Discovery of a Gut-Restricted JAK Inhibitor for the Treatment of Inflammatory Bowel Disease

Article abstract of DOI:10.1021/acs.jmedchem.9b01439

To identify Janus kinase (JAK) inhibitors that selectively target gastrointestinal tissues with limited systemic exposures, a class of imidazopyrrolopyridines with a range of physical properties was prepared and evaluated. We identified compounds with low intrinsic permeability and determined a correlation between permeability and physicochemical properties, clogP and tPSA, for a subset of compounds. This low intrinsic permeability translated into compounds displaying high colonic exposure and low systemic exposure after oral dosing at 25 mg/kg in mouse. In a mouse PK/PD model, oral dosing of lead compound 2 demonstrated dose-dependent inhibition of pSTAT phosphorylation in colonic explants post-oral dose but low systemic exposure and no measurable systemic pharmacodynamic activity. We thus demonstrate the utility of JAK inhibitors with low intrinsic permeability as a feasible approach to develop gut-restricted, pharmacologically active molecules with a potential advantage over systemically available compounds that are limited by systemic on-target adverse events.

Full text of DOI:10.1021/acs.jmedchem.9b01439

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Article
Discovery of a Gut-Restricted JAK Inhibitor for
the Treatment of Inflammatory Bowel Disease
Kristi A. Leonard, Lisa A. Madge, Paul John Krawczuk, Aihua Wang, Kevin D. Kreutter, Genesis
M. Bacani, Wenying Chai, Russell C Smith, Mark S. Tichenor, Michael C Harris, Ravi Malaviya,
Mark Seierstad, Marguerite E. Johnson, Jennifer D. Venable, Suzie Kim, Gavin C. Hirst, Ashok S.
Mathur, Tadimeti S. Rao, James Patrick Edwards, Michele C. Rizzolio, and Tatiana Koudriakova
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01439 • Publication Date (Web): 05 Mar 2020
Downloaded from pubs.acs.org on March 9, 2020
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Discovery of a Gut-Restricted JAK Inhibitor for the Treatment of Inflammatory Bowel
Disease
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Kristi A. Leonard , Lisa A. Madge , Paul J. Krawczuk , Aihua Wang , Kevin D. Kreutter ,
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Genesis M. Bacani , Wenying Chai , Russell C. Smith , Mark S. Tichenor , Michael C. Harris ,
Ravi Malaviya , Mark Seierstad , Marguerite E. Johnson , Jennifer D. Venable , Suzie Kim ,
Gavin C. Hirst , Ashok S. Mathur , Tadimeti S. Rao , James P. Edwards , Michele C. Rizzolio ,
Tatiana Koudriakova
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1Janssen Research and Development, 1400 McKean Road, Spring House, PA 19477, United
States
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Janssen Research and Development, 3210 Merryfield Row, San Diego, CA 92121, United
States
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Contributions by K.K. and G.H. were performed while employed at Janssen Research and
Development. K.K. current address is Nimbus Therapeutics, 130 Prospect St, Suite 301
Cambridge, MA 02139, United States
ABSTRACT: To identify JAK inhibitors that selectively target gastrointestinal tissues with
limited systemic exposures, a class of imidazopyrrolopyridines with a range of physical properties
were prepared and evaluated. We identified compounds with low intrinsic permeability and
determined a correlation between permeability and physicochemical properties, clogP and tPSA,
for a subset of compounds. This low intrinsic permeability translated into compounds displaying
high colonic exposure and low systemic exposure after oral dosing at 25 mg/kg in mouse. In a
mouse PK/PD model, oral dosing of lead compound 2 demonstrated dose-dependent inhibition of
pSTAT phosphorylation in colonic explants post oral dose but low systemic exposure and no
measurable systemic pharmacodynamic activity. We thus demonstrate the utility of JAK
inhibitors with low intrinsic permeability as a feasible approach to develop gut-restricted,
pharmacologically active molecules with potential advantage over systemically available
compounds that are limited by systemic on-target adverse events.
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INTRODUCTION
Inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC),
are characterized by recurrent intestinal inflammation, disruption of the epithelial barrier and
microbial dysbiosis. The excessive inflammatory response in the gastrointestinal (GI) tract is
mediated by inflammatory cytokines such as TNFα, IFN-γ, IL-1, IL-6, IL-12, IL-21 and IL-23 that
exert their effects on cells of the innate and adaptive immune system including T and B
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lymphocytes, epithelial cells, macrophages and dendritic cells (DC) . The Janus kinase (JAK)
family, JAK1, JAK2, JAK3 and Tyk2, are non-receptor tyrosine kinases that play a pivotal role in
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the response to many such cytokines . Following receptor ligation, associated JAK homo- or
heterodimers are phosphorylated and activated enabling the subsequent recruitment,
phosphorylation and activation of the signal transducers and activators of transcription (STAT)
family of transcription factors. Phosphorylated STATs (pSTATs) translocate to the nucleus and
induce gene transcription of several chemokines, cytokines and proteases implicated in the
pathogenesis of IBD1.
Genetic studies in patients with IBD have identified polymorphisms in several proteins associated
with the JAK/STAT pathway such as IL-23R, IL-12B, JAK2, Tyk2 and STAT3 as risk factors for
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IBD . Since the JAK/STAT pathway is common to multiple inflammatory cytokine responses
(Table 1), it offers an attractive therapeutic target to prevent the excessive inflammatory response
associated with IBD. In support of this data, several JAK inhibitors such as tofacitinib and
filgotinib are currently in clinical development for IBD6.
Cytokines
IL-2, -4, -7, -9, -15, -21
IFN
JAKs
JAK1/JAK3
JAK1/JAK2
JAK1/Tyk2
JAK2/Tyk2
IL-10, IFN
IL-12, IL-23
IL-6, -11, -13, -27, -31, -35
EPO, GM-CSF, IL-3, IL-5, TPO
JAK1/Tyk2 or JAK1/JAK2
JAK2/JAK2
Table 1. Cytokines that signal through JAK homo- and heterodimers2.
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Tofacitinib is approved in the United States for the treatment of rheumatoid arthritis (RA) and UC7,
8. Tofacitinib was also in clinical development for the treatment of CD; however, further
development for this indication was discontinued due to failure to achieve significant clinical
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efficacy in a 4 week, Phase 2 clinical trial in moderate to severe CD . Despite positive evidence of
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target engagement based on biomarker analysis, it is currently unclear whether lack of efficacy of
tofacitinib in CD relates to clinical study design, mechanistic differences between UC and CD or
dose limiting systemic adverse events (AEs) that prevent adequate exposure to intestinal tissue1, 9,
10.
Common to both Phase 2 and Phase 3 IBD clinical trials with tofacitinib, systemic adverse
events (AEs) included decreased hemoglobin, decreased absolute neutrophil count (ANC),
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elevated total cholesterol (low and high density lipid) and infection . Such AEs are consistent
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with those observed following tofacitinib treatment in RA patients and is consistent with JAK2
dependent inhibition of EPO, TPO and granulocyte monocyte colony stimulating factor (GM-CSF)
and/or JAK1 dependent inhibition of IL-611, 12. Based on results of an ongoing post marketing
safety trial with tofacitinib, the FDA recently approved a black box warning regarding increased
risk of blood clots and death with utility of tofacitinib at 10 mg with twice daily dosing. In the
ongoing trial, dosing at 10 mg was stopped but continues to be evaluated at 5 mg twice daily dose.
In patients with UC, the FDA recommends tofacitinib at 5 mg twice daily and restriction to those
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patients who have failed or are intolerant to TNF blockers .
There are several potential approaches to overcome systemic AEs driven by JAK inhibition. One
such approach would be to develop orally available inhibitors that show less activity against JAK2.
Such an approach is utilized by JAK1 selective inhibitors such as filgotinib and upadacitinib
currently in Phase 3 clinical trial for CD and UC. Despite the theoretical safety advantage of a
JAK1 selective molecule, recent approval of upadacitinib for RA at a dose of 15 mg twice daily
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also has a black box warning from the FDA pertaining to risk of thrombosis . An alternative
approach would be to restrict exposure of the inhibitor to the GI tract to maximize the intestinal
tissue exposure of a JAK inhibitor while sparing systemic exposure that can lead to on-target side
effects.
Our objective was to develop a JAK inhibitor with similar cellular potency to tofacitinib, yet with
exposure limited to the intestinal tract. We hypothesized that improved efficacy in both UC and
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CD could be driven by increased intestinal exposure while preventing dose-limiting side effects
that are associated with systemic pan JAK inhibition.
Several gut tissue targeting strategies have been employed to increase intestinal tissue exposure
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while reducing systemic exposure . One common approach is to use a formulation to release the
compound at different regions of the GI tract16-18. Another approach is to modify the
physicochemical properties of the molecules to limit systemic exposure. Compounds can be made
with high cLogP that are rapidly metabolized or with high molecular weight and polar surface
areas that limit intestinal permeability16-18. An alternative strategy is to use a prodrug that is
converted to an active metabolite in the intestines.
RESULTS AND DISCUSSION
We envisaged developing potent, cell active JAK inhibitors with physicochemical properties that
lead to limited systemic exposure after oral dosing but maintain the ability to inhibit cytokine
signaling locally in the intestinal tissues. We set out to identify molecules with low intrinsic
permeability as determined in a Madin Darby canine kidney cell with MDR1 gene [the gene
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encoding for the efflux protein P-glycoprotein (P-gp)] . This (MDCK-MDR1) assay focuses
specifically on intrinsic permeability as measured by apical to basolateral permeability rate in the
presence of a P-gp inhibitor, elacridar [(A-B (+P-gp inhibitor)]20, 21. It is important to note that in
order to target JAK kinases, which are expressed in inflammatory cells within the lamina propria,
some degree of uptake into the GI tract is required to achieve the desired pharmacological effects.
Molecules with intrinsic permeability that is too low would result in decreased systemic exposure
but would also lead to decreased penetration to the target tissue and lack of efficacy. Therefore,
finding a balance in lowering permeability while maintaining cell and tissue-based activity was
essential.
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CN
Enzyme IC50 (nM) MDCK-MDR1 permeability
Mouse plasma (25 mg/kg p.o.)
1.18 M @ 0.5hr
(Papp x 10-6 cm/s)
C2
JAK1 <0.2
JAK2 <0.2
JAK3 12
N
N
A->B (+P-gp inhibitor) 22.1
Tyk2 0.89
N
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Compound 1
Figure 1. Enzyme potency (JAK1, JAK2, JAK3, and Tyk2), intrinsic permeability in MDCK-
MDR1 permeability assay, and systemic exposure at 0.5 hours after oral dosing in mouse of
Compound 1.
To identify JAK inhibitors with appropriate physicochemical properties that would limit systemic
exposure while maintaining cell permeability and the ability to penetrate intestinal tissue,
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compound 1 was used as a starting point. As shown in Figure 1, compound 1 is a potent pan-
JAK inhibitor with high intrinsic permeability. This permeability results in compound 1 having
systemic exposure of 1.18 M measured 30 minutes post oral dose at 25 mg/kg in mice. Our
approach focused on making changes to the portion of the molecule with a vector toward the
solvent exposed region of the JAK proteins. We hypothesized that changes to this portion of the
molecule would have a high probability of maintaining sufficient enzyme inhibition activity, while
allowing for the evaluation of analogs with a wide range of physicochemical properties that could
lead to intestinal tissue-targeting properties. Based on the crystal structure of similar molecules
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from this series , we proposed that the C2 of the imidazole would be the most effective position
to build chemical diversity, and that any loss in JAK enzyme potency or isoform selectivity would
be due to dynamic interactions not described by the static crystal structure. The structural
modifications made at the C2-position of the imidazole are shown in Figure 2 (this position is in
the solvent exposed region; see supplemental Figure S1).
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OH
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n = 15
n = 79
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R = see supplemental file
n = number of compounds in each subset
Figure 2. Analogues of compound 1 synthesized to evaluate a range of physicochemical properties
through substitution at the C2-position of the imidazole. See Supplemental Material for details.
By varying the moiety at the C2-position, a set of 187 compounds was obtained with a range of
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physical properties including cLogP (0.22-5.73), H-bond donors (1-4), tPSA (70-149Å ) and
molecular weight (MW) (323-579), as shown in Figure 3. This broad range of properties obtained
allowed us to assess specific features responsible for lowering the intrinsic permeability of these
molecules.
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Figure 3. Range of properties of analogs from Figure 2 including cLogP, tPSA, H-bond donors,
and MW that create a diverse set of physicochemical properties.
Within this set of analogs, trends were not sufficiently robust to consistently predict the
physicochemical properties that were responsible for low intrinsic permeability, with the exception
that four of the compounds with four H-bond donors all had low intrinsic permeability (defined
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here as AB [+P-gp inhibitor] <2 Papp x10 cm/s). However, among a subset of 94 analogs
containing -CH CONRR and -CH NRCOR substitution at C2-position of the imidazole moiety, a
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clear trend of low permeability was observed which correlated to cLogP and tPSA (see Figure 4
and supplemental Table S2. For the corresponding analysis of permeability in relation to cLogP
and tPSA properties for all other analogues, see Table S1). The parameters determined to correlate
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to low intrinsic permeability of <2 Papp x10 cm/s within this series are cLogP <2.5, and tPSA >99
Å . Compounds with tPSA <99Å were more likely to have intrinsic permeability >2 P x10-6
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cm/s.
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Figure 4. Correlation between instrinsic permeability [measured by MDCK-MDR1 (AB +P-gp
inhibitor)] and clogP and tPSA (logarithmic scale used on y-axis) of compounds with -
CH CONRR and -CH NRCOR substituents at the C2-imidazole position.
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An analysis of the JAK1 enzyme potency of these molecules demonstrated that substitution at C2
of the imidazole generally had a detrimental impact on JAK1 inhibition (see Supplementary
Material). However, most molecules were no more than 10- to 15-fold less potent than compound
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, and 60% of these compounds inhibited JAK1 with single-digit nM potency. Also, the isoform
selectivity across the JAK-family was generally preserved (see supplemental Figures S3-S5). This
level of in vitro potency, combined with the analysis of the physicochemical properties, allowed
us to select molecules for in vivo evaluation.
To support the selection of compounds for in vivo mouse pharmacokinetic/pharmacodynamic
(PK/PD) studies, we chose compounds with a range of intrinsic permeability values and measured
plasma exposures following a single 25 mg/kg oral dose in mice. Plasma exposures were
determined at 0.5 hours (approximate systemic Tmax) in addition to measurement at 4 hours when
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colon exposure was also determined (approximate colon Tmax - data not shown). To support
selection for further in vivo studies, compounds that achieved systemic concentrations below 100
nM at 0.5 and 4 hours were considered for further profiling. Compounds were prioritized based on
this threshold of 100 nM such that systemic exposures were expected to be less than the whole
blood IC . As shown in Figure 5, the results of the screen established a correlation between plasma
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exposures and intrinsic permeability. Compounds with intrinsic permeability less than 1.5 Papp x10-
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cm/s were more likely to have low systemic exposure (defined as <100 nM) at 0.5 hours. This
correlation demonstrates that, for this subset of compounds, calculated physicochemical properties
(cLogP and tPSA) can be used to identify compounds that achieve low intrinsic permeability, and
this low intrinsic permeability translates into compounds with low systemic exposure in vivo.
There are a number of publications establishing correlations between physicochemical properties
and permeability. The Lipinski “rule of five” is one method of assessing the likelihood that a small
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molecule will be orally bioavailable based on calculated physiochemical properties . Compounds
having a MW <500, cLogP less than five, five or fewer hydrogen bond donors, and ten or fewer
hydrogen bond acceptors have a higher probability of oral bioavailability. A more recent analysis
by Paolini et al. identified a calculated centroid in the same physicochemical parameters of 617
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oral drugs at MW = 316, cLogP = 2.3, 2 hydrogen bond donors and 4 hydrogen bond acceptors .
The physicochemical properties of the JAK inhibitors in Figure 5 range from 2-3 H-bond donors,
1
03-128 tPSA, 389-490 MW and 0.24-2.79 cLogP, suggesting that many of these compounds
would have been expected to be in good physicochemical space for permeability and
bioavailability based on known criteria for assessing these parameters.
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9
0
Figure 5. A comparison of mouse plasma exposure at 0.5 hours following oral dosing to intrinsic
permeability [MDCK-MDR1 AB (+P-gp inhibitor)] (logarithmic scale used on y-axis).
The permeability, and enzyme and cellular potency, and plasma exposure after p.o. dosing in
mice of select compounds from Figure 4 are shown in Table 2. These data show that many
molecules with low intrinsic permeability and low systemic exposure can maintain good cell
potency in IL-2-, IFNα-, and GM-CSF-stimulated peripheral blood mononuclear cells (PBMCs)
(compounds 2-9). However, we did obtain compounds that lost a significant amount of potency
in PBMCs compared to their enzyme activity, as exemplified by compounds 10 and 11.
Although we were unable to determine a direct correlation of permeability and cell activity
(compare, for example, entried 3 & 11, Table 2), empirically we were able to successfully
discover molecules with potent cell activity and modest absorption after oral administration. All
compounds tested had low but quantifiable levels of systemic exposure at 0.5 hours; all but
compound 3 had quantifiable plasma levels at 4 hours. At this 4 hour timepoint, exposures in the
colon tissue were also measured and in every case were found to be high (1335-16717ng/g). We
also assessed metabolic stability in mouse liver microsomes expressed as extraction ratios (E.R.)
for all compounds which demonstrates metabolism is not a major contributor to reduced
systemic exposure. This is exemplified by compounds 1 and 2 that have similar extraction ratios
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(
0.34 vs 0.37, respectively) but have a large difference in exposure at 0.5hr (1.18 µM vs 0.032
µM, respectively) which correlates to intrinsic permeability.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
R
MDCK-MDR1
Permeability
Liver
Micro-
somes
Enzyme IC50
(nM)
PBMC Cell IC50
(nM)
N
Mouse PK Exposure
N
-6
(cm/s x 10 )
N
N
H
Plasma Plasma
Colon
4.0 hr
(ng/g)
IL-2/
GM-CSF
IFNα/
A->B Papp
Mouse
E.R.
Cmpd.
R-group
JAK1 JAK2 JAK3 Tyk2
0.5 h
(µM)
4.0 hr
(µM)
pSTAT5 / pSTAT5 pSTAT4
(+P-gp Inh)
3.22
tofa
NA
1.3
5.0
<0.2
8.6
8.5
12
92
31
96
24
0.89
7.4
6.7
NT
22
54
36
38
NT
83
26
NT
60
NT
65
0.57
0.34
0.37
0.79
0.37
9.6
0.17
0.29
7660
895
1
2
3
4
<0.2
0.37
0.30
0.14
22.1
<0.478; 1.08
0.516
1.18
Me
O
0.032
0.0033
0.036
0.008
BLOQ
0.008
8592
2460
4448
N
HO
H
N
O
2.1
2.6
148
90
N
H
O
N
6.6
2.3
0.542
N
H
O
N
5
0.14
6.1
106
2.7
47
243
NT
0.898
0.67
0.012
0.005
2627
N
O
O
6
7
8
9
0.22
0.25
0.92
0.49
1.2
5.6
5.6
22
98
69
4.8
3.4
15
9.7
7.1
9.4
2.6
103
409
69
39
39
NT
NT
NT
NT
NT
1.24
2.05
>0.9
0.67
0.67
>0.9
0.34
NT
0.11
0.35
2.1
0.012
0.011
0.068
0.009
NT
12008
7413
16717
1335
NT
N
H
N
O
F3C
N
H
O
221
174
74
126
28
2.49
N
H
O
N
H
9.2
7.2
2.8
14
4.64
0.39
NT
F
HO
O
O
1
1
0
1
11
399
1962
0.828
0.947
N
H
H2N
0.54
56
4.9
NT
NT
NT
N
H
O
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1
1
2
2
2
2
2
2
2
2
2
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3
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5
5
5
6
BLOQ=below limit of quantification; NT=not tested; NA=not applicable; tofa=tofacitinib
Table 2. Enzyme, cell-based data, mouse extraction ratios and intrinsic permeability data, as well
as, mouse PK exposures after single dose of 25 mg/kg for tofacitinib and compounds 1-11.
0
1
2
3
4
5
6
7
8
9
0
1
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6
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8
9
0
1
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8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
Based on cell potency, low systemic exposures (0.5 and 4 hours post dose), and high colonic
exposure (4 hours post dose) following a 25 mg/kg dose in mouse, compound 2 was chosen for
further profiling in vitro and in vivo. Head-to-head comparison studies with tofacitinib and
compound 2 demonstrated that the potency of compound 2 is similar to tofacitinib in PBMC
assays measuring either STAT4 or STAT5 phosphorylation in response to a range of cytokine
stimulants (Table 3). Compound 2 had comparable potency to tofacitinib in an IFNα-stimulated
STAT4 (54 nM and 31 nM, respectively) and an IL-12-stimulated STAT4 (257 nM and 440 nM,
respectively) assay. In contrast, compound 2 was 5-6-fold less potent in GM-CSF-stimulated
pSTAT5 (303 nM and 51 nM, respectively) and IL-2-stimulated pSTAT5 (61 nM and 12 nM,
respectively) assays.
Compound 2 (IC , nM)
tofacitinib (IC , nM)
n
5
0
50
PBMC assay
Mean (95% CI)
61 (46-80)
Mean (95% CI)
12 (9-17)
IL-2/pSTAT5
IFNα/pSTAT4
IL-12/pSTAT4
GM-CSF/pSTAT5
3
5
2
3
54 (28-108)
31 (20-49)
257 (153-432)
303 (160-576)
440 (201-961)
51 (36-72)
Table 3. Head-to-head comparison studies of compound 2 with tofacitinib in PBMC assays
stimulated with cytokines IL-2, IFNα, IL-12, and GM-CSF. CI = confidence interval. N=2-5
independent experiments.
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1
Chemistry. The route used to synthesize these JAK inhibitors corresponded to the type of linker
attached to the 2-position of the imidazole. Compounds 2-7, 9-11, containing the -CH CONRR
2
linker, were accessed using the synthetic sequence outlined in Scheme 1. Chloro-
pyrrolopyridine intermediate 12 reacted with 2-((1r,4r)-4-aminocyclohexyl)acetonitrile
hydrochloride in the presence of DIPEA in DMA at 80 °C to give intermediate 13 in 95% yield.
A subsequent nitro reduction of 13 generated diamino compound 14 by passing a solution of 13
in 1:1 THF and MeOH through a 10% Pd/C cartridge at 80 °C in a continuous-flow
hydrogenation reactor, Thales Nano H-Cube®. Condensation of diamino compound 14 with
ethyl 3-ethoxy-3-iminopropanoate in EtOH at 90 °C afforded the versatile ester 15. From this
key intermediate, a variety of amines were attached by aminolysis of the ester 15 via heating in
DMA to give the amide, followed by removal of the sulfonyl protecting group by treatment with
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
KOH in water and dioxane to give the final -CH CONRR compounds. Alternatively, ester 15 can
2
be saponified with NaOH to give the sodium salt 15a, followed by PyBrOP coupling conditions
with amines.
N
N
N
O
Cl
N+
HN
N
-O
H N
2
O
N+
HN
N
(b)
H N
2
N
S
-O
N
O
N
S
(a)
O
O
N
S
O
12
O
O
14
13
N
O
O
R2
N
N
R1
O
O
N
(d)
N
O
N
O
NH
or
N
(e)
(c)
N
N
S
O
N
N
O
H
15
2-7, 9-11
Scheme 1. Reagents and conditions: a) DIPEA, DMA, 80 °C; b) Pd/C, 1 atm H (via H-Cube),
2
THF/MeOH (1:1), 80 °C; c) EtOH, 90 °C; d) (1) R R NH, DMA, 125 °C, (2) 3 M KOH in
1
2
1
Compounds presented in this article were also described in the following patent application
publications: US2018/0170931A1 and WO2018/112382A1 (both published Jun. 21, 2018)
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
water, dioxane, 80 °C; e) (1) 1 M NaOH in water, MeOH/THF (1:1), (2) R R NH, PyBrOP,
1
2
DIPEA, DMF.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
O
N
O
N
O
N
O
HN
O
H2N
N
O
N+
HN
N
HN
O
OH
N
(b, c)
N
N
O
N
-O
H
N
N
N
S
N
S
N
(d)
(
a)
O
N
O
N
O
H
N
N
O
H
17
16
13
8
Scheme 2. Reagents and conditions: a) Na S O , DMSO/MeOH/water, 100 °C; b) 3 M KOH in
2
2
4
water, dioxane, 80 °C; c) 4 M HCl in dioxane, DCM; d) HATU, DIPEA, DCM.
Compound 8, with the -CH NHCOR linker, was synthesized using the route shown in Scheme 2.
2
In a single pot, intermediate nitro 13 and tert-butyl (2-oxoethyl)carbamate were combined
directly using sodium hydrosulfite to afford the tricyclic intermediate 16. After removal of the
sulfonyl and Boc protecting groups, HATU promoted amidation with cyclopropylacetic acid
afforded compound 8.
To determine whether compound 2 achieved systemic activity in vivo, mice were dosed orally
with either vehicle (20% HPβCD), compound 2 (25 mg/kg) or tofacitinib (25 mg/kg). At 0.5
hours post dose, serum drug levels were determined. Observed mean serum levels of compound
2
and tofacitinib were 5.1 ± 1.6 nM and 2166 ± 314nM, respectively. To confirm that the low
level of exposure of compound 2 had no measurable systemic activity, whole blood taken 0.5
hours post oral dose was stimulated with IFNα. This cytokine stimulation induced a significant
increase in pSTAT3 and pSTAT4 in whole blood from vehicle treated mice and the response was
significantly inhibited by tofacitinib. Corresponding with the low systemic exposure observed
for compound 2 post oral dose, no inhibition of IFNα-induced pSTAT4 (Figure 6) or pSTAT3
(data not shown) could be observed in blood. Compound 2 and tofacitinib inhibited IFNα-
induced pSTAT3 response when directly added to murine whole blood with an average IC of
5
0
299 (95% CI 121-733) nM and 112 (95% CI 51-249) nM, respectively (data not shown).
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1
1
50
00
##
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
4
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5
6
0
1
2
3
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5
6
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8
9
0
1
2
3
4
5
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8
9
0
1
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3
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2
3
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9
0
1
2
3
4
5
6
7
8
9
0
*
*
unstimulated
IFNa stimulated
5
0
0
e
b
l
i
2
c
n
i
iti
d
h
n
e
c
u
v
a
o
f
p
o
t
m
o
c
Figure 6: Comparative activity of compound 2 and tofacitinib in mouse whole blood post
oral dose. Mice were dosed orally with compound 2 and tofacitinib at 25 mg/kg. Control mice
received equal volume of 20% of 2-hydroxypropyl-β-cyclodextrin (HPCD) in the similar
fashion. After 0.5 hours, animals were sacrificed, and their blood was collected and stimulated
with IFNα as described in Methods. pSTAT4 levels were measured following 30 minutes of
stimulation and expressed as a percentage of the IFN-induced pSTAT4 response observed
relative to the vehicle treated group. Data are presented as mean ± SEM; N= 4 mice/group.
Statistics derived from ANOVA analysis of pSTAT4 raw Relative Luminescent Values (RLU)
and are applicable to data presented as percent inhibition. **P < 0.001 vs. cytokine simulated
vehicle. ##P<0.001 vs unstimulated vehicle.
To demonstrate target engagement in the colon which is a unique tissue-based assay, we
examined the effect of orally administered compound 2 on the pSTAT3 response elicited by a
cocktail of pro-inflammatory cytokines (IL-22, IL-6, IFNα). We selected this cytokine cocktail
based on its ability to elicit a robust response in gut tissue when given exogenously to murine
colonic explants in culture. Robust increases in colonic pSTAT3 were observed in explant tissue
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
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6
collected from mice pre-dosed with vehicle (HPβCD) (Figure 7A). Tofacitinib (25 mg/kg p.o.),
used as a control, demonstrated inhibition of both baseline (unstimulated) (87.3% inhibition) and
IL-22/IL-6/IFNα stimulated (91.2% inhibition) pSTAT3 (Figure 7A). Compound 2 inhibited
baseline and IL-22/IL-6/IFNα-induced pSTAT3 in a dose-dependent manner (Figure 7A). Basal
pSTAT3 was inhibited by 36.7, 66.2, 71.3 and 83.8% at 0.5, 2.5, 5 and 25 mg/kg doses of
compound 2, respectively. Similarly, compound 2 inhibited IL-22/IL-6/IFNα stimulated
pSTAT3 by 5.5, 62.0, 68.1 and 89.8% in 0.5, 2.5, 5 and 25 mg/kg dosed mice, respectively.
Thus, compound 2 demonstrates robust target engagement in the colon tissue ex vivo through the
inhibition of the IL-22/IL-6/IFNα-induced pSTAT3 response at doses with limited systemic
exposure.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7: Dose response of compound 2 effect on basal and IL-22/IL-6/IFNα-induced
pSTAT3 in mouse colon explants. Mice were dosed orally with compound 2 or tofacitinib at
indicated doses. Control mice received equal volume of 20% HPβCD (Veh) in the similar
fashion. At 4 hours post dose, animals were sacrificed, and colon tissue was harvested as
detailed in the Methods Section. (A) pSTAT3 levels were measured in the proximal colon
explants following ex vivo stimulation with IL-22/IL-6/IFNα cytokine cocktail for 1 hour.
Control explants (no stimulation) were treated with media alone. Data are presented as mean ±
SEM; N = 5 mice/group. **=p<0.01; ****=p<0.00001. ns= not significant (B) Colonic tissue
levels of compound 2 from all mice dosed 0.5-25mg/kg were measured by liquid
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chromatography-triple quadrupole mass spectrometry (LC MS/MS) method and corresponding
inhibition of IL-22/IL-6/IFNα responses for each explant plotted.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Finally, to establish PK/PD relationship, terminal colonic exposure of compound 2 was
measured. Colonic exposure of compound 2 increased in dose-dependent fashion and correlated
with the inhibition of the cytokine-induced pSTAT3 response, as illustrated in Figure 7B.
According to this linear relationship, more than 50% of the pSTAT3 response in tissue can be
inhibited at colonic concentrations greater than 897 ng/g of tissue.
CONCLUSION
In conclusion, correlations between the physicochemical properties (cLogP and tPSA) of a series
of imidazopyrrolopyridines and their measured intrinsic permeability were derived. The
correlations developed from these imidazopyrrolopyridines allowed for the identification of gut-
restricted pan-JAK inhibitors that maintained cellular activity despite their low intrinsic
permeabilities. Compound 2 demonstrated dose-dependent pharmacological activity in a PK/PD
study, demonstrating that it is a GI tract restricted JAK inhibitor that selectively inhibited
cytokine responses in colonic tissue and not in plasma. Compound 2 is therefore a candidate
therapeutic for IBD indications that would benefit from selective inhibition of cytokine responses
driven by the JAK/STAT pathway in the GI tract.
EXPERIMENTAL SECTION
General Experimental. Anhydrous solvents and reagents were used as supplied by the
manufacturer. Unless otherwise stated, reaction mixtures were magnetically stirred at room
temperature (rt). Where solutions are “dried,” they are generally dried over a drying agent such as
Na SO or MgSO . Concentrated refers to concentrated using a rotary evaporator under reduced
2
4
4
pressure. Unless specified otherwise, normal-phase flash column chromatography (FCC) was
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2
2
2
2
2
2
2
2
2
2
3
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5
6
performed on silica gel (SiO ). The calculated (calcd) mass corresponds to the exact mass. HRMS
2
was obtained on an Agilent Technologies 6230 series TOF-MSD, using ESI in positive mode.
Nuclear magnetic resonance (NMR) spectra were obtained on Bruker model DRX spectrometers.
Chemical shifts (δ) are expressed in parts per million, relative to internal tetramethylsilane;
coupling constants (J) are in hertz (Hz). The following abbreviations are used to describe peak
patterns when appropriate: s (singlet), d (doublet), t (triplet), q (quartet), qt (quintet), m (multiplet),
app (apparent), and br (broad). HPLC−MS chromatograms and spectra were obtained using one
of the following methods: (1) Agilent 1200 HPLC and G6100 system on X-Bridge ShieldRP18
0
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0
1
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0
1
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0
(
50x2.1mm 5μm) and a gradient system of 0.05% NH OH in H O/CH CN, 100:0 to 5:95 over 7.5
4 2 3
min, then 100:0 for 2.5 min at temperature 40 °C; (2) Agilent 1200 HPLC and G6100 system on
Phenomenex Luna-C18 (50×2mm, 5μm) and a gradient system of 0.1% TFA in H O/0.05% TFA
2
in CH CN, 100:0 to 15:85 over 7.5 min, then 100:0 for 2.5 min at temperature 50 °C; or (3) Agilent
3
1100 HPLC and G1367A system on X-Bridge C18 (100x3mm, 3.5uM) and a gradient system of
2
0 mM NH OH in H O/CH CN 90:10 over 2 min, then 0:100 for 1 min at a flow rate of 2.4
4 2 3
mL/min at temperature 45 °C. All compounds tested were of a minimum of 95% purity as
determined by HPLC. None of the prepared compounds contain PAINS moieties25, 26
.
Chemistry: Preparation of intermediates 13-17.
Intermediate 13. 2-((1r,4r)-4-((5-Nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-
yl)amino)cyclohexyl)acetonitrile. Step A: tert-butyl N-[(1r,4r)-4-
Hydroxymethyl)cyclohexyl]carbamate. To a 20-L 4-necked round-bottom flask purged and
(
maintained with an inert atmosphere of nitrogen, was placed (1r,4r)-4-[[(tert-
butoxy)carbonyl]amino]cyclohexane-1-carboxylic acid (1066 g, 4.38 mol, 1.00 equiv) and THF
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(
10 L). This was followed by the addition of BH -Me S (10 mol/L) (660 mL) dropwise with
3
2
o
o
stirring at -10 C over 1 h. The resulting solution was stirred for 3 h at 15 C. This reaction was
performed three times in parallel and the reaction mixtures were combined. The reaction was then
quenched by the addition of 2 L of methanol. The resulting mixture was concentrated under
1
1
1
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0
vacuum.
This
resulted
in
3000
g
(99.6%)
of
tert-butyl
N-[(1r,4r)-4-
(hydroxymethyl)cyclohexyl]carbamate as a white solid. MS (ESI): mass calcd. for C H NO ,
12 23
3
+
1
2
29.32; m/z found, 215.2 [M-tBu+MeCN+H] ; H NMR: (300 MHz, CDCl ): δ 4.40 (s, 1H), 3.45
3
(d, J = 6.3 Hz, 2H), 3.38 (s, 1H), 2.05-2.02 (m, 2H), 1.84-1.81 (m, 2H), 1.44 (s, 10H), 1.17-1.01
(m, 4H).
Step B: tert-butyl N-[(1r,4r)-4-[(methanesulfonyloxy)methyl]cyclohexyl]carbamate. To a 20-L
4-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was
placed tert-butyl N-[(1r,4r)-4-(hydroxymethyl)cyclohexyl]carbamate (1000 g, 4.36 mol, 1.00
equiv.), dichloromethane (10 L), pyridine (1380 g, 17.5 mol, 4.00 equiv.). This was followed by
o
the addition of MsCl (1000 g, 8.73 mol, 2.00 equiv.) dropwise with stirring at -15 C. The resulting
o
solution was stirred overnight at 25 C. This reaction was performed parallel for 3 times. The
reaction was then quenched by the addition of 2 L of water. The water phase was extracted with
ethyl acetate (1x9 L). The organic layer was separated and washed with 1 M HCl (3x10 L),
NaHCO (aq.) (2x10 L), water (1x10 L) and brine (1x10 L). The mixture was dried over anhydrous
3
sodium sulfate, filtered and concentrated under vacuum. This resulted in 3300 g (82%) of tert-
butyl N-[(1r,4r)-4-[(methanesulfonyloxy)methyl]cyclohexyl]carbamate as a white solid. LC-MS:
+
1
MS (ESI): mass calcd. for C H NO S, 307.15; m/z found 292.1, [M-tBu+MeCN+H] ; H NMR:
1
3
25
5
(300 MHz, CDCl ): δ 4.03 (d, J = 6.6 Hz, 2H), 3.38 (s, 1H), 3.00 (s, 3H), 2.07-2.05 (m, 2H), 1.87-
3
1
.84 (m, 2H), 1.72-1.69 (m, 1H), 1.44 (s, 9H), 1.19-1.04 (m, 4H).
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Step C: tert-butyl N-[(1r,4r)-4-(cyanomethyl)cyclohexyl]carbamate. To a 10-L 4-necked round-
bottom
[(methanesulfonyloxy)methyl]cyclohexyl]carbamate (1100 g, 3.58 mol, 1.00 equiv.), DMSO
5500 mL), NaCN (406 g, 8.29 mol, 2.30 equiv.). The resulting solution was stirred for 5 h at 90
flask
was
placed
tert-butyl
N-[(1r,4r)-4-
0
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2
3
4
5
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8
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4
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8
9
0
(
oC. This reaction was performed parallel 3 times. The reaction was then quenched by the addition
of 15 L of water/ice. The solids were collected by filtration. The solids were washed with water (3
x
10 L). This resulted in 2480
g
(97%) of tert-butyl N-[(1r,4r)-4-
(
cyanomethyl)cyclohexyl]carbamate as a white solid. MS (ESI): mass calcd. for C H N O ,
1
3
22
2
2
+
1
2
38.17; m/z found 224 [M-tBu+MeCN+H] ; H NMR: (300 MHz, CDCl ): δ 4.39 (s, 1H), 3.38 (s,
3
1H), 2.26 (d, J = 6.9 Hz, 2H), 2.08-2.04 (m, 2H), 1.92-1.88 (m, 2H), 1.67-1.61 (m, 1H), 1.44 (s,
H), 1.26-1.06 (m, 4H).
9
Step D: 2-[(1r,4r)-4-aminocyclohexyl]acetonitrile hydrochloride. To a 10-L round-bottom flask
was placed tert-butyl N-[(1r,4r)-4-(cyanomethyl)cyclohexyl]carbamate (620 g, 2.60 mol, 1.00
equiv.), and 1,4-dioxane (2 L). This was followed by the addition of a solution of HCl in 1, 4-
o
dioxane (5 L, 4 mol/L) dropwise with stirring at 10 C. The resulting solution was stirred overnight
o
at 25 C. This reaction was performed for 4 times. The solids were collected by filtration. The solids
were washed with 1, 4-dioxane (3x3 L), ethyl acetate (3x3 L) and hexane (3x3 L). This resulted in
1
,753 g (96%) of 2-[(1r,4r)-4-aminocyclohexyl]acetonitrile hydrochloride as a white solid. MS
+
1
(
ESI): mass calcd. for C H N , 138.12; m/z found 139.25, [M+1] ; H NMR: (300 MHz, DMSO-
8 14 2
d6): δ 8.14 (s, 3H), 2.96-2.84 (m, 1H), 2.46 (d, J = 6.3 Hz, 2H), 1.98 (d, J = 11.1 Hz, 2H), 1.79 (d,
J = 12.0 Hz, 2H), 1.64-1.49 (m, 1H), 1.42-1.29 (m, 2H), 1.18-1.04 (m, 2H).
Step
E:
2-((1r,4r)-4-((5-Nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-
yl)amino)cyclohexyl)acetonitrile. To a 1000 mL round bottom flask containing 2-((1r,4r)-4-
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aminocyclohexyl)acetonitrile hydrochloride (29.10 g, 166.6 mmol) was added DMA (400 mL).
The resulting suspension was treated with 4-chloro-5-nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-
b]pyridine (51.53 g, 152.6 mmol), followed by DIPEA (63.0 mL, 366 mmol). The reaction mixture
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2
2
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3
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4
4
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4
4
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4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
was placed under N and heated at 80 °C for 4 h. The crude reaction mixture was cooled to room
2
temperature and slowly poured into a vigorously stirred 2 L flask containing 1.6 L water. The
resulting suspension was stirred for 15 minutes at room temperature, then filtered and dried for 16
h in a vacuum oven with heating at 70 °C to provide the title compound (63.37 g, 95% yield) as a
+
1
yellow solid. MS (ESI): mass calcd. for C H N O S, 439.1; m/z found, 440.1 [M+H] . H NMR
2
1
21
5
4
(
500 MHz,CDCl ): δ 9.10 (s, 1H), 8.99 (d, J = 7.8 Hz, 1H), 8.23 - 8.15 (m, 2H), 7.66 - 7.59 (m,
3
2H), 7.56 - 7.49 (m, 2H), 6.67 (d, J = 4.2 Hz, 1H), 3.95 - 3.79 (m, 1H), 2.38 (d, J = 6.2 Hz, 2H),
.32 - 2.21 (m, 2H), 2.08 - 1.98 (m, 2H), 1.88 - 1.76 (m, 1H), 1.60 - 1.32 (m, 4H).
2
Intermediate 14. 2-((1r,4r)-4-((5-Amino-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-
yl)amino)cyclohexyl)acetonitrile. 2-((1r,4r)-4-((5-Nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-
b]pyridin-4-yl)amino)cyclohexyl)acetonitrile (13, 58.60 g, 133.3 mmol) was dissolved in
THF/MeOH (1:1, 4800 mL). The mixture was passed through a continuous-flow hydrogenation
reactor, such as a Thales Nano H-Cube®, at 10 mL/min with 100 % hydrogen (atmospheric
pressure, 80 °C) through a 10% Pd/C cartridge then concentrated to dryness to provide a purple
solid. The resulting purple solid was suspended in EtOAc (400 mL) and was stirred vigorously
at room temperature for 5 minutes. The solid was filtered off and the solid was re-suspended in
2
00 mL MeOH and stirred vigorously at room temperature for 3 h. The solid was filtered off to
separate the solid from the MeOH. The solvent was removed in vacuo and the residue was
purified by flash column chromatography to provide the title compound (54.12 g, 99% yield).
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+
1
MS (ESI): mass calcd. for C H N O S, 409.2; m/z found, 410.2 [M+H] . H NMR (400 MHz,
2
1
23
5
2
CDCl ) δ 8.10 - 8.03 (m, 2H), 7.76 (s, 1H), 7.51 - 7.43 (m, 1H), 7.43 - 7.34 (m, 3H), 6.44 (d, J =
3
4.2 Hz, 1H), 4.61 (d, J = 8.5 Hz, 1H), 3.65 - 3.51 (m, 1H), 2.74 (s, 2H), 2.26 (d, J = 6.4 Hz, 2H),
0
1
2
3
4
5
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8
9
0
2
.19 - 2.05 (m, 2H), 1.97 - 1.86 (m, 2H), 1.76 - 1.59 (m, 1H), 1.33 - 1.12 (m, 4H).
Intermediate 15. Ethyl 2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-
dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate. To a 1000 mL round bottom flask
containing a stir bar and 2-((1r,4r)-4-((5-amino-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-
yl)amino)cyclohexyl)acetonitrile (14, 58.31 g, 142.4 mmol) was added ethyl 3-ethoxy-3-
iminopropanoate (60.51 g, 309.3 mmol), followed by EtOH (600 mL, dried over 3Å molecular
sieves for 48 h). A reflux condenser was attached to the reaction flask, the reaction was purged
with N , and was heated at 90 °C for 9 h. The reaction mixture was cooled to room temperature
2
and left to stand for 30 h where the product crystallized out as brown needles. The solids were
broken up with a spatula and the reaction mixture was transferred to a 2 L flask. Water (1.4 L)
was added slowly via separatory funnel with vigorous stirring. The suspension was stirred for 30
minutes, after addition of the water was complete. The brown needles were isolated by filtration
and then dried by pulling air through the filter for 1 h. The product was transferred to a 500 mL
flask and treated with EtOAc (200 mL). A small quantity of seed crystals was added which
induced the formation of a white solid precipitate. The suspension was stirred for 30 minutes at
room temperature, filtered, rinsed with EtOAc (25 mL), and dried under vacuum to provide the
product as a white solid (48.65 g, 68% yield). MS (ESI): mass calcd. for C H N O S, 505.2;
2
6
27
5
4
+
1
m/z found, 506.2 [M+H] . H NMR (400 MHz, CDCl ) δ 8.85 (s, 1H), 8.28 - 8.19 (m, 2H), 7.84
3
(
d, J = 4.0 Hz, 1H), 7.61 - 7.53 (m, 1H), 7.52 - 7.43 (m, 2H), 6.84 (d, J = 4.1 Hz, 1H), 4.32 (s,
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H), 4.20 (q, J = 7.1 Hz, 2H), 4.09 (s, 2H), 2.44 (d, J = 6.2 Hz, 2H), 2.40 - 2.27 (m, 2H), 2.16 (d,
J = 13.3 Hz, 2H), 2.12 - 1.96 (m, 3H), 1.54 - 1.38 (m, 2H), 1.27 (t, J = 7.1 Hz, 3H).
1
1
1
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2
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Intermediate 15a. Sodium 2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-
dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate. To a solution of ethyl 2-(1-((1r,4r)-
4
-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-
b]pyridin-2-yl)acetate (15, 9.50 g, 18.8 mmol) in MeOH (30 mL) and THF (30 mL) was added
sodium hydroxide (aqueous) (56.4 mL, 56.4 mmol, 1 M) and was stirred at room-temperature for
14 hours. The solvent was removed under reduced pressure at room-temperature to provide title
compound (7 g) as brown solid, which was used for next step without further purification. MS
+
1
(ESI): mass calcd. for C H N NaO , 359.1; m/z found, 337.9 [M+H-Na] . H NMR
18 18 5 2
(400MHz, CD OD) δ 8.52 - 8.47 (m, 1H), 7.85 - 7.81 (m, 2H), 7.46 - 7.41 (m, 4H), 6.85 - 6.81
3
(
m, 1H), 4.60 - 4.46 (m, 1H), 3.96 (s, 2H), 2.59 - 2.49 (m, 4H), 2.19 - 2.05 (m, 6H), 1.56 - 1.43
m, 2H) (a 1:1 mixture of the title compound and benzenesulfonic acid).
(
Intermediate 17. 2-((1r,4r)-4-(2-(Aminomethyl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-
yl)cyclohexyl)acetonitrile hydrochloride. Step A: tert-Butyl ((1-((1r,4r)-4-
(
cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-
-yl)methyl)carbamate (16) and 2-((1r,4r)-4-(2-(aminomethyl)-6-(phenylsulfonyl)imidazo[4,5-
d]pyrrolo[2,3-b]pyridin-1(6H)-yl)cyclohexyl)acetonitrile. A solution of 2-((1r,4r)-4-((5-nitro-1-
phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)amino)cyclohexyl)acetonitrile (13, 1.50 g, 3.41
2
(
mmol), tert-butyl (2-oxoethyl)carbamate (1.47 g, 7.85 mmol), sodium hydrosulfite (2.97 g, 17.1
mmol), DMSO (3 mL), methanol (10 mL), and water (5 mL) was stirred for 2.5 h at 100 °C. A
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mixture of two products, tert-butyl ((1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-
dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)methyl)carbamate (16) and 2-((1r,4r)-4-(2-
(aminomethyl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)cyclohexyl)acetonitrile, were
found. The reaction mixture was used to the next step directly.
0
1
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Step
B:
tert-Butyl
((1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-
dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)methyl)carbamate (16). (Boc) O (1.04 g, 4.78
2
mmol) was added to a mixture of tert-butyl ((1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-
(
phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)methyl)carbamate and 2-
(1r,4r)-4-(2-(aminomethyl)-6-(phenylsulfonyl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-
(
yl)cyclohexyl)acetonitrile (36 mL reaction mixture, 6.826 mmol), Na CO (2.17 g, 20.5 mmol),
2
3
and methanol (50 mL) and was stirred for 2 hours at 12 °C. The reaction was concentrated under
vacuum to dryness and the aqueous layer was diluted with water (30 mL) and extracted with ethyl
acetate (50 mL × 2). The combined organic extracts were dried over anhydrous Na SO , filtered,
2
4
and concentrated to dryness. The residue was purified by flash column chromatography to provide
1
the title compound (2.4 g, 63% over two steps) as a light yellow solid. H NMR (400MHz, CDCl ):
3
 8.83 (s, 1H), 8.23 (d, J = 7.6 Hz, 2H), 7.84 (d, J = 4.0 Hz, 1H), 7.60 - 7.52 (m, 1H), 7.52 - 7.44
(
m, 2H), 6.83 (d, J = 4.0 Hz, 1H), 5.36 (br s, 1H), 4.67 (d, J = 5.6 Hz, 2H), 4.65 - 4.59 (m, 1H),
.40 (d, J = 6.4 Hz, 2H), 2.37 - 2.22 (m, 2H), 2.20 - 2.09 (m, 2H), 2.04 - 1.87 (m, 3H), 1.53 - 1.39
(m, 11H).
Step C:
2
tert-Butyl
((1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-
d]pyrrolo[2,3-b]pyridin-2-yl)methyl)carbamate. Aqueous KOH (4.37 mL, 13.1 mmol) was added
to a solution consisting of tert-butyl ((1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-
1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)methyl)carbamate (16, 2.40 g, 4.37 mmol)
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and dioxane (25 mL) and was stirred for 2 hours at 80 °C. The reaction mixture was adjusted to
pH = 7-8 with 1 M HCl and the volatile solvents were removed under vacuum. The aqueous phase
was diluted with water (20 mL) and extracted with ethyl acetate (50 mL × 2). The combined
organic extracts were dried over anhydrous Na SO , filtered, and concentrated to dryness to
1
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provide the title compound (2 g, 100% yield) as a light yellow solid, which was used in the next
step without further purification. MS (ESI): mass calcd. for C H N O , 408.2; m/z found, 409.1
22 28
6
2
+
1
[
M+H] . H NMR (400MHz, DMSO-d ):  11.86 (s, 1H), 8.51 (s, 1H), 7.56 - 7.49 (m, 1H), 7.47
6
(t, J = 2.8 Hz, 1H), 6.74 - 6.68 (m, 1H), 4.66 - 4.58 (m, 1H), 4.53 (d, J = 5.6 Hz, 2H), 2.58 (d, J =
5.6 Hz, 2H), 2.41 - 2.28 (m, 2H), 2.08 - 1.94 (m, 3H), 1.93 - 1.83 (m, 2H), 1.54 - 1.23 (m, 11H).
Step D: 2-((1r,4r)-4-(2-(Aminomethyl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-
yl)cyclohexyl)acetonitrile hydrochloride (17). HCl/dioxane (10 mL, 40 mmol, 4 M) was added to
a solution of tert-butyl ((1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-
d]pyrrolo[2,3-b]pyridin-2-yl)methyl)carbamate (2.0 g, 4.9 mmol) in CH Cl (20 mL) at 0 °C and
2
2
was stirred for 30 minutes at 10 °C. The reaction was concentrated to dryness to provide the title
compound (1.7 g, 100% yield) as a pale white solid, which was used without further purification.
MS (ESI): mass calcd. for C H ClN , 308.2; m/z found, 308.9 [M+H] , 1H NMR (400 MHz,
+
1
7
20
6
DMSO-d ) δ 12.53 (s, 1H), 8.88 (s, 1H), 8.86 (br. s, 3H), 7.67 (t, J = 3.2 Hz, 1H), 6.93 (s, 1H),
6
4
.71 - 4.63 (m, 2H), 4.60 - 4.42 (m, 1H), 2.60 (d, J = 6.0 Hz, 2H), 2.36 - 2.21 (m, 2H), 2.12 - 1.93
(m, 5H), 1.56 - 1.42 (m, 2H).
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Synthesis of Compounds 2-11.
Compound 2. 2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-
d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide
0
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Step A: 2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-
d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide. To ensure dry starting
material,
ethyl
2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-
dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate (15) was heated under vacuum at 50 °C
for 18 h prior to the reaction. In a 1 L flask, ethyl 2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-
(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate (15, 52.59 g, 104.0
mmol) was suspended in DMA (50 mL). 1-Amino-2-methylpropan-2-ol (50 mL) was added and
the reaction was heated to 110 °C for 45 minutes, then to 125 °C for 5 hours. The reaction was
cooled to room temperature and diluted with EtOAc (800 mL). The organic layer was extracted
3
6
-times (1 L water plus 50 mL brine). The aqueous layers were back extracted with EtOAc (2 ×
00 mL). The combined organic layers were dried over anhydrous MgSO , filtered, concentrated
4
to dryness, and then dried for 3 days under vacuum to provide the title compound (65.9 g, 98%
yield) as a yellow foam. The product was taken to the next step with no further purification. MS
+
1
(
ESI): mass calcd. for C H N O S, 548.22; m/z found, 549.2 [M+H] . H NMR (400 MHz,
28 32 6 4
CDCl ): δ 8.76 (s, 1H), 8.26 - 8.19 (m, 2H), 7.84 (d, J = 4.1 Hz, 1H), 7.60 - 7.53 (m, 1H), 7.50 -
3
7.44 (m, 2H), 6.84 (d, J = 4.2 Hz, 1H), 4.76 - 4.61 (m, 1H), 3.97 (s, 2H), 3.45 (s, 1H), 3.27 (d, J =
5
4
.9 Hz, 2H), 2.41 (d, J = 6.5 Hz, 2H), 2.38 - 2.25 (m, 2H), 2.23 - 2.12 (m, 2H), 2.09 - 1.94 (m,
H), 1.48 (qd, J = 13.6, 4.0 Hz, 2H), 1.21 (s, 6H).
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Step
B:
2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-
b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide.
(Cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-
-yl)-N-(2-hydroxy-2-methylpropyl)acetamide (65.90 g, 102.1 mmol) was added to a 1 L flask
2-(1-((1r,4r)-4-
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containing a stir bar. Dioxane (300 mL) was added, followed by KOH in water (3 M, 150 mL).
The reaction was heated at 80 °C for 2 h. The reaction was cooled to room temperature and the
solvent volume was reduced to about 200 mL on a rotovap. The residue was treated with 100 mL
water plus 100 mL brine and then extracted 2 times with 10% MeOH in CH Cl (1000 ml). The
2
2
organic layers were combined, dried over anhydrous MgSO , filtered, and concentrated to dryness
4
to provide a yellow solid. The solid was suspended in 200 mL CH Cl , stirred vigorously for 30
2
2
minutes, and then filtered. The solid was rinsed with 100 mL CH Cl , dried by pulling air through
2
2
the filter, and then further dried under vacuum at room temperature for 16 h to provide the title
compound (42.59 g, 89% yield) as a white solid. MS (ESI): mass calcd. for C H N O , 408.23;
2
2
28
6
2
+
1
m/z found, 409.2 [M+H] . H NMR (600 MHz, DMSO-d ):  11.85 (s, 1H), 8.50 (s, 1H), 8.21 –
6
8
.10 (m, 1H), 7.49 – 7.43 (m, 1H), 6.74 – 6.65 (m, 1H), 4.53 – 4.42 (m, 2H), 4.07 (s, 2H), 3.08 (d,
J = 6.0 Hz, 2H), 2.58 (d, J = 6.1 Hz, 2H), 2.41 – 2.28 (m, 2H), 2.09 – 1.92 (m, 5H), 1.42 – 1.31
m, 2H), 1.09 (s, 6H).
(
Compound 3. 2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-
d]pyrrolo[2,3-b]pyridin-2-yl)-N-(1-isobutylpiperidin-4-yl)acetamide
A
mixture of ethyl 2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-
dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate (15, 300 mg, 0.59 mmol) and 1-
isobutylpiperidin-4-amine (976 mg, 5.93 mmol) was heated at 125 °C in the microwave for 22 hr.
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The reaction was cooled to room temp, then 1,4-dioxane (2.4 mL) and 3N KOH (1.6 mL) aqueous
solution were added. The vial was heated at 80 °C for 2 h in the microwave, then purified by basic
HPLC using a Xbridge Prep OBD C 50 mm x 100 mm, 5 m column (eluent = 0-100% aq
18
0
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NH OH/ACN (10 min)) to yield the title compound (111 mg, 39% yield). MS (ESI): mass calcd.
4
+
1
for C H N O, 475.31; m/z found, 476.3 [M+H] . H NMR (500 MHz, CD OD):  8.54 (s, 1H),
27 37
7
3
7
.49 (d, J = 3.5 Hz, 1H), 6.86 (d, J = 3.5 Hz, 1H), 4.54 (s, 1H), 4.11 – 4.04 (m, 1H), 3.75 – 3.64
m, 1H), 2.89 (d, J = 11.8 Hz, 2H), 2.63 – 2.54 (m, 3H), 2.19 – 1.78 (m, 13H), 1.69 – 1.44 (m,
H), 0.97 – 0.88 (m, 7H).
(
4
Compound 4. N-(2-Cyanoethyl)-2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-
dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetamide
To a solution of sodium 2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-
d]pyrrolo[2,3-b]pyridin-2-yl)acetate (15a, 400 mg, 1.11 mmol) and 3-aminopropanenitrile (320
mg, 2.24 mmol) in DMF (5 mL) was added PyBOP (870 mg, 1.67 mmol) and DIPEA (0.60 mL,
3
.5 mmol). The reaction mixture was stirred at room temperature for 40 h. After removal of the
DMF in vacuo, the residue was purified by flash column chromatography using 50-100% ethyl
acetate in heptane. The collected fractions were concentrated in vacuo to a small volume and white
solid which had precipitated out was filtered off, washed with 10% MeOH in CH Cl , and dried
2
2
to provide to provide the title compound (75 mg, 17% yield). The filtrate was concentrated to
dryness and purified by reverse phase-HPLC using a Varian Pursuit XR 5 Diphenyl 100 mm x 30
s
mm column (eluent 10 – 90% CH CN in water, 0.1% TFA) to provide a clear oil. This material
3
was dissolved in 10% MeOH in CH Cl , passed through three 500 mg columns of SILICYCLE
2
2
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SPE-R66030B-03P Carbonate (SiliaBond acid scavenger solid phase extraction cartridge) to
remove the TFA and eluted with 10% MeOH in CH Cl to provide an additional fraction of the
2
2
title compound (88 mg, 20% yield). The two fractions were combined to provide the final product
1
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(
163 mg, 37% yield) as a white solid. MS (ESI): mass calcd. for C H N O, 389.20; m/z found,
21 23 7
+
1
3
90.3 [M+H] . H NMR (400 MHz, CD OD): δ 8.53 (s, 1H), 7.48 (d, J = 3.0 Hz, 1H), 6.84 (d, J
3
=
-
3.5 Hz, 1H), 4.51 (br s, 1H), 4.12 (s, 2H), 3.50 (t, J = 6.6 Hz, 2H), 2.71 (t, J = 6.6 Hz, 2H), 2.45
2.63 (m, 4H), 2.03 - 2.19 (m, 5H), 1.44 - 1.57 (m, 2H).
Compound 5. 2-((1r,4r)-4-(2-(2-(4-Morpholinopiperidin-1-yl)-2-oxoethyl)imidazo[4,5-
d]pyrrolo[2,3-b]pyridin-1(6H)-yl)cyclohexyl)acetonitrile
A solution of sodium 2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-
d]pyrrolo[2,3-b]pyridin-2-yl)acetate (15a, 200 mg, 0.557 mmol), 4-(piperidin-4-yl)morpholine
(94.8 mg, 0.557 mmol ), DIPEA (144 mg, 1.11 mmol), and DMF (5 mL) was stirred at 0 °C for 1
h. Then PyBrOP (311 mg, 0.668 mmol) was added and stirred at room-temperature overnight.
The mixture was quenched with 10 mL water and was purified by preparative acidic HPLC using
a Boston Green ODS 150 mm x30 mm, 5m column (eluent: water (0.05% HCl)-ACN from 0%
to 30%, v/v) and then by preparative basic HPLC using a Kromasil 150 mm x 25 mm, 10m
column (eluent: water (0.05% ammonia hydroxide v/v)-ACN from 10% to 40%, v/v) to provide
the title compound (25 mg, 9% yield) as a white solid. MS (ESI): mass calcd. for C H N O ,
2
7
35
7
2
+
1
4
89.29; m/z found, 490.3 [M+H] . H NMR (400MHz, DMSO-d ):  11.85 (br s, 1H), 8.51 - 8.47
6
(m, 1H), 7.48 - 7.44 (m, 1H), 6.72 - 6.68 (m, 1H), 4.42 - 4.29 (m, 2H), 4.25 (d, J = 8.4 Hz, 2H),
4
.16 - 4.07 (m, 1H), 3.59 - 3.52 (m, 4H), 3.15 - 3.05 (m, 1H), 2.69 – 2.63 (m, 1H), 2.58 (d, J = 6.0
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