Identification of C-2 Hydroxyethyl Imidazopyrrolopyridines as Potent

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Identification of C‑2 Hydroxyethyl Imidazopyrrolopyridines as Potent JAK1 Inhibitors with Favorable Physicochemical Properties and High Selectivity over JAK2 Mark Zak,*,† Christopher A. Hurley,¶ Stuart I. Ward,¶ Philippe Bergeron,† Kathy Barrett,‡ Mercedesz Balazs,◆ Wade S. Blair,‡ Richard Bull,¶ Paroma Chakravarty,# Christine Chang,‡ Peter Crackett,¶ Gauri Deshmukh,§ Jason DeVoss,◆ Peter S. Dragovich,† Charles Eigenbrot,○ Charles Ellwood,¶ Simon Gaines,¶ Nico Ghilardi,∥ Paul Gibbons,† Stefan Gradl,† Peter Gribling,◆ Chris Hamman,† Eric Harstad,⊥ Peter Hewitt,¶ Adam Johnson,‡ Tony Johnson,¶ Jane R. Kenny,§ Michael F. T. Koehler,† Pawan Bir Kohli,‡ Sharada Labadie,† Wyne P. Lee,◆ Jiangpeng Liao,+ Marya Liimatta,‡ Rohan Mendonca,† Raman Narukulla,¶ Rebecca Pulk,† Austin Reeve,¶ Scott Savage,▽ Steven Shia,○ Micah Steffek,○ Savita Ubhayakar,§ Anne van Abbema,‡ Ignacio Aliagas,† Barbara Avitabile-Woo,¶ Yisong Xiao,+ Jing Yang,+ and Janusz J. Kulagowski¶ †

Departments of Discovery Chemistry, ‡Biochemical and Cellular Pharmacology, §Drug Metabolism and Pharmacokinetics, Immunology, ⊥Safety Assessment, #Small Molecule Pharmaceutical Sciences, ▽Small Molecule Process Chemistry, ○Structural Biology, ◆Translational Immunology, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States ¶ Argenta, 8/9 Spire Green Centre, Flex Meadow, Harlow, Essex CM19 5TR, United Kingdom + WuXi AppTec Co., Ltd., 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, P. R. China ∥

S Supporting Information *

ABSTRACT: Herein we report on the structure-based discovery of a C-2 hydroxyethyl moiety which provided consistently high levels of selectivity for JAK1 over JAK2 to the imidazopyrrolopyridine series of JAK1 inhibitors. X-ray structures of a C-2 hydroxyethyl analogue in complex with both JAK1 and JAK2 revealed differential ligand/protein interactions between the two isoforms and offered an explanation for the observed selectivity. Analysis of historical data from related molecules was used to develop a set of physicochemical compound design parameters to impart desirable properties such as acceptable membrane permeability, potent whole blood activity, and a high degree of metabolic stability. This work culminated in the identification of a highly JAK1 selective compound (31) exhibiting favorable oral bioavailability across a range of preclinical species and robust efficacy in a rat CIA model.

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INTRODUCTION The Janus kinases (JAK1, JAK2, JAK3, and TYK2) are intracellular protein tyrosine kinases with essential roles in immune function,1 inflammation,2 and hematopoiesis.3 Targeted inhibition of the JAKs has shown promise against associated hematopoietic and immunologic diseases, as evidenced by recent approvals of the multi-JAK inhibitors (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)3-cyclopentylpropanenitrile (INCB018424)4 and 1 (CP690,550)5 (see Table 1) for treatment of myelofibrosis and rheumatoid arthritis (RA), respectively.6 Despite the clinical success of these agents, there may be benefit in identifying inhibitors with greater specificity for a single JAK isoform, particularly for immunologic diseases such as RA. As previously described, we believe selective JAK1 inhibition will be sufficient © XXXX American Chemical Society

to treat RA while minimizing JAK2 inhibition could improve the therapeutic index by avoiding anemia.7 As such, we wished to identify potent and orally bioavailable small molecule inhibitors of JAK1, with selectivity over JAK2, as potential therapies for RA and other immunologic disorders.8,9 Our group has previously reported on the discovery and optimization of the imidazopyrrolopyridine series of JAK1 inhibitors, including the observation that a methyl group at the C-2 position may lead to improvements in JAK1 selectivity over JAK2.7 One particularly striking example of the C-2 methyl effect is exemplified by the matched pair comparison of compounds 2 and 3 (Figure 1a). As depicted in Figure 1b, the Received: April 4, 2013

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dx.doi.org/10.1021/jm4004895 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Figure 1. (a) Matched pair analysis of C-2 H (2) and C-2 Me (3) imidazopyrrolopyridines. (b) X-ray structure of 3 in complex with JAK1 (gray, PDB 4EHZ) overlaid with a key residue difference with JAK2 (green, D939 from structure 4F09). Superscript letters indicate the following: (a) arithmetic mean of at least three separate runs (n ≥ 3); (b) the average coefficient of variation was less than 0.3 times the mean for biochemical assays;13 (c) JAK2 Ki/JAK1 Ki; (d) apparent permeability in MDCK transwell culture. A:B, apical-to-basolateral.

however, offered a dramatic breakthrough in that it exhibited greater JAK1 selectivity than compound 3 while maintaining biochemical and cell-based potency equivalent to 1. Further branching to give tertiary alcohol 9 was poorly tolerated from a potency perspective, while homologation of the secondary alcohol to produce compounds 10 and 11 led to no improvement relative to parent compounds 7 and 8. Finally, chiral diols 12 and 13 were found to maintain potency but lose selectivity compared to 7 and 8. Excited by the highly ligand efficient and predictably selective nature of the C-2 substituted hydroxyls, we evaluated the most promising analogues in additional assays. The effect of plasma proteins and other blood components on cell-based JAK1 inhibition was assessed by comparing the potencies of multiple compounds in the standard low serum cell-based assay (IL-6 pSTAT3) to the potencies in human whole blood.13 As shown in Table 2, compound 1 maintained potent JAK1 cell-based inhibition in human whole blood, with no potency shift between the two assay formats. By contrast, several of the other compounds tested were much less potent in human whole blood, with compounds 6 and 11 notably shifting by greater than 10-fold. Examination of the limited data set in Table 2 indicated that there was a relationship between cLogD7.414 and the magnitude of potency loss in human whole blood for the imidazopyrrolopyridine series. The more lipophilic analogues 6, 8, and 11 showed larger shifts than the less lipophilic analogues 5, and 13. Additional relationships between the physical or structural properties of compounds and experimental outcomes in relevant assays are also noted in Table 2. MDCK permeability was very good for the monohydroxylated compounds but dropped slightly for the diol 13, consistent with that compound’s significant increase in TPSA.15 All compounds were found to show good to moderate in vitro stability in the presence of human liver microsomes (HLMs), however stability in human hepatocytes varied widely.16 A relationship was noted between the degree of branching surrounding the alcohol and human hepatocyte stability. The primary alcohols 5 and 6 were found to be highly unstable in hepatocytes, while the secondary and tertiary alcohols 8 and 9 exhibited moderate and very good stability, respectively. We believe the contrasting hepatocyte stability was related to the alcohols’ abilities to form phase two conjugates. Indeed, hepatocyte metabolism identification studies on analogues related to primary alcohol 5 and secondary alcohol 8 revealed significant glucuronidation of the

amplification of JAK1 selectivity may potentially be explained by the differential proximity of the methyl group in compound 3 to a key residue difference between JAK1 (E966, 3.4 Å) and JAK2 (D939, 5.1 Å). Despite this intriguing observation, the C2 methyl group did not lead to consistently high levels of JAK1 selectivity. The nature of the group extending beneath the Ploop also played an important, yet poorly understood role, and groups other than the 4-piperidine present in 3 generally reduced JAK1 selectivity. Unfortunately, molecules containing the selectivity-enhancing 4-piperidine group possessed characteristics not compatible with orally administered therapeutics, including relatively weak target potency and poor membrane permeability. As part of our continued investigation of the imidazopyrrolopyridine series, we wished to discover molecules with JAK1 selectivity equal to or greater than 3 with improved MDCK permeability and JAK1 potency.

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RESULTS AND DISCUSSION With the goal of achieving greater and more consistent selectivity for JAK1 over JAK2, we prepared a series of C-2 substituted alcohols (5−13, Table 1) designed to engage in differential H-bonding interactions between E966 (JAK1) and D939 (JAK2). Alcohols were chosen due to their modest impact on parameters such as molecular weight, TPSA, and basicity relative to other potential H-bonding groups. We believed these attributes would allow for molecules to contain a broad spectrum of moieties extending beneath the P-loop while maintaining physicochemical parameters consistent with orally dosed therapeutics.10 The initial SAR exploration of C-2 alcohols was carried out in combination with a cyclohexyl Ploop group due to improved potency and MDCK permeability relative to corresponding 4-piperidine analogues. The marketed drug 1 and the C-2 methyl imidazopyrrolopyridine 47 are also included in Table 1 for comparison. Compound 4 exhibited levels of JAK1 potency and ligand efficiency11,12 comparable to 1, yet only modest improvement to JAK1 vs JAK2 selectivity in both biochemical and cell-based assays. Gratifyingly, the hydroxylated analogue 5 maintained equivalent potency with improved JAK1 selectivity, supporting the hypothesis that a hydrogen bond donor at the C-2 position could better exploit the E966−D939 residue difference. The straight chain homologated analogue 6 was slightly less potent and selective, while the (S)-isomer (7) of the branched homologue subtly improved JAK1 selectivity relative to 5 but suffered from notably reduced potency. The corresponding (R)-isomer (8), B



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Table 1. JAK1 Potency and Selectivity of 1 and C-2 Substituted Imidazopyrrolopyridines 4−13

a Ligand efficiency11 = (−1.4log Ki)/(n heavy atoms). bLigand-lipophilicity efficiency12 = (−log Ki) − (cLogP). cArithmetic mean of at least three separate runs (n ≥ 3).13 dThe average coefficient of variation was less than 0.3 times the mean for biochemical assays. eJAK2 Ki/JAK1 Ki. fJAK1 cellbased assay.13 gThe average coefficient of variation was less than 0.5 times the mean for cell-based assays. hJAK2 cell-based assay.13 i(EPO pSTAT5 EC50)/(IL-6 pSTAT3 EC50). jNot applicable. kSingle enantiomers. Known stereochemistry. lSingle enantiomers. Stereochemistry inferred from potency relationships between 7 and 8.

Table 2. Additional Data for 1 and C-2 Substituted Imidazopyrrolopyridines ex

IL-6 pSTAT3 EC50 (nM)a,b

whole blood EC50 (nM)a,c

whole blood shiftd

1 5 6 8 9 11 13

53 64 130 47 330 84 69

43 313 1500 330

0.8 4.9 12 7.0

1600 220

19 3.2

cLogD7.4e

TPSA (Å2)f

MDCK Papp A:B (× 10−6 cm/s)g

1.5 1.7 2.1 2.1 2.6 2.6 0.9

88 66 66 66 66 66 86

24 23 23 19 22 16

HLM, HHep CLH (mL min−1 kg−1)h 5.6, 5.3, 4.5, 5.8, 7.6, 1.8,

19 17 11 1.7 12 6.8

a Arithmetic mean of at least three separate runs (n ≥ 3). bJAK1 cell-based assay.13 The average coefficient of variation was less than 0.5 times the mean for cell-based assays. cJAK1 cell-based assay (IL-6 pSTAT3) run in the presence of whole blood.13 d(whole blood EC50)/(IL-6 pSTAT3 EC50). e See ref 14. fSee ref 15. gApparent permeability in MDCK transwell culture. A:B, apical-to-basolateral. Mean of at least two separate runs (n ≥ 2). h Hepatic clearance in the presence of human liver microsomes and human hepatocytes.16 Mean of at least two separate runs (n ≥ 2).

C



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Figure 2. Defining the ideal calculated property space of C-2 H and Me imidazopyrrolopyridines.a All compounds analyzed were synthesized prior to the imidazopyrrolopyridines in Tables 1, 2, and 4. Superscript letters indicate the following: (a) compound structures and tabulated data are listed in the Supporting Information; (b) (whole blood EC50)/(IL-6 pSTAT3 EC50);13 (c) see ref 14; (d) oral bioavailability in the rat; (e) apparent permeability in MDCK transwell culture; A:B, apical-to-basolateral; (f) in vivo clearance in the rat after IV administration; (g) see ref 15; (h) experimental hepatic clearance in the presence of human liver microsomes;16 (i) prospectively calculated probability of being stable in the presence of human liver microsomes.17

former but minimal glucuronidation of the latter (data not shown). Although greater branching led to better agreement between the two in vitro metabolic stability assay formats, no compound became significantly more stable in the presence of human hepatocytes than in the presence of liver microsomes. Therefore, a high degree of HLM stability was desired as a prerequisite to acceptable human hepatocyte stability. The emerging trends noted in the limited data set presented in Table 2 prompted us to perform more extensive properties analyses on existing compounds to ascertain whether an ideal range of calculated properties could be identified to balance low whole blood shift with acceptable membrane permeability and HLM stability. We have previously reported on C-2 H and methyl substituted imidazopyrrolopyridine compounds (Figure 2, R = H or Me),7,9 many of which were synthesized prior to initiation of the current work. As shown in Figure 2a, analysis of this historical data set revealed that compounds with cLogD7.4 greater than 2 possessed a high probability of exhibiting large whole blood shifts (>5×), whereas compounds with cLogD7.4

50%) or moderate (30−50%) bioavailability categories, while compounds possessing MDCK Papp (A:B) below than that value were often found to cluster in the low bioavailability class (%F < 30%). TPSA and cLogD7.4 were found to be useful computational predictors of acceptable MDCK permeability. Figure 2c shows that compounds with TPSA values of >90 Å2 or cLogD7.4 3 × 10−6 cm/s) than those with TPSA 0.5. As expected, high in vivo clearance could also erode oral bioavailability. Indeed, the compound in the bottom right quadrant of Figure 2b possessed extremely good MDCK D



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unlikely. As we have previously reported,7,9 many N-substituted 4-piperidine P-loop groups exhibited high levels of JAK1 potency in the imidazopyrrolopyridine series and, given the ease of synthesis, appeared to offer an attractive means to tune physicochemical properties. Unfortunately, we found it difficult to identify substituted 4-piperidines with acceptable profiles when combined with the C-2 (R)-hydroxyethyl selectivity group. For example, compound 14 (Table 4) fell within the desired calculated properties ranges but suffered from a large whole blood shift. A related analogue (15) possessed reasonable JAK1 potency in the biochemical and whole blood assays but fell outside the ideal cLogD7.4 and TPSA ranges and suffered from low MDCK permeability. Although multiple other N-substituted 4-piperidine compounds were synthesized (data not shown), none balanced acceptable JAK1 potency with low whole blood shift and acceptable in vitro and in vivo DMPK properties. The much more synthetically complex gemdifluoro piperidine 16 was somewhat more promising in balancing reasonable JAK1 potency and selectivity with the other experimental parameters listed in Table 4. N-substituted 3-piperidine groups were not successful (e.g., 17 and 18), and similar to the 4-piperidine isomers, it was not possible to balance potency with the various other calculated and experimental parameters. Tetrahydropyran (THP) P-loop groups possessed ideal calculated properties, however, the 4regioisomer 19 and the (R)-epimer of the 3-regioisomer (20) were disappointingly weak biochemical JAK1 inhibitors. In contrast, the (S)-epimer of the 3-THP regioisomer (21) was far more biochemically active, as well as being highly selective for JAK1 over JAK2 (>25× and >50× in biochemical and cellbased assays, respectively) and possessing good MDCK permeability and excellent metabolic stability. In addition, a very low whole blood shift allowed 21 to be the first C-2 hydroxyethyl compound to exhibit a whole blood JAK1 cellular EC50 of 15× biochemical, >25× cell-based) and excellent metabolic stability with very potent whole blood JAK1 inhibition, comparable to that of 1 (Table 2). The 2,2-difluoroethyl analogue 27 was also highly potent and even more selective than 26, but it suffered from reduced MDCK permeability. The cis 1,4-diamino cyclohexane 28 suffered from reduced potency and decreased metabolic stability relative to the trans version 26. Unlike the matched pair of cis and trans cyclohexanes 26 and 28, the corresponding cyclopentanes 29 and 30 exhibited similar

permeability yet exhibited low bioavailability (presumably due to very high in vivo CL). In an effort to control in vivo CL, we wished to identify compounds with a high degree of metabolic stability in the presence of HLMs. We evaluated the ability of a computational model trained on a set of experimental HLM data to prospectively predict the probability of HLM stability of new compounds in the imidazopyrrolopyridine series.17 Figure 2d shows the correlation between the model’s predicted probability of HLM stability and the experimental CLH values obtained by incubating compounds with HLMs. Indeed, the model indicated that imidazopyrrolopyridine compounds with calculated probabilities 15 mL min−1 kg−1) or high− moderate (10−15 mL min−1 kg−1) HLM CLH. By contrast, calculated probabilities of >0.5 resulted in an enriched proportion of molecules with good metabolic stability, i.e., low (0.5

balance MDCK Pappd with whole blood shifte MDCK Pappd human liver microsome stabilityf

a

See ref 14. bSee ref 15. cProspectively calculated probability of being stable in the presence of human liver microsomes.17 dApparent permeability in MDCK transwell culture; A:B, apical-to-basolateral. e (whole blood EC50)/(IL-6 pSTAT3 EC50).13 fExperimental in vivo stability in the presence of human liver microsomes.

cLogD7.4 range of 0.5−2.0 was desirable. Compounds with lower values often suffered from poor MDCK permeability, whereas analogues with higher values typically exhibited large whole blood potency shifts. An additional determinant of MDCK permeability was TPSA, with values of >90 Å2 often associated with unacceptable permeability. Finally, compounds with calculated HLM stability probability 35× biochemical, >60× cell-based). The corresponding cis isomer 32 was slightly more potent, although at the cost of reduced JAK1 selectivity. The singly and doubly homologated trans isomers 33 and 34 were extremely potent in all biochemical and cell-based measures of the JAK1 inhibition (including in human whole blood), although JAK1 selectivity was reduced relative to 31. One final note on the inhibitors in Table 4 is that a high degree of ligand efficiency was maintained throughout the optimization effort, with the majority of sub-5 nM Ki compounds maintaining LE ≥ 0.45 and LLE ≥ 7.0. As shown in Figure 3, X-ray crystal structures of the highly JAK1 selective compound 31 were generated in complex with

both JAK1 and JAK2. Compound 31 adopts broadly similar binding modes in the presence of each JAK isoform, with the pyrrolopyridine moiety contacting the hinge residues as expected, and the free lone pair of the imidazole ring interacting with a crystallographic water. Also similar between the two structures, the cyano group of the ligand does not interact directly with the P-loop but rather extends beneath it and likely participates in polar interactions with nearby crystallographic waters. An important difference between the two structures is observed in the region surrounding the key JAK1 (E966) vs JAK2 (D939) residue difference targeted by the C-2 hydroxy moiety. In the case of JAK1, the C-2 hydroxyethyl group forms a favorable direct H-bonding interaction with E966 at an optimal distance of 2.8 Å. The distance between the ligand and the corresponding residue in JAK2 (D939) is 4.3 Å, too far for a direct interaction. To satisfy the H-bonding partners of the ligand and D939, a bridging, and presumably entropically disfavored, water molecule is incorporated into the inhibitor− protein complex. We believe the suboptimal interaction between the C-2 hydroxyethyl group of the ligand and D939 of the protein produces the observed reduction in JAK2 potency and, hence, the excellent JAK1 selectivity. Although the physicochemical properties of compound 31 allowed it to exhibit greater whole blood potency than the G



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Figure 4. (a−c) X-ray structures of compounds 33, 34, and 1, respectively, in complex with JAK1. PDB codes and resolutions are as follows: 4IVC, 2.4 Å (a); 4IVD, 1.9 Å (b); 3EYG,18 1.9 Å (c). A key residue difference between JAK1 (E966) and JAK2 (D939) is highlighted in green. Notable crystallographic waters are highlighted as red spheres. Hydrogen-bonding interactions are denoted as dashed lines. (d) Superposition of the ligands from the X-ray structures of 1,18 31, 33, and 34 in complex with JAK1.

In vivo DMPK and associated data for notable C-2 hydroxyethyl compounds are depicted in Table 5. All compounds possessed uniformly high levels of pH 2 solubility, while pH 7.4 solubility was more variable and appeared to be influenced by lipophilicity. Indeed, the compounds with the lowest cLogD7.4 values (21 and 31) possessed the best pH 7.4 solubility. Additionally, all compounds tested possessed low to moderate plasma protein binding across species. Similar to human hepatocytes, the in vitro rat and dog hepatocyte CLH values were found to be in the low to moderate range. The compounds were generally less stable in the presence of monkey hepatocytes, with typical in vitro CLH values in the moderate to high range. The in vivo PK profiles of the tricyclic compounds generally compared favorably with 1. Moderate to high IV clearance was observed in the rat, yet oral exposure was better than expected, with the ratio of experimental bioavailability (F) to maximum theoretical bioavailability (Fmax),20 typically larger than one. In vivo dog clearance was typically moderate, although compounds 21 and 34 fell into the low and high ranges, respectively. Similar to the rat, dog oral exposure was generally greater than would be expected on the basis of IV clearance (i.e., the ratio of F to Fmax was typically >1). A definitive explanation for the unexpectedly good oral exposure in rat and dog has not been established to date. The compounds profiled in monkey PK exhibited moderate IV clearance and lower F/Fmax ratios than in other species. Indeed, the oral bioavailabilities of compounds 21 and 31 were very

unsubstituted cyclohexane 8, the cyano group was found to be detrimental to intrinsic JAK1 biochemical potency (JAK1 Ki: 31 = 1.9 nM, 8 = 0.8 nM). By comparison, both the singly and doubly homologated nitriles 33 and 34 exhibited improved JAK1 biochemical inhibition relative to the unsubstituted cyclohexane 8. Crystal structures of 33 and 34 in complex with JAK1 were generated (Figure 4a,b) and compared to those of both 31 (Figure 3a) and the published structure of 1 (Figure 4c).18 All structures revealed very similar interactions with the hinge region of JAK1, with the imidazopyrrolopyridine (31, 33, 34) or pyrrolopyrimidine (1) cores contacting E957 and L959 and interacting with a crystallographic water molecule. The interaction between the hydroxyethyl moieties of 31, 33, and 34 and E966 were likewise found to be very similar. However, the overlay of the four JAK1 structures revealed a notable difference between the highly potent biochemical inhibitors (1, 33, 34) and the less potent inhibitor 31 (Figure 4d). The favorable interactions between the nitrile of 1 and the P-loop have previously been described.18,19 Nitriles 33 and 34 adopt conformations comparable to 1, extending up toward the Ploop and likely participating in similar favorable interactions. In contrast, and as noted above, the nitrile of 31 does not interact directly with the P-loop, but instead extends in an approximately perpendicular direction below it. This difference in nitrile placement may explain the reduced biochemical potency of 31 relative to 1, 33, and 34. H



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Table 5. In Vivo PK Data for 1 and Selected C-2 (R)-Hydroxyethyl Imidazopyrrolopyridines a

Ex

aq sol, pH 2, pH 7.4

MDCK Papp A:B, B:A (× 10−6 cm/s)b

1j

speciesc

PPBd (%)

Hep CLHe (mL min−1 kg−1)

R D M

CLf (mL min−1 kg−1)

T1/2f (h)

AUCg (μM·h)

CMaxg (μM)

Fh (%)

Fmaxi (%)

F/Fmax

62 19 18

0.6 1.2 2.1

0.58 1.9 2.0

0.28 1.3 1.0

27 78 48

11 39 59

2.5 2.0 0.8

21

>4000, >4000

7.6, 8.6

R D M

39 61 54

2.3 4.9 21

22 2.1 19

1.5 5.7 1.1

10.7 65.7 4.3

5.2 9.8 1.8

197 111 70

69 93 56

2.9 1.2 1.3

26

3900, 130

6.7, 10

R D M

56 55 65

11 2.3 26

36 13

0.5 1.7

1.4 9.9

0.96 2.1

54 139

49 58

1.1 2.4

29

>4000, 70

10, 18

R D M

49 59 56

14 9.8 27

55 14

1.1 1.2

1.0 9.0

0.98 3.0

61 131

29 55

2.1 2.4

31

>4000, 1900

2.7, 4.7

R D M

38 47 60

3.7 3 2.4

60 16 18

0.5 2.2 1.7

0.94 8.3 3.9

0.42 2.1 0.63

52 119 65

14 48 59

3.7 2.5 1.1

33

>4000, 130

3.5, 4.9

R D M

63 63 70

15 12 25

38 17

0.5 1.8

0.93 2.5

49 183

54 45

0.9 4.1

34

>4000, 16

4.7, 9.3

R D M

68 66 76

28 11 36

126 29 29

0.6 1.3 0.8

0.22 1.3 0.07

50 125 8

0 6 33

20 0.2

1.3 11.2

0.42 4.3 0.26

Thermodynamic solubility (reported in μM) of solid powder in aqueous buffer. bApparent permeability in MDCK transwell culture. A:B = apicalto-basolateral, B:A = basolateral-to-apical. Mean of at least two separate runs (n ≥ 2). cAbbreviations as follows: R = rat, D = dog, M = monkey. d Plasma protein binding. eIn vitro hepatic clearance in the presence of hepatocytes.16 Mean of at least two separate runs (n ≥ 2). fIn vivo parameters after IV dosing. CL = clearance; T1/2 = half-life. Compounds 21, 26, 29, 31, 33, and 34 were formulated as solutions in PEG400/citrate buffer (pH 5) and dosed at 0.4 mg/kg. Data are the average from three separate animals. gIn vivo parameters after PO dosing. AUC = area under the curve, CMax = maximum concentration. Compounds 21, 26, 29, 31, 33, and 34 were formulated as suspensions in MCT and dosed at 2 mg/kg. Data are the average from three separate animals. hExperimental oral bioavailability. iTheoretical maximum achievable oral bioavailability.20 jAll values obtained or calculated from ref 5. PO parameters scaled to 2 mg/kg. a

close to what would be predicted based on IV clearance (F/Fmax ∼ 1). Compound 21 possessed the best rat and dog in vivo PK of all compounds tested, with IV clearance and oral exposure values superior to those reported for 1. Monkey PK, however, was distinctly different and all IV and oral parameters were less favorable than in the rat or dog. It is notable that of all species tested, 21 was least stable in monkey hepatocytes and further that monkey hepatocytes were very predictive of in vivo clearance. It is possible that the moderate monkey PK parameters may be due at least in part to the intrinsically poorer metabolic stability of 21 in the monkey relative to other species. The most JAK1 selective compound in this work (31) exhibited moderate, yet consistent PK across all species tested, and compared favorably to the preclinical PK reported for 1. The off target kinase activities of multiple C-2 hydroxyethylcontaining compounds were assessed in a panel of 285 kinases (Table 6). Each compound was tested at a low (0.01−0.15 μM) and high (0.1−1.0 μM) concentration. The concentrations were chosen based on biochemical potency against JAK1, with more potent compounds tested at lower concentrations. The JAK family kinases were included as controls in every panel tested and, as expected, each compound demonstrated virtually

complete (>90%) inhibition of JAK1 at both concentrations tested. All compounds appeared to be relatively selective for the JAK family of kinases over the broader kinome at the lower concentration. However, at the higher concentration, the compounds began to differentiate significantly, with 29 and 31 showing the highest levels of selectivity. Indeed, at a 1 μM test concentration (≥500 × JAK1 Ki), the only non-JAK kinases inhibited by >50% were LRRK2 (72%) by compound 29 and CSF1R (75%) and the V561D mutant form of PDGFRα (55%) by compound 31. Other off-target activities of these compounds are summarized in Table 7. In biochemical assays, all compounds other than 1 were much less potent against JAK3 than JAK1, while the differential between TYK2 and JAK1 inhibition was smaller. No cellular measure of JAK3 inhibition independent from JAK1 was available, however, cellular TYK2 inhibition was measured in the IL-12 pSTAT4 assay. There was a consistently larger cell shift between the biochemical and cell-based measures of TYK2 inhibition relative to JAK1, thus compounds exhibited greater cellular selectivity for JAK1 vs TYK2 than would be expected based on their biochemical potencies. hERG inhibition was not a concern for the majority of the compounds I



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Table 6. Inhibition of a 285-Member Kinase Panel by Imidazopyrrolopyridine JAK1 Inhibitorsa

a

Off-target kinase inhibition assessed by SelectScreen Kinase Profiling Services (Invitrogen-Life Technologies, Madison, WI, USA). Each column contains 285 cells (one kinase per cell). The cells are colored as follows: green, < 50% inhibition; yellow, 50−75% inhibition; red, >75% inhibition; white, not tested. Identities of all kinases tested and corresponding % inhibition data may be found in the Supporting Information.

tested, with typical IC50 values of >100 μM being observed in a functional patch clamp assay. The nitrile-containing analogues 31, 33, and 34 showed consistent, yet weak reversible inhibition of CYP3A4 using a midazolam probe (IC50 = 6.7−8.8 μM), however, the remaining isoforms were not inhibited up to the highest concentration tested (10 μM). Finally, none of the compounds tested in Table 7 showed time dependent inhibition of any CYP isoform tested (3A4, 2C9, 2D6, 2C19, 1A2). Because of multiple favorable features including high selectivity for JAK1 over the other JAK family members and the broader kinome, potent inhibition of cell-based JAK1 activity in human whole blood, high aqueous solubility, and consistent cross-species preclinical PK comparing favorably with 1, compound 31 was further progressed to a collagen induced arthritis (CIA) efficacy study in the rat. As shown in Figure 5a, compound 31 exhibited approximately dose

proportional increases in plasma exposure when dosed orally in rats over a range of 20−500 mg/kg. These oral exposures were also close to dose proportional with the low dose rat PK study (2 mg/kg PO, Table 5). The oral exposures reported in Figure 5a did not change appreciably between day 10 of the CIA efficacy study, when the first daily dose of 31 was administered, and day 20 of the study, when the eleventh and final daily dose of 31 was delivered. The results from the CIA efficacy model are shown in Figure 5b. Collagen-immunized animals receiving only the corn oil vehicle exhibited marked increases in ankle thicknesses due to arthritic disease (gray line), while robust suppression of inflammation was observed even in the group treated with the lowest dose of 31 (20 mg/kg). The mid-dose of 31 (100 mg/kg) fully suppressed inflammation and maintained ankle ̈ animals untreated with thicknesses equivalent to those of naive collagen. Full suppression of inflammation was likewise J



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Scheme 1. Preparation of Dianiline Intermediate 38a

Table 7. Other Off-Target Inhibition of Selected Analogues

ex

JAK3 Ki (nM)a

TYK2 Ki (nM)a

IL-12 pSTAT4 EC50 (nM)b

hERG IC50 (μM)c

1 21 26 29 31 33 34

0.4 110 76 53 280 5.9 7.8

7.3 31 3.4 20 12 0.4 1.7

710 >5800 310 2200 5600 100 980

>100 >100 >100 >100 >100 20

reversible CYP (IC50, μM) 3A4(mid)d, othere >10, >10 >10, >10 >10, >10 7.9, >10 8.8, >10 6.7, >10

Reagents and conditions: (i) DIPEA, i-PrOH, 80 °C; (ii) H2, Pd/C, EtOH, THF, 50 °C.

a

further reduced with sodium borohydride to produce homologated alcohol 6b. Enantiopure secondary alcohols 7a and 8a were accessed by engaging 38 with the imidates derived from (R)-(+)-lactamide or (S)-(−)-lactamide, respectively. Oxidation of the secondary alcohol 7a and reaction of the resulting ketone (9a) with methylmagnesium bromide afforded tertiary alcohol 9b. Removal of the phenylsulfonyl protecting groups present in 5a, 6b, 7a, 8a, and 9b under basic hydrolysis conditions afforded the corresponding final products 5−9. Treatment of dianiline 38 with triethyl orthoformate led to C-2 unsubsituted imidazole 39, which could be selectively deprotonated with (tmp)2Mg·2LiCl24 and trapped by either propionaldehyde or 2-(benzyloxy)acetaldehyde to provide racemic secondary alcohols 10a and 12a, respectively. Removal of the phenylsulfonamide protecting groups afforded free azaindoles 10b and 12b, which were further separated into their corresponding enantiopure components (10b → 10 + 11, 12b → 12c + 13c) via chiral preparative SFC. Removal of the benzyl groups present in 12c and 13c under standard conditions afforded diols 12 and 13. As shown in Scheme 3, the aryl chloride present in 35 was similarly displaced with additional amines25 14a−17a, 19a− 24a, 26a, and 28a−34a, then the nitro groups in the resulting products were reduced to the corresponding anilines to provide the c series of dianiline intermediates. Reaction of the dianilines with the imidate derived from (R)-(+)-lactamide provided the d series of (R)-alcohols. The phenylsulfonamide moieties present in 15d, 16d, 19d−24d, and 31d−34d were removed under hydrolytic conditions to provide final products 15, 16, 19−24, and 31−34, whereas intermediates 14d, 17d, 26d, and 28d−30d were further manipulated as described in Scheme 4. As illustrated in Scheme 4, the Boc groups of 14d, 17d, 26d, and 28d−30d were removed to liberate the corresponding free

Arithmetic mean of at least three separate runs (n ≥ 3). The average coefficient of variation was less than 0.3 times the mean for biochemical assays.13 The biochemical JAK3 and TYK2 Ki values of the remaining compounds in this work are reported in the Supporting Information. bTYK2 cell-based assay.13 The average coefficient of variation was less than 0.5 times the mean for cell-based assays. cPatch clamp assay conducted by ChanTest. dReversible CYP3A4 inhibition measured in HLMs with midazolam as LCMS/MS probe. eOther CYP isoforms tested (LCMS/MS probe used): 3A4 (testosterone), 2C9 (warfarin), 2D6 (dextromethorphan), 2C19 (mephenytoin), 1A2 (phenacetin). a

observed in the high dose group (500 mg/kg of 31). Given its selectivity profile against the JAK family and the broader kinome, it is likely that the robust CIA efficacy demonstrated by 31 is driven predominantly by JAK1 inhibition. As such, these CIA results appear to support the hypothesis that selective JAK1 inhibitors could provide treatments for patients suffering from rheumatoid arthritis and other immunologic disorders.

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CHEMISTRY The preparation of analogues 5−13 commenced with synthesis of dianiline intermediate 38 (Scheme 1).21 SNAr displacement of the activated aryl chloride 359 by cyclohexylamine 36, followed by reduction of the resulting aryl nitro 37, produced dianiline 38. As shown in Scheme 2, dianiline 38 was cyclized under multiple conditions to form various C-2 substituted imidazoles. Treatment of the dianiline 38 with the imidate formed by the action of Meerwein’s reagent22 on 2-hydroxyacetamide produced the primary alcohol 5a.23 Coupling of 38 to ethyl 3-chloro-3-oxopropanoate, followed by dehydrative cyclization, yielded the ethyl ester-substituted imidazole 6a, which was

Figure 5. (a) Rat plasma exposures of 31 on days 10 and 20 of the CIA efficacy study.a (b) Mean ankle thicknesses of rats either treated or not treated with 31 in the CIA efficacy study.a Superscript letter indicates the following: (a) 31 was formulated in corn oil and dosed orally once daily at the indicated doses for 11 days. The first and last doses of 31 were administered on study days 10 and 20, respectively. K



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Scheme 2. Preparation of C-2 Hydroxyl Substituted Imidazopyrrolopyridine Analogues 5−13a

Reagents and conditions: (i) 2-hydroxyacetamide, Et3(O+)BF4−, THF,23 EtOH, 25−70 °C; (ii) NaOH, H2O, EtOH or EtOH/THF, 25−50 °C; (iii) TEA, ethyl 3-chloro-3-oxopropanoate, DCM, 25 °C, then AcOH, reflux; (iv) NaBH4, EtOH, 50 °C; (v) (R)-(+)-lactamide, Et3(O+)BF4−, THF, EtOH, 25−70 °C; (vi) (S)-(−)-lactamide, Et3(O+)BF4−, THF,23 EtOH, 25−70 °C; (vii) Dess−Martin periodinane, DCM, 25 °C; (viii) MeMgBr, THF, −10 to 25 °C; (ix) triethyl orthoformate, 82 °C; (x) (tmp)2Mg·2LiCl, propionaldehyde, THF, 0−25 °C; (xi) preparative chiral SFC; (xii) (tmp)2Mg·2LiCl, 2-(benzyloxy)acetaldehyde, THF, 0−25 °C; (xiii) Pd(OH)2/C, MeOH, EtOH, H2, 50 psi, 25 °C. a

amino groups (e series of intermediates). Alkylation or acylation of the exposed amines and phenylsulfonamide hydrolysis delivered final compounds 14, 17, 18, and 26−30.

potency was observed in human whole blood. Analysis of historical data generated in the C-2 H9 and methyl7 imidazopyrrolopyridine series indicated that maintaining cLogD7.4 95% for all final compounds as assessed by LCMS. Further details on the LCMS conditions used for individual compounds may be found in the Supporting Information. Cyclohexyl-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)methanol (5a). Triethyloxonium tetrafluoroborate (3.81 g, 201 mmol) was added to a solution of 2hydroxyacetamide (1.51 g, 201 mmol) in THF (100 mL) at 25 °C.23 The reaction mixture was stirred at 25 °C for 2 h. After concentrating

selectivity relative to the broader kinome, with 29 and 31 being particularly discriminating (Table 6). Finally, compound 31 was found to be highly efficacious in a rat CIA efficacy model (Figure 5), supporting the hypothesis that selective JAK1 inhibition may be a viable method to treat RA and other immunologic disorders.

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EXPERIMENTAL SECTION

General. Unless otherwise indicated, all reagents and solvents were purchased from commercial sources and used without further purification. Moisture or oxygen sensitive reactions were conducted under an atmosphere of argon or nitrogen gas. Unless otherwise stated, 1H NMR spectra were recorded at 298−300 K using Varian Unity Inova or Bruker Avance DRX400 instruments operating at the indicated frequencies. Chemical shifts are expressed in ppm relative to M



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Scheme 4. Preparation of C-2 (R)-Hydroxyethyl Imidazopyrrolopyridine Analogues 14, 17, 18, and 26−30a

a Reagents and conditions: (i) HCl/1,4-dioxane or TFA/H2O, 23 °C; (ii) 2,2,2-trifluoroethyl trifluoromethanesulfonate, TEA, DCM, DMF, 23 °C; (iii) NaOH, H2O, EtOH or EtOH/THF or MeOH/THF, 25−60 °C; (iv) EDC, 4-DMAP, DIPEA, 2-cyanoacetic acid (for 17) or 3,3,3trifluoropropanoic acid (for 18), 23 °C; (v) 2,2,-difluoroethyl trifluoromethanesulfonate, TEA, DCM, DMF, 23 °C.

were dried over MgSO4, filtered, and concentrated under vacuum. The resulting solid was triturated with diethyl ether (2 × 15 mL), then dried under vacuum to afford the title product as an off-white solid (26.2 mg, 18%). 1H NMR (400 MHz, DMSO-d6) δ 11.83 (s, 1H), 8.53 (s, 1H), 7.47 (s, 1H), 6.74 (s, 1H), 5.67 (s, 1H), 4.78 (d, J = 4.4 Hz, 2H), 4.75−4.61 (m, 1H), 2.39−2.20 (m, 2H), 2.02−1.87 (m, 4H), 1.86−1.74 (m, 1H), 1.62−1.37 (m, 3H). LCMS (method LCMS1, ESI): RT = 5.81 min, m/z = 271.0 [M + H]+. Ethyl 2-(1-Cyclohexyl-6-(phenylsulfonyl)-1,6dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate (6a). TEA (450 mg, 2.97 mmol) and ethyl 3-chloro-3-oxopropanoate (327 mg, 3.24 mmol) were added to a solution of 387 (1.0 g, 2.7 mmol) in DCM (15 mL). The reaction mixture was stirred at 25 °C for 1 h, then concentrated under vacuum. Acetic acid (10 mL) was added to the residue, and the mixture was heated at reflux for 16 h. The reaction mixture was then cooled to 25 °C and concentrated under vacuum. The residue was purified by column chromatography on silica gel, eluting with EtOAc/heptane to afford 210 mg (16%) of

under vacuum, the resulting oil was dissolved in EtOH (50 mL), then added to a mixture of 387 (2.92 g, 78.8 mmol) in EtOH (100 mL) and stirred at 75 °C for 80 min. The reaction mixture was cooled to 25 °C and allowed to stand for 2 h. The resulting precipitate was collected by filtration and purified by column chromatography on silica gel, eluting with EtOAc/heptane to afford 2.93 g (91%) of the title compound as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.67 (s, 1H), 8.14 (d, J = 8.0 Hz, 2H), 7.98 (s, 1H), 7.72−7.68 (m, 1H), 7.65−7.57 (m, 2H), 7.12 (s, 1H), 5.74−5.68 (m, 1H), 4.82−4.77 (m, 2H), 4.74− 4.64 (m, 1H), 2.19−2.07 (m, 2H), 1.96−1.88 (m, 4H), 1.79−1.74 (m, 1H), 1.56−1.44 (m, 3H). (1-Cyclohexyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)methanol (5). First, 1.0 mL of a 1 N aqueous solution of NaOH was added to a mixture of 5a (224 mg, 0.54 mmol) in EtOH and THF (5 mL each). The reaction mixture was stirred at 50 °C for 1 h, then was cooled to 25 °C and poured into 100 mL of a half saturated aqueous solution of NaHCO3. The aqueous layer was extracted with EtOAc (2 × 100 mL), then the combined organic layers N



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the title compound as a yellow solid. This material was used in the next step without further purification or characterization. 2-(1-Cyclohexyl-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5d]pyrrolo[2,3-b]pyridin-2-yl)ethanol (6b). Sodium borohydride was added to a solution of 6a (210 mg, 0.45 mmol) in EtOH (5 mL). The reaction mixture was stirred at 50 °C for 30 min, then cooled to 25 °C and concentrated under vacuum. The residue was purified by column chromatography on silica gel, eluting with EtOAc/heptane to give the title compound (151 mg, 79%) as a gray solid. LCMS (method LCMS2, ESI): RT = 1.37 min, m/z = 425.1 [M + H]+. 2-(1-Cyclohexyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)ethanol (6). Sodium hydroxide (8 mL of a 2 N aqueous solution) was added to a solution of 6b (151 mg, 0.35 mmol) in EtOH (4 mL). The reaction mixture was stirred at 25 °C for 16 h, then adjusted to pH ∼7 with HCl (2 N aqueous solution) and extracted with EtOAc (2 × 30 mL). The combined organic extracts were washed with water and brine (10 mL each), dried over MgSO4, filtered, and concentrated. The residue was purified by column chromatography on silica gel, eluting with EtOAc/heptane to give the title compound (42 mg, 39%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 11.80 (s, 1H), 8.49 (s, 1H), 7.46 (s, 1H), 6.72 (s, 1H), 4.88 (t, J = 5.2 Hz, 1H), 4.56−4.43 (m, 1H), 3.87−3.81 (br, 2H), 3.13 (t, J = 6.7 Hz, 2H), 2.36−2.24 (m, 2H), 2.03−1.83 (m, 4H), 1.81−1.76 (m, 1H), 1.64− 1.39 (m, 3H). LCMS (method LCMS3, ESI): RT = 3.17 min, m/z = 285.1 [M + H]+. (S)-1-(1-Cyclohexyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)ethanol (7). Synthesized by methods similar to those used for 8, substituting (S)-(−)-lactamide for (R)-(+)-lactamide. Analytical chiral SFC (method SFC1): RT = 0.57 min. 1H NMR and LCMS match 8. (R)-1-(1-Cyclohexyl-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)ethanol (8a). Triethyloxonium tetrafluoroborate (0.569 g, 3.00 mmol) was added to a solution of (R)(+)-lactamide (267 mg, 3.00 mmol) in THF (25 mL) at 25 °C.23 The resulting suspension was stirred at 25 °C for 2 h, then was concentrated under reduced pressure to afford an oil. This material was dissolved in EtOH (10 mL), and the resulting solution was added to to a solution of 387 (370 mg, 1.00 mmol) in EtOH (4 mL) at 25 °C. The reaction mixture was heated at 75 °C for 1 h, then was cooled to 25 °C and was partitioned between half-saturated aqueous NaHCO3 (100 mL) and EtOAc (2 × 100 mL). The organic layers were dried over MgSO4 and filtered, and the filtrate was concentrated under vacuum. Purification of the resulting solid by column chromatography on silica gel (gradient: 0−8% CH3OH in DCM) afforded the title compound (202 mg, 95%) as a gray solid. LCMS (method LCMS4, ESI): RT = 0.99 min, m/z = 425.3 [M + H]+. (R)-1-(1-Cyclohexyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3b]pyridin-2-yl)ethanol (8). Sodium hydroxide (1 mL of a 1 N aqueous solution) was added to a solution of 8a (203 mg, 0.48 mmol) in a 1:1 mixture of THF and EtOH (10 mL) at 25 °C. The reaction mixture was stirred at 50 °C for 1 h, then was cooled to 25 °C and was partitioned between half-saturated aqueous NaHCO3 (100 mL) and EtOAc (2 × 125 mL). The combined organic layers were dried over MgSO4 and filtered, and the filtrate was concentrated under reduced pressure. Purification of the residue by preparative HPLC (column: Gemini-NX, 5 cm × 10 cm, 10 μm; detection:, UV 254 nm; mobile phase A, water with 0.1% NH4OH; mobile phase B, CH3CN; flow rate, 120 mL/min; gradient 5−95% B over 15 min) afforded the title compound (46 mg, 34%) as an off-white solid. Analytical chiral SFC (method SFC1): RT = 0.54 min. 1H NMR (400 MHz, DMSO-d6) δ 11.84 (s, 1H), 8.55 (s, 1H), 7.47 (s, 1H), 6.74 (s, 1H), 5.66 (d, J = 6.5 Hz, 1H), 5.12 (p, J = 6.4 Hz, 1H), 4.91−4.76 (m, 1H), 2.41−2.22 (m, 2H), 2.02−1.84 (m, 4H), 1.80 (s, 1H), 1.64 (d, J = 6.4 Hz, 3H), 1.58− 1.42 (m, 3H). LCMS (method LCMS5, ESI): RT = 6.56 min, m/z = 285.3 [M + H]+. 1-(1-Cyclohexyl-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5d]pyrrolo[2,3-b]pyridin-2-yl)ethanone (9a). Dess−Martin periodinane (1.2 g, 2.8 mmol) was added to a solution of 7a (300 mg, 0.71 mmol) in DCM (5 mL). The reaction mixture was stirred for 30 min at 25 °C, then diluted with an additional 50 mL of DCM. The organic

layer was washed with saturated aqueous NaHCO3 solution, dried over MgSO4, and concentrated under vacuum to give the title compound (199 mg, 66%) as an off-white solid. 2-(1-Cyclohexyl-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5d]pyrrolo[2,3-b]pyridin-2-yl)propan-2-ol (9b). To a solution of compound 9a (198 mg, 0.47 mmol) in THF (5 mL) was added methylmagnesium bromide (1 mL of 1.0 M solution in dibutyl ether) at −10 °C. The reaction mixture was warmed to 25 °C and stirred at that temperature for 30 min. Water (10 mL) was added, and the mixture was extracted with EtOAc (2 × 10 mL). The combined extracts were washed with water and brine (10 mL each), dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography on silica gel, eluting with EtOAc/heptane to afford 200 mg (97%) of the title compound as a gray solid. LCMS (method LCMS2, ESI): RT = 1.43 min, m/z = 439.2 [M + H]+. 2-(1-Cyclohexyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)propan-2-ol (9). NaOH (2 N aqueous solution, 10 mL) was added to a solution of 9b (200 mg, 0.46 mmol) in EtOH (5 mL). The reaction mixture was stirred at 25 °C for 16 h, then the pH was adjusted to ∼7 with 2 N aqueous HCl solution. The mixture was extracted with EtOAc (2 × 15 mL). The combined organic layers were washed with water and brine (10 mL each), dried over MgSO4, filtered, and concentrated under vacuum. The residue was purified by column chromatography on silica gel, eluting with EtOAc/heptane to afford 90 mg of the title compound as a white solid (69%). 1H NMR (400 MHz, DMSO-d6) δ 11.83 (s, 1H), 8.53 (s, 1H), 7.47 (s, 1H), 6.74 (s, 1H), 5.74 (s, 1H), 5.43 (t, J = 12.3 Hz, 1H), 2.50 (s, 6H), 2.37 (dd, J = 23.4, 11.2 Hz, 2H), 2.04−1.85 (m, 4H), 1.84−1.76 (m, 1H), 1.57−1.37 (m, 3H). LCMS (method LCMS3, ESI): RT = 3.47 min, m/z = 299.1 [M + H]+. 1-Cyclohexyl-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridine (39). A suspension of 387 (7.80 g, 21.1 mmol) in triethyl orthoformate (48 mL) was heated at 82 °C for 16 h. The reaction mixture was cooled to 25 °C and concentrated under vacuum. The residue was purified by column chromatography on silica gel, eluting with EtOAc/heptane to afford 5.34 g (68%) of the title compound as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.72 (s, 1H), 8.46 (s, 1H), 8.15−8.10 (m, 2H), 7.97 (d, J = 4.0 Hz, 1H), 7.73− 7.66 (m, 1H), 7.64−7.55 (m, 2H), 7.18 (d, J = 4.0 Hz, 1H), 4.64−4.49 (m, 1H), 2.15−2.03 (m, 2H), 1.91−1.68 (m, 5H), 1.67−1.51 (m, 2H), 1.36−1.21 (m, 1H). LCMS (method LCMS6, ESI): RT = 2.27 min, m/z = 381.3 [M + H]+. (±)-1-(1-Cyclohexyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3b]pyridin-2-yl)propan-1-ol (10b). 2,2,6,6-Tetramethylpiperidinemagnesium chloride lithium chloride complex (1 M solution in THF, 677 μL) was added to a solution of 39 (200 mg, 0.50 mmol) in THF (4 mL) at 0 °C. After stirring for 5 min at 0 °C, the reaction mixture was removed from the ice bath and stirred for 1 h at 25 °C. The reaction mixture was then added to a stirred solution of propionaldehyde (97 μL, 1.35 mmol) in THF (1 mL) and left to stir for 16 h. EtOH (5 mL) and sodium hydroxide (1 N aqueous solution, 5 mL) were then added, and the reaction mixture was stirred at 50 °C for 2 h. The reaction mixture was concentrated under vacuum and purified by column chromatography eluting with EtOAc/heptane to afford the racemic title compound (10b) as a white solid (18.1 mg, 12%). (S)-1-(1-Cyclohexyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)propan-1-ol (10) and (R)-1-(1-Cyclohexyl-1,6dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)propan-1-ol (11). 10b was separated into its individual enantiomers by preparative chiral SFC (Lux Cellulose-1 column, 21.2 mm × 150 mm, 5 μm; detection, UV 220 nm; mobile phase A, CO2; mobile phase B, MeOH containing 0.1% NH4OH; flow rate, 70 mL/min; gradient, isocratic; A:B = 80:20). Isolation and concentration of the appropriate fractions afforded two products with the following characteristics. 10: white solid. Analytical chiral SFC (method SFC2): RT = 0.48 min. 1H NMR and LCMS match 11. 11: white solid. Analytical chiral SFC (method SFC2) RT = 0.45 min. 1H NMR (400 MHz, DMSO-d6) δ 11.85 (s, 1H), 8.54 (s, 1H), 7.47 (t, J = 3.0 Hz, 1H), 6.73 (s, 1H), 5.73−5.66 (m, 1H), 4.98−4.63 (m, 2H), 2.38−2.26 (m, 2H), 2.07−2.00 (m, 1H), O



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DMSO-d6) δ 11.82 (s, 1H), 8.55 (s, 1H), 7.49 (t, J = 3.0 Hz, 1H), 6.90 (s, 1H), 5.68 (d, J = 6.6 Hz, 1H), 5.13 (t, J = 6.5 Hz, 1H), 4.93−4.81 (m, 1H), 3.38−3.30 (m, 2H), 3.21−3.11 (s, 2H), 2.66−2.55 (m, 4H), 1.93−1.81 (m, 2H), 1.63 (d, J = 6.4 Hz, 3H). LCMS (method LCMS7, ESI): RT = 3.02 min, m/z = 368.3 [M + H]+. (R)-3-(4-(2-(1-Hydroxyethyl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)piperidin-1-yl)propanenitrile (15). 15 was synthesized by methods similar to those used for 8, substituting 3(4-aminopiperidin-1-yl)propanenitrile (15a)26 for cyclohexylamine (36). 1H NMR (400 MHz, DMSO-d6) δ 11.81 (s, 1H), 8.55 (s, 1H), 7.40 (t, J = 3.0 Hz, 1H), 7.00 (br s, 1H), 5.69 (d, J = 6.7 Hz, 1H), 5.13 (p, J = 6.5 Hz, 1H), 4.94−4.81 (m, 1H), 3.18−3.11 (m, 2H), 2.82−2.67 (m, 4H), 2.67−2.55 (m, 2H), 2.25 (t, J = 11.9 Hz, 2H), 1.94−1.84 (m, 2H), 1.63 (d, J = 6.4 Hz, 3H). LCMS (method LCMS7, ESI): RT = 1.38 min, m/z = 339.2 [M + H]+. ( 1R ) - 1 - (1 - (1 - E t h y l - 3, 3 - d i fl u o r o p i p e r i d i n - 4 - yl ) - 1 , 6 dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)ethanol (16). A diastereomeric mixture of 16 and its 4-piperidine epimer was synthesized by methods similar to those used for 8, substituting (±)-1ethyl-3,3-difluoropiperidin-4-amine (16a)27 for cyclohexylamine (36). The diastereomers were separated by preparative chiral SFC (Lux Cellulose-1 column: 21.2 mm × 250 mm, 5 μm; detection, UV 220 nm; mobile phase A, CO2; mobile phase B, MeOH containing 0.1% NH4OH; flow rate, 70 mL/min; gradient, isocratic; A:B = 85:15) to provide 16 (later eluting peak) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 11.74 (s, 1H), 8.58 (s, 1H), 7.41 (t, J = 3.1 Hz, 1H), 6.74−6.62 (m, 1H), 5.75 (d, J = 7.4 Hz, 1H), 5.46−5.28 (m, 1H), 5.04 (p, J = 6.6 Hz, 1H), 3.38 (t, J = 11.8 Hz, 1H), 3.22−3.13 (m, 1H), 3.06 (qd, J = 12.4, 4.6 Hz, 1H), 2.69−2.53 (m, 3H), 2.33 (t, J = 11.7 Hz, 1H), 2.00−1.89 (m, 1H), 1.68 (d, J = 6.3 Hz, 3H), 1.11 (t, J = 7.1 Hz, 3H). LCMS (method LCMS1, ESI): RT = 2.96 min, m/z = 350.4 [M + H]+. (R)-tert-Butyl 3-(2-((R)-1-hydroxyethyl)-6-(phenylsulfonyl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)piperidine-1-carboxylate (17d). 17d was synthesized by methods similar to those used for 8a, substituting (R)-tert-butyl 3-aminopiperidine-1-carboxylate (17a) for cyclohexylamine (36). 1H NMR (400 MHz, DMSOd6) δ 8.72 (s, 1H), 8.14 (d, J = 7.6 Hz, 2H), 7.96 (d, J = 3.8 Hz, 1H), 7.75−7.58 (m, 3H), 7.21 (d, J = 3.7 Hz, 1H), 5.85−5.75 (m, 1H), 5.19−5.08 (m, 1H), 4.99−4.86 (m, 1H), 4.27−4.17 (m, 1H), 4.15− 3.98 (m, 1H), 2.36−2.21 (m, 1H), 2.06−1.97 (m, 1H), 1.90 (br d, J = 12.1 Hz, 1H), 1.62 (d, J = 6.2 Hz, 3H), 1.51 (s, 3H), 1.38 (br s, 9H). (R)-1-(1-((R)-Piperidin-3-yl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)ethanol (17f). 17d (8.09 g, 15.4 mmol) was stirred in a 4.0 M solution of hydrogen chloride in 1,4-dioxane (150 mL) for 3 h. The volatiles were removed under vacuum and the solids were washed with Et2O (3 × 100 mL) and isolated by filtration. The resulting hygroscopic white powder was dissolved in a 1.0 M solution of sodium hydroxide in water (100 mL) and EtOH (100 mL) and heated to 60 °C for 8 h. The crude reaction mixture was then concentrated onto silica gel and purified by column chromatography, eluting with a gradient of 0% to 20% 2 M NH3 in MeOH in DCM, to provide the title compound as an off-white solid (2.70 g, 61%). 1H NMR (400 MHz, DMSO-d6) δ 11.83 (s, 1H), 8.54 (s, 1H), 7.48 (s, 1H), 6.84 (s, 1H), 5.66 (d, J = 6.5 Hz, 1H), 5.17−5.04 (m, 1H), 4.86 (s, 1H), 3.43−3.36 (m, 1H), 3.17 (d, J = 5.1 Hz, 1H), 3.10−2.94 (m, 2H), 2.75 (t, J = 11.9 Hz, 1H), 2.46−2.38 (m, 1H), 2.03−1.80 (m, 2H), 1.70−1.57 (m, 4H). 3-((R)-3-(2-((R)-1-Hydroxyethyl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)piperidin-1-yl)-3-oxopropanenitrile (17). EDC (77 mg, 0.40 mmol) was added to a solution of 17f (60 mg, 0.21 mmol), 4-dimethylaminopyridine (10 mg, 0.08 mmol), DIPEA (80 μL, 0.46 mmol), and 2-cyanoacetic acid (34 mg, 0.40 mmol) in DMF (1 mL). The reaction mixture was stirred at 23 °C for 16 h, then diluted with EtOAc (10 mL). The organic layer was washed successively with 1 M aqueous HCl solution, saturated aqueous NaHCO3 solution, and brine (1 × 3 mL each), then dried over Na2SO4, filtered, and concentrated under vacuum. The residue was purified by preparative HPLC (column: Gemini-NX, 5 cm × 10 cm, 10 μm; detection, UV 254 nm; mobile phase A, water with 0.1%

2.00−1.92 (m, 3H), 1.88 (d, J = 11.2 Hz, 2H), 1.83−1.78 (m, 1H), 1.56−1.43 (m, 3H), 0.98 (t, J = 7.4 Hz, 3H). LCMS (method LCMS3, ESI): RT = 3.46 min, m/z = 299.1 [M + H]+. (±)-2-(Benzyloxy)-1-(1-cyclohexyl-6-(phenylsulfonyl)-1,6dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)ethanol (12a). A 1 M solution of 2,2,6,6-tetramethylpiperidinemagnesium chloride lithium chloride complex in THF (16.8 mL, 16.8 mmol) was added to a solution of 39 (5.34 g, 14.0 mmol) in anhydrous THF (100 mL) at 0 °C. The reaction mixture was stirred for 1 h at 0 °C, then added to a solution of 2-(benzyloxy)acetaldehyde (3.95 mL, 28.1 mmol) in THF (35 mL) at 0 °C. The mixture was stirred for 1 h at 0 °C, then slowly warmed to 25 °C. Stirring was continued for an additional 14 h, then a saturated aqueous solution of NH4Cl (50 mL) was added. The mixture was extracted with EtOAc (3 × 75 mL), then the combined organic layers were dried over MgSO4, filtered, and concentrated under vacuum. The residue was purified by column chromatography eluting with EtOAc/heptane to afford 6.56 g (88%) of the title compound as a foamy off-white solid. LCMS (method LCMS6, ESI): RT = 2.71 min, m/z = 531.3 [M + H]+. (±)-2-(Benzyloxy)-1-(1-cyclohexyl-1,6-dihydroimidazo[4,5d]pyrrolo[2,3-b]pyridin-2-yl)ethanol (12b). To a solution of 12a (6.56 g, 12.4 mmol) in EtOH (50 mL) was added a 1 M solution of aqueous sodium hydroxide (24.7 mL, 24.7 mmol). The reaction mixture was stirred at 23 °C for 16 h, then a 1 M aqueous solution of HCl (25 mL) was added. The mixture was concentrated to dryness under vacuum, then an additional 100 mL of EtOH was added. The mixture was filtered, then the filtrate was concentrated under vacuum to afford the crude title product as an off-white solid. (R)-2-(Benzyloxy)-1-(1-cyclohexyl-1,6-dihydroimidazo[4,5d]pyrrolo[2,3-b]pyridin-2-yl)ethanol (12c) and (S)-2-(Benzyloxy)-1-(1-cyclohexyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)ethanol (13c). The crude residue obtained in the previous step was purified and separated into its individual enantiomers by preparative chiral SFC (column: Lux Cellulose-1, 3 cm × 25 cm, 5 μm; detection, UV 220 nm; mobile phase A, CO2; mobile phase B, MeOH containing 0.1% dimethylamine; flow rate, 200 mL/min; gradient, isocratic; A:B = 70:30). First-eluting peak (13c): off-white solid (2.12 g, 44%). 1H NMR (400 MHz, DMSO-d6) δ 11.82 (s, 1H), 8.53 (s, 1H), 7.46 (t, J = 2.9 Hz, 1H), 7.38−7.25 (m, 5H), 6.73 (s, 1H), 5.92 (d, J = 6.6 Hz, 1H), 5.17 (q, J = 6.3 Hz, 1H), 4.91− 4.69 (m, 1H), 4.61 (s, 2H), 4.05 (dd, J = 10.1, 4.6 Hz, 1H), 3.93 (dd, J = 10.1, 7.4 Hz, 1H), 2.39−2.21 (m, 2H), 1.98−1.74 (m, 5H), 1.59− 1.38 (m, 3H). LCMS (method LCMS5, ESI): RT = 10.7 min, m/z = 391.1 [M + H]+. Second-eluting peak (12c): off-white solid (1.53 g, 32%). 1H NMR and LCMS match 13c. (R)-1-(1-Cyclohexyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3b]pyridin-2-yl)ethane-1,2-diol (12). 12c was converted to 12 by the same method used to convert 13c to 13. The 1H NMR and LCMS of 12 match those of 13. (S)-1-(1-Cyclohexyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)ethane-1,2-diol (13). To a solution of 13c (2.1 g, 5.3 mmol) in a 1:1 mixture of MeOH:EtOH (50 mL) was added 20% palladium hydroxide on carbon (371 mg). The reaction mixture was pressurized to 50 psi with hydrogen gas, then shaken on a Parr apparatus for 48 h. The reaction mixture was filtered through Celite, then the filtrate was concentrated under vacuum. The residue was purified by reverse phase HPLC (Gemini-NX column, 30 mm × 100 mm, 5 μm; detection, UV 230 nm; mobile phase A, water with 0.1% NH4OH; mobile phase B, CH3CN; flow rate, 60 mL/min; gradient 5− 50% B over 10 min) to afford 253 mg (17%) the title product as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 11.82 (s, 1H), 8.54 (s, 1H), 7.46 (t, J = 3.0 Hz, 1H), 6.74 (dd, J = 3.5, 1.9 Hz, 1H), 5.76 (d, J = 6.4 Hz, 1H), 4.94 (q, J = 6.2 Hz, 1H), 4.82 (t, J = 6.0 Hz, 2H), 3.99−3.91 (m, 1H), 3.89−3.82 (m, 1H), 2.41−2.19 (m, 2H), 2.02− 1.68 (m, 5H), 1.60−1.39 (m, 3H). LCMS (method LCMS5, ESI): RT = 10.7 min, m/z = 301.1 [M + H] +. (R)-1-(1-(1-(2,2,2-Trifluoroethyl)piperidin-4-yl)-1,6dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)ethanol (14). 14 was synthesized by methods similar to those used for 26, substituting tert-butyl 4-aminopiperidine-1-carboxylate (14a) for tertbutyl (trans-4-aminocyclohexyl)carbamate (26a). 1H NMR (400 MHz, P



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NH4OH; mobile phase B, CH3CN; flow rate, 120 mL/min; gradient 5−95% B over 15 min) to afford 50 mg (68%) of the title compound as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 11.91 (s, 1H), 8.57 (d, J = 6.4 Hz, 1H), 8.32 (s, 1H), 7.49 (s, 1H), 6.80 (d, J = 13.8 Hz, 1H), 5.25−5.06 (m, 1H), 5.00−4.80 (m, 1H), 4.68−4.45 (m, 1H), 4.25−4.04 (m, 2H), 3.94−3.75 (m, 2H), 3.03−2.89 (m, 1H), 2.12− 1.89 (m, 2H), 1.86−1.71 (m, 1H), 1.71−1.59 (m, 4H). LCMS (method LCMS8, ESI): RT = 2.70 min, m/z = 353.1 [M + H]+. 3,3,3-Trifluoro-1-((R)-3-(2-((R)-1-hydroxyethyl)imidazo[4,5d]pyrrolo[2,3-b]pyridin-1(6H)-yl)piperidin-1-yl)propan-1-one (18). 18 was synthesized by methods similar to those used for 17, substituting 3,3,3-trifluoropropanoic acid for 2-cyanoacetic acid. 1H NMR (400 MHz, DMSO-d6) δ 11.92 (s, 1H), 8.58 (d, J = 8.4 Hz, 1H), 7.50 (s, 1H), 6.81 (d, J = 15.8 Hz, 1H), 5.85−5.70 (m, 1H), 5.24−5.06 (m, 1H), 4.98−4.79 (m, 1H), 4.73−4.51 (m, 1H), 4.04− 3.91 (m, 1H), 3.86−3.66 (m, 2H), 2.93 (t, J = 14.1 Hz, 1H), 2.70− 2.56 (m, 1H), 2.12−1.92 (m, 3H), 1.72−1.57 (m, 4H). LCMS (method LCMS3, ESI): RT = 3.05 min, m/z = 396.1 [M + H]+. (R)-1-(1-(Tetrahydro-2H-pyran-4-yl)-1,6-dihydroimidazo[4,5d]pyrrolo[2,3-b]pyridin-2-yl)ethanol (19). 19 was synthesized by methods similar to those used for 21, substituting tetrahydro-2Hpyran-4-amine (19a) for (S)-tetrahydro-2H-pyran-3-amine (21a). 1H NMR (400 MHz, DMSO-d6) δ 11.85 (s, 1H), 8.57 (s, 1H), 7.50 (s, 1H), 6.76 (s, 1H), 5.78−5.63 (m, 1H), 5.22−5.05 (m, 2H), 4.17−4.10 (m, 2H), 3.57 (t, J = 12.0 Hz, 2H), 2.70−2.53 (m, 2H), 1.92−1.80 (m, 2H), 1.65 (d, J = 6.4 Hz, 3H). LCMS (method LCMS3, ESI): RT = 2.65 min, m/z = 287.1 [M + H]+. (R)-1-(1-((R)-Tetrahydro-2H-pyran-3-yl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)ethanol (20). 20 was synthesized by methods similar to those used for 21, substituting (R)tetrahydro-2H-pyran-3-amine (20a) for (S)-tetrahydro-2H-pyran-3amine (21a). 1H NMR (400 MHz, DMSO-d6) δ 11.88 (s, 1H), 8.56 (s, 1H), 7.50 (s, 1H), 6.86 (s, 1H), 5.72−5.64 (m, 1H), 5.19−5.09 (m, 1H), 5.04−4.95 (m, 1H), 4.10 (t, J = 11.2 Hz, 1H), 4.02−3.91 (m, 2H), 3.70 (t, J = 11.8 Hz, 1H), 2.68−2.50 (m, 1H), 2.11−2.03 (m, 1H), 1.96−1.80 (m, 2H), 1.65 (br s, 3H). LCMS (method LCMS3, ESI): RT = 2.72 min, m/z = 287.1 [M + H]+. (S)-5-Nitro-1-(phenylsulfonyl)-N-(tetrahydro-2H-pyran-3-yl)1H-pyrrolo[2,3-b]pyridin-4-amine (21b). DIPEA (130 mL, 760 mmol) was added to a solution of (S)-tetrahydro-2H-pyran-3-amine (21a,28 40.7 g, 296 mmol) and 359 (100 g, 296 mmol) in isopropyl alcohol (1.0 L). The mixture was stirred at an internal temperature of 75 °C for 4 h. The mixture was then cooled slowly to 23 °C and allowed to stand for 16 h. The precipitate was collected by filtration and washed with cold isopropyl alcohol (100 mL). The solid was dried in a vacuum oven at 50 °C for 24 h to afford 114 g (96%) of the title compound as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 9.21 (d, J = 8.0 Hz, 1H), 9.11 (s, 1H), 8.19 (dd, J = 7.6, 1.6 Hz, 2H), 7.65−7.58 (m, 2H), 7.52 (t, J = 7.7 Hz, 2H), 6.75 (d, J = 4.1 Hz, 1H), 4.18−4.07 (m, 1H), 3.98 (dd, J = 11.5, 3.0 Hz, 1H), 3.81−3.65 (m, 2H), 3.57 (dd, J = 11.6, 6.5 Hz, 1H), 2.19−2.06 (m, 1H), 1.93−1.78 (m, 2H), 1.75−1.62 (m, 1H). LCMS (method LCMS9, ESI): RT = 1.63 min, m/z = 403.2 [M + H]+. (S)-1-(Phenylsulfonyl)-N 4-(tetrahydro-2H-pyran-3-yl)-1Hpyrrolo[2,3-b]pyridine-4,5-diamine (21c). Palladium on carbon (10%, 30.0 g, 28.2 mmol) was added to a solution of 21b in EtOAc (2.5 L). The mixture was stirred at 23 °C under an atmosphere of hydrogen gas for 48 h, then filtered through Celite and washed with 10% MeOH in DCM (1 L). The filtrate was concentrated to afford 106 g (100%) of the title compound as an off-white solid. LCMS (method LCMS9, ESI): RT = 1.13 min, m/z = 373.2 [M + H]+. (R)-1-(6-(Phenylsulfonyl)-1-((S)-tetrahydro-2H-pyran-3-yl)1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)ethanol (21d). (R)-(+)-lactamide (33.5 g, 376 mmol) was added to a suspension of triethyloxonium tetrafluoroborate (73.6 g, 376 mmol) in THF (800 mL).23 The reaction mixture was stirred for 1 h at 23 °C, during which time it became a clear solution. The solution was then added to a solution of 21c (70.0 g, 188 mmol) in EtOH (1.5 L), and the reaction mixture was stirred at 67 °C for 2 h. The solution was then cooled to room temperature, and EtOAc (500 mL) was added. The mixture was allowed to stand for 16 h, then the resulting

precipitate was collected by filtration, washed with cold EtOAc (50 mL), and dried under vacuum for 16 h to afford 60.9 g (80%) of the title compound as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.71 (s, 1H), 8.18−8.09 (m, 2H), 7.99 (d, J = 4.0 Hz, 1H), 7.74−7.67 (m, 1H), 7.65−7.57 (m, 2H), 7.26 (d, J = 4.1 Hz, 1H), 5.82 (d, J = 6.4 Hz, 1H), 5.14 (p, J = 6.3 Hz, 1H), 5.08−4.94 (m, 1H), 3.95 (d, J = 8.3 Hz, 3H), 3.68 (t, J = 11.2 Hz, 1H), 2.46−2.30 (m, 1H), 2.07 (d, J = 11.7 Hz, 1H), 1.95−1.75 (m, 2H), 1.62 (d, J = 6.3 Hz, 3H). LCMS (method LCMS9, ESI): RT = 1.34 min, m/z = 427.2 [M + H]+. 1H (R)-1-(1-((S)-Tetrahydro-2H-pyran-3-yl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)ethanol (21). To a suspension of 21d (40.0 g, 93.8 mmol) in EtOH (50 mL) was added an aqueous solution of sodium hydroxide (9 M, 30 mL, 270 mmol). The reaction mixture was stirred for 1 h at 60 °C, then cooled to room temperature. The majority of the EtOH was removed under vacuum, then the remaining aqueous solution was cooled to 0 °C. The pH was adjusted to ∼7 with 37% aqueous hydrochloric acid solution. The resulting solid was removed by filtration. The filtrate was concentrated under vacuum, the residue was taken up in a 1:1 mixture of EtOH:EtOAc (200 mL), and the solid was removed with another filtration. The filtrate was concentrated, and the residue was dissolved in MeOH (150 mL) and treated with macroporous triethylammonium methylpolystyrene carbonate resin to remove the remaining phenylsulfonic acid. The resin was removed by filtration and washed with MeOH (30 mL). The filtrate was concentrated and the residue crystallized from hot CH3CN. The resulting solid was collected by filtration, washed with cold CH3CN, and then taken up in water (15 mL). The water was removed under vacuum to afford 10.6 g (40%) of the title compound as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 11.87 (s, 1H), 8.55 (s, 1H), 7.49 (d, J = 3.3 Hz, 1H), 6.85 (d, J = 3.4 Hz, 1H), 5.89−5.56 (m, 1H), 5.14 (q, J = 6.4 Hz, 1H), 5.08−4.94 (m, 1H), 4.11 (t, J = 11.1 Hz, 1H), 4.02−3.92 (m, 2H), 3.69 (td, J = 11.9, 2.5 Hz, 1H), 2.63− 2.51 (m, 1H), 2.14−2.03 (m, 1H), 1.97−1.74 (m, 2H), 1.63 (d, J = 6.4 Hz, 3H). LCMS (method LCMS1, ESI): RT = 3.60 min, m/z = 287.0 [M + H]+. trans-4-(2-((R)-1-Hydroxyethyl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)cyclohexanol (22). 22 was synthesized by methods similar to those used for 21, substituting trans-4-aminocyclohexanol (22a) for (S)-tetrahydro-2H-pyran-3-amine (21a). 1H NMR (400 MHz, DMSO-d6) δ 11.81 (s, 1H), 8.54 (s, 1H), 7.46 (d, J = 3.4 Hz, 1H), 6.66 (d, J = 3.5 Hz, 1H), 5.65 (br s, 1H), 5.12 (q, J = 6.4 Hz, 1H), 4.88−4.69 (m, 2H), 3.86−3.74 (m, 1H), 2.46−2.32 (m, 2H), 2.10−2.01 (m, 2H), 1.95−1.83 (m, 2H), 1.63 (d, J = 6.4 Hz, 3H), 1.53−1.39 (m, 2H). LCMS (method LCMS1, ESI): RT = 7.14 min, m/z = 301.2 [M + H]+. cis-4-(2-((R)-1-Hydroxyethyl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)cyclohexanol (23). 23 was synthesized by methods similar to those used for 21, substituting cis-4-aminocyclohexanol (23a) for (S)-tetrahydro-2H-pyran-3-amine (21a). 1H NMR (400 MHz, DMSO-d6) δ 11.71 (s, 1H), 8.52 (s, 1H), 7.41 (s, 2H), 5.63 (br s, 1H), 5.11 (q, J = 6.5 Hz, 1H), 4.97−4.74 (m, 2H), 4.03 (s, 1H), 2.79−2.64 (m, 2H), 1.94−1.83 (m, 2H), 1.72−1.55 (m, 7H). LCMS (method LCMS1, ESI): RT = 7.18 min, m/z = 301.2 [M + H]+. (1R,3R)-3-(2-((R)-1-Hydroxyethyl)imidazo[4,5-d]pyrrolo[2,3b]pyridin-1(6H)-yl)cyclohexanol (24). 24 was synthesized by methods similar to those used for 21, substituting (1R,3R)-3aminocyclohexanol (24a)29 for (S)-tetrahydro-2H-pyran-3-amine (21a). 1H NMR (400 MHz, DMSO-d6) δ 11.80 (s, 1H), 8.52 (s, 1H), 7.45 (t, J = 2.6 Hz, 1H), 6.66 (d, J = 3.0 Hz, 1H), 5.77−5.45 (m, 1H), 5.40−5.24 (m, 1H), 5.14−5.02 (q, J = 6.5 Hz, 1H), 4.68 (br s, 1H), 4.22 (br s, 1H), 2.48−2.40 (m, 1H), 2.35−2.19 (m, 1H), 2.01− 1.74 (m, 4H), 1.74−1.63 (m, 2H), 1.61 (d, J = 6.5 Hz, 3H). LCMS (method LCMS1, ESI): RT = 3.87 min, m/z = 301.1 [M + H]+. (R)-1-(1-(trans-4-Methoxycyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)ethanol (25).27 1H NMR (400 MHz, DMSO-d6) δ 11.85 (s, 1H), 8.54 (s, 1H), 7.47 (t, J = 3.0 Hz, 1H), 6.69 (m, 1H), 5.66 (d, J = 6.7 Hz, 1H), 5.12 (m, 1H), 4.84 (m, 1H), 3.54 (m, 4H), 2.39 (m, 2H), 2.24 (m, 2H), 1.94 (m, 2H), 1.63 Q



Article

(d, J = 6.5 Hz, 3H), 1.40 (m, 2H). LCMS (method LCMS11, ESI): RT = 2.34 min, m/z = 315.5 [M + H]+. (R)-1-(1-(trans-4-Aminocyclohexyl)-6-(phenylsulfonyl)-1,6dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)ethanol (26e). 26d was synthesized by methods similar to those used for 31d, substituting tert-butyl (trans-4-aminocyclohexyl)carbamate (26a) for trans-4-aminocyclohexanecarbonitrile trifluoroacetic acid salt (31a). 26d (2.00 g, 3.71 mmol) was then added to a mixture of trifluoroacetic acid (10 mL) and water (1 mL). The reaction mixture was stirred at 23 °C for 1 h, then concentrated under vacuum. The residue was taken up in MeOH and purified by Isolute SCX column chromatography (gradient: MeOH to 2 M NH3 in MeOH) to give 1.59 g (98%) of the title compound as an off-white solid. LCMS (method LCMS10, ESI): RT = 1.81 min, m/z = 440.3 [M + H]+. (R)-1-(1-(trans-4-((2,2,2-Trifluoroethyl)amino)cyclohexyl)1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)ethanol (26). 26e (200 mg, 0.46 mmol) was dissolved in DCM (2 mL) and DMF (2 mL). TEA (254 μL, 1.84 mmol) was added, followed by 2,2,2-trifluoroethyl trifluoromethanesulfonate (131 μL, 0.91 mmol). The reaction mixture was stirred for 16 h at 23 °C. Water (10 mL) was added and the mixture was extracted with DCM (3 × 5 mL), and the combined organic extracts were dried (Na2SO4), filtered, and concentrated under vacuum. The residue was dissolved in MeOH (2 mL) and THF (2 mL), and 2 M aqueous NaOH (2 mL) was added. The reaction mixture was stirred at 23 °C for 2 h, then concentrated under vacuum. Then 1 M aqueous HCl solution (4 mL) was added to the residue, and the mixture was concentrated under vacuum. Purification by column chromatography on silica gel (gradient: DCM to 8% (2 M NH3 in MeOH) in DCM) followed by trituration with CH3CN gave 77 mg (44%) of the title compound as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 11.82 (s, 1H), 8.54 (s, 1H), 7.47 (t, J = 3.0 Hz, 1H), 6.71−6.63 (dd, J = 3.5, 1.9 Hz, 1H), 5.64 (d, J = 6.5 Hz, 1H), 5.12 (p, J = 6.5 Hz, 1H), 4.89−4.74 (m, 1H), 3.42− 3.31 (m, 2H), 2.92−2.74 (m, 1H), 2.46−2.29 (m, 3H), 2.07 (d, J = 12.2 Hz, 2H), 1.98−1.83 (m, 2H), 1.63 (d, J = 6.3 Hz, 3H), 1.40−1.22 (m, 2H). LCMS (method LCMS1, ESI): RT = 2.57 min, m/z = 382.4 [M + H]+. (R)-1-(1-(trans-4-((2,2-Difluoroethyl)amino)cyclohexyl)-1,6dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)ethanol (27). 27 was synthesized by methods similar to those used for 26, substituting 2,2-difluoroethyl trifluoromethanesulfonate for 2,2,2trifluoroethyl trifluoromethanesulfonate. 1H NMR (400 MHz, DMSO-d6) δ 11.81 (s, 1H), 8.54 (s, 1H), 7.46 (t, J = 2.9 Hz, 1H), 6.69 (t, J = 2.5 Hz, 1H), 6.01 (tt, J = 56.6, 4.3 Hz, 1H), 5.64 (d, J = 6.2 Hz, 1H), 5.12 (p, J = 6.5 Hz, 1H), 4.90−4.74 (m, 1H), 3.01 (t, J = 16.2 Hz, 2H), 2.89−2.72 (m, 1H), 2.38 (q, J = 12.8 Hz, 2H), 2.12 (d, J = 12.0 Hz, 2H), 2.01−1.83 (m, 3H), 1.63 (d, J = 6.4 Hz, 3H), 1.37−1.19 (m, 2H). LCMS (method LCMS1, ESI): RT = 2.20 min, m/z = 364.4 [M + H]+. (R)-1-(1-(cis-4-((2,2,2-Trifluoroethyl)amino)cyclohexyl)-1,6dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)ethanol (28). 28 was synthesized by methods similar to those used for 26, substituting tert-butyl (cis-4-aminocyclohexyl)carbamate (28a)30 for tert-butyl (trans-4-aminocyclohexyl)carbamate (26a). 1H NMR (400 MHz, DMSO-d6) δ 11.65 (s, 1H), 8.51 (s, 1H), 7.64 (s, 1H), 7.27 (t, J = 3.0 Hz, 1H), 5.62 (d, J = 6.0 Hz, 1H), 5.11 (p, J = 6.4 Hz, 1H), 4.81 (tt, J = 12.3, 4.2 Hz, 1H), 3.43−3.32 (m, 2H), 3.00 (s, 1H), 2.79−2.58 (m, 3H), 1.99 (d, J = 13.2 Hz, 2H), 1.72−1.52 (m, 7H). LCMS (method LCMS1, ESI): RT = 8.43 min, m/z = 382.2 [M + H]+. (R)-1-(1-((1S,3R)-3-((2,2,2-Trifluoroethyl)amino)cyclopentyl)1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)ethanol (29). 29 was synthesized by methods similar to those used for 26, substituting tert-butyl ((1R,3S)-3-aminocyclopentyl)carbamate (29a)31 for tert-butyl (trans-4-aminocyclohexyl)carbamate (26a). 1H NMR (400 MHz, DMSO-d6) δ 11.80 (s, 1H), 8.54 (s, 1H), 7.42 (t, J = 3.0 Hz, 1H), 6.96 (dd, J = 3.4, 1.9 Hz, 1H), 5.65 (d, J = 6.5 Hz, 1H), 5.38−5.23 (m, 1H), 5.12 (p, J = 6.5 Hz, 1H), 3.43−3.33 (m, 1H), 3.30−3.21 (m, 2H), 2.81 (td, J = 7.7, 5.0 Hz, 1H), 2.42 (td, J = 12.0, 11.3, 6.6 Hz, 1H), 2.32 (dt, J = 13.2, 6.8 Hz, 1H), 2.24−2.00 (m, 3H),

1.93−1.80 (m, 1H), 1.62 (d, J = 6.5 Hz, 3H). LCMS (method LCMS1, ESI): RT = 7.54 min, m/z = 368.2 [M + H]+. (R)-1-(1-((1S,3S)-3-((2,2,2-Trifluoroethyl)amino)cyclopentyl)1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)ethanol (30). 30 was synthesized by methods similar to those used for 26, substituting tert-butyl ((1S,3S)-3-aminocyclopentyl)carbamate (30a)32 for tert-butyl (trans-4-aminocyclohexyl)carbamate (26a). 1H NMR (400 MHz, DMSO-d6) δ 11.83 (s, 1H), 8.55 (s, 1H), 7.45 (t, J = 3.0 Hz, 1H), 6.49 (dd, J = 3.5, 1.8 Hz, 1H), 5.60 (d, J = 6.6 Hz, 1H), 5.52 (p, J = 9.5 Hz, 1H), 5.10 (p, J = 6.5 Hz, 1H), 3.71−3.62 (m, 1H), 3.32−3.20 (m, 2H), 2.64−2.56 (m, 1H), 2.48−2.11 (m, 4H), 2.06− 1.96 (m, 1H), 1.71−1.62 (m, 1H), 1.64 (d, J = 6.4 Hz, 3H). LCMS (method LCMS1, ESI): RT = 2.58 min, m/z = 368.1 [M + H]+. trans-4-((5-Nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)amino)cyclohexanecarbonitrile (31b). A mixture of trans-4-aminocyclohexanecarbonitrile trifluoroacetic acid salt33 (31a, 6.36 g, 26.7 mmol), 359 (8.21 g, 24.3 mmol) and DIPEA (12.5 mL, 73.0 mmol) in isopropyl alcohol (200 mL) was heated at reflux for 3 h. The cooled mixture was filtered and the precipitate washed with isopropyl alcohol (100 mL) to afford 9.04 g (87%) of the title compound as a bright-yellow solid. 1H NMR (400 MHz, CDCl3) δ 9.11 (s, 1H), 9.07 (d, J = 7.7 Hz, 1H), 8.20 (m, 2H), 7.63 (m, 2H), 7.53 (m, 2H), 6.67 (d, J = 4.2 Hz, 1H), 4.06 (m, 1H), 2.66 (m, 1H), 2.24 (m, 4H), 1.83 (m, 2H), 1.58 (m, 2H). LCMS (method LCMS11, ESI): RT = 4.15 min, m/z = 426.0 [M + H]+. trans-4-((5-Amino-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)amino)cyclohexanecarbonitrile (31c). A suspension of 31b (5.00 g, 11.8 mmol), iron powder (−325 mesh, 2.59 g, 47.0 mmol), and ammonium chloride (3.74 g, 70.5 mmol) in a mixture of EtOH and water (3:1, 400 mL) was mechanically stirred at reflux for 4 h. The cooled mixture was filtered through Celite. The filtrate was concentrated under vacuum to remove the majority of the EtOH, then the residual aqueous layer was extracted with DCM (3 × 100 mL). The combined extracts were washed with water and brine (100 mL each), dried over Na2SO4, filtered, and concentrated under vacuum. Purification by diethyl ether trituration afforded 4.51 g (97%) of the title compound as a light-gray solid. LCMS (method LCMS11, ESI): RT = 2.69 min, m/z = 396.1 [M + H]+. trans-4-(2-((R)-1-Hydroxyethyl)-6-(phenylsulfonyl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)cyclohexanecarbonitrile (31d). A suspension of triethyloxonium tetrafluoroborate (5.36 g, 28.2 mmol) and (R)-(+)-lactamide (2.85 g, 32.0 mmol) in dry THF (40 mL) was stirred at 23 °C under a nitrogen atmosphere for 2 h.23 The mixture was concentrated under vacuum and stirred in absolute EtOH (70 mL) for 10 min. 31c (4.00 g, 10.1 mmol) was added, and the reaction mixture was heated at reflux for 2 h. The cooled mixture was concentrated under vacuum, and the residue was triturated with EtOAc to afford 4.50 g (99%) of the title compound as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.71 (s, 1H), 8.13 (m, 2H), 7.94 (d, J = 4.1 Hz, 1H), 7.69 (m, 1H), 7.61 (m, 2H), 7.25 (m, 1H), 5.74 (d, J = 6.7 Hz, 1H), 5.14 (m, 1H), 4.85 (m, 1H), 3.23 (m, 1H), 2.20 (m, 4H), 1.85 (m, 4H), 1.62 (d, J = 6.4 Hz, 3H). LCMS (method LCMS11, ESI): RT = 3.33 min, m/z = 450.3 [M + H]+. trans-4-(2-((R)-1-Hydroxyethyl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)cyclohexanecarbonitrile (31). A solution of 31d (1.20 g, 2.67 mmol) in MeOH:THF (1:1, 150 mL) was treated with a 1 M solution of aqeuous sodium hydroxide (20 mL) and stirred at 23 °C for 3 h. The organic solvent was removed under vacuum, and the aqueous residue was extracted with EtOAc (3 × 20 mL). The combined organic extracts were washed with water and brine (20 mL each), dried over Na2SO4, filtered, and concentrated under vacuum. Purification by column chromatography on silica gel (gradient: 0−10% [2 M NH3 in MeOH] in EtOAc) with further purification by reverse phase HPLC (XBridge Prep C18 OBD, 19 mm × 250 mm, 5 μm; detection, UV 220 and 254 nm; mobile phase A, water with 0.1% NH4OH; mobile phase B, CH3CN with 0.1% NH4OH; flow rate, 18 mL/min; gradient 5−75% B over 20 min) afforded 149 mg (18%) of the title compound as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 11.82 (s, 1H), 8.54 (s, 1H), 7.44 (t, J = 2.9 Hz, 1H), 6.79 (t, J = 2.5 Hz, 1H), 5.63 (d, J = 6.0 Hz, 1H), 5.13 (p, J = 6.3 Hz, 1H), 4.90−4.76 R



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(m, 1H), 3.21−3.05 (m, 1H), 2.41−2.18 (m, 4H), 2.01−1.90 (m, 2H), 1.90−1.71 (m, 2H), 1.63 (d, J = 6.4 Hz, 3H). LCMS (method LCMS1, ESI): RT = 4.48 min, m/z = 310.1 [M + H]+. cis-4-(2-((R)-1-Hydroxyethyl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)cyclohexanecarbonitrile (32). 32 was synthesized by methods similar to those used for 31, substituting cis-4aminocyclohexanecarbonitrile trifluoroacetic acid salt (31a)33 for trans4-aminocyclohexanecarbonitrile trifluoroacetic acid salt (32a). 1H NMR (400 MHz, DMSO-d6) δ 11.82 (s, 1H), 8.55 (s, 1H), 7.45 (t, J = 3.0 Hz, 1H), 7.18 (s, 1H), 5.65 (d, J = 6.8 Hz, 1H), 5.14 (p, J = 6.6 Hz, 1H), 4.89−4.76 (m, 1H), 3.34 (br s, 1H), 2.60−2.46 (m, 2H), 2.23− 2.10 (m, 2H), 2.01−1.81 (m, 4H), 1.64 (d, J = 6.4 Hz, 3H). LCMS (method LCMS1, ESI): RT = 4.42 min, m/z = 310.1 [M + H]+. 2-(trans-4-(2-((R)-1-Hydroxyethyl)imidazo[4,5-d]pyrrolo[2,3b]pyridin-1(6H)-yl)cyclohexyl)acetonitrile (33). 33 was synthesized by methods similar to those used for 31, substituting 2-(trans-4aminocyclohexyl)acetonitrile (33a)27 for trans-4-aminocyclohexanecarbonitrile trifluoroacetic acid salt (31a). 1H NMR (400 MHz, DMSO-d6) δ 11.82 (s, 1H), 8.54 (s, 1H), 7.45 (t, J = 2.9 Hz, 1H), 6.72 (t, J = 2.4 Hz, 1H), 5.66 (d, J = 6.2 Hz, 1H), 5.13 (p, J = 6.5 Hz, 1H), 4.90−4.76 (m, 1H), 2.59 (d, J = 5.8 Hz, 2H), 2.45−2.30 (m, 2H), 2.14−1.88 (m, 5H), 1.63 (d, J = 6.3 Hz, 3H), 1.49−1.29 (m, 2H). LCMS (method LCMS1, ESI): RT = 4.86 min, m/z = 324.1 [M + H]+. 3-(trans-4-(2-((R)-1-Hydroxyethyl)imidazo[4,5-d]pyrrolo[2,3b]pyridin-1(6H)-yl)cyclohexyl)propanenitrile (34). 34 was synthesized by methods similar to those used for 31, substituting 3-(trans4-aminocyclohexyl)propanenitrile trifluoroacetic acid salt (34a)27 for trans-4-aminocyclohexanecarbonitrile trifluoroacetic acid salt (31a). 1 H NMR (400 MHz, DMSO-d6) δ 11.82 (s, 1H), 8.54 (s, 1H), 7.47 (t, J = 3.0 Hz, 1H), 6.68 (t, J = 2.5 Hz, 1H), 5.64 (d, J = 5.5 Hz, 1H), 5.12 (p, J = 6.5 Hz, 1H), 4.90−4.73 (m, 1H), 2.61 (t, J = 7.3 Hz, 2H), 2.43−2.29 (m, 2H), 2.05−1.86 (m, 4H), 1.76−1.56 (m, 6H), 1.31− 1.14 (m, 2H). LCMS (method LCMS1, ESI): RT = 6.13 min, m/z = 338.1 [M + H]+.

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ACKNOWLEDGMENTS We thank Michael Hayes, Mengling Wong, Baiwei Lin, Deven Wang, Yutao Jiang, and Yanzhou Liu for purification and analytical support; Daniel Hascall, Grady Howes, Gigi Yuen, Garima Porwal, and Mandy Yim for compound management support; Hoa Le and Qin Yue for ADME support; Emile Plise and Jonathan Cheng for MDCK data; Erlie Delarosa for hepatocyte stability; Ning Liu for microsomal stability studies; Suzanne Tay for TDI studies; Jasleen Sodhi for reversible CYP inhibition measurement; Quynh Ho for plasma protein binding measurement; Wei Jia, Po-Chang (Tom) Chiang, and Amy Sambrone for formulations support; Zhiyu Huang for collation of data from the rat CIA study.

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ABBREVIATIONS USED ADME, absorption, distribution, metabolism, and excretion; AUC, area under the curve; Bn, benzyl; Boc, tert-butyl carbamate; brine, a saturated aqueous solution of sodium chloride; CH3CN, acetonitrile; CIA, collagen induced arthritis; CL, clearance; CLH, hepatic clearance, CLint, intrinsic clearance; cLogD7.4, calculated log of partition coefficient between octanol and pH7.4 aqueous buffer; Cmax, maximum concentration; CYP, cytochrome P450; D, aspartic acid; DCM, dichloromethane; DEA, diethylamine, DIPEA, N,N-diisopropylethylamine; DMF, dimethylformamide; DMSO, dimethylsulfoxide; E, glutamic acid; EC 50, half-maximal effective concentration; EPO, erythropoietin; EtOAc, ethyl acetate; EtOH, ethanol; ex, example; F, oral bioavailability; Fmax, theoretical maximum achievable oral bioavailability; h, hour; HBD, hydrogen-bond donor; hERG, human ether-a-go-go-related gene; HLM, human liver microsome; HPLC, high performance liquid chromatography; IL-6, interleukin-6; i-PrOH, isopropyl alcohol; IV, intravenous; JAK, Janus kinase; JAK1 biochemical selectivity index, JAK2 Ki ÷ JAK1 Ki; JAK1 cellular selectivity index, EPOpSTAT5 EC50 ÷ IL6-pSTAT3 EC50; Ki, inhibition constant; LCMS, liquid chromatography−mass spectrometry; LE, ligand efficiency; LLE, ligand-lipophilicity efficiency; MCT, methyl cellulose/Tween; MDCK, Madin−Darby canine kidney; MeOH, methanol; min, minute; Papp, apparent permeability; PEG400, polyethylene glycol 400; Ph, phenyl; PK, pharmacokinetics; PO, by mouth; PPB, plasma protein binding; PSI, pounds per square inch; RA, rheumatoid arthritis; RT, retention time; SAR, structure−activity relationships; SFC, supercritical fluid chromatography; STAT, signal transducer and activator of transcription; (tmp)2Mg·2LiCl, 2,2,6,6tetramethylpiperidinemagnesium chloride lithium chloride complex; TEA, triethylamine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TPSA, topological polar surface area; UV, ultraviolet

ASSOCIATED CONTENT

S Supporting Information *

Synthetic procedures for 16a, 33a, 34a, and 25; identities of kinases in Table 6 and % inhibition by 21, 26, 29, 31, 33, and 34; biochemical JAK3 and TYK2 Ki values of 1−34; tabulated compound structures and data used to produce the graphs in Figure 2. Details of: in vitro and in vivo ADME experimental procedures, JAK enzymatic and cellular assays (low serum and whole blood), in vitro hERG assay, CIA efficacy study of 31 in the rat, protein expression/purification, and crystallographic methods and procedures for 31 (in complex with JAK1 and JAK2) and 33 and 34 (in complex with JAK1), thermodynamic solubility methods, and analytical LCMS and SFC conditions. This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes

PDB numbers: 4EHZ for 2 complexed with JAK1, 4F09 for 2 complexed with JAK2, 4IVB for 31 complexed with JAK1, 4IVA for 31 complexed with JAK2, 4IVC for 33 complexed with JAK1, 4IVD for 34 complexed with JAK1.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Phone: 650-467-4533. E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. S



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Stewart, K. D.; Hajduk, P. J. Cheminformatic Tools for Medicinal Chemists. J. Med. Chem. 2010, 53, 4830−4841. (o) Rogers, D.; Hahn, M. Extended-Connectivity Fingerprints. J. Chem. Inf. Model. 2010, 50, 742−754. (p) Ortwine, D. F.; Aliagas, I. Physicochemical and DMPK in silico models: facilitating their use by medicinal chemists. Mol. Pharmaceutics 2013, 10, 1153−1161. (18) Williams, N. K.; Bamert, R. S.; Patel, O.; Wang, C.; Walden, P. M.; Wilks, A. F.; Fantino, E.; Rossjohn, J.; Lucet, I. S. Dissecting specificity in the Janus kinases: the structures of JAK-specific inhibitors complexed to the JAK1 and JAK2 protein tyrosine kinase domains. J. Mol. Biol. 2009, 387, 219−232. (19) Chrencik, J. E.; Patny, A.; Leung, I. K.; Korniski, B.; Emmons, T. L.; Hall, T.; Weinberg, R. A.; Gormley, J. A.; Williams, J. M.; Day, J. E.; Hirsch, J. L.; Kiefer, J. R.; Leone, J. W.; Fischer, H. D.; Sommers, C. D.; Huang, H. C.; Jacobsen, E. J.; Tenbrink, R. E.; Tomasselli, A. G.; Benson, T. E. Structural and thermodynamic characterization of the TYK2 and JAK3 kinase domains in complex with CP-690550 and CMP-6. J. Mol. Biol. 2010, 400, 413−433. (20) The maximum achievable oral bioavailability (Fmax) is the product of the fraction absorbed (Fa), the fraction escaping intestinal metabolism (Fg), and the fraction escaping hepatic metabolism (Fh), as expressed by the equation: Fmax = Fa·Fg·Fh. Assuming a best case scenario of no intestinal wall metabolism, and complete absorption of the oral dose (Fg and Fa = 1), the equation is simplified to Fmax = Fh. Because Fh = 1 − CL/Qh, where CL is clearance and Qh is hepatic blood flow, it is apparent that the maximum achievable bioavailability should be controlled by clearance according to the equation: Fmax = 1 − CL/Qh. Hepatic blood flows of 70, 31, and 44 mL min−1 kg−1 were used for rat, dog, and cyno Fmax calculations, respectively. See: Rautio, J.; Kumpulainen, H.; Heimbach, T.; Oliyai, R.; Oh, D.; Järvinen, T.; Savolainen, J. Prodrugs: design and clinical applications. Nature Rev. Drug Discovery 2008, 7, 255−270. (21) Detailed experimental procedures were previously published in ref 7. (22) Meerwein, H. Triethyloxonium fluoroborate. Org. Synth. 1973, 5, 1080. (23) (a) In some instances when the imidate formation with triethyloxonium tetrafluoroborate was performed in THF, polymerization of the solvent occurred to form a viscous mass and the reaction could not be continued. Similar observations were previously reported: Vartak, A. P.; Crooks, P. A. Org. Process Res. Dev. 2009, 13, 415. (b) Imidate formation can also be carried out in DCM: Kiessling, A. J.; McClure, C. K. Synth. Commun. 1997, 27, 923. (24) Clososki, G. C.; Rohbogner, C. J.; Knochel, P. Direct Magnesiation of Polyfunctionalized Arenes and Heteroarenes Using (tmp)2Mg·2LiCl. Angew. Chem., Int. Ed. 2007, 46, 7681−7684. (25) All amines were either purchased from commercial sources, synthesized as described in the references in the Experimental Section or synthesized by the methods in the Supporting Information. (26) Broo, A.; Holm, P.; Judkins, R.; Li, L.; Lindstedt-Alstermark, E.; Sandberg, P.; Swanson, M.; Weidolf. L.; Mrickmann, K. Non-anilinic derivatives of isothiazol-3(2H)-one 1,2-dioxides as Liver X receptor modulators. WO2006073366, 2006. (27) Prepared as described in the Supporting Information. (28) Savage, S.; Babu, S.; Zak, M.; Mao, Z.; Cao, J.; Ge, Y.; Ma, D.; Jiang, G. Kilogram synthesis of (S)-3-aminopyran from L-glutamic acid. Synlett 2013, 24, 987−990. (29) Brocklehurst, C. E.; Laumen, K.; La Vecchia, L.; Shaw, D.; Vögtle, M. Diastereoisomeric salt formation and enzyme-catalyzed kinetic resolution as complementary methods for the chiral separation of cis-/trans-enantiomers of 3-aminocyclohexanol. Org. Process Res. Dev. 2011, 15, 294−300. (30) Sekiguchi, Y.; Kanuma, K.; Omodera, K.; Busujima, T.; Tran, T.; Han, S.; Casper, M.; Kramer, B. A.; Semple, G.; Zou, N. Quinoline, tetrahydroquinoline and pyrimidine derivatives as MCH antagonist. EP1464335, 2004. (31) Sekiguchi, Y.i; Kanuma, K.; Omodera, K.; Busujima, T.; Tran, T.; Han, S.; Casper, M.; Kramer, B. A. Preparation of novel quinazolines as MCH receptor antagonists. WO2004087680, 2004. U



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(32) Thompson, W.; Young, S. D.; Phillips, B. T.; Munson, P.; Whitter, W.; Liverton, N.; Dieckhaus, C.; Butcher, J.; Mccauley, J. A.; Mcintyre, C. J.; Layton, M. E.; Sanderson, P. E. 4-Cycloalkylaminopyrazolopyrimidine NMDA/NR2B antagonists. US20050054658. 2005. (33) Aahlin, K.; Arvidsson, P. I.; Besidski, Y.; Nilsson, L. I. Preparation of cyano group containing isoindoline derivatives for treating pain disorders. WO2009145719, 2009.

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