Discovery and optimization of C-2 methyl imidazopyrrolopyridines as potent and orally bioavailable JAK1 inhibitors with selectivity over JAK2

Article abstract of DOI:10.1021/jm300628c

Herein we report the discovery of the C-2 methyl substituted imidazopyrrolopyridine series and its optimization to provide potent and orally bioavailable JAK1 inhibitors with selectivity over JAK2. The C-2 methyl substituted inhibitor 4 exhibited not only improved JAK1 potency relative to unsubstituted compound 3 but also notable JAK1 vs JAK2 selectivity (20-fold and >33-fold in biochemical and cell-based assays, respectively). Features of the X-ray structures of 4 in complex with both JAK1 and JAK2 are delineated. Efforts to improve the in vitro and in vivo ADME properties of 4 while maintaining JAK1 selectivity are described, culminating in the discovery of a highly optimized and balanced inhibitor (20). Details of the biological characterization of 20 are disclosed including JAK1 vs JAK2 selectivity levels, preclinical in vivo PK profiles, performance in an in vivo JAK1-mediated PK/PD model, and attributes of an X-ray structure in complex with JAK1.

Full text of DOI:10.1021/jm300628c

Article
pubs.acs.org/jmc
Discovery and Optimization of C-2 Methyl Imidazopyrrolopyridines
as Potent and Orally Bioavailable JAK1 Inhibitors with Selectivity
over JAK2
Mark Zak,*,† Rohan Mendonca,† Mercedesz Balazs,× Kathy Barrett,‡ Philippe Bergeron,† Wade S. Blair,‡
Christine Chang,‡ Gauri Deshmukh,§ Jason DeVoss,× Peter S. Dragovich,† Charles Eigenbrot,∞
Nico Ghilardi,∥ Paul Gibbons,† Stefan Gradl,† Chris Hamman,† Emily J. Hanan,† Eric Harstad,⊥
Peter R. Hewitt,○ Christopher A. Hurley,○ Tian Jin,● Adam Johnson,‡ Tony Johnson,○ Jane R. Kenny,§
Michael F. T. Koehler,† Pawan Bir Kohli,‡ Janusz J. Kulagowski,○ Sharada Labadie,† Jiangpeng Liao,●
Marya Liimatta,‡ Zhonghua Lin,× Patrick J. Lupardus,∞ Robert J. Maxey,○ Jeremy M. Murray,∞
Rebecca Pulk,† Madeleine Rodriguez,× Scott Savage,# Steven Shia,∞ Micah Steffek,∞ Savita Ubhayakar,§
Mark Ultsch,∞ Anne van Abbema,‡ Stuart I. Ward,○ Ling Xiao,● and Yisong Xiao●
†
‡
§
Departments of Discovery Chemistry, Biochemical and Cellular Pharmacology, Drug Metabolism and Pharmacokinetics,
⊥
#
×
∥Immunology, Safety Assessment, Small Molecule Process Chemistry, ∞Structural Biology, and 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 the discovery of the C-2
methyl substituted imidazopyrrolopyridine series and its
optimization to provide potent and orally bioavailable JAK1
inhibitors with selectivity over JAK2. The C-2 methyl
substituted inhibitor 4 exhibited not only improved JAK1
potency relative to unsubstituted compound 3 but also notable
JAK1 vs JAK2 selectivity (20-fold and >33-fold in biochemical
and cell-based assays, respectively). Features of the X-ray
structures of 4 in complex with both JAK1 and JAK2 are
delineated. Efforts to improve the in vitro and in vivo ADME properties of 4 while maintaining JAK1 selectivity are described,
culminating in the discovery of a highly optimized and balanced inhibitor (20). Details of the biological characterization of 20 are
disclosed including JAK1 vs JAK2 selectivity levels, preclinical in vivo PK profiles, performance in an in vivo JAK1-mediated PK/
PD model, and attributes of an X-ray structure in complex with JAK1.
The JAKs are activated in specific patterns by different
cytokines that play essential roles in immune function,9
inflammation,10 and hematopoiesis.11 For example, members
of the γ common (γc) subfamily, namely, interleukins IL-2, IL-
4, IL-7, IL-9, IL-15, and IL-21, activate JAK1 and JAK3 but
never JAK2 or TYK2.12 The importance of the γc cytokines to
the immune system is highlighted by the observation of SCID
(severe combined immunodeficiency) when loss of function
mutations occur in the γc chain or JAK3.13 Another large
subfamily of cytokines shares the glycoprotein 130 (gp130)
signal transducing subunit and includes IL-6, IL-11, IL-27, and
several other cytokines. Signaling by these cytokines always
involves JAK1 activation, and JAK2 and TYK2 are also
consistently engaged.14 IL-6 is heavily implicated in immune
INTRODUCTION
■
The Janus protein tyrosine kinases (JAK1,1 JAK2,2 JAK3,3 and
TYK24) are key intracellular mediators of helical cytokine5
signaling pathways. The cytokine ligands signal to cells by
interacting with the extracellular portions of homo- or
heterodimeric cell surface transmembrane receptors,6 which
are further engaged with JAKs in their cytoplasmic domains.
Upon ligand binding, these receptors induce activation of the
associated JAKs, a mandatory initial step for all downstream
signaling events, since the receptors themselves have no
intrinsic kinase activity. JAK activation results in a cascade of
phosphorylation and recognition events that culminate in the
phosphorylation, dimerization, and nuclear translocation of one
or several signal transducer and activator of transcription
(STAT)7 proteins. Once inside the nucleus, the STATs
modulate gene transcription and expression.8
Received: May 5, 2012
© XXXX American Chemical Society
A
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response, and excessive stimulation of this pathway is linked to
various autoimmune and chronic inflammatory conditions.15
Finally, the receptor for erythropoietin (EPO) represents a
subfamily of homodimeric receptors that also includes the
receptors for prolactin, thrombopoietin, and growth hormone.
The EPO pathway activates JAK2 exclusively16 and is essential
to red blood cell formation or erythropoiesis.17
Each JAK isoform is employed by multiple cytokine
pathways, and by extension, the biological activities of many
cytokines may be abrogated by inhibition of a single JAK
isoform. Since several of these cytokine activities regulate
immune function, targeted inhibition of JAKs offers an
attractive opportunity for therapeutic intervention against
rheumatoid arthritis (RA) and other immunologic disorders.
A number of small molecule JAK inhibitors have been evaluated
in clinical trials for the treatment of RA, including tofacitinib/
CP-690,550 (1)18−21 and ruxolitinib/INCB018424 (2).22−24
The preclinical characteristics of both molecules, including the
JAK isoform potency and selectivity, have been extensively
profiled by other groups.25,26 Consistent with these previously
reported results, our biochemical assays show that 1 inhibits
JAK1, JAK2, and JAK3 to an approximately equal extent and is
less potent against TYK2, while 2 is a JAK1, JAK2, and TYK2
inhibitor with reduced potency against JAK3 (Figure 1). The
show significant activity in both cell-based assays. Each
molecule’s IL6-pSTAT3 EC50 is less than 55 nM and is within
2-fold of its corresponding EPO-pSTAT5 value, indicating
potent inhibition of both JAK1 and JAK2 function in TF-1
cells.
Both 1 and 2 have demonstrated clinical efficacy in patients
suffering from RA.20,21,23a,24 Although suppression of JAK3 and
TYK2 activity may contribute to these favorable clinical
outcomes, several lines of evidence point to JAK1 inhibition
playing a critical and potentially dominant role. The first
evidence comes from the divergent in vitro potency profiles of
these compounds against the individual Janus kinase family
members. Compounds 1 and 2 have considerably reduced
potency against either TYK2 or JAK3, respectively, demon-
strating that significant suppression of those kinase activities is
likely not a prerequisite to observing clinical modulation of
immune function. The second piece of evidence comes from
the biology surrounding the proinflammatory cytokine IL-6.
Although IL-6 activates three Janus family members (JAK1,
JAK2, and TYK2), knockout studies in mice show that JAK1 is
especially important to signal transduction. Indeed, after IL-6
stimulation, cells from JAK1 deficient mice show profound
reductions in downstream signaling compared to wild type,
whereas cells deficient in TYK2 or JAK2 maintain robust IL-6
responsiveness.28−30 The clinical success of the biological agent
tocilizumab31 against RA is therefore consistent with the
hypothesis that selective JAK1 inhibition will lead to
therapeutic immune modulation. Tocilizumab is a neutralizing
antibody directed against the IL-6 receptor α chain and is
believed to exert its therapeutic effects by inhibiting the activity
of the JAK1 dependent cytokine IL-6. The final evidence
supporting JAK1 as an appropriate target for immune
modulation comes from recent work suggesting that JAK1
rather than JAK3 kinase function is dominant in driving the
activity of the immunorelevant γc cytokines.32
On the basis of the above lines of evidence, we believed that
a selective JAK1 inhibitor would provide efficacy against RA.
We further believed that an ideal RA therapy would minimize
inhibition of JAK2. JAK2 plays an integral role in the EPO
signaling pathway necessary for red blood cell formation, and
its inactivation leads to anemia in animal models.30,33
Additionally, hemoglobin reduction and anemia have been
noted in human patients enrolled in clinical trials of
compounds 1 and 2, both potent inhibitors of JAK2
activity.20,34 The authors describing the results of one such
trial note that hemoglobin reductions at higher doses of
tofacitinib (>5 mg b.i.d.) may be the result of effects on
hematopoiesis due to JAK2 inhibition.20a On the basis of the
published clinical results, we believed that even subtle
selectivity for JAK1 over JAK2, on the order of 5- to 10-fold,
could widen the therapeutic index for JAK1 mediated anti-RA
effects relative to JAK2 dependent anemia. We therefore
embarked on a program to identify potent and orally
bioavailable small molecule inhibitors of JAK1, with selectivity
over JAK2,35 as potential therapies for RA and other
immunologic disorders.
Figure 1. Inhibition of biochemical and cell-based JAK activity by 1
and 2. Superscript letters indicate the following: (a) biochemical
assays; (b) arithmetic mean of at least three separate runs (n ≥ 3),
where on average, the coefficients of variation were less than 0.3 times
the mean for biochemical assays and less than 0.5 times the mean for
cell-based assays; (c) cell-based measure of JAK1 inhibition; (d) cell-
based measure of JAK2 inhibition.
JAK1 and JAK2 biochemical results are supported by our cell-
based measures of both JAK1 and JAK2 potency. Phosphor-
ylation of STAT3 in TF-1 cells following IL-6 stimulation is
driven predominantly by JAK1 kinase activity and forms the
basis of our cellular assessment of JAK1 inhibition (IL6-
pSTAT3 assay).27 The corresponding assessment of cellular
JAK2 inhibition is also carried out in TF-1 cells but relies on
measurement of phospho-STAT5 levels following EPO
stimulation (EPO-pSTAT5 assay).27 Compounds 1 and 2
RESULTS AND DISCUSSION
■
The discovery of the imidazopyrrolopyridine 3 (Figure 2)
represented an important initial step in our JAK1 program.36,37
As previously reported,36 3 was found to be a moderately
potent biochemical inhibitor of JAK1 (Ki = 46 nM) with
reduced potency for JAK2 (Ki = 250 nM), resulting in a
B
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recalcitrant permeability. As such, a series of analogues (5−9)
40
with increased log D7.2 was synthesized. Additionally, as a
means to further enhance permeability, these initial derivatives
were all designed to contain only a single hydrogen-bond donor
(HBD), one less than in compound 4.
As hoped, increasing the log D7.2 of the single HBD-
containing analogues resulted in MDCK permeability improve-
ments to the moderate (6 and 9) or high (5, 7, and 8) range
(Table 1). A trend in the opposite direction was identified for
human hepatocyte stability, with the most lipophilic com-
pounds 5 and 7 exhibiting the worst metabolic stability.
Fortunately, compound 6 balanced moderate permeability with
excellent stability in human hepatocytes, demonstrating that an
optimal range of physicochemical properties existed in this
series. The initial series of analogues (5−9) also highlighted the
fact that JAK1 biochemical and cell-based potency could be
maintained or improved relative to 4 with a wide array of 4-
piperidine replacements. The JAK1 selectivity indexes,
however, were all significantly eroded by replacing the 4-
piperidine group resident in 4 with any of the HBD-lacking R-
groups listed in Table 1. In fact, the three compounds with
improved cellular potency (5, 6, and 7) all exhibited cell-based
JAK1 selectivity indexes of less than 5-fold. These findings
indicated that combining the C-2 methyl group with a HBD-
containing R-group could be important to JAK1 selectivity. As
such, focus was shifted to preparing additional analogues of 4
with R-groups containing HBD moieties.
Figure 2. Structure of compound 3 and inhibition of biochemical and
cell-based JAK1/JAK2 activity. Superscript letters indicate the
following: (a) biochemical assays; (b) arithmetic mean of eight
separate runs (n = 8), where on average, the coefficients of variation
were less than 0.3 times the mean for biochemical assays; (c)
Ki(JAK2)/Ki(JAK1); (d) cell-based measure of JAK1 inhibition; (e)
arithmetic mean of three separate runs (n = 3), where on average, the
coefficients of variation were less than 0.5 times the mean for cell-
based assays; (f) cell-based measure of JAK2 inhibition; (g) see ref 38;
(h) EC50(EPO-pSTAT5)/EC50(IL6-pSTAT3).
biochemical JAK1 selectivity index (Ki(JAK2)/Ki(JAK1)) of
5.4-fold. Compound 3 also showed modest potency in the cell-
based measurement of JAK1 inhibition (IL6-pSTAT3 EC50
=
1400 nM). Similar to the biochemical assay results, 3 was less
potent in the cell-based measure of JAK2 inhibition (EPO-
pSTAT5 EC50 > 8800 nM). Since its low potency precluded
definitive assessment of EC50 in the JAK2-driven EPO-pSTAT5
assay,38 it was not possible to report the unequivocal cell-based
JAK1 selectivity index of 3 (EC50(EPO-pSTAT5)/EC50(IL6-
pSTAT3)), beyond stating that it was greater than 6.3-fold.
Intrigued by the emerging JAK1 selectivity displayed by
compound 3, we wished to synthesize related analogues with
similar selectivity but improved potency.
In an attempt to balance optimal properties, lipophilicity
restraints were put in place in the subsequent design of
40
compounds 10−24, with log D7.2 values higher than that of
compound 4 (permeability considerations) but lower than
those of compounds 5 and 7 (metabolic stability concerns)
targeted for the majority of analogues. The 3-substituted
piperidine 10 was found to be more lipophilic than the
corresponding 4-substituted analogue (4), presumably because
of closer proximity of the basic center to the electron-
withdrawing tricyclic system. However, 10 displayed a large loss
in biochemical JAK1 potency with only minimal gain in MDCK
permeability relative to 4. Increasing log D7.2 and modulating
the basicity of the 4-piperidine group with fluorine atoms
proved to be a more fruitful line of investigation. The trans-
monofluoro analogue 11 displayed superior biochemical and
cell-based JAK1 potency relative to its cis counterpart 12 and
the starting point compound 4. Additionally, compound 11 had
much improved MDCK permeability relative to 4, and although
JAK1 vs JAK2 selectivity was somewhat diminished, reasonable
JAK1 biochemical and cell-based indexes were maintained (5.3-
fold and 15-fold, respectively). The difluorinated piperidine 13
also had improved MDCK permeability relative to 4. Among
the trans-1,4-diaminocyclohexanes examined (14−17), only the
most lipophilic analogue (compound 17) was found to exhibit
high levels of MDCK permeability. Unfortunately the
promising MDCK permeability of compound 17 came at the
expense of reduced stability in human hepatocytes relative to
compound 4. Although a related example (compound 14)
displayed suboptimal permeability, it was modestly enhanced
relative to 4. Biochemical and cell-based JAK1 potency were
also improved, while excellent human hepatocyte stability was
maintained. These findings, coupled with respectable JAK1
selectivity indexes (biochemical, 6.2-fold; cell based, 15-fold),
allowed 14 to remain of interest for further progression.
Compounds 15 and 16, bearing N-sulfonylmethyl and N-acetyl
moieties, respectively, both lost significant potency in the cell-
During the optimization campaign of inhibitor 3, a very
promising C-2 methyl substituted analogue 4 was identified. As
shown in Table 1, compound 4 exhibited notably increased
biochemical inhibition of JAK1 (Ki = 10 nM), with only a
minimal gain against JAK2 (Ki = 200 nM) relative to the des-
methyl analogue 3. The larger increase in JAK1 relative to JAK2
inhibition of compound 4 resulted in another positive outcome,
namely, increased JAK1 selectivity (biochemical JAK1
selectivity indexes: 3, 5.4-fold; 4, 20-fold). Also of note, the
C-2 methyl group’s amplification of biochemical JAK1
inhibition was mirrored in the cell-based measure of JAK1
potency (IL6-pSTAT3 EC50: compound 3, 1400 nM;
compound 4, 250 nM). The weak potency of both compounds
3 and 4 in the cellular measure of JAK2 inhibition (EPO-
pSTAT5 EC50: compound 3, >8800 nM; compound 4, >8200
nM)38 precluded a definitive conclusion on the impact of the
C-2 methyl group on the cell-based JAK1 selectivity index.
However, it is likely that the cell-based JAK1 selectivity index of
compound 4 (>33-fold) was greater than that of 3 (>6.3-fold).
Despite the promising selectivity profile and increased JAK1
inhibition relative to its C-2 unsubstituted counterpart,
compound 4 was still not an ideal development candidate. A
further increase in cell-based potency was desired, and
furthermore, 4 displayed poor apparent permeability as
measured by flux through MDCK cells in transwell culture
(A/B Papp = 0.4 × 10−6 cm/s). The low permeability elicited
concern that 4 would be poorly absorbed through the intestine
after oral dosing, thus potentially limiting its oral bioavailability
(vide infra). The topological polar surface area (TPSA)39 of
compound 4 was relatively low (58 Å2), pointing toward a very
low measured log D7.2 (0.1) as the probable explanation for the
C
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Table 1. In Vitro Biological and Physical Properties of Imidazopyrrolopyridine Analogues
a
Unless otherwise indicated, all compounds are single isomers. Where the possibility of chirality exists, data from only the most potent enantiomer
b
are shown. Biochemical assays. For brevity, only JAK1 and JAK2 data are reported. JAK3 and TYK2 inhibition data may be found in the Supporting
Information. Unless otherwise noted, all biochemical and cell-based assay results are reported as the arithmetic mean of at least three separate runs
c
(n = 3). On average, the coefficients of variation were less than 0.3 times the mean for biochemical assays and less than 0.5 times the mean for cell-
d
e
f
g
h
based assays. Ki(JAK2)/Ki(JAK1). Cell-based JAK1 assay. Cell-based JAK2 assay. EC50(EPO-pSTAT5)/EC50(IL6-pSTAT3). Measured log of
distribution coefficient between octanol and aqueous pH 7.2 buffer. Topological polar surface area.39 Apparent permeability in MDCK transwell
culture. A/B, apical-to-basolateral. In vitro stability in cryopreserved human hepatocytes. Arithmetic mean of 11 runs (n = 11) See ref 38.
Absolute stereochemistry assigned arbitrarily. Not determined. Relative stereochemistry known but absolute stereochemistry assigned arbitrarily.
n = 2. n = 1. Racemic mixture.
i
j
k
l
m
n
o
p
q
r
s
D
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based assays. A series of hydroxylated cyclohexanes (18−22)
and cyclopentanes (23 and 24) as replacements for the 4-
piperidine group of 4 was subsequently examined. Similar to
previous examples, the regio- and stereochemistries of the
hydroxyl substituents were important in modulating potency
and selectivity. The trans-3-hydroxy analogue 20 struck the best
balance among the hydroxylated derivatives and exhibited
excellent biochemical and cell-based potency, acceptable JAK1
selectivity indexes (biochemical, 6.7-fold; cell-based, 18-fold),
and greatly improved membrane permeability (MDCK A/B,
7.0 × 10−6 cm/s) relative to the starting point 4. 20 also
exhibited a high degree of stability in human hepatocytes. By
comparison, the remaining cyclohexanol isomers (18, 19, 21,
and 22) and cyclopentanol analogues (23 and 24) lost varying
degrees of JAK1 potency and selectivity.
The biochemical and cell-based measures of JAK1 and JAK2
inhibition led to similar conclusions about JAK1 vs JAK2
selectivity of the compounds in Table 1. Although there was a
trend toward subtly increased selectivity in the cellular context,
less than a 2-fold difference between biochemical and cell-based
measures of selectivity for any given compound was common.
Even in the most extreme case (compound 19) only a 3.4-fold
difference was observed between the biochemical and cell-based
JAK1 selectivity indexes. Furthermore, the compounds with the
greatest levels of cell-based selectivity (4, 11, 14, and 20) all
exhibited biochemical JAK1 selectivity indexes of greater than
5-fold, which reflected statistically significant differences in
those compounds’ JAK1 relative to JAK2 Ki values.41 While
compound 4 was undeniably the most selective JAK1 inhibitor
listed in Table 1, a number of better balanced molecules with
greatly increased chances of progression as orally delivered drug
candidates were identified. Notably, compounds 11, 14, and 20
all offered improvements in both JAK1 potency and
permeability relative to 4 while retaining cell-based JAK1
selectivity indexes of 15-fold or more. Both compound 4 and a
number of optimized analogues were progressed to in vivo rat
PK studies to evaluate the impact of MDCK apparent
permeability on oral exposure.
The in vivo rat PK parameters of reference compound 1 have
been previously reported.19,42 As summarized in Table 2,
compound 1 exhibited relatively high clearance after intra-
venous (iv) delivery, with oral bioavailability (F) greater than 2-
fold higher than the predicted theoretical maximum (Fmax).43
The compounds in the current work all exhibited similarly high
in vivo clearance in the rat, while exposure and bioavailability
varied widely after oral dosing. Two compounds (11 and 20)
exhibited a similar profile to compound 1 in that oral
bioavailability was substantially better than would be expected
based on iv clearance.43 It is unclear how the unexpectedly high
oral exposure was achieved or whether the same mechanism
was responsible for each compound.
Despite possessing an incomplete understanding of why
bioavailability for certain compounds was so good in the face of
high clearance, several observations allowed us to optimize oral
exposure. In vitro MDCK apparent permeability did indeed
appear to play an important role in influencing the in vivo oral
PK parameters, with the low permeability compounds (4 and
14) exhibiting the poorest oral bioavailability and exposure. A
combination of at least moderate permeability coupled with
high solubility at pH 7.4 appeared to be the best in vitro
predictor of good in vivo oral exposure. Compound 17
exhibited the best MDCK permeability but worst pH 7.4
solubility relative to the other compounds tested and exhibited
E
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Figure 3. Exposure vs time profiles after iv and oral doses of compound 20 in the dog and relevant in vitro and in vivo parameters. Superscript letters
indicate the following: (a) total plasma concentration of 20 after intravenous dosing (0.4 mg/kg), where the sample formulated is as a solution in
PEG400/citrate buffer (pH5.0) and data are the average from three separate animals; (b) total plasma concentration of 20 after oral dosing (2 mg/
kg), where the sample is formulated as a suspension in MCT and data are the average from three separate animals; (c) unbound plasma
concentration of 20 after oral dosing (2 mg/kg), [20]unbound = [20]total(1 − (PPB/100)); (d) in vitro IL6-pSTAT3 EC50 measured in TF-1 cells (87
nM); (e) dog plasma protein binding; (f) in vitro stability in cryopreserved dog hepatocytes; (g) theoretical maximum of achievable oral
bioavailability (see ref 43 for calculation, and a hepatic blood flow of 30.9 mL min−1 kg−1 in the dog is assumed); (h) amount of time unbound drug
plasma concentration following 2 mg/kg po dose remains in excess of in vitro IL6-pSTAT3 EC50.
Table 3. In Vitro Measures of Either JAK1 Inhibition or Off-Target Activities of Compound 20
reversible CYP
inhibition, IC50
(μM)
evidence of
time-dependent CYP
inhibition?
hERG:
JAK3
TYK2
no. of
f
f
IL6-pSTAT3 whole
% inhibition at
Ki
Ki
non-JAK
% inhibition
h
a
e
h
compd blood, EC50 (nM)
10 μM
(nM)
(nM)
kinases
at 0.1 μM
b
c
cd
,
g
g
20
160
>10
no
8.4
22
6.2
170
→
→
→
<50
i
6
j
50−75
>75
2
a
b
In vitro IL6-pSTAT3 cell-based assay conducted in TF-1 cells in the presence of human whole blood. Arithmetic mean of eight separate runs with
c
eight separate blood donors. CYP isoforms tested (LCMS/MS probes used): 3A4 (testosterone and midazolam), 2C9 (warfarin), 2D6
(dextromethorphan), 2C19 (mephenytoin), 1A2 (phenacetin). Absence of shift in AUC of the CYP inhibition curve of 20 with and without
NADPH preincubation. Patch clamp assay conducted by ChanTest. Biochemical assays. Arithmetic mean of six separate runs. Off-target kinase
inhibition assessed by SelectScreen Kinase Profiling Services (Invitrogen-Life Technologies, Madison, WI, U.S.). JAK1 was included as a positive
control and exhibited >95% inhibition at the concentration of 20 tested (0.1 μM). Non-JAK kinases exhibiting 50−75% inhibition at 0.1 μM 20:
CSF1R, MAP2K1, MINK1, cRAF, RSK3, RSK4. Non-JAK kinases exhibiting >75% inhibition at 0.1 μM 20: SGK3, LRRK2.
d
e
f
g
h
i
j
intermediate levels of oral exposure and bioavailability. By
comparison, 11 and 20 balanced moderate permeability with
excellent pH 7.4 solubility and achieved high levels of oral
bioavailability and exposure. To help benchmark the potential
biological relevance of these oral exposures, unbound plasma
concentrations following oral dosing were compared to the
corresponding in vitro EC50 values in the IL6-pSTAT3 cell-
based assay. The oral exposure profiles of compounds 4 and 14
were not sufficient to reach even transient maximum
concentrations of free drug in excess of the corresponding
IL6-pSTAT3 EC50 values. The greatly improved permeability of
17 relative to 4 and 14 translated to significantly better oral
exposure, and consequently, unbound plasma concentrations of
this compound exceeded the IL6-pSTAT3 EC50 for a portion of
the dosing interval (0.5 h). The optimized R-groups of
compounds 11 and 20 resulted in yet greater improvements
in vitro and in vivo PK parameters are shown in Figure 3. As
observed in other species, 20 exhibited low dog plasma protein
binding (50%) as well as excellent in vitro hepatocyte stability
(t1/2 > 8.0 h). Gratifyingly, compound 20 also exhibited
favorable in vivo PK characteristics in the dog, with low iv
clearance (5.8 mL min−1 kg−1) observed. The low iv clearance,
coupled with good MDCK permeability, and high aqueous
solubility were all consistent with the excellent oral
bioavailability observed for compound 20. Indeed, the
experimental bioavailability (95%) was only marginally higher
than the theoretical Fmax of 81%.43 The oral exposure achieved
by compound 20 translated to free plasma concentrations in
excess of the corresponding in vitro IL6-pSTAT3 EC50 value
for 15 h after a single oral dose of 2 mg/kg. The excellent in
vivo PK profile of compound 20 in the dog provided further
impetus for additional preclinical characterization.
As shown in Table 3, compound 20 was evaluated in a
number of additional in vitro assays measuring either inhibition
of JAK1 or off-target activities. Compound 20 exhibited low
human plasma protein binding (40% bound) and did not
distribute preferentially into red blood cells and thus
maintained potent activity in an in vitro TF-1 cell-based
measure of JAK1 inhibition conducted in the presence of
human whole blood (IL6-pSTAT3 whole blood EC50 = 160
nM). Compound 20 also exhibited negligible reversible or
time-dependent inhibition of cytochrome P450s (CYP) and
was thus considered to be at low risk for causing CYP-mediated
drug−drug interactions. Risk of in vivo QT interval
to oral PK parameters, with notable increases to AUC, Cmax
,
and bioavailability relative to 4. The oral PK profiles of 11 and
20, coupled with their potent cell-based inhibition of JAK1
activity, were sufficient to maintain unbound plasma concen-
trations in excess of IL6-pSTAT3 EC50 for 4.0 and 5.6 h,
respectively. Despite the relatively high iv clearance of
compound 20 in the rat, we were encouraged by the observed
level of target coverage after a single and relatively modest oral
dose (2 mg/kg) and thus subjected it to further in vivo
characterization.
The plasma concentration vs time curves after both oral and
iv administration of compound 20 in the dog as well as relevant
F
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prolongation was also predicted to be low because of the lack of
a significant effect in an in vitro hERG patch clamp assay (8.4%
inhibition at 10 μM 20). The ability of 20 to inhibit the
activities of the remaining two members of the JAK family was
determined in biochemical assays: JAK3 Ki = 22 nM and TYK2
Ki = 6.2 nM. Finally, inhibitor 20 was found to have high
selectivity for the JAK family within the broader kinome, as
revealed by the results of a screen against a panel of non-JAK
kinases at 0.1 μM (>50-fold over its JAK1 Ki). Of the 178
kinases tested, most (170 kinases) were inhibited to only a
minimal (<50%) extent while just eight were inhibited at a
more potent level (six at 50−75% inhibition, two at >75%
inhibition).
To further assess the biological activity of 20, its ability to
inhibit JAK1-mediated cytokine signaling was examined in vivo
in a PK/PD experiment in the mouse. As shown in Figure 4,
Figure 5. X-ray crystal structure of 3 in complex with JAK2 (PDB
code 4F08). The ligand 3 is shown in orange. Hydrogen-bond
interactions with the hinge residues L932 and E930 are shown as
dashed black lines. The side chains differing between JAK1 and JAK2
are highlighted in green. The corresponding JAK1 side chains are
noted in Figure 6a. The structure is of insufficient resolution (2.8 Å) to
observe bound waters around the ligand.
site of JAK2, with the tricyclic core interacting with the kinase
hinge region and forming hydrogen bond contacts with E930
(3.1 Å) and L932 (3.1 Å). The piperidine extended beneath the
P-loop but did not form any direct polar interactions with the
protein. The piperidine moiety further adopted a chair
conformation, placing the tricyclic core in an equatorial
position, and was rotated by approximately 90° relative to the
plane of tricyclic system.
The X-ray structures of compound 4 in complex with both
JAK1 and JAK2 are shown in Figure 6a and Figure 6b,
respectively. The general features of both structures were
similar, although subtle differences did exist. The ligand’s
interactions with the hinge backbone were virtually identical in
both isoforms, with the pyridine N engaging either the L959
(JAK1) or L932 (JAK2) amide NH and with the pyrrole NH
pairing with the E957 (JAK1) or E930 (JAK2) carbonyl
oxygen. As previously observed with compound 3, the
piperidine group extending beneath the JAK P-loops adopted
a chair conformation and oriented the tricyclic core in an
equatorial disposition. Also similar to compound 3, the free
piperidine NH of 4 did not directly contact the JAK proteins;
however, the crystal structures of 4 were of sufficient resolution
to reveal its association with two crystallographic water
molecules residing beneath the P-loops of JAK1 and JAK2. In
the case of JAK1, these waters were tightly associated with the
piperidine moiety of the ligand at distances of 2.9 and 2.8 Å,
respectively, while the association was looser in JAK2, 3.2 and
3.3 Å. The weaker interactions between the ligand and the
waters residing beneath the JAK2 P-loop compared to their
JAK1 counterparts likely reflect a slightly less optimal fit for
compound 4 in the JAK2 ATP binding pocket and may be a
contributing factor to the JAK1 vs JAK2 selectivity observed for
compound 4.
Figure 4. Design and results of an IL-6 induced pSTAT3 PK/PD
study of compound 20 in the mouse(c). (a) Measured 1 h after dosing
of 20. (b) Measured 30 min after dosing of IL-6. (c) Five animals were
used in each of the control and dosing arms.
mice were pretreated with ascending oral doses of compound
20 ranging from 3 to 100 mg/kg, followed by a 1 μg
intraperitoneal (ip) injection of IL-6 cytokine 30 min later. An
additional 30 min after IL-6 administration, concentrations of
compound 20 and pSTAT3 were measured in plasma and snap-
frozen spleens, respectively. As further demonstrated in Figure
4, the increasing plasma concentrations achieved with
ascending oral doses of 20 in the mouse were associated with
decreasing pSTAT3 levels in the spleens of IL-6 treated
animals. Indeed, at a dose of 100 mg/kg 20, pSTAT3
expression was reduced to levels comparable to those of
control animals untreated with IL-6, indicating that robust in
vivo suppression of JAK1-mediated signaling was achieved.
In an attempt to understand the intriguing effect of the C-2
methyl substituent on JAK1 potency and selectivity, X-ray
crystal structures of several imidazopyrrolopyridines in the
presence of JAK1 and/or JAK2 were generated. As shown in
Figure 5, the first structure generated was that of compound 3
in complex with JAK2. The inhibitor occupied the ATP-binding
A third crystallographic water molecule was observed to be
associated with inhibitor 4 in the X-ray crystal structure in
complex with JAK2. As shown in Figure 6b, this water molecule
interacted with the available imidazole N lone pair of
compound 4 at a distance of 2.8 Å. A similar interaction was
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Figure 6. (a) X-ray structure of 4 in complex with JAK1 (PDB code 4EHZ). (b) X-ray structure of 4 in complex with JAK2 (PDB code 4F09).
Compound 4 is depicted in orange. Hydrogen bonds to the ligand are depicted as dashed black lines. Backbone hinge atoms contacting 4 are
highlighted (E957 and L959 for JAK1; E930 and L932 for JAK2). Notable crystallographic waters are denoted as red spheres (waters modeled into
electron density peaks of >1.5σ). A molecule of ethylene glycol (teal) is observed in part a. The double-headed black arrows highlight the proximity
of the C-2 methyl group of the ligand to a key residue difference between JAK1 (E966) and JAK2 (D939). The resolutions of the X-ray structures are
2.2 Å for JAK1 (a) and 2.4 Å for JAK2 (b).
observed in the X-ray structure of 4 in complex with JAK1
(Figure 6a), where the hydrogen bond-accepting capacity of the
imidazole unit was satisfied by a molecule of ethylene glycol, a
component of the crystallization solution that presumably
replaced a water molecule in the crystal lattice.
crystallographically determined conformations of 3 in complex
with JAK2 and of 4 in complex with both JAK1 and JAK2 were
all found to be virtually identical. We therefore concluded that
the C-2 methyl substituent was unlikely to have a large impact
upon the bound conformations of ligands in either JAK1 or
JAK2.
The residue differences in the ATP-binding sites of JAK1 and
JAK2 are highlighted in green in Figure 6a and Figure 6b.
Compound 4 did not generally approach any of these closely
enough to provide a clear justification for the observed isoform
selectivity. One exception, however, was the E966 (JAK1) →
D939 (JAK2) residue difference. In the case of JAK2, the C-2
methyl group was too far from the D939 side chain (5.1 Å) for
an interaction to take place. By contrast, in JAK1, the C-2
methyl group of 4 approached a carboxylate O-atom of the
E966 side chain within 3.4 Å, a distance similar to the
combined van der Waals radii of the two moieties.44 The close
approach of the C-2 methyl group to the JAK1 E966 side chain
was suggestive of a potential interaction between the two
groups and could be a further contributing factor to the JAK1
vs JAK2 selectivity profile of compound 4.
To ascertain whether the methyl group impacted unbound
conformations, quantum mechanical (QM) calculations45 were
performed using two aqueous solvent models. As shown in
Figure 8, these calculations identified two distinct local
minimum energy conformations of unbound 3 and 4. One
local minimum (out) oriented the piperidine ring junction H
atom away from the tricyclic core and was equivalent to the
bound conformations observed experimentally in the X-ray
structures (Figure 7). The other local minimum (in) rotated
the piperidine moiety by 180° such that the ring junction H
atom pointed toward the tricyclic core. Both QM models
indicated that the experimentally observed bound conformation
for compound 3 (3out) was higher in energy than its rotational
isomer 3in by 0.8 kcal/mol. Additional QM calculations using
the same solvent models indicated that addition of the methyl
group to the C-2 position of the tricyclic core (compound 4)
reversed the bias of the unbound ligand such that 4out was
predicted to be 0.9−1.0 kcal/mol lower in energy than the
rotational isomer 4in. The preference of ligand 4 for the bound
conformation could contribute to its greater potency against
JAK1 and JAK2 relative to compound 3. The conformational
bias of compound 4 toward the bound conformation does not,
however, offer additional insight into why it is more JAK1
selective than compound 3. We consider it likely that the
observed potency and selectivity profiles are influenced by
multiple factors including the ligands’ interactions with
crystallographic waters, the close approach of C-2 methyl
substituted analogues to E966 in JAK1, and the different
conformational preferences of the unbound ligands.
In order to further understand the effect of the C-2 methyl
group on JAK potency, the bound conformations of ligands 3
and 4 were examined in more detail. As shown in Figure 7, the
As shown in Figure 9, the X-ray structure of compound 20 in
complex with JAK1 displayed several similar attributes to the
corresponding structure of 4 (Figure 6a). The pyrrolopyridine
moiety present in 20 anchored it to the L959 and E957 hinge
residues of the ATP-binding site. The C-2 methyl group
Figure 7. Bound conformations of 3 in complex with JAK2 (green)
and of 4 in complex with JAK1 (pink) and JAK2 (yellow) determined
by X-ray crystallography. For clarity, the structures of the proteins have
been omitted.
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Figure 8. Relative energies of out vs in conformations of compounds 3 and 4 calculated with aqueous solvent models.45
in complex with JAK2 was not generated, it is likely that a
similar interaction takes place between 20 and JAK2 as well.
CHEMISTRY
■
As shown in Scheme 1, preparation of the C-2 methylimida-
zopyrrolopyridine analogues 4−24 commenced with SNAr
substitution of activated aryl chloride 2536 with amines 5a, 7a−
14a, and 18a−24a to deliver nitro anilines 5b, 7b−14b, and
18b−24b. Reduction of the nitro groups under catalytic
hydrogenation or reducing metal conditions led to dianilines
5c, 7c−14c, and 18c−24c. Two subsequent general methods
were then used to effect conversion to the corresponding C-2
methylimidazoles (intermediates d). The dianilines c could be
treated with triethyl orthoacetate in the presence of acid to
promote cyclization and dehydration and afford the corre-
sponding C-2 methyl imidazoles d. Alternatively, it was possible
to access these imidazoles by engaging the dianiline precursors
c with an in situ generated imidate reagent produced by the
action of triethyloxonium tetrafluoroborate on acetamide. After
imidazole formation, the d series of intermediates was subjected
to basic hydrolysis conditions to remove the phenylsulfonamide
protecting groups liberating the corresponding free indoles
10e−14e and 5, 7−9, and 18−24. The N-benzyl group
protecting intermediate 10e was removed under transfer
hydrogenolysis conditions at ambient atmospheric pressure to
afford N-unsubstituted 3-piperidine 10. The benzyl group
resident in difluoropiperidine 13e was found to be more
resilient toward deprotection, and recourse to a high pressure,
continuous flow H-Cube reactor was necessary to effect its
removal to afford 13. The Boc groups protecting monofluor-
opiperidines 11e and 12e and 4-aminocyclohexane 14e were
removed with acid to provide final compounds 11 and 12 and
intermediate 14f, respectively. 14f was converted to final
products 14, 15, 16, and 17 by reaction of the primary amine
with acrylonitrile, methanesulfonyl chloride, acetic anhydride,
and methyl chloroformate, respectively. A portion of N-benzyl
protected piperidine 5, prepared above, was converted to free
piperidine 4 via transfer hydrogenolysis conditions under
ambient atmospheric pressure. Finally, a portion of 4 was
converted to 6 by alkylation of the free amine of the piperidine
group with acrylonitrile.
Figure 9. X-ray structure of 20 in complex with JAK1 (PDB code
4EI4). Compound 20 is depicted in orange. Hydrogen bonds to the
ligand are depicted as dashed black lines. The backbone hinge atoms
contacting 20 are highlighted (E957 and L959). An additional residue
(R1007) forming a hydrogen bond with 20 is shown in white. Notable
crystallographic waters are denoted as red spheres (waters modeled
into electron density peaks of >1.5σ). The double-headed black arrow
highlights the proximity of the C-2 methyl group of the ligand to a key
residue difference between JAK1 (E966) and JAK2 (D939). The
resolution of the X-ray structure is 2.2 Å.
resident in 20 approached the E966 side chain within 3.5 Å,
again potentially contributing to the biochemical JAK1 vs JAK2
selectivity profile. The free imidazole lone pair of 20 was
satisfied by an interaction with a crystallographic water
observed at a distance of 2.9 Å from the ligand. Reminiscent
of the piperidine moiety of compound 4, the substituted
cyclohexane ring present in 20 extended beneath the P-loop of
JAK1 in a chair conformation, adopting the out rotational
isomer and placing the tricyclic core in an equatorial
disposition. As further demonstrated by Figure 9, a water
molecule was observed beneath the P-loop in a location similar
to one of the crystallographic waters associating with the
piperidine moiety of 4. However, unlike 4, there was no
hydrogen bond interaction between this water molecule and
ligand 20. The water was instead pushed into the back pocket
by ∼1 Å, presumably because of the hydrophobic environment
created by the cyclohexane. Additionally, the axial hydroxyl
group pendent to the cyclohexane ring of 20 formed a
hydrogen bond with the backbone carbonyl oxygen of a residue
conserved between JAK1 and JAK2 (R1007 in JAK1) at a
distance of 2.7 Å. Although the corresponding X-ray structure
CONCLUSION
■
The C-2 methyl substituted imidazopyrrolopyridine analogue 4
was found to exhibit not only increased JAK1 inhibition but
also increased JAK1 vs JAK2 selectivity relative to the
unsubstituted analogue 3. Differences observed between the
I
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a
Scheme 1. Preparation of C-2 Methylimidazopyrrolopyridine Analogues
a
Reagents and conditions: (i) DIPEA, i-PrOH, 65−120 °C; (ii) Fe, NH4Cl, MeOH, H2O, reflux; (ii′) H2, Pd/C, EtOH, THF, 50 °C; (iii) triethyl
orthoacetate, p-TsOH, toluene, reflux; (iii′) acetamide, Et3(O+)BF4 , THF, EtOH, 25 → 70 °C; (iv) NaOH, H2O, MeOH or EtOH, 50 °C; (v)
−
Pd(OH)2, ammonium formate, MeOH, 75 °C; (v′) H-Cube, Pd(OH)2 cartridge, 50 psi, 80 °C; (vi) TFA, H2O, 25 °C; (vii) acrylonitrile, EtOH, 75
b
°C; (viii) MsCl, DIPEA, THF, 25 °C; (ix) Ac2O, DIPEA, THF, 25 °C (x) methyl chloroformate, DIPEA, THF, 25 °C. Relative stereochemistry
c
d
e
between substituents on piperidine, cyclohexane, or cyclopentane rings. Not applicable. Racemic mixture. Single enantiomer, (S)-stereochemistry.
f
g
Single enantiomer, (R,R)-stereochemistry Enantiomers separated by preparative chiral SFC.
X-ray structures of compound 4 in complex with JAK1 and
JAK2 suggested hypotheses for the observed selectivity profile:
(1) the close approach of the C-2 methyl group of 4 to E966 in
JAK1 but not D939 in JAK2; (2) the tighter association
between ligand 4 and crystallographic waters under the P-loop
of JAK1 compared with the corresponding waters in JAK2.
Additionally, an analysis comparing the QM-calculated solution
conformations of imidazopyrrolopyridines 3 and 4 to their
bound X-ray structures in complex with JAK1 and/or JAK2
indicated that the C-2 methyl group’s enforcement of the
bound conformation likely contributed to the increased
potency of 4. Despite the increased JAK1 potency and
selectivity of compound 4, suboptimal membrane permeability
limited its plasma exposure after oral administration in the rat
and precluded its progression as an orally administered
therapeutic. As such, an optimization campaign was undertaken
in the C-2 methylimidazopyrrolopyridine series aimed at
improving the permeability and oral exposure of 4 while
maintaining measurable selectivity for JAK1 over JAK2.
Compound 20, the end result of this optimization effort,
possessed a favorable balance of in vitro properties, leading to
promising levels of oral exposure in the rat despite relatively
high iv clearance. In the dog, excellent oral exposure of 20 was
observed, consistent with low iv clearance, good MDCK
permeability, and high aqueous solubility. Furthermore,
compound 20 exhibited potent inhibition in an in vitro cell-
based measure of JAK1 activity in the presence of human whole
blood (whole blood IL6-pSTAT3 EC50 = 160 nM) while
maintaining significant cell-based selectivity for JAK1 over
JAK2 (JAK1 cell-based selectivity index of 18-fold). The
combination of favorable oral PK characteristics and potent
whole blood activity allowed 20 to exert a substantial effect in
an in vivo IL6-stimulated pSTAT3 PK/PD study in the mouse.
On the basis of its balanced suite of favorable physicochemical
properties, its potency and selectivity for JAK1, and promising
in vivo PK profile, compound 20 was selected for further
progression in preclinical in vitro and in vivo safety studies.
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
1
stated, H and 13C 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 an internal standard, tetramethylsilane (0.00 ppm). The
following abbreviations are used: br = broad signal, s = singlet, d =
doublet, dd = doublet of doublets, t = triplet, q = quartet, m =
multiplet. Unless otherwise indicated, all 13C peaks are singlets.
Purification by silica gel chromatography was carried out using either
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hand-packed glass columns or Teledyne Isco CombiFlash systems with
prepacked cartridges. Some basic compounds were purified by capture
on SCX (strong cation exchange) sulfonic acid cartridges, followed by
elution with methanolic ammonia. Racemic mixtures of final
compounds were separated into individual enantiomers by chiral
SFC under the indicated conditions. Chemical and chiral (where
applicable) purities were >95% for all final compounds as assessed by
LCMS and chiral SFC (supercritical fluid) analysis, respectively.
Further details on the analytical conditions used for individual
compounds may be found in the Supporting Information. High
resolution mass spectrometry (HRMS) data were collected on a
Waters time of flight (LCT Premier XE) instrument using electrospray
ionization.
was triturated with diethyl ether (10 mL) to give 137 mg (70%) of the
title compound as a white solid. H NMR (400 MHz, DMSO-d6) δ:
1
11.78 (s, 1H), 8.46 (s, 1H), 7.51 (t, J = 3.0 Hz, 1H), 7.41 (m, 4H),
7.28 (t, J = 7.1 Hz, 1H), 6.96 (s, 1H), 4.48 (m, 1H), 3.62 (s, 2H), 3.04
(d, J = 11.2 Hz, 2H), 2.62 (s, 3H), 2.57 (m, 2H), 2.23 (t, J = 11.7 Hz,
2H), 1.88 (d, J = 12.4 Hz, 2H). 13C NMR (125 MHz, DMSO-d6) δ:
149.2, 144.2, 138.4, 134.6, 134.1, 132.0, 128.6, 128.3, 126.9, 123.6,
104.3, 99.5, 62.0, 54.9, 52.3 29.5, 14.4. LCMS (method LCMS2, ESI):
tR = 1.83 min, m/z = 346.1 [M + H]+. HRMS [M + H]+ expected,
346.2032; found, 346.2061.
2-Methyl-1-(piperidin-4-yl)-1,6-dihydroimidazo[4,5-d]-
pyrrolo[2,3-b]pyridine (4). A mixture of 5 (1.70 g, 4.92 mmol),
palladium hydroxide (20 wt % on carbon, 492 mg, 707 μmol), and
ammonium formate (4.44 g, 70.6 mmol) in MeOH (120 mL) was
heated at 75 °C for 3 h. The cooled mixture was filtered through Celite
and concentrated to dryness under vacuum. The residue obtained was
triturated with EtOAc (10 mL) to afford the crude title compound as a
beige solid (1.1 g, 87%). Then 200 mg of the crude product was
purified by preparative HPLC (column, Phenomenex Gemini C18,
21.2 cm × 250 cm, 5 μm; detection, UV 254 nm; mobile phase A,
water containing 0.1% NH4OH; mobile phase B, CH3CN containing
0.1% NH4OH; flow rate of 18 mL/min; gradient 5−30% B over 15
min) and then by an Isolute SCX-2 column chromatography (gradient,
MeOH to 2 M NH3 in MeOH) to afford 78 mg of the pure title
compound as a white solid. 1H NMR (400 MHz, DMSO-d6) δ: 11.73
(s, 1H), 8.45 (s, 1H), 7.43 (t, J = 3.0 Hz, 1H), 6.96 (s, 1H), 4.48 (s,
1H), 3.16 (d, J = 12.2 Hz, 2H), 2.71 (t, J = 12.2 Hz, 2H), 2.62 (s, 3H),
2.38 (m, 3H), 1.79 (d, J = 12.1 Hz, 2H). 13C NMR (125 MHz,
DMSO-d6) δ: 149.2, 144.3, 134.6, 134.1, 132.2, 123.3, 104.2, 100.1,
53.4, 45.6, 30.6, 14.5. LCMS (method LCMS2, ESI): tR = 0.68 min,
m/z = 256.1 [M + H]+. HRMS [M + H]+ expected, 256.1562; found,
256.1574.
N-(1-Benzylpiperidin-4-yl)-5-nitro-1-(phenylsulfonyl)-1H-
pyrrolo[2,3-b]pyridin-4-amine (5b). A mixture of 4-chloro-5-nitro-
1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (25)36 (500 mg, 1.48
mmol), 1-benzylpiperidin-4-amine (5a) (337 mg, 1.78 mmol), DIPEA
(380 μL, 2.22 mmol), and i-PrOH (7.4 mL) was heated at 120 °C for
12 min using microwave irradiation. The cooled mixture was diluted
with DCM (30 mL), loaded onto diatomaceous earth, and purified by
chromatography on silica gel (gradient 0−50% EtOAc in DCM) to
1
afford 499 mg (69%) of the title product as a yellow foam. H NMR
(400 MHz, CDCl3) δ: 9.10 (m, 2H), 8.19 (d, J = 7.9 Hz, 2H), 7.60 (m,
2H), 7.51 (t, J = 7.8 Hz, 2H), 7.30 (m, 5H), 6.69 (d, J = 4.2 Hz, 1H),
4.00 (m, 1H), 3.57 (s, 2H), 2.85 (m, 2H), 2.31 (m, 2H), 2.11 (m, 2H),
1.79 (m, 2H). LCMS (method LCMS1, ESI) tR = 3.35 min, m/z =
492.6 [M + H]+.
N4-(1-Benzylpiperidin-4-yl)-1-(phenylsulfonyl)-1H-pyrrolo-
[2,3-b]pyridine-4,5-diamine (5c). A mixture of 5b (442 mg, 899
μmol), iron powder (∼325 mesh, 201 mg, 3.60 mmol), and
ammonium chloride (289 mg, 5.39 mmol) was suspended in a
mixture of MeOH and water (3:1, 12 mL) and was heated at reflux for
4 h. The cooled mixture was filtered through Celite, washing with
MeOH (50 mL). The filtrate was concentrated under vacuum, and the
resulting residue was partitioned between a saturated aqueous sodium
bicarbonate solution (25 mL) and DCM (25 mL). The layers were
separated. Then the aqueous phase was re-extracted with DCM (2 ×
25 mL). The combined organic extracts were washed with brine (25
mL), dried over sodium sulfate, filtered, and concentrated under
vacuum to afford a dark pink residue. Purification by column
chromatography on silica gel (gradient 0−10% MeOH in DCM)
afforded 321 mg (77%) of the title compound as an off-white solid.
LCMS (LCMS1, ESI): tR = 2.55 min, m/z = 462.5 [M + H]+.
1-(1-Benzylpiperidin-4-yl)-2-methyl-6-(phenylsulfonyl)-1,6-
dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridine (5d). A mixture of
5c (325 mg, 0.704 mmol), triethyl orthoacetate (394 μL, 2.16 mmol),
and p-toluenesulfonic acid monohydrate (20.0 mg, 0.115 mmol) in
toluene (10 mL) was heated to reflux for 20 h. After the mixture was
cooled to room temperature, EtOAc (25 mL) was added. The organic
phase was washed with a saturated aqueous solution of sodium
hydrogen carbonate, water, and brine (1 × 20 mL each), then dried
over sodium sulfate, filtered, and concentrated to dryness under
vacuum. Purification of the residue by column chromatography on
silica gel (gradient 0−50% EtOAc in DCM) gave 301 mg (88%) of the
title compound as an off-white solid. 1H NMR (400 MHz, DMSO-d6)
δ: 8.79 (s, 1H), 8.23 (d, J = 7.8 Hz, 2H), 7.85 (d, J = 4.0 Hz, 1H), 7.54
(t, J = 7.4 Hz, 1H), 7.46 (t, J = 7.7 Hz, 2H), 7.39 (m, 5H), 7.31 (m,
1H), 4.36 (m, 1H), 3.62 (s, 2H), 3.13 (m, 2H), 2.66 (s, 3H), 2.58 (m,
2H), 2.21 (t, J = 11.7 Hz, 2H), 1.85 (m, 2H). LCMS (method
LCMS1, ESI): tR = 3.16 min, m/z = 486.4 [M + H]+.
3-(4-(2-Methylimidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)-
piperidin-1-yl)propanenitrile (6). Compound 4 was treated with
conditions similar to those used to convert 14f to 14 (vide infra), to
1
produce the title compound as a white solid. H NMR (400 MHz,
DMSO-d6) δ: 11.73 (s, 1H), 8.45 (s, 1H), 7.42−7.35 (m, 1H), 6.94 (s,
1H), 4.48 (m, 1H), 3.12 (d, J = 11.3 Hz, 2H), 2.81−2.66 (m, 4H),
2.62 (s, 3H), 2.59−2.52 (m, 2H), 2.29 (t, J = 11.0 Hz, 2H), 1.90 (d, J
= 8.3 Hz, 2H). LCMS (method LCMS2, ESI) tR = 1.06 min, m/z =
309.1 [M + H]+.
N-Cyclohexyl-5-nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]-
pyridin-4-amine (7b). A mixture of 2536(10.0 g, 29.6 mmol),
cyclohexylamine (7a) (3.40 mL, 30.0 mmol), and DIPEA (11.0 mL,
65.0 mmol) in i-PrOH (150 mL) was heated at 80 °C for 14 h. The
mixture was then cooled to 25 °C and stirred for an additional 6 h. A
yellow solid precipitated from solution and was collected by vacuum
filtration, then washed with i-PrOH (1 × 30 mL) and air-dried to
1
afford the title compound as a yellow solid (10.45 g, 90%). H NMR
(400 MHz, DMSO-d6) δ: 8.93 (s, 1H), 8.91 (s, 1H), 8.13 (d, J = 7.6
Hz, 2H), 7.82 (d, J = 4.1 Hz, 1H), 7.77 (t, J = 7.4 Hz, 1H), 7.66 (t, J =
7.8 Hz, 2H), 6.99 (d, J = 4.2 Hz, 1H), 4.12−4.01 (m, 1H), 2.02−1.93
(m, 2H), 1.76−1.54 (m, 3H), 1.55−1.38 (m, 4H), 1.33−1.20 (m, 1H).
LCMS (method LCMS3, ESI): tR = 1.33 min, m/z = 401.2 [M + H]+.
N4-Cyclohexyl-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]-
pyridine-4,5-diamine (7c). A suspension of 7b (10.0 g, 25.0 mmol)
and palladium on carbon (2.7 g, 10%, wet, Degussa, E101 NE/W) in a
3:1 mixture of THF and EtOH (200 mL) was stirred under a
hydrogen atmosphere (balloon) at 50 °C for 13 h. The reaction
mixture was cooled to 25 °C and then was filtered through Celite. The
filtrate was concentrated under reduced pressure to afford the crude
title product (9.61 g, 100%) as a rose foam. LCMS (method LCMS3,
ESI): tR = 0.83 min, m/z = 371.2 [M + H]+. This material was used in
subsequent reactions without additional purification or character-
ization.
1-(1-Benzylpiperidin-4-yl)-2-methyl-1,6-dihydroimidazo-
[4,5-d]pyrrolo[2,3-b]pyridine (5). A mixture of 5d (275 mg, 566
μmol) in MeOH (20 mL) was treated with a 1 M aqueous NaOH
solution (5 mL) and stirred at room temperature for 18 h. The
majority of the MeOH was removed under vacuum, and the resulting
suspension was extracted with EtOAc (3 × 20 mL). The combined
organic extracts were washed with water and brine (1 × 20 mL each),
dried over sodium sulfate, filtered, and concentrated to dryness under
vacuum. Purification of the residue by column chromatography on
silica gel (gradient 0−10% MeOH in DCM) afforded a residue which
1-Cyclohexyl-2-methyl-6-(phenylsulfonyl)-1,6-
dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridine (7d). p-Toluenesul-
fonic acid monohydrate (0.429 g, 2.26 mmol) was added to a solution
of 7c (1.04 g, 2.82 mmol) and triethyl orthoacetate (1.29 mL, 7.05
mmol) in toluene (10 mL) at 25 °C. The reaction mixture was heated
K
dx.doi.org/10.1021/jm300628c | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
at 105 °C for 13 h, then was cooled to 25 °C and partitioned between
half-saturated aqueous NaHCO3 solution (100 mL) and a 1:1 mixture
of EtOAc and heptane (2 × 125 mL). The organic layers were dried
over MgSO4, filtered, and the filtrate was concentrated under reduced
pressure. Purification of the resulting solid by column chromatography
on silica gel (gradient 0−8% MeOH in DCM) afforded the title
product (0.572 g, 52%) as a beige solid. 1H NMR (400 MHz, CDCl3)
δ: 8.80 (s, 1H), 8.23 (d, J = 7.8 Hz, 2H), 7.80 (d, J = 4.0 Hz, 1H), 7.55
(t, J = 7.4 Hz, 1H), 7.47 (t, J = 7.7 Hz, 2H), 7.26 (s, 1H), 6.90 (br s,
1H), 4.43−4.25 (m, 1H), 2.67 (s, 3H), 2.30−2.11 (m, 2H), 2.11−1.99
(m, 3H), 1.99−1.81 (m, 2H), 1.57−1.41 (m, 2H). LCMS (method
LCMS3, ESI): tR = 0.99 min, m/z = 395.2 [M + H]+.
amino-3-fluoropiperidine-1-carboxylate (11a46) (878 mg, 4.02 mmol),
and DIPEA (1.65 mL, 9.50 mmol) in i-PrOH (10 mL) was stirred
overnight at 82 °C, then cooled to room temperature. An off-white
solid precipitated from solution and was collected by filtration and
washed with water to yield 1.62 g (78%) of the title compound. This
1
material was used in the next step without further purification. H
NMR (400 MHz, DMSO-d6) δ: 8.96−8.89 (m, 2H), 8.13 (d, J = 7.4,
2H), 7.86−7.83 (m, 1H), 7.76 (t, J = 8.0 Hz, 1H), 7.65 (t, J = 8.0 Hz,
2H), 7.20−7.15 (m, 1H), 4.88−4.69 (m, 1H), 4.57−4.41 (m, 1H),
4.18−4.04 (m, 1H), 3.88−3.74 (m, 1H), 3.26−3.02 (m, 2H), 2.08−
1.96 (m, 1H), 1.81−1.69 (m, 1H), 1.42 (s, 9H). LCMS (method
LCMS5, ESI): tR = 1.22 min, m/z = 520.0 [M + H]+.
1-Cyclohexyl-2-methyl-1,6-dihydroimidazo[4,5-d]pyrrolo-
[2,3-b]pyridine (7). Sodium hydroxide (10 mL of a 1.0 M aqueous
solution) was added to a solution of 7d (0.574 g, 1.46 mmol) in
MeOH (20 mL) at 25 °C. The reaction mixture was stirred at 50 °C
for 14 h, then was cooled to 25 °C, and the majority of MeOH was
removed under vacuum. The mixture was then partitioned between
water (100 mL) and EtOAc (2 × 200 mL). The combined organic
layers were dried over MgSO4, 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 containing 0.1%
NH4OH; mobile phase B, CH3CN; flow rate of 18 mL/min; gradient
5−95% B over 15 min) afforded the title compound (0.181 g, 49%) as
( )-trans-tert-Butyl 4-(5-Amino-1-(phenylsulfonyl)-1H-
pyrrolo[2,3-b]pyridin-4-ylamino)-3-fluoropiperidine-1-carbox-
ylate (11c). 10% palladium on activated carbon (0.025 g, 0.023
mmol) was added to 11b (0.618 g, 1.19 mmol) in EtOAc (10 mL),
and the mixture was stirred at 50 °C under an atmosphere of hydrogen
(balloon) for 16 h. The mixture was then filtered through
diatomaceous earth and the filtrate concentrated under vacuum to
give 540 mg (92%) of the title compound as an off-white solid. This
material was used immediately in the next step without further
purification. LCMS (method LCMS5, ESI): tR = 0.88 min, m/z =
490.0 [M + H]+.
(
) - t r a n s - t e r t - B u t y l 3 - F l u o r o - 4 - ( 2 - m e t h y l - 6 -
(phenylsulfonyl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)-
piperidine-1-carboxylate (11d). A mixture of 11c (0.540 g, 1.10
mmol) and triethyl orthoacetate (1.01 mL, 5.52 mmol) in acetic acid
(5 mL) was heated at 120 °C for 10 min. The mixture was then cooled
to room temperature and concentrated under vacuum. Purification of
the residue by column chromatography on silica gel (gradient 0−100%
(20% MeOH/EtOAc) in heptane) gave 476 mg (84%) of the title
1
an off-white solid. H NMR (400 MHz, DMSO-d6) δ: 11.77 (s, 1H),
8.45 (s, 1H), 7.45 (t, J = 2.9 Hz, 1H), 6.71 (s, 1H), 4.53−4.35 (m,
1H), 2.62 (s, 3H), 2.34−2.18 (m, 2H), 1.92 (t, J = 13.8 Hz, 4H), 1.78
(d, J = 11.3 Hz, 1H), 1.64−1.36 (m, 3H). 13C NMR (125 MHz,
DMSO-d6) δ: 149.2, 144.2, 134.6, 134.2, 132.0, 123.3, 104.0, 99.6,
54.6, 30.1, 25.1, 24.4, 14.4. LCMS (method LCMS4, ESI): tR = 3.26
min, m/z = 255.1 [M + H]+. HRMS [M + H]+ expected, 255.1610;
found, 255.1676.
1
compound as an off-white solid. H NMR (400 MHz, DMSO-d6) δ:
8.66 (s, 1H), 8.16 (d, J = 7.3 Hz, 2H), 7.94 (d, J = 3.7 Hz, 1H), 7.71 (t,
J = 7.4 Hz, 1H), 7.62 (t, J = 7.8 Hz, 2H), 6.48 (br, 1H), 5.17−5.08 (m,
1H), 5.02−4.93 (m, 2H), 4.48−4.35 (m, 1H), 4.19−4.07 (m, 1H),
3.22−3.05 (m, 1H), 2.63 (s, 3H), 2.31−2.19 (m, 1H), 2.19−2.08 (m,
1H), 1.49 (s, 9H). LCMS (method LCMS5, ESI): tR = 1.07 min, m/z
= 514.0 [M + H]+.
2-Methyl-1-(tetrahydro-2H-pyran-4-yl)-1,6-dihydroimidazo-
[4,5-d]pyrrolo[2,3-b]pyridine (8). 8 was synthesized by methods
similar to those used for 7, substituting 4-aminotetrahydropyran (8a)
1
for cyclohexylamine (7a). H NMR (400 MHz, DMSO-d6) δ: 11.82
(s, 1H), 8.47 (s, 1H), 7.48 (d, J = 3.1 Hz, 1H), 6.72 (d, J = 3.1 Hz,
1H), 4.75 (m, 1H), 4.11 (dd, J = 11.5, 4.7 Hz, 2H), 3.60 (t, J = 11.3
Hz, 2H), 2.65 (s, 3H), 2.60−2.49 (m, 2H), 1.87 (dd, J = 12.6, 4.3 Hz,
2H). LCMS (method LCMS4, ESI): tR = 2.55 min, m/z = 257.2 [M +
H]+.
(
)-trans-tert-Butyl 3-Fluoro-4-(2-methylimidazo[4,5-d]-
pyrrolo[2,3-b]pyridin-1(6H)-yl)piperidine-1-carboxylate (11e).
Sodium hydroxide (1 M aqueous solution, 5 mL) was added to a
mixture of 11d (0.476 g, 0.927 mmol) in EtOH (5 mL) and
tetrahydrofuran (8 mL). The reaction mixture was heated at 50 °C for
2 h. The mixture was then cooled to room temperature and
concentrated under vacuum. Purification of the residue by column
chromatography on silica gel (gradient 0−100% (20% MeOH/EtOAc)
in heptane) gave 238 mg (69%) of the title compound as an off-white
solid. 1H NMR (400 MHz, DMSO-d6) δ: 11.93 (s, 1H), 8.51 (s, 1H),
7.42 (s, 1H), 6.19 (s, 1H), 5.28−5.21 (m, 1H), 5.12−5.08 (m, 1H),
4.98−4.87 (m, 1H), 4.48−4.35 (m, 1H), 4.19−4.07 (m, 1H), 3.22−
3.05 (m, 2H), 2.62 (s, 3H), 2.19−2.08 (m, 1H), 1.52 (s, 9H). LCMS
(method LCMS5, ESI): tR = 0.14 min, m/z = 374.0 [M + H]+.
trans-1-(4-Fluoropiperidin-3-yl)-2-methyl-1, 6-
dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridine (11). To a mixture
of 11e (238 mg, 0.637 mmol) in dioxane (5 mL) was added HCl (4 M
solution in dioxane, 1 mL). The reaction mixture was stirred at room
temperature for 2 h, then concentrated to dryness under vacuum to
give the crude racemic title product. 11 was separated from ent-11 by
preparative chiral SFC (column, Chiralpak IC, 21.2 mm × 250 mm, 5
μm; detection, UV 220 nm; mobile phase A, CO2; mobile phase B,
MeOH containing 0.1% DEA; flow rate of 50 mL/min; gradient,
isocratic, A/B = 70:30). Isolation and concentration of the appropriate
fractions afforded two products with the following characteristics. For
11, white solid (36.5 mg, 21%). Analytical chiral SFC (method SFC2):
tR = 0.89 min. 1H NMR (400 MHz, DMSO-d6) δ: 11.83 (s, 1H), 8.49
(s, 1H), 7.47 (s, 1H), 6.86 (s, 1H), 5.34−5.10 (m, 1H), 4.71−4.52 (m,
1H), 3.59−3.41 (m, 1H), 3.15−3.02 (m, 1H), 2.83−2.64 (m, 3H),
2.60 (s, 3H), 2.07−1.93 (m, 1H). 13C NMR (125 MHz, DMSO-d6) δ:
150.1, 144.3, 134.8, 134.2, 131.3, 123.8, 103.9, 99.6, 88.5 (d, J = 176.7
Hz), 57.8 (d, J = 17.7 Hz), 49.6 (d, J = 22.3 Hz), 44.3, 30.6 (d, J = 5.2
2-Methyl-1-(tetrahydro-2H-pyran-3-yl)-1,6-dihydroimidazo-
[4,5-d]pyrrolo[2,3-b]pyridine (9). ( )-9 was synthesized by
methods similar to those used for 7 and substituting ( )-3-
aminotetrahydropyran (9a) for cyclohexylamine (7a). 9 was separated
from ent-9 by preparative chiral SFC (column, Chiralpak AD, 2.12 cm
× 25 cm, 5 μm; detection, UV 220 nm; mobile phase A, CO2; mobile
phase B, MeOH; flow rate of 50 mL/min; gradient, isocratic, A/B =
75:25). Isolation and concentration of the appropriate fractions
afforded two products with the following characteristics. For 9,
1
analytical chiral SFC (method SFC1) tR = 1.12 min. H NMR (400
MHz, DMSO-d6) δ: 11.81 (s, 1H), 8.46 (s, 1H), 7.47 (s, 1H), 6.81 (d,
J = 3.2 Hz, 1H), 4.61 (m, 1H), 4.01 (m, 3H), 3.75−3.56 (m, 1H), 2.65
(s, 3H), 2.56−2.46 (m, 1H, overlaps with DMSO-d6), 2.09 (d, J = 14.7
Hz, 1H), 1.92 (m, 2H). LCMS (method LCMS4, ESI): tR = 2.64 min,
m/z = 257.1 [M + H]+. For ent-9, analytical chiral SFC (method
1
SFC1) tR = 1.28 min. H NMR and LCMS data match those of 9.
(S)-2-Methyl-1-(piperidin-3-yl)-1,6-dihydroimidazo[4,5-d]-
pyrrolo[2,3-b]pyridine (10). 10 was synthesized by methods similar
to those used for 4 and 5 and substituting (S)-1-benzylpiperidin-3-
amine (10a) for 1-benzylpiperidin-4-amine (5a). 1H NMR (400 MHz,
DMSO-d6) δ: 11.76 (s, 1H), 8.45 (s, 1H), 7.45 (t, J = 3.0 Hz, 1H),
6.86−6.76 (m, 1H), 4.49 (s, 1H), 3.10−2.92 (m, 2H), 2.74−2.65 (m,
1H), 2.63 (s, 3H), 2.45−2.30 (m, 2H), 2.01 (d, J = 12.1 Hz, 1H), 1.85
(d, J = 12.3 Hz, 1H), 1.77−1.60 (m, 1H). LCMS (method LCMS4,
ESI): tR = 2.05 min, m/z = 256.1 [M + H]+.
( )-trans-tert-Butyl 3-Fluoro-4-(5-nitro-1-(phenylsulfonyl)-
1H-pyrrolo[2,3-b]pyridin-4-ylamino)piperidine-1-carboxylate
(11b). A mixture of 2536 (1.77 g, 5.23 mmol), ( )-trans-tert-butyl 4-
L
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Journal of Medicinal Chemistry
Article
Hz), 14.2. LCMS (method LCMS6, ESI): tR = 3.70 min, m/z = 274.0
[M + H]+. HRMS [M + H]+ expected, 274.1468; found, 274.1463. For
ent-11, white solid (31.3 mg, 18%). Analytical chiral SFC (method
were washed with brine (1 × 50 mL), then dried over sodium sulfate,
filtered, and concentrated to dryness under vacuum. The resulting
residue was triturated (DCM, 10 mL) and air-dried to afford 3.08 g
1
1
SFC2): tR = 1.24 min. H NMR and LCMS data match those for 11.
(71%) of the title compound as a pale yellow solid. H NMR (400
cis-1-(3-Fluoropiperidin-4-yl)-2-methyl-1,6-dihydroimidazo-
[4,5-d]pyrrolo[2,3-b]pyridine (12). ( )-12 was synthesized by
methods similar to those used for ( )-11 and substituting ( )-cis-tert-
butyl 4-amino-3-fluoropiperidine-1-carboxylate (12a46) for ( )-trans-
tert-butyl 4-amino-3-fluoropiperidine-1-carboxylate (11a). 12 was
separated from ent-12 by preparative chiral SFC (column, Chiralpak
AD, 2.12 cm × 25 cm, 5 μm; detection, UV 220 nm; mobile phase A,
CO2; mobile phase B, EtOH containing 0.1% DEA; flow rate of 40
mL/min; gradient, isocratic, A/B = 65:35). Isolation and concen-
tration of the appropriate fractions afforded two products with the
following characteristics. For 12, white solid. Analytical chiral SFC
MHz, DMSO-d6) δ: 11.80 (s, 1H), 8.45 (s, 1H), 7.49 (m, 1H), 6.95
(d, J = 7.3 Hz, 1H), 6.64 (d, J = 3.3 Hz, 1 H), 4.42 (m, 1H), 3.56 (m,
1H), 2.63 (s, 3H), 2.35 (m, 2H), 1.96 (m, 4H), 1.51 (m, 2H), 1.42 (s,
9H). LCMS (method LCMS7, ESI): tR = 2.18 min, m/z = 370.0 [M +
H]+.
trans-4-(2-Methylimidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-
yl)cyclohexanamine (14f). To a slurry of 14e (3.08 g, 8.34 mmol)
and water (150 mL) was added trifluoroacetic acid (25 mL) at room
temperature. The mixture was stirred for 2 h and then concentrated to
dryness under vacuum. Purification of the residue by flash
chromatography (SCX-2, gradient, MeOH to 2 M NH3 in MeOH)
afforded the title compound as a colorless oil (2.24 g, 100%) which
was used directly in the next step without further purification. LCMS
(method LCMS7, ESI): tR = 0.55 and 0.67 min, m/z = 270.1 [M +
H]+.
trans-3-(4-(2-Methylimidazo[4,5-d]pyrrolo[2,3-b]pyridin-
1(6H)-yl)cyclohexylamino)propanenitrile (14). A mixture of 14f
(2.24 g, 8.34 mmol) and acrylonitrile (2.75 mL, 41.7 mmol) in EtOH
(100 mL) was heated to 80 °C for 2 h. After cooling to room
temperature, the reaction mixture was concentrated to dryness under
vacuum. The residue was triturated with diethyl ether and EtOAc (10
mL each) and air-dried. Purification of the resulting solid by column
chromatography on silica gel (gradient 0−20% [2 M NH3 in MeOH]
in DCM) followed by trituration with acetonitrile (3 mL) afforded 750
mg (28%) of the title compound as a white solid. 1H NMR (400 MHz,
DMSO-d6) δ: 11.77 (s, 1H), 8.45 (s, 1H), 7.44 (t, J = 3.0 Hz, 1H),
6.70 (s, 1Hs), 4.45 (m, 1H), 2.87 (m, 2H), 2.77 (m, 1H), 2.62 (m,
5H), 2.33 (m, 2H), 2.09 (d, J = 12.5 Hz, 2 H), 1.92 (m, 3H), 1.37 (m,
2H). 13C NMR (125 MHz, DMSO-d6) δ: 149.3, 144.2, 134.6, 134.2,
123.5, 120.2, 104.0, 99.6, 54.3, 42.0, 31.8, 28.8, 18.4, 14.4. LCMS
(method LCMS2, ESI): tR = 1.24 min, m/z = 323.2 [M + H]+. HRMS
[M + H]+ expected, 323.1984; found, 323.1999.
1
(method SFC3): tR = 1.34 min. H NMR (400 MHz, DMSO-d6) δ:
11.68 (s, 1H), 8.44 (s, 1H), 7.38 (s, 1H), 6.86 (s, 1H), 4.90 (s, 1H),
4.78 (s, 1H), 3.25−2.94 (m, 4H), 2.86−2.73 (m, 2H), 2.65 (s, 3H),
1.81 (br, 1H). LCMS (method LCMS4, ESI): tR = 0.75 min, m/z =
274.1 [M + H]+. For ent-12, white solid. Analytical chiral SFC
(method SFC3): tR = 1.91 min. 1H NMR and LCMS data match those
of 12.
(
)-1-(3,3-Difluoropiperidin-4-yl)-2-methyl-1,6-
dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridine (13). ( )-1-(1-
Benzyl-3,3-difluoropiperidin-4-yl)-2-methyl-1,6-dihydroimidazo[4,5-
d]pyrrolo[2,3-b]pyridine (13e) was synthesized by methods similar to
those used for 5 and substituting ( )-1-benzyl-3,3-difluoropiperidin-4-
amine (13a47) for 1-benzylpiperidin-4-amine (5a). A solution of 13e
(69.0 mg, 0.181 mmol) in THF (6 mL) was hydrogenated on a
ThalesNano H-Cube continuous-flow hydrogenation reactor (1 mL/
min, 50 psi, 80 °C, one pass) using a 20% Pd(OH)2/C CatCart. The
effluent was collected and concentrated to dryness under vacuum. The
residue was purified by preparative HPLC (column, Gemini-NX, 3 cm
× 10 cm, 10 μm; detection, UV 254 nm; mobile phase A, water
containing 0.1% NH4OH; mobile phase B, CH3CN containing 0.1%
NH4OH; flow rate of 20 mL/min; gradient 5−95% B over 15 min) to
afford 25.0 mg (47%) of the title compound as a white solid. 1H NMR
(400 MHz, DMSO-d6, 360 K) δ: 11.16 (br, 1H), 8.43 (s, 1H), 7.30 (d,
J = 3.5 Hz, 1H), 6.80 (t, J = 3.3 Hz, 1H), 5.01 (br, 1H), 3.30 (t, J =
13.3 Hz, 1H), 3.23−3.04 (m, 2H), 2.94−2.82 (m, 2H), 2.63 (s, 3H),
2.62−2.54 (m, 1H), 2.04 (br, 1H). LCMS (method LCMS4, ESI): tR
= 1.98 min, m/z = 292.0 [M + H]+.
trans-N-(4-(2-Methylimidazo[4,5-d]pyrrolo[2,3-b]pyridin-
1(6H)-yl)cyclohexyl)methanesulfonamide (15). Methanesulfonyl
chloride (24 μL, 0.31 mmol) was added to a solution of 14f (67.5 mg,
0.251 mmol) and triethylamine (105 μL, 0.75 mmol) in DCM (2 mL)
at room temperature, and the reaction mixture was stirred for 16 h.
The reaction mixture was loaded directly onto a silica gel column,
eluting with a gradient of 0−10% MeOH in EtOAc. The appropriate
fractions were collected and concentrated to dryness under vacuum.
The residue was triturated with water (1 mL), then heated at 40 °C
under vacuum for 16 h to afford the title compound as a white solid
trans-tert-Butyl 4-(2-Methyl-6-(phenylsulfonyl)imidazo[4,5-
d]pyrrolo[2,3-b]pyridin-1(6H)-yl)cyclohexylcarbamate (14d).
trans-tert-Butyl 4-(5-amino-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]-
pyridin-4-ylamino)cyclohexylcarbamate (14c) was synthesized by
methods similar to those used for 7c, substituting trans-tert-butyl 4-
aminocyclohexylcarbamate (14a) for cyclohexylamine (7a). A mixture
of 14c (10.0 g, 20.6 mmol), triethyl orthoacetate (15.0 mL, 82.4
mmol), and acetic acid (120 mL) was heated to reflux for 20 min.
After cooling to room temperature, the mixture was concentrated to
dryness under vacuum. The residue was dissolved in EtOAc (150 mL),
and the organic layer was washed with aqueous saturated sodium
hydrogen carbonate solution (3 × 50 mL) and brine (50 mL). The
organic layer was dried over sodium sulfate, filtered, and concentrated
to dryness under vacuum. Purification of the residue by column
chromatography on silica gel (gradient, DCM to neat EtOAc to 5%
MeOH in EtOAc) and trituration (diethyl ether, 20 mL) provided
1
(44.1 mg, 51%). H NMR (400 MHz, DMSO-d6) δ: 11.78 (s, 1H),
8.45 (s, 1H), 7.47 (t, J = 3.0 Hz, 1H), 7.16 (d, J = 7.0 Hz, 1H), 6.72
(dd, J = 3.5, 1.9 Hz, 1 H), 4.43 (m, 1H), 3.46 (m, 1H), 3.02 (s, 3H),
2.63 (s, 3H), 2.37 (m, 2H), 2.13 (d, J = 12.6 Hz, 2H), 1.92 (d, J = 12.3
Hz, 2 H), 1.61 (t, J = 12.8 Hz, 2H). LCMS (method LCMS2, ESI) tR
= 2.00 min, m/z = 348.1 [M + H]+.
trans-N-(4-(2-Methylimidazo[4,5-d]pyrrolo[2,3-b]pyridin-
1(6H)-yl)cyclohexyl)acetamide (16). Acetic anhydride (21 μL, 0.22
mmol) was added to a mixture of 14f (54.3 mg, 0.202 mmol) and
DIPEA (44 μL, 0.25 mmol) in DCM (2 mL). The reaction mixture
was stirred for 1 h at room temperature, then concentrated to dryness
under vacuum. The residue was purified by column chromatography
on silica gel (gradient 0−10% [2 M NH3 in MeOH] in DCM) to give
1
8.30 g (79%) of the title compound as a gray solid. H NMR (400
1
the title compound (50.2 mg, 87%) as a white solid. H NMR (400
MHz, CDCl3) δ: 8.80 (s, 1H), 8.23 (m, 2H), 7.81 (d, J = 4.1 Hz, 1H),
7.51 (m, 3H), 6.86 (br s, 1H), 4.50 (m, 1H), 4.35 (m, 1H), 3.74 (m,
1H), 2.68 (s, 3H), 2.34 (m, 4H), 1.98 (m, 2H), 1.48 (s, 9H), 1.43 (m,
2H). LCMS (method LCMS7, ESI): tR = 3.29 min, m/z = 510.2 [M +
H]+.
trans-tert-Butyl 4-(2-Methylimidazo[4,5-d]pyrrolo[2,3-b]-
pyridin-1(6H)-yl)cyclohexylcarbamate (14e). Aqueous sodium
hydroxide solution (29.4 mL, 2.0 M) was added to a solution of
14d (6.00 g, 11.8 mmol) in MeOH (150 mL) and THF (150 mL).
The reaction mixture was stirred at room temperature for 5 h, then
extracted with DCM (3 × 75 mL). The combined organic extracts
MHz, DMSO-d6) δ: 11.80 (s, 1H), 8.46 (s, 1H), 7.88 (d, J = 7.4 Hz,
1H), 7.49 (t, J = 3.0 Hz, 1H), 6.65 (dd, J = 3.4, 1.9 Hz, 1H), 4.48 (m,
1H), 3.83 (m, 1H), 2.64 (s, 3H), 2.36 (m, 2H), 2.98 (m, 4H), 1.84 (s,
3H), 1.52 (m, 2H). LCMS (method LCMS2, ESI) tR = 1.89 min, m/z
= 312.1 [M + H]+.
trans-Methyl 4-(2-Methylimidazo[4,5-d]pyrrolo[2,3-b]-
pyridin-1(6H)-yl)cyclohexylcarbamate (17). 17 was synthesized
by methods similar to those used for 16 and substituting methyl
1
chloroformate for acetic anhydride. H NMR (400 MHz, DMSO-d6)
δ: 11.80 (s, 1H), 8.45 (s, 1H), 7.49 (t, J = 3.0 Hz, 1H), 7.25 (s, 1H),
M
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6.64 (s, 1H), 4.45 (s, 1H), 3.56 (m, 4H), 2.63 (s, 3H), 2.35 (m, 2H),
2.04 (m, 2H), 1.94 (m, 2H), 1.52 (m, 2H). 13C NMR (125 MHz,
DMSO-d6) δ: 155.9, 149.3, 144.2, 134.7, 134.1, 131.9, 123.6, 103.9,
99.3, 59.7, 53.6, 51.1, 48.5, 31.2, 14.0. LCMS (method LCMS2, ESI) tR
= 2.18 min, m/z = 328.3 [M + H]+. HRMS [M + H]+ expected,
328.1773; found, 328.1761.
for 24 h, the solid was isolated by filtration and dried under vacuum for
18 h to afford the title compound as a white solid (14.7 g, 93%). H
1
NMR (400 MHz, DMSO-d6) δ: 11.77 (s, 1H), 8.44 (s, 1H), 7.43 (s,
1H), 6.67 (s, 1H), 4.79 (overlapping s and br s, 2H), 4.21 (s, 1H), 2.61
(s, 3H), 2.48−2.17 (m, 2H), 2.06−1.84 (m, 3H), 1.78 (d, J = 12.2 Hz,
1H), 1.74−1.58 (m, 2H). 13C NMR (125 MHz, DMSO-d6) δ: 149.1,
144.2, 134.6, 131.9, 123.5, 104.0, 99.5, 64.6, 50.0, 36.9, 30.9, 29.8, 19.1,
14.4. LCMS (method LCMS4, ESI): tR = 2.71 min, m/z = 271.1 [M +
H]+. HRMS [M + H]+ expected, 271.1559; found, 271.1596.
trans-4-(2-Methylimidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-
yl)cyclohexanol (18). 18 was synthesized by methods similar to
those used for 20 (vide infra) and substituting trans-4-amino-
cyclohexanol (18a) for (1R,3R)-3-aminocyclohexanol (20a). 1H
NMR (400 MHz, DMSO-d6) δ: 11.75 (s, 1H), 8.44 (s, 1H), 7.44
(t, J = 2.9 Hz, 1H), 6.65 (s, 1H), 4.76 (d, J = 3.9 Hz, 1H), 4.46 (br s,
1H), 3.77 (br s, 1H), 2.62 (s, 3H), 2.38−2.26 (m, 2H), 2.03 (d, J =
11.1 Hz, 2H), 1.88 (d, J = 11.2 Hz, 2H), 1.67−1.46 (m, 2H). LCMS
(method LCMS4, ESI) tR = 2.42 min, m/z = 271.1 [M + H]+.
cis-4-(2-Methylimidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-
yl)cyclohexanol (19). 19 was synthesized by methods similar to
those used for 20 (vide infra) and substituting cis-4-aminocyclohexanol
cis-3-(2-Methylimidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-
yl)cyclohexanol (21). ( )-21 was synthesized by methods similar to
those used for 20 and substituting ( )-cis-3-aminocyclohexanol
(21a48) for (1R,3R)-3-aminocyclohexanol (20a). 21 was separated
from ent-21 by preparative chiral SFC (column, Phenomenex Lux
Cellulose-2, 21.2 mm × 250 mm, 5 μm; detection, UV 220 nm; mobile
phase A, CO2; mobile phase B, EtOH containing 0.1% triethylamine;
flow rate of 50 mL/min; gradient, isocratic, A/B = 70:30). Isolation
and concentration of the appropriate fractions afforded two products
with the following characteristics. For 21, white solid. Analytical chiral
SFC (method SFC4): tR = 0.85 min. 1H NMR (400 MHz, DMSO-d6)
δ: 11.78 (s, 1H), 8.45 (s, 1H), 7.46 (t, J = 2.9 Hz, 1H), 6.69 (s, 1H),
4.87 (d, J = 4.9 Hz, 1H), 4.49 (br s, 1H), 3.74−3.63 (m, 1H), 2.61 (s,
3H), 2.28−2.02 (m, 3H), 1.97 (br d, J = 9.9 Hz, 1H), 1.89 (br d, J =
13.3 Hz, 1H), 1.83 (br d, J = 9.9 Hz, 1H), 1.59−1.27 (m, 2H). 13C
NMR (125 MHz, DMSO-d6) δ: 149.2, 144.2, 134.7, 134.2, 131.8,
123.5, 103.9, 99.7, 67.8, 52.9, 48.5, 34.2, 29.0, 21.7, 14.4. LCMS
(method LCMS4, ESI) tR = 2.63 min, m/z = 271.1 [M + H]+. HRMS
[M + H]+ expected, 271.1559; found, 271.1571. For ent-21, white
1
(19a) for (1R,3R)-3-aminocyclohexanol (20a). H NMR (400 MHz,
DMSO-d6) δ: 11.66 (s, 1H), 8.43 (s, 1H), 7.39 (d, J = 2.3 Hz, 1H),
7.36 (s, 1H), 4.87 (s, 1H), 4.41 (br, 1H), 4.03 (s, 1H), 2.76−2.61 (m,
2H), 2.61 (s, 3H), 1.88 (d, J = 13.2 Hz, 2H), 1.70 (t, J = 13.5 Hz, 2H),
1.61 (d, J = 11.7 Hz, 2H). LCMS (method LCMS4, ESI) tR = 2.54
min, m/z = 271.1 [M + H]+.
(1R,3R)-3-(5-Amino-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]-
pyridin-4-ylamino)cyclohexanol (20c). (1R,3R)-3-Aminocyclohex-
anol (20a,48 8.91 g, 76.3 mmol) was added to a stirred suspension of
2536 (24.5 g, 72.6 mmol) and DIPEA (13.3 mL, 76.3 mmol) in MeOH
(120 mL) at room temperature. The reaction mixture was heated to 65
°C and stirred at that temperature for 3 h. The mixture was then
cooled to room temperature, and 10% palladium on carbon (7.6 g, 3.6
mmol) was added. The reaction mixture was pressurized to 100 psi
with hydrogen and stirred at room temperature for 18 h. The reaction
mixture was filtered, and the filtrate was concentrated to dryness under
vacuum to afford the title compound as a thick yellow oil. LCMS
(method LCMS8, ESI): tR = 2.17 min, m/z = 387.1 [M + H]+. This
material was used immediately without further purification or
characterization in the next step.
1
solid. Analytical chiral SFC (method SFC4): tR = 0.71 min. H NMR
and LCMS data match those for 21.
trans-2-(2-Methylimidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-
yl)cyclohexanol (22). ( )-22 was synthesized by methods similar to
those used for 20 and substituting ( )-cis-2-aminocyclohexanol (22a)
for (1R,3R)-3-aminocyclohexanol (20a). 22 was separated from ent-22
by preparative chiral SFC (column, Chiralpak AD, 2.12 mm × 25 mm,
5 μm; detection, UV 220 nm; mobile phase A, CO2; mobile phase B,
MeOH; flow rate of 50 mL/min; gradient, isocratic, A/B = 60:40).
Isolation and concentration of the appropriate fractions afforded two
products with the following characteristics. For 22, white solid.
(1R,3R)-3-(2-Methyl-6-(phenylsulfonyl)imidazo[4,5-d]-
pyrrolo[2,3-b]pyridin-1(6H)-yl)cyclohexanol (20d). Triethyloxo-
nium tetrafluoroborate (22.8 g, 116 mmol) was added to a slurry of
acetamide (7.11 g, 120 mmol) in tetrahydrofuran (THF) (125 mL).
The reaction mixture was stirred for 1 h at room temperature. Then a
solution of 20c (31.0 g, 80.2 mmol) in EtOH (220 mL) was added.
The reaction mixture was heated to 70 °C and stirred at that
temperature for 2 h. The reaction mixture was then cooled to room
temperature, and water (150 mL) was added. The mixture was
extracted with 2-methyltetrahydrofuran (2 × 250 mL). Then the
combined organic layers were dried over sodium sulfate, filtered, and
concentrated to dryness. Ethanol (200 mL) was added, and the
mixture was warmed to 50 °C. After a solution formed, the mixture
was then cooled to room temperature, at which point a white solid
precipitated. The solid was isolated by filtration, then dried under
vacuum to afford the title compound (24.0 g, 73%) as a faintly yellow
1
Analytical chiral SFC (method SFC5): tR = 0.74 min. H NMR (400
MHz, DMSO-d6) δ: 11.72 (s, 1H), 8.45 (s, 1H), 7.42 (s, 1H), 6.58 (s,
1H), 4.83 (d, J = 5.5 Hz, 1H), 4.28−4.16 (m, 1H), 4.14−4.06 (m,
1H), 2.58 (s, 3H), 2.41−2.30 (m, 1H), 2.10 (br d, J = 12.1 Hz, 1H),
1.95−1.73 (m, 3H), 1.65−1.38 (m, 3H). LCMS (method LCMS4,
ESI) tR = 2.87 min, m/z = 271.0 [M + H]+. For ent-22, white solid.
1
Analytical chiral SFC (method SFC5): tR = 0.52 min. H NMR and
LCMS data match those of 22.
trans-3-(2-Methylimidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-
yl)cyclopentanol (23). An amount of 1.6 g (3.1 mmol) of the N-tert-
butyl carbamate protected precursor to ( )-trans-3-aminocyclopenta-
nol (23a) was synthesized as previously described,49 then treated with
a 4 M solution of HCl in EtOAc (15 mL). The reaction mixture was
stirred for 3 h at room temperature and then concentrated to dryness
under vacuum. The crude residue was used immediately without
further purification or characterization in the next step. ( )-23 was
synthesized by methods similar to those used for 20 and substituting
( )-trans-3-aminocyclopentanol (23a) for (1R,3R)-3-aminocyclohex-
anol (20a). 23 was separated from ent-23 by preparative chiral SFC
(column, Chiralpak AD, 30 mm × 250 mm, 5 μm; detection, UV 220
nm; mobile phase A, CO2; mobile phase B, MeOH containing 0.2%
DEA; flow rate of 50 mL/min; gradient, isocratic, A/B = 70:30).
Isolation and concentration of the appropriate fractions afforded two
products with the following characteristics. For 23, white solid.
1
solid. H NMR (400 MHz, CDCl3) δ: 8.79 (s, 1H), 8.22 (d, J = 8.2
Hz, 2H), 7.78 (d, J = 4.0 Hz, 1H), 7.58−7.41 (m, 3H), 6.90 (s, 1H),
4.48−4.42 (m, 1H), 3.99−3.85 (m, 1H), 2.68 (s, 3H), 2.41−1.48 (m,
8H). LCMS (method LCMS8, ESI): tR = 2.52 min, m/z = 411.1 [M +
H]+.
(1R,3R)-3-(2-Methylimidazo[4,5-d]pyrrolo[2,3-b]pyridin-
1(6H)-yl)cyclohexanol (20). An aqueous 1 M solution of sodium
hydroxide (168 mL, 168 mmol) was added to a slurry of 20d (24.0 g,
58.5 mmol) in EtOH (240 mL) at room temperature. The reaction
mixture was stirred at 50 °C for 18 h, then cooled to room
temperature. The majority of the EtOH was removed under vacuum.
Then the residual mixture was extracted with methyl ethyl ketone (2 ×
250 mL). The combined organic layers were washed with brine (1 ×
100 mL), then dried over magnesium sulfate and filtered. The filtrate
was concentrated to dryness and then i-PrOH (50 mL) was added to
the residue. After the resulting slurry was stirred at room temperature
1
Analytical chiral SFC (method SFC6): tR = 6.26 min. H NMR (400
MHz, DMSO-d6) δ: 11.81 (s, 1H), 8.46 (s, 1H), 7.43 (s, 1H), 6.43 (s,
1H), 5.28−5.21 (m, 1H), 4.92 (s, 1H), 4.51 (s, 1H), 2.61 (s, 3H),
2.49−2.32 (m, 2H), 2.28−2.12 (m, 2H), 2.05−1.97 (m, 1H), 1.81−
1.71 (m, 1H). 13C NMR (125 MHz, DMSO-d6) δ: 149.7, 144.1, 134.7,
134.1, 132.0, 123.7, 104.3, 97.9, 70.1, 54.8, 33.6, 27.8, 14.6. LCMS
(method LCMS9, ESI) tR = 0.77 min, m/z = 257.1 [M + H]+. HRMS
N
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[M + H]+ expected, 257.1402; found, 257.1413. For ent-23, white
solid. Analytical chiral SFC (method SFC6): tR = 6.48 min. H NMR
and LCMS data match those of 23.
ABBREVIATIONS USED
■
1
ADME, absorption, distribution, metabolism, and excretion; aq,
aqueous; ATP, adenosine triphosphate; AUC, area under the
curve; b.i.d., twice daily; Bn, benzyl; Boc, tert-butyl carbamate;
CL, clearance; Cmax, maximum concentration; CYP, cyto-
chrome P450; D, aspartic acid; DEA, diethylamine; DIPEA,
N,N-diisopropylethylamine; E, glutamic acid; EC50, half-
maximal effective concentration; ent, enantiomer; 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; HPLC, high
performance liquid chromatography; HRMS, high resolution
mass spectrometry; IL-6, interleukin-6; i-PrOH, isopropyl
alcohol; iv, intravenous; ip, intraperitoneal; JAK, Janus kinase;
Ki(JAK2)/Ki(JAK1), JAK1 biochemcal selectivity index;
EC50(EPO-pSTAT5)/EC50(IL6-pSTAT3), JAK1 cellular selec-
tivity index; Ki, inhibition constant; L, leucine; LCMS, liquid
chromatography−mass spectrometry; log D7.2, log of partition
coefficient between octanol and pH 7.2 aqueous buffer; MCT,
methylcellulose/Tween; MDCK, Madin−Darby canine kidney;
MeOH, methanol; MF, myelofibrosis; min, minute; MsCl,
methanesulfonyl chloride; N, asparagine; Papp, apparent
permeability; PEG400, polyethylene glycol 400; PK, pharma-
cokinetics; PK/PD, pharmacokinetic/pharmakodynamic; po,
by mouth; PPB, plasma protein binding; psi, pounds per square
inch; p-TsOH, p-toluenesulfonic acid; R, arginine; RA,
rheumatoid arthritis; S, serine; SFC, supercritical fluid
chromatography; STAT, signal transducer and activator of
transcription; QM, quantum mechanical; TFA, trifluoroacetic
acid; THF, tetrahydrofuran; TPSA, topological polar surface
area; UV, ultraviolet; Y, tyrosine
cis-3-(2-Methylimidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-
yl)cyclopentanol (24). The N-tert-butyl carbamate protected
precursor to ( )-cis-3-aminocyclopentanol (24a) was synthesized as
previously described.49 The tert-butyl carbamate protecting group was
removed as described above for example 23. ( )-24 was synthesized
by methods similar to those used for 20 and substituting ( )-cis-3-
aminocyclopentanol (24a) for (1R,3R)-3-aminocyclohexanol (20a).
24 was separated from ent-24 by preparative chiral SFC (column,
Chiralpak AD, 30 mm × 250 mm, 20 μm; detection, UV 220 nm;
mobile phase A, CO2; mobile phase B, MeOH containing 0.2% DEA;
flow rate of 50 mL/min; gradient, isocratic, A/B = 75:25). Isolation
and concentration of the appropriate fractions afforded two products
with the following characteristics. For 24, white solid. Analytical chiral
SFC (method SFC6): tR = 6.65 min. 1H NMR (400 MHz, DMSO-d6)
δ: 11.72 (s, 1H), 8.43 (s, 1H), 7.42−7.40 (m, 1H), 7.10−7.09 (m,
1H), 5.10 (d, J = 5.4 Hz, 1H), 4.89−4.85 (m, 1H), 4.38−4.35 (m,
1H), 2.60 (s, 3H), 2.49−2.41 (m, 2H), 2.22−2.14 (m, 1H), 1.98−1.91
(m, 2H), 1.88−1.83 (m, 1H). LCMS (method LCMS9, ESI) tR = 0.86
min, m/z = 256.9 [M + H]+. For ent-24: white solid. Analytical chiral
SFC (method SFC6): tR = 6.80 min. 1H NMR and LCMS data match
those for 24.
ASSOCIATED CONTENT
■
S
* Supporting Information
Details of analytical LCMS and SFC methods; experimental
log D measurement procedures; thermodynamic solubility
measurement procedures; in vitro and in vivo ADME
experimental procedures; experimental details for IL6-induced
pSTAT3 PK/PD study of compound 20 in the mouse; protein
expression/purification and crystallographic methods and
procedures for 3 (in complex with JAK2), 4 (in complex
with JAK1 and JAK2), 20 (in complex with JAK1); details of
JAK biochemical and cell-based assays; details of in vitro hERG
assay; details of QM calculations and methods; biochemical
inhibition of JAK3 and TYK2 by compounds 3−24. This
material is available free of charge via the Internet at http://
pubs.acs.org.
REFERENCES
■
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Accession Codes
PDB codes are as follows: 4F08 for 3 complexed with JAK2;
4EHZ for 4 complexed with JAK1; 4F09 for 4 complexed with
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: 650-467-4533. E-mail: mzak@gene.com.
Notes
The authors declare no competing financial interest.
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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,
and Garima Porwal 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; Suzanne Tay for TDI
studies; Jasleen Sodhi for reversible CYP inhibition measure-
ment; Quynh Ho for plasma protein binding measurement;
Paroma Chakravarty and Wei Jia for formulations support.
O
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potent compounds achieve >50% inhibition in some, but not all, of the
replicate runs. In these cases, we report EC50 > X, where X is the
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noncalculable. For example, compound 3 displayed replicate EC50
Q
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