90 (s, 1H), 4.73 (d, J = 11.9 Hz, 1H), 4.07 (d, J = 12.4 Hz, 1H), 3.82 (s, 3H), 3.18–3.12 (m, 2H), 2.87 (dt, J = 13.6, 6.7 Hz, 1H), 2.70–2.47 (m, 4H), 2.16–2.04 (m, 1H), 2.00–1.90 (m, 1H), 1.60–1.45 (m, 1H), 1.30–1.10 (m, 7H). MS (ES+) m/z 478 (M+H).

3-Cyano-N-(3-(1-isobutyrylpiperidin-4-yl)-1,4-dimethyl-1H-pyrrolo[2,3-b]pyridin-5-yl)-4-methylbenzamide (69d)

3-Cyano-4-methylbenzoic acid, 38 mg (26%). 1H NMR (400 MHz, CD3OD) δ 8.32 (s, 1H), 8.18 (dd, J = 8.0, 1.2 Hz, 1H), 8.11 (s, 1H), 7.61 (d, J = 8.0 Hz, 1H), 7.23 (s, 1H), 4.69 (d, J = 12.4 Hz, 1H), 4.18 (d, J = 12.4 Hz, 1H), 3.81 (s, 3H), 3.48–3.26 (m, 2H), 3.03–2.98 (m, 1H), 2.81–2.75 (m, 1H), 2.64 (s, 6H), 2.19–2.09 (m, 2H), 1.67–1.53 (m, 2H), 1.14 (d, J = 7.2 Hz, 3H), 1.11 (d, J = 6.4 Hz, 3H). MS (ES+) m/z 458 (M+H).

PAINS

All compounds reported with biological activity were electronically filtered for structural attributes consistent with classification as pan assay interference compounds (PAINS) and were found to be negative. (50) Compound 3 has undergone broad enzyme and receptor profiling and been found not to be promiscuous (4/81 targets with % inhibition >50%, 10 μM concentration) (see Supporting Information, Table S2).

Torsional Energy Calculations

All quantum mechanical calculations were performed with the PetaChem software (41) utilizing the B3LYP density functional and the 6-31G* basis set in gas phase. Dihedral scans were performed using a series of constrained optimizations, whereby the specified dihedral angle was kept frozen at a given value, with full optimization of the remaining geometric parameters subject to the given dihedral constraint. Total energies for a given system were then converted into relative energies (kcal/mol) for graphical depiction.

Gal4-RORC2 Luciferase Reporter Assay

Neuro2A cells (murine neuroblastoma cell line obtained from HPACC) were transiently transfected with a mammalian expression vector (pM) containing Gal4-RORC2 LBD and a Gal4-responsive reporter gene containing firefly luciferase (5xGAL4UAS-Luc3). Gal4-RORC2 LBD is active in the transfected Neuro2a cells in the absence of any added stimuli, resulting in a robust luciferase response signal. The growth medium was composed of MEM EBS without l-glutamine, 10% (v/v) FBS, 2 mM l-glutamine and 1× nonessential amino acid (NEAA). The seeding medium was composed of MEM EBS without l-glutamine or phenol red, 4% (v/v) FBS, 2 mM l-glutamine, 1× NEAA, 1% penicillin (10 000 U/mL)/streptomycin (10,000 μg/mL). The assay medium was composed of MEM EBS without l-glutamine or phenol red, 4% (v/v) FBS, 2 mM l-glutamine, 1× NEAA, 1% penicillin (10 000 U/mL)/streptomycin (10 000 μg/mL). Neuro2A cells were cultured in growth medium in humidified chambers at 37 °C and 5% CO2 using standard tissue culture procedures. On day one of the assay, cells were seeded and transfected. Neuro2A cells were suspended in seeding medium and mixed with plasmids and transfection reagent which were dissolved in OptiMEM I reduced serum medium (Invitrogen), and then seeded to 384-well plates (Corning, Black, Clear bottom) in 40 μL/well containing 12 500 cells, 17.25 ng Gal4-Luc3, 5.75 ng either empty pM vector (no receptor control wells) or pM-Gal4RORgamma-LBD, and 0.11 μL Lipofectamine2000. On day two of the assay, the cells were treated with compounds. Compounds were serially diluted in a 96-well polypropylene plate with assay medium containing 0.5% (v/v) DMSO at 5-times final assay concentration. Compounds (10 μL) or 0.5% DMSO in assay medium for no compound control wells were transferred from the dilution plate to the 384-format cell plate such that final assay volume was 50 μL and final DMSO concentration was 0.1% (v/v). Cells were incubated for 20–24 h in humidified chambers at 37 °C and 5% CO2. On day three of the assay, luminescence was measured. SteadyLite Plus reagent (10 μL) (PerkinElmer) was added to each well. The cell plates were incubated at room temperature for 15 min in the dark before reading of luminescence on a MicroBeta Trilux (Wallac). IC50 values were determined by the nonlinear regression analysis of dose–response curves.
Similar procedures were followed for the Gal4-RORA and Gal4-RORB luciferase reporter assays employing the corresponding constructs.

RORC2 TR-FRET Coactivator Recruitment Assay

The assay employed N-terminally Six-Histidine-tagged-RORC2 ligand binding domain (6-His-RORC2 LBD), expressed in E. coli and purified by affinity chromatography, and biotin-coactivator peptide SRC1-2 (biotin-aminohexanoic acid-CPSSHSSLTERHKILHRLLQEGSPS-NH2; SEQ ID NO: 1) containing the LXXLL consensus domain which is responsible for receptor binding. The assay was carried out in black polystyrene, 384-well plates in a total assay volume of 50.5 μL. The assay buffer contained 50 mM TRIS-HCL pH 7.5, 1 mM NaCl, 2 mM MgCl2, 0.5 mg/mL bovine serum albumin, and 5 mM dithiothreitol. The final concentration of reagents was 6.3 nM RORC2 LBD, 200 nM SRC1-2, 50 nM streptavidin APC, 1 nM europium-labeled anti-His antibody, and varying concentrations of compounds such that final concentration of DMSO was 1% (v/v). Compound (0.5 μL at 100× final concentration in DMSO) or DMSO was dispensed followed by 50 μL mixture of the other assay components including receptor (test wells) or excluding receptor (control wells for maximal inhibition). Assay mixtures were incubated at room temperature for 3 h and then read using an EnVision 2100 Multilabel Reader (PerkinElmer Life Sciences) at Excitation Filter 320, Emission Europium Filter 615, Emission APC Filter 665, and Dichroic Mirror D400/D630. The TR-FRET signal was determined by calculating the ratio of 665 nm by 615 nm. IC50 values of compounds were determined by the nonlinear regression analysis of dose–response curves.
Similar procedures were followed for the RORA and RORB TR-FRET coactivator recruitment assays employing the corresponding constructs.

Estimation of Compound Dissociation Rate Using TR-FRET Coactivator Recruitment Assay

A modification of the coactivator peptide recruitment assay was used to estimate the off-rate of inverse agonists. RORC2 LBD, SRC1-2, and compound (at approximately its IC75 concentration) were incubated for 3 h as indicated above. Then, 3 μM RORC2 agonist 3,5-dibromo-4-(3-isopropyl-4-methoxyphenoxy)phenyl)(1-(ethoxymethyl)-1H-imidazol-2-yl)methanol (EC50 = 9.5 nM, see Supporting Information for synthesis) was added, and the TR-FRET signal was monitored during the subsequent 9.5 h. Apparent T1/2 values of compounds were determined by analysis of the progress curves.

[3H]-25-Hydroxycholesterol Scintillation Proximity Assay (SPA)

The receptor was enzymatically biotinylated at its N-terminal region and attached to streptavidin coated YSi scintillation proximity assay (SPA) beads prior to assay. This complex was circulated using a peristaltic pump prior to dispensing into the assay plate (96-well). The final assay cocktail contained: 4 μg/well YSi beads, 2.35–2.62 nM [3H]-25-hydroxycholesterol (KD = 7.7 nM, PerkinElmer; #NET674250UC), 9% propylene glycol and 1% DMSO (or equivalent concentration of compound). The assay buffer consisted of 50 mM HEPES, 150 mM NaCl, 5 mM MgCl, 0.01% BSA, and 1 mM TCEP. To prevent nonspecific binding to plastic, [3H]-25-hydroxycholesterol was diluted in buffer containing 36% propylene glycol. The assay was incubated overnight at room temperature with shaking (400–480 rpm) followed by centrifugation at 2000 rpm for 3 min and measurement in a scintillation counter (Trilux Microbeta; PerkinElmer). IC50 values of compounds were determined by the nonlinear regression analysis of dose inhibition curves.

IL-17 Production Assay in Human Th17 Cells

Human CD4+ T cells were purified from buffy coats from healthy donors (Massachusetts General Hospital) by negative selection according to the following procedure: mixing 25 mL of blood with 1 mL of Rosette Sep CD4+ T cell enrichment cocktail (StemCell Technologies) followed by application of a layer of 14 mL Ficoll Paque Plus (Amersham GE Healthcare) and subsequent centrifugation at 1200g for 20 min at room temperature. The Ficoll layer was then harvested and washed with phosphate saline buffer containing 2% (v/v) fetal bovine serum and cells were resuspended with RPMI medium containing 10% (v/v) fetal bovine serum and 10% (v/v) DMSO, frozen and kept in liquid nitrogen until used. On the first day of the assay, a vial containing 107 CD4+ T cells was thawed rapidly in a 37 °C water bath, immediately transferred into 20 mL X-Vivo 15 medium (Lonza), and spun for 6 min at 300g. The supernatant was discarded, and the resulting pellet was resuspended at 106 cells/mL in 10 mL fresh X-Vivo 15 medium, followed by storage overnight in a humidified chamber at 37 °C and 5% CO2. Serial dilutions of compounds were prepared at 10× final concentration in X-Vivo15 medium containing 3% (v/v) DMSO. On the second day of the assay, a 384-well tissue culture plate was coated with 10 μg/mL anti-hCD3 (eBioscience) at 50 μL/well. After 2 h at 37 °C, the supernatant was discarded and the coated plates are kept in a sterile tissue culture hood. Cytokine plus anti-CD28 cocktail was prepared by mixing 25 ng/mL hIL-6 (Peprotech), 5 ng/mL hTGFbeta1 (Peprotech), 12.5 ng/mL IL-1beta (Peprotech), 25 ng/mL hIL-21 (Pfizer in-house preparation), 25 ng/mL hIL-23 (R&D Systems), and 1 μg/mL anti-hCD28 (eBioscience) in X-Vivo 15 medium. The cytokine plus anti-CD28 cocktail with CD4+ cells was prepared such that the cocktail was diluted 10-fold and density is 0.22 × 106 cells/mL. The mixture was dispensed at 90 μL (20 000 cells) per well in the anti-hCD3 coated plate prepared as noted above and then was incubated 1 h at 37 °C. Compounds (10 μL per well, 10× ) were added (final DMSO = 0.3%), and the cells were incubated in a humidified chamber at 37 °C and 5% CO2 for 6 days. On day six of the assay, production of IL-17A in 10 μL of the supernatant was determined by sandwich ELISA using 384w hIL-17 MSD plates (Meso Scale Discovery) following the manufacturer’s protocol. Measurement was carried out in a Sector Imager 6000. Signal units from the instrument were converted to pg/mL using a calibration curve with known amounts of IL-17A. IC50 values of test compounds were determined by the nonlinear regression analysis of dose–response curves. Cell viability in the assay plate was determined by CellTiter-Glo method (Promega) following the manufacturer’s protocol.

RORC2 Half-Life in Human T Cells

Human CD4+ cells were subjected to Th17 differentiation as described above. On day 4, cells were metabolically labeled with methionine/cysteine-free culture medium containing 35S l-methionine and 35S l-cysteine (obtained from PerkinElmer) at 0.1 mCi/mL for 30 min (“pulse”) followed by washing and continuing the incubation with original culture medium (“chase”). Cell samples were collected at the indicated time points. Nuclear extracts were prepared and immunoprecipitated with anti-RORC2 monoclonal antibody (MABF81, Millipore) and subjected to SDS gel electrophoresis followed by autoradiography. The RORC2 band was identified by its position in the gel relative to protein standards, and its presence in the extract was also confirmed by immunoblotting of a portion of the extracts prior to immunoprecipitation. The RORC2 band was quantified by densitometry and plotted against time after pulse. Data were fitted to a single phase decay exponential equation.

IL-17 Production Assay in Mouse Th17 Cells

Mouse CD4+ T cells were prepared from mouse splenocytes by negative selection using magnetic beads coated with antibodies that capture unwanted cells using Mouse CD4+ EasySep kit from StemCell Technologies and following the manufacturer’s protocol. Each mouse spleen (from 6–8 week-old Balb/c mice, Jackson Laboratories) was minced and mixed with 10 mL phosphate buffer saline containing 2% (v/v) fetal bovine serum and then filtered through a 70-μm cell strainer. The resulting filtrate containing splenocytes was collected into a 50 mL tube and centrifuged at 300g at room temperature for 10 min, and then resuspended at 108 cells/mL with RoboSep buffer. Then 8 mL of the cell suspension were transferred to a 14 mL polystyrene tube, and rat serum was added at 50 μL/mL of cell suspension. The following reagents were added in order: EasySep mouse CD4+ T cells enrichment cocktail (50 μL/mL of cell suspension), EasySep biotin selection cocktail (100 μL/mL of cell suspension) and EasySep D2Magnetic Particles (100 μL nanoparticle suspension/mL of cell suspension). Each addition was followed by manual mixing and incubation at 2–8 °C for 15, 15, and 5 min, respectively. Then a magnetic separation was carried out by placing the tube into Silver EasySep magnet (StemCell Technologies) for 5 min following by decantation of CD4+ cells.
CD4+ cells were suspended with X-Vivo 15 medium containing 50 μM β-mercaptoethanol at 106 cell/mL and placed in a tissue culture incubator at 37 °C overnight. The next day the compound plate was prepared by dispensing 1 μL/well of compound 66 dissolved in 100% DMSO at 2000-fold final concentrations in the assay or DMSO only for control wells. X-Vivo 15 medium was then added to the compound plate at 199 μL/well. CD4+ cells were activated with antimouse CD3 and antimouse CD28 and induced to differentiate into Th17 cells with a mixture of cytokines. The assay plate was prepared as follows. A cytokine cocktail at 10-fold final concentration was prepared by mixing the following reagents in X-Vivo 15 medium: human TGFβ (5 ng/mL), mouse IL-6 (20 ng/mL), mouse IL-1β (20 ng/mL), and mouse IL-23 (40 ng/mL). Then, 6.75 mL cytokine cocktail was mixed with 47.25 mL CD4+ cells (adjusted to a cell density of 7.1 × 105 cells/mL resulting in 50 000 cells/well in the assay), and the mixture was dispensed at 80 μL/well to a tissue culture 384-well plate. Then, Mouse T-Activator CD3/CD28 beads (5 × 106 beads/mL) were added at 10 μL/well. The assay plate was then incubated at 37 °C for 1 h in a tissue culture incubator. Various concentrations of compound 66 were transferred from the compound plate to the assay plate at 10 μL/well, and the assay plate was placed in a tissue culture incubator at 37 °C for 4 days. Determination of IL-17 in the supernatant was carried out by an electrochemiluminescence assay system from Meso Scale Diagnostics following the manufacturer’s protocol as described above. Cell viability in the assay plate was determined by CellTiter-Glo method (Promega) following the manufacturer’s protocol.

Gene Expression in Human Lymphocytes

Human CD4+ cells were prepared, differentiated and treated with compound 66 as described above. At the end of the 6-day incubation, cells were lysed and RNA purified with solid-phase extraction using RNeasy kit from Qiagen following the manufacturer’s protocol. Cells were mixed with 150 μL Buffer RLT in a 96 well plate, and then 150 μL 70% ethanol was added. The resulting cell lysate was transferred to a RNeasy 96 well plate that contains silica-based resin to which RNA binds. The plate was washed three times with Buffer RPE by centrifugation at 5600g for 4 min at room temperature. To elute RNA, 45–70 μL RNase-free water was added to each well followed by centrifugation at 5600g for 4 min at room temperature. RNA was stored at −70 °C. RNA concentration, and purity was assessed spectrophotometrically. Quantitative PCR (qPCR) was performed using TaqMan primer/probe chemistry. Reverse transcription was carried out in a thermocycler with 5 to 10 ng/μL RNA and buffer and enzyme mix from TaqMan Fast Cells kit in a final volume of 20 μL with two consecutive incubations at 37 and 95 °C for 60 and 5 min, respectively. PCR amplification was carried out using the Biomark HD platform (Fluidigm). Gene expression values were normalized to expression levels of housekeeping genes (RPLP0 and B2M) prior to calculating relative expression compared with DMSO vehicle.

Nuclear Receptor Selectivity

The activity of compound 66 on nuclear receptors in vitro was evaluated by a trans-FACTORIAL system (Attagene Inc.) which is a multiplexed cell-based assay technology that measures reporter RNA levels upon transfection of 48 chimeric constructs composed of nuclear receptor ligand binding domain and Gal4 DNA binding domain.

In Vivo Pharmacokinetic and Skin Inflammation Model

All procedures performed on animals were in accordance with regulations and established guidelines and were reviewed and approved by the Pfizer Institutional Animal Care and Use Committee.
For pharmacokinetic studies in rats, compound 66 was dosed to male Wistar Han rats as an IV bolus in 10% DMSO, 50% PEG400 and 40% water; orally by gavage as a suspension of crystalline solid in 0.5% methyl cellulose; or orally by gavage as a suspension of 25% spray dried dispersion (HPMCAS-H polymer) in 0.5% methyl cellulose, 0.5% HPMCAS-HF and 20 mM Tris pH 7.4 buffer. For pharmacokinetic studies in dogs, male Beagle dogs were dosed with an IV bolus of compound 66 in 10% DMSO, 40% PEG400, 30% glycerol formal, 1% N-methyl-2-pyrrolidone, and 19% phosphate buffered saline or orally by gavage as a suspension of 25% spray dried dispersion (HPMCAS-H polymer) in 0.5% methyl cellulose, 0.5% HPMCAS-HF, and 20 mM Tris pH 7.4 buffer. For pharmacokinetic studies in mice, female Balb/c mice were dosed with compound 66 as an IV bolus in 10% DMSO, 50% PEG400, and 40% water or orally by gavage as a suspension of 25% spray dried dispersion (HPMCAS-H polymer) in 0.5% methyl cellulose, 0.5% HPMCAS-HF, and 20 mM Tris pH 7.4 buffer. Blood was collected over a 24 h time course, and following centrifugation, the resulting plasma samples were precipitated with acetonitrile and analyzed for test compound concentration using an LC-MS/MS procedure. PK parameters were calculated from plasma concentration–time curves using noncompartmental analysis in Watson LIMS.
The imiquimod-induced skin inflammation model was performed with 8–10 week old female Balb/c mice, purchased from Taconic Farms, Germantown, NY. Commercially available 5% imiquimod cream (Sandoz, Princeton, NJ) was applied to the shaved back skin and left ear for three consecutive days (days 1–3). Mice were dosed with vehicle or compound 66 as a spray dried dispersion (25%, HPMCAS-H polymer) once daily by oral gavage in 0.5% methylcellulose, 0.5% HPMCAS-HF and 20 mM Tris pH 7.4 buffer for 5 days (days 1–5). Alternatively, mice were dosed by intraperitoneal injection with either anti-p40 Ab, anti-IL-17A Ab, or isotype control (20 mg/kg) on days 1 and 3. At the end of the study (day 5), ear thickness was measured in triplicate using an engineer’s micrometer (Mitutoyo, IL) as a means of assessing swelling and epidermal hyperplasia. Also, plasma samples were collected for exposure, and ear tissues samples were collected for IL-17A protein measurements. Ears were homogenized and protein was measured using the Pierce BCA protein kit (Thermo Fisher, Rockford, IL). Samples were normalized for protein (100 μg), and then murine IL-17A cytokine was measured with the R&D Quantikine ELISA kit.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00392.

  • Synthetic procedures and characterization data for intermediates 62bd and agonist used in TR-FRET dissociation rate assay; receptor expression, purification, and crystallography procedures; broad screening data for compound 3; and gene transcriptional analysis with compound 66 (PDF)

  • Molecule formula strings (CSV)

Accession Codes

PDB accession codes 6CN5 and 6CN6. Authors will release the atomic coordinates and experimental data upon article publication.

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Author
  • Authors
    • Mattias Wennerstål - Karo Bio AB (now Karo Pharma AB), 111 48 Stockholm, Sweden
    • Jennifer Alley - Inflammation and Immunology Research and , Pfizer Inc., Cambridge, Massachusetts 02139, United States
    • Martin Bengtsson - Karo Bio AB (now Karo Pharma AB), 111 48 Stockholm, Sweden
    • James R. Blinn - Worldwide Medicinal Chemistry and , Pfizer Inc., St. Louis, Missouri 63017, United States
    • Charles W. Bolten - Inflammation and Immunology Research and , Pfizer Inc., St. Louis, Missouri 63017, United States
    • Timothy Braden - Inflammation and Immunology Research and , Pfizer Inc., St. Louis, Missouri 63017, United States
    • Tomas Bonn - Karo Bio AB (now Karo Pharma AB), 111 48 Stockholm, Sweden
    • Bo Carlsson - Karo Bio AB (now Karo Pharma AB), 111 48 Stockholm, Sweden
    • Nicole Caspers - Medicine Design, Pfizer Inc., Groton, Connecticut 06340, United States
    • Ming Chen - Medicine Design, Pfizer Inc., Groton, Connecticut 06340, United States
    • Chulho Choi - Medicine Design, Pfizer Inc., Groton, Connecticut 06340, United States
    • Leon P. Collis - Inflammation and Immunology Research and , Pfizer Inc., Cambridge, Massachusetts 02139, United States
    • Kimberly Crouse - Inflammation and Immunology Research and , Pfizer Inc., Cambridge, Massachusetts 02139, United States
    • Mathias Färnegårdh - Karo Bio AB (now Karo Pharma AB), 111 48 Stockholm, Sweden
    • Kimberly F. Fennell - Medicine Design, Pfizer Inc., Groton, Connecticut 06340, United States
    • Susan Fish - Inflammation and Immunology Research and , Pfizer Inc., Cambridge, Massachusetts 02139, United States
    • Andrew C. Flick - Medicine Design, Pfizer Inc., Groton, Connecticut 06340, United States
    • Annika Goos-Nilsson - Karo Bio AB (now Karo Pharma AB), 111 48 Stockholm, Sweden
    • Hjalmar Gullberg - Karo Bio AB (now Karo Pharma AB), 111 48 Stockholm, Sweden
    • Peter K. Harris - Inflammation and Immunology Research and , Pfizer Inc., St. Louis, Missouri 63017, United States
    • Steven E. Heasley - Medicine Design, Pfizer Inc., Groton, Connecticut 06340, United States
    • Martin Hegen - Inflammation and Immunology Research and , Pfizer Inc., Cambridge, Massachusetts 02139, United States
    • Alexander E. Hromockyj - Inflammation and Immunology Research and , Pfizer Inc., St. Louis, Missouri 63017, United States
    • Xiao Hu - Inflammation and Immunology Research and , Pfizer Inc., St. Louis, Missouri 63017, United States
    • Bolette Husman - Karo Bio AB (now Karo Pharma AB), 111 48 Stockholm, Sweden
    • Tomasz Janosik - Karo Bio AB (now Karo Pharma AB), 111 48 Stockholm, Sweden
    • Peter Jones - Medicine Design and , Pfizer Inc., Cambridge, Massachusetts 02139, United States
    • Neelu Kaila - Medicine Design and , Pfizer Inc., Cambridge, Massachusetts 02139, United States
    • Elisabet Kallin - Karo Bio AB (now Karo Pharma AB), 111 48 Stockholm, Sweden
    • Björn Kauppi - Karo Bio AB (now Karo Pharma AB), 111 48 Stockholm, Sweden
    • James R. Kiefer - Worldwide Medicinal Chemistry and , Pfizer Inc., St. Louis, Missouri 63017, United States
    • John Knafels - Medicine Design, Pfizer Inc., Groton, Connecticut 06340, United States
    • Konrad Koehler - Karo Bio AB (now Karo Pharma AB), 111 48 Stockholm, Sweden
    • Lars Kruger - Karo Bio AB (now Karo Pharma AB), 111 48 Stockholm, Sweden
    • Ravi G. Kurumbail - Medicine Design, Pfizer Inc., Groton, Connecticut 06340, United States
    • Robert E. Kyne - Medicine Design, Pfizer Inc., Groton, Connecticut 06340, United States
    • Wei Li - Inflammation and Immunology Research and , Pfizer Inc., Cambridge, Massachusetts 02139, United States
    • Joakim Löfstedt - Karo Bio AB (now Karo Pharma AB), 111 48 Stockholm, Sweden
    • Scott A. Long - Worldwide Medicinal Chemistry and , Pfizer Inc., St. Louis, Missouri 63017, United States
    • Carol A. Menard - Medicine Design, Pfizer Inc., Groton, Connecticut 06340, United States
    • Scot Mente - Medicine Design and , Pfizer Inc., Cambridge, Massachusetts 02139, United States
    • Dean Messing - Medicine Design and , Pfizer Inc., Cambridge, Massachusetts 02139, United States
    • Marvin J. Meyers - Worldwide Medicinal Chemistry and , Pfizer Inc., St. Louis, Missouri 63017, United States
    • Lee Napierata - Inflammation and Immunology Research and , Pfizer Inc., Cambridge, Massachusetts 02139, United States
    • Daniel Nöteberg - Karo Bio AB (now Karo Pharma AB), 111 48 Stockholm, Sweden
    • Philippe Nuhant - Medicine Design, Pfizer Inc., Groton, Connecticut 06340, United StatesOrcidhttp://orcid.org/0000-0001-7816-7474
    • Matthew J. Pelc - Worldwide Medicinal Chemistry and , Pfizer Inc., St. Louis, Missouri 63017, United States
    • Michael J. Prinsen - Inflammation and Immunology Research and , Pfizer Inc., St. Louis, Missouri 63017, United States
    • Patrik Rhönnstad - Karo Bio AB (now Karo Pharma AB), 111 48 Stockholm, Sweden
    • Eva Backström-Rydin - Karo Bio AB (now Karo Pharma AB), 111 48 Stockholm, Sweden
    • Johnny Sandberg - Karo Bio AB (now Karo Pharma AB), 111 48 Stockholm, Sweden
    • Maria Sandström - Karo Bio AB (now Karo Pharma AB), 111 48 Stockholm, Sweden
    • Falgun Shah - Medicine Design and , Pfizer Inc., Cambridge, Massachusetts 02139, United States
    • Maria Sjöberg - Karo Bio AB (now Karo Pharma AB), 111 48 Stockholm, Sweden
    • Aron Sundell - Karo Bio AB (now Karo Pharma AB), 111 48 Stockholm, Sweden
    • Alexandria P. Taylor - Medicine Design, Pfizer Inc., Groton, Connecticut 06340, United States
    • Atli Thorarensen - Medicine Design and , Pfizer Inc., Cambridge, Massachusetts 02139, United States
    • John I. Trujillo - Medicine Design, Pfizer Inc., Groton, Connecticut 06340, United States
    • John D. Trzupek - Medicine Design and , Pfizer Inc., Cambridge, Massachusetts 02139, United States
    • Ray Unwalla - Medicine Design and , Pfizer Inc., Cambridge, Massachusetts 02139, United StatesOrcidhttp://orcid.org/0000-0002-5789-7336
    • Felix F. Vajdos - Medicine Design, Pfizer Inc., Groton, Connecticut 06340, United States
    • Robin A. Weinberg - Inflammation and Immunology Research and , Pfizer Inc., St. Louis, Missouri 63017, United States
    • David C. Wood - Inflammation and Immunology Research and , Pfizer Inc., St. Louis, Missouri 63017, United States
    • Li Xing - Medicine Design and , Pfizer Inc., Cambridge, Massachusetts 02139, United States
    • Edouard Zamaratski - Karo Bio AB (now Karo Pharma AB), 111 48 Stockholm, Sweden
    • Christoph W. Zapf - Medicine Design and , Pfizer Inc., Cambridge, Massachusetts 02139, United States
    • Yajuan Zhao - Inflammation and Immunology Research and , Pfizer Inc., Cambridge, Massachusetts 02139, United States
    • Anna Wilhelmsson - Karo Bio AB (now Karo Pharma AB), 111 48 Stockholm, Sweden
    • Gabriel Berstein - Inflammation and Immunology Research and , Pfizer Inc., Cambridge, Massachusetts 02139, United States
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We would like to thank Suvit Thaisrivongs, James Clark, and Johan Lund for their leadership of the collaboration; Philip McGurk, Mark Farmery, and Sergio Rotstein for collaboration operational support; Lisa Thomasco, Dale McLeod, Sandra Gordon, and Birgit Partola for compound management support; Karen Bois, Danielle Portelada, and Marina Shalaeva for log D measurements; Catherine Ambler for formulation support; Arthur Wittwer for screening guidance; and Xin Yang for coordinating pharmacokinetic studies. This research used resources at the Industrial Macromolecular Crystallography Association Collaborative Access Team (IMCA-CAT) beamline 17-ID, supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with Hauptman-Woodward Medical Research Institute, and resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.

Abbreviations Used

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ADME

absorption, distribution, metabolism, and excretion

AUC

area under the curve

CL

clearance

DIPEA

N,N-diisopropylethylamine

DMAP

4-(dimethylamino)pyridine

DMF

N,N-dimethylformamide

DMSO

dimethyl sulfoxide

ESP

electrostatic potential

F

fraction absorbed

Fu

fraction unbound

HATU

1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

HEK

human embryonic kidney

HLM

human liver microsomes

HOBT

1-hydroxybenzotriazole

HPMCAS

hydroxypropyl methylcellulose acetate succinate

IL-17

interleukin-17

LBD

ligand binding domain

LIPE

lipophilic efficiency

NMP

N-methyl-2-pyrrolidone

ROR

retinoic acid receptor-related orphan receptor

SDD

spray-dried dispersion

SPA

scintillation proximity assay

T1/2

half-life

TBTU

O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate

Th17

T helper 17

THF

tetrahydrofuran

TR-FRET

time-resolved fluorescence resonance energy transfer

Vdss

volume of distribution at steady state

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  • Abstract

    Scheme 1

    Scheme 1. Synthesis of Screening Hit 3 and 1-Methylindole Analogue 4a

    aReagents and conditions: (a) DIPEA, TBTU, 3-cyanobenzoic acid, DMF; (b) HCl, dioxane; (c) DIPEA, cyclopentane carbonyl chloride, DMF; (d) NaH, THF; CH3I.

    Scheme 2

    Scheme 2. Representative Approach to 1-Methylindole Analogues, Including 7, 8a, and 8ba

    aReagents and conditions: (a) benzoyl chloride or 4-cyanopicolinoyl chloride, triethylamine, CH2Cl2; (b) HCl, dioxane, MeOH; (c) cyclopentanecarbonyl chloride, triethylamine, CH2Cl2; or DIPEA, TBTU, DMF, cyclohexanecarboxylic acid, or cyclopentanecarboxylic acid, 40 °C.

    Scheme 3

    Scheme 3. Synthesis of 1-Ethylindole Analogue 11a

    aReagents and conditions: (a) 4-cyanopicolinoyl chloride, triethylamine, CH2Cl2; (b) NaOH, EtI, Aliquat 336, CH2Cl2; (c) HCl, dioxane; (d) cyclopentanecarbonyl chloride, triethylamine, CH2Cl2.

    Scheme 4

    Scheme 4. Synthesis of Pyrrolo[2,3-b]pyridine Analogues 17 and 18a

    aReagents and conditions: (a) KOH, I2, DMF; K2CO3, CH3I; (b) 12, PdCl2(PPh3)2, K2CO3, DME, EtOH, H2O, 120 °C; (c) NH4HCO2, Pd, THF:NMP (9:1), 150 °C; (d) pyridine, CH2Cl2; 3-cyanobenzoyl chloride or 4-cyanopicolinoyl chloride.

    Scheme 5

    Scheme 5. Synthesis of Representative Pyrrolo[3,2-b]pyridine, Pyrrolo[2,3-c]pyridine, and Pyrrolo[3,2-d]pyrimidine Scaffold Analoguesa

    aReagents and conditions: (a) N-iodosuccinimide, DMF; (b) Bu4NBr, NaOH, CH3I, CH2Cl2; (c) 12, (PPh3)4Pd, K2CO3, DMF:H2O (8:1), 100 °C; (d) NH4HCO2, Pd/C, DMF:NMP (5:1), 150 °C; (e) triethylamine, 4-cyanopicolinoyl chloride, CH2Cl2; (f) NaHCO3, Pd/C, H2, EtOH; (g) 12, PdCl2(PPh3)2, K2CO3, DMF:EtOH:H2O (4:1:1), 120 °C; (h) Pd(OAc)2, BINAP, dioxane; Ph2CNH, NaOt-Bu, 140 °C; (i) NH4HCO2, Pd/C, THF:NMP (5:1), 150 °C.

    Scheme 6

    Scheme 6. Synthesis of 1,4-Dimethylindole Analogue 36a

    aReagents and conditions: (a) KOH, I2, DMF; K2CO3, CH3I; (b) 12, Pd EnCat TPP30, K2CO3, DME:EtOH:H2O (4:1:1), 70 °C; (c) NH4HCO2, Pd/C, EtOH, 85 °C; d) triethylamine, 3-cyanobenzoyl chloride, CH2Cl2.

    Scheme 7

    Scheme 7. Synthesis of 5-Methylpyrrolo[2,3-b]pyridine Scaffold Intermediate 43a

    aReagents and conditions: (a) CH3MgBr, Pd(dppf)Cl2, toluene, 80 °C; (b) Bu4NBr, KOH, CH2Cl2; PhSO2Cl; (c) tetramethylammonium nitrate, (CF3CO)2O, CH2Cl2; (d) K2CO3, morpholine, MeOH, 65 °C; (e) K2CO3, CH3I, DMF; (f) N-iodosuccinimide, DMF.

    Scheme 8

    Scheme 8. Synthesis of 5-Isopropylpyrrolo[2,3-b]pyridine Scaffold Intermediate 49a

    aReagents and conditions: (a) 4,4,5,5-tetramethyl-2-(prop-1-en-2-yl)-1,3,2-dioxaborolane, Pd(PPh3)4, K2CO3, DMF:H2O (10/1), 120 °C; (b) Pd/C, H2, EtOH; c) tetrabutylammonium nitrate, (CF3CO)2O, CH2Cl2, 0 °C; (d) NaOH, THF; (e) KOH, I2, DMF; K2CO3, CH3I.

    Scheme 9

    Scheme 9. Synthesis of 5-Methoxypyrrolo[2,3-b]pyridine Scaffold Intermediate 53a

    aReagents and conditions: (a) tetramethylammonium nitrate, (CF3CO)2O, CH2Cl2; (b) NaOMe, MeOH, reflux; (c) NaH, CH3I, DMF; (d) N-iodosuccinimide, DMF.

    Scheme 10

    Scheme 10. Synthesis of 5-Trifluoromethylpyrrolo[2,3-b]pyridine Scaffold Intermediate 58a

    aReagents and conditions: (a) CH3C(O)Cl, NaI, CH3CN, 100 °C; NaOH, MeOH; (b) (Bu4N)2SO4, NaOH, CH2Cl2, 0 °C; PhSO2Cl; (c) tetramethylammonium nitrate, (CF3CO)2O, CH2Cl2; (d) methyl 2,2-difluoro-2-(fluorosulfonyl)acetate, CuI, DMF, 100 °C; (e) KOH, I2, 2-MeTHF:EtOH (2:1); K2CO3, CH3I.

    Scheme 11

    Scheme 11. Synthesis of 5-Substituted Pyrrolo[2,3-b]pyridine Analogues 63al and 6466a

    aReagents and conditions: (a) tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate, K2CO3, Pd(PPh3)4, DME:EtOH:H2O, 80 °C; (b) (X = Me) triethylamine, Pd/C, H2, MeOH; (X = iPr) NH4HCO2, Pd/C, EtOH; or (X = OCH3, CF3) Pd(OH)2, H2, MeOH; (c) 3-cyanobenzoyl chloride or 3-cyanobenzoic acid, various conditions; (d) CF3CO2H, CH2Cl2; or HCl, dioxane; (e) RCO2H or RC(O)Cl, various conditions.

    Scheme 12

    Scheme 12. Synthesis of 5-Methylpyrrolo[2,3-b]pyridine Analogues 69ada

    aReagents and conditions: (a) HCl, dioxane, MeOH; (b) triethylamine, isobutyryl chloride, CH2Cl2; (c) triethylamine, Pd/C, H2, MeOH; (d) substituted 3-cyanobenzoic acid, DIPEA, HATU, CH2Cl2.

    Figure 1

    Figure 1. RORC2 active conformation His479–Tyr502–Phe506 triplet latch (3KYT). (37)

    Figure 2

    Figure 2. RORC2 LDB cocrystal structure with compound 8b (6CN5). Representation of ligand binding site with respect to receptor structure (H12 disordered and not shown).

    Figure 3

    Figure 3. RORC2 LDB cocrystal structure with compound 8b (6CN5). (a) Representation of piperidine amide portion of ligand binding site: residues in gray depict cocrystal structure with compound 8b, and residues in yellow depict RORC2 active conformation (3KYT). (b) Representation of methylindole binding pocket with receptor surface displayed. (c) Representation of benzamide indole portion of ligand binding site.

    Figure 4

    Figure 4. Calculated torsional energy profile (B3LYP density functional, 6-31G* basis set) (41) for model systems representing rotation of (a) the 5-benzamide substituent (C4–C5–N–C dihedral) and (b) the 3-piperidine substituent (C2–C3–C–H dihedral) to the indole ring. R substituent is hydrogen (blue), methyl (green), trifluoromethyl (red), methoxy (orange), or isopropyl (brown). Dihedral angle observed in bound conformation represented by dashed line.

    Figure 5

    Figure 5. Overlay of bound conformation for compound 8b (orange) with cocrystal structure of RORC2 LBD with compound 36 (green with gray residues) (6CN6).

    Figure 6

    Figure 6. Effect of compound 66 (n = 3 replicates) against 48 nuclear receptors in HepG2 cells at 1 μM concentration and 24 h time point. HepG2 cells were transiently transfected with optimized trans-FACTORIAL library. Post-transfection (24 h), cells were washed and supplied with fresh low serum (1% FBS, charcoal stripped) culture medium and treated with inducer for 24 h. Profile of the trans-FACTORIAL activities was determined as fold of induction values versus vehicle-treated (DMSO) control cells. Graph shows average fold-induction data plotted in logarithmic scale.

    Figure 7

    Figure 7. Unbound plasma concentrations of compound 66 following last dosing in the mouse imiquimod-induced skin inflammation model. Compound dosed at 10 mg/kg (blue), 30 mg/kg (green), and 100 mg/kg (red) once a day by oral gavage. For reference, the respective mouse IL-17 IC50 and IC80 (4 × IC50) adjusted for fraction unbound in the in vitro assay media (Fu = 0.62) are depicted (IC50 = 20 nM, IC80 = 80 nM).

    Figure 8

    Figure 8. End of study magnitude of ear swelling (Δ μm) (a) and protein levels of IL-17A (pg/mL) (b) in the ear following mouse imiquimod-induced skin inflammation model when treated with compound 66, anti-IL-17 Ab (20 mg/kg), or anti-p40 Ab (20 mg/kg) (mean ±95% CI).

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00392.

    • Synthetic procedures and characterization data for intermediates 62bd and agonist used in TR-FRET dissociation rate assay; receptor expression, purification, and crystallography procedures; broad screening data for compound 3; and gene transcriptional analysis with compound 66 (PDF)

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