Synthesis and Evaluation of 1 H -Pyrrolo[2,3- b ]pyridine Derivatives as Novel Immunomodulators Targeting Janus Kinase 3

Abstract

Janus kinases (JAKs) have been known to play crucial roles in modulating a number of inflammatory and immune mediators. Here, we describe a series of 1H-pyrrolo[2,3-b]pyridine derivatives as novel immunomodulators targeting JAK3 for use in treating immune diseases such as organ transplantation. In the chemical modification of compound 6, the introduction of a carbamoyl group to the C5-position and substitution of a cyclohexylamino group at the C4-position of the 1H-pyrrolo[2,3-b]pyridine ring led to a large increase in JAK3 inhibitory activity. Compound 14c was identified as a potent, moderately selective JAK3 inhibitor, and the immunomodulating effect of 14c on interleukin-2-stimulated T cell proliferation was shown. Docking calculations and WaterMap analysis of the 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives were conducted to confirm the substituent effects on JAK3 inhibitory activity. Key words.

Full text

© 2015 The Pharmaceutical Society of Japan
Vol. 63, No. 5 341Chem. Pharm. Bull. 63, 341–353 (2015)
Regular Article
Synthesis and Evaluation of 1H-Pyrrolo[2,3 -b]pyridine Derivatives as
Novel Immunomodulators Targeting Janus Kinase 3
Yutaka Nakajima,* Takashi Tojo, Masataka Morita, Keiko Hatanaka, Shohei Shirakami,
Akira Tanaka, Hiroshi Sasaki, Kazuo Nakai, Koichiro Mukoyoshi, Hisao Hamaguchi,
Fumie Takahashi, Ayako Moritomo, Yasuyuki Higashi, and Takayuki Inoue*
Drug Discovery Research, Astellas Pharma Inc.; 21 Miyukigaoka, Tsukuba, Ibaraki 305– 8585, Japan.
Received January 14, 2015; accepted February 24, 2015; advance publication released online March 14, 2015
Janus kinases (JAKs) have been known to play crucial roles in modulating a number of inflammatory
and immune mediators. Here, we describe a series of 1H-pyrrolo[2,3-b]pyridine derivatives as novel im-
munomodulators targeting JAK3 for use in treating immune diseases such as organ transplantation. In the
chemical modification of compound 6, the introduction of a carbamoyl group to the C5 -position and substi-
tution of a cyclohexylamino group at the C4-position of the 1H-pyrrolo[2,3 -b]pyridine ring led to a large
increase in JAK3 inhibitory activity. Compound 14c was identified as a potent, moderately selective JAK3 in-
hibitor, and the immunomodulating effect of 14c on interleukin-2-stimulated T cell proliferation was shown.
Docking calculations and WaterMap analysis of the 1H-pyrrolo[2,3 -b]pyridine-5-carboxamide derivatives
were conducted to confirm the substituent effects on JAK3 inhibitory activity.
Key words Janus kinase 3 inhibitor; immunomodulator; docking calculation; WaterMap
Janus kinases (JAKs) are cytoplasmic protein tyrosine ki-
nases with four known members (JAK1, JAK2, JAK3, and
TYK2) which play important roles in cytokine-mediated
signal transduction.1–5) Cytokines bind to their respective
receptors associated with JAKs and induce JAK activation,
following phosphorylation of the receptors. Activated JAKs
subsequently phosphorylate signal transducers and activa-
tors of transcription proteins (STATs) in cytoplasm, which
are dimerized to translocate to the nucleus and activate gene
transcription to promote cytokine-responsive gene expression.
In the JAK family, JAK3 is specifically associated with the
common γc subunit of cytokine receptors of interleukins (ILs),
such as IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, all of which
are involved in differentiation, proliferation, and survival
of T cells.1–5) The role of JAK3 was studied by analysis of
severe combined immunodeficiency patients, showing JAK3
gene mutation and decreased expression of JAK3 protein.6,7)
Further, JAK3 knockout mice exhibited immunodeficiency
with remarkably reduced numbers of functional T cells, and
no other profound phenotype was observed.8,9) While other
members of JAKs are ubiquitously expressed in whole body,
expression of JAK3 is limited to hematopoietic cells.10, 11)
Therefore, the effects of JAK3 inhibition are suspected to be
limited to immune system. JAK1 is associated with IL-2 re-
ceptors and is involved in regulating the function of T cells in
concert with JAK3. In addition, JAK1 is also involved in the
signaling pathways of IL-6 and interferon (IFN)-γ for inflam-
matory responses.1–5) JAK2 participates in differentiation and
proliferation of erythrocytes, neutrophils, and thrombocytes
by mediating signaling of hematopoietic growth factors such
as erythropoietin, colony-stimulating factor, and thrombopoi-
etin.1–5)
A number of laboratories have attempted to develop JAK
inhibitors.12 –14) Pfizer’s group discovered tofacitinib (com-
pound 1, Fig. 1) as a pan-JAK inhibitor15,16) which proved
effective in various animal models17,18) and was recently ap-
proved for use in treating rheumatoid arthritis (RA). Incyte’s
group discovered the JAK1 and JAK2 inhibitor baricitinib
(compound 2, Fig. 1),16) which is in phase 3 clinical trials for
the treatment of RA. JAK inhibitors presently attract a great
deal of attention with regard to their potential therapeutic ap-
plication for inflammatory and immune diseases such as RA,
psoriasis, and organ transplant rejection, and several com-
pounds have been advanced to the clinical stage.19–2 1)
For the treatment of organ transplantation, calcineurin in-
hibitors (CNIs), such as tacrolimus and cyclosporin A, have
been used as a standard immunosuppressive therapy to in-
hibit IL-2 production and the following T cell activation, and
achieved high efficacy to prevent acute transplant rejection.2 2)
However, the long-term use of CNIs is associated with side
effects such as nephrotoxicity and neurotoxicity.23–25)
In our own research on novel immunomodulators, we main-
ly focused on JAK3 inhibition, as targeting JAK3 may offer
novel and safe immunomodulating regimen due to the effect
on IL-2-dependent T cell proliferation and the limited JAK3
expression on lymphoid cells. In compound screening, JAK1
inhibitory activity was evaluated in addition to JAK3 inhibito-
* To whom correspondence should be addressed. e-mail: yutaka.nakajima@astellas.com; inoue_eck@mii.maruho.co.jp
Fig. 1. Chemical Struct ures of JAK Inhibitors

342 Vol. 63, No. 5 (2015)
Chem. Pharm. Bull.
ry activity, as JAK3 and JAK1 are known to regulate the IL-2
signaling pathway in concert. JAK2 inhibitory activity was
also evaluated, as JAK2 inhibition may be related to adverse
hematopoietic effects such as anemia.26) In our laborator y, we
paid attention to hydrogen bond interaction with the hinge
region of the ATP-binding site, and a number of heteroaryl
compounds were synthesized. Among these compounds, 1H-
pyr rolo[2,3-b] pyridine derivative 6 (Table 1) was identified as
the initial template compound to show JAK inhibitory activity.
We focused on the structure of 6 and attempted to enhance
inhibitory activity toward JAK3, as the 1H-py rrolo[2,3-b]-
pyridine ring mimicked the pyrrolopyrimidine scaffold of 1
and 2. Here, we report findings from a structure–activity rela-
tionship (SAR) study of a series of 1H-pyrrolo[2,3-b] pyridine
derivatives on JAK3 inhibitory activity as novel immuno-
modulators.
Chemistry
As shown in Chart 1, commercially available compound
3 was treated with 2-(trimethylsilyl) ethoxymethyl (SEM)
chloride, followed by a Pd-catalyzed coupling reaction with
N-methylcyclohexylamine to achieve insertion of an amino
group to the C4-position. Deprotection of the SEM group of
compound 5 was conducted by treatment with trifluoroacetic
acid (TFA), followed by alkalization in the presence of 1,2-di-
aminoethane to afford the desired compound 6.
General synthetic routes of the 1H-pyrrolo[2,3-b] pyrid ine-5-
carboxamide derivatives are shown in Charts 2–4. As shown
in Chart 2, protection of compound 3 by treatment of triiso-
propylsilyl (TIPS) chloride and NaH gave compound 7, which
was subjected to ortho-lithiation by sec-Bu Li, followed by
addition of ethyl chloroformate to introduce an ester group at
the C5-posit ion.27 ) Subsequent deprotection of the TIPS group
using tetra-n-butylammonium fluoride (TBAF) afforded 8.
Conversion of the ethyl ester group of 8 to a carbamoyl group
was carried out by basic hydrolysis and subsequent amida-
tion using carbonyldiimidazole (CDI) and aqueous ammonia,
thereby yielding the carboxamide intermediate 10. Nucleo-
philic substitution at the C4-position with a variety of amines
under microwave irradiation afforded the desired compounds
11a–k. The reaction is convenient for conversion of C4-amino
group of the 1H-pyrrolo[2,3-b] pyridine-5-carboxamide deriva-
tives.
Chart 3 shows alternative synthetic routes of the 1H-
pyr rolo[2,3-b] pyridine-5-carboxamide derivatives. The in-
termediate 8 was reacted with a variety of amines under
Table 1. SARs of C5-Substitutent of 1H-Pyrrolo[2,3-b]pyridine Derivatives
Compd. R1 R2 h JAK3
IC50
a) (nM)
h JAK1
IC50
a) (nM)
h JAK2
IC50
a) (nM)
Rat T cell
IC50
b) (nM)
6H Me 1100 2900 1800 NTd)
11a CONH2Me 1600 10000 5300 2400
14a CONH2H 14 55 50 120
15a H 85 230 57 350
15b H 3400 5000 2000 3200
15c H 1200 570 640 NTd)
1c)0.8 3.7 3.1 23
a) IC50 values are the average of duplicate experiments except for compound 1. b) Inhibitory effect on IL-2-stimulated T cell proliferation using rat spleen cells (n=2).
c) IC50 values of JAK assays are the average of four experiments. d) NT=not tested.
Reagents and condition s: (a) SEMCl, NaH, DM F, 0°C; (b) N-Methylcyclohexylamine, Pd(OAc)2, 2-(di- tert-butylphosphino)biphenyl, Cs2CO3, 110°C; (c) 1) TFA, CH2Cl2,
room temperature, 2) 1
M NaOH, 1,2-diaminoethane, CH2Cl2, room tempe rature.
Chart 1

Vol. 63, No. 5 (2015) 343
Chem. Pharm. Bull.
microwave irradiation to give corresponding C4-substituted
compounds 12a–c, after which the ester groups were hydro-
lyzed to give carboxylic acids 13a–c. Condensation of 13a–c
with aqueous ammonia using 1-hydroxybenzotriazole (HOBt)
and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)
prepared the desired compounds 14a–c.
As shown in Chart 4, carboxylic acid in Chart 3 is avail-
able for the conversion of the amide moiety at the C5-position.
Compound 13a was condensed with several amines using
HOBt and EDC to afford the N-substituted-1H-pyrrolo[2,3-b]-
Reagents and condition s: (a) TIPSCl, NaH, DMF, 5°C; (b) 1) sec-BuLi, ethyl chloroformate, THF, −78°C, 2) TBAF, THF, room tempe rature; (c) 1 M NaOH, EtOH, 60°C;
(d) CDI, DMF, room temperature , then 28% NH4OH, ro om temperat ure; (e) Amines, DIPEA, n-BuOH or NM P, microwave, 150–160°C.
Chart 2
Reagents and condition s: (a) Amines, DIPEA, n-BuOH , microwave, 160°C; (b) 2 M NaOH, EtOH, reflux; (c) HOBt, EDC, DM F, 60 °C, the n 28% NH4OH, room temp era-
ture.
Chart 3

344 Vol. 63, No. 5 (2015)
Chem. Pharm. Bull.
pyridine-5-carboxamide derivatives 15a–c.
Results and Discussion
Our synthesized compounds were evaluated for inhibitory
activity toward human JAK3, and IC50 values were calculated.
JAK1 and JAK2 inhibitory activity were also investigated for
JAK selectivity. To evaluate the cellular immunomodulating
effect, the inhibitory effect on IL-2-stimulated T cell prolifera-
tion using rat spleen cells was tested.
As shown in Table 1, our initial compound, 1H-pyrrolo[2,3-
b] pyridine analogue 6 showed potential inhibitory activity
toward JAKs (JAK3, JAK1, and JAK2 IC50=1100, 2900, and
180 0 nM, respectively). The ligand binding pocket of JAK3 is
known to be comprised of a hinge region in the ATP-binding
site and a spatial cavity surrounded by hydrophobic amino
acid residues.28) In order to improve JAK3 inhibitory activ-
ity, interaction with the JAK3 binding pocket is important.
We therefore performed docking calculations of our com-
pounds to human JAK3 (PDB code: 3LXK2 8)), indicating
that the 1H-pyrrolo[2,3-b] pyridine scaffold was located near
the hinge region and the C4-substituent was directed to the
hydrophobic cavity (Fig. 2). Given that 6 had a space allow-
ing a substituent in C5-position, introduction of a carbamoyl
group was investigated. Results showed that 1H-pyrrolo[2,3-
b] pyridine-5-carboxamide derivative 11a maintained moderate
JAK3 inhibitory activity (IC50=160 0 n M), equipotent to 6. On
Reagents and condition s: (a) Amines, HOBt, EDC, DM F, 55°C.
Chart 4
Fig. 2. Predicted Binding Mode of Compound 6 to Human JAK3 (PDB
Code: 3LXK)
Fig. 3. Metabolic Pathway of 14c in Rat Liver Microsomes
Fig. 4. (A) Predicted Binding Mode of Compound 14c to Human JAK3
(PDB Code: 3LXK, Orange: 14c , Green: 1). (B) 14c with WaterMap Un-
favorable Water Molecules (ΔG=>2.0 kcal /mol)

Vol. 63, No. 5 (2015) 345
Chem. Pharm. Bull.
the other hand, when an N-methyl group at the C4-position
of 6 was deleted to solve the steric hindrance with C5-
carbamoyl group, compound 14a achieved an over 100-fold
increase in JAK3 inhibitory activity (IC50=14 n M) compared
to 11a. Although JAK1 and JAK2 inhibitory activity of 14a
were also increased, several-fold selectivity for JAK3 was
maintained. We then investigated the acceptability of modify-
ing the carbamoyl group. Given that N-methyl carboxamide
analogue 15a maintained moderate JAK3 inhibitory activity
(IC50=85 nM), introduction of other substituted groups was
examined. However, 15b and 15c showed substantial de-
creases in JAK3 inhibitor y activity (IC50=3400 and 1200 nM,
respectively), indicating intolerance to introduction of bulky
aliphatic or aromatic substituted carbamoyl groups. Therefore,
1H-pyrrolo[2,3-b] pyridine-5-carboxamide was deemed a po-
tential scaffold for development of a JAK inhibitor. Of note,
14a showed moderate cellular inhibition of IL-2-stimulated rat
T cell proliferation (IC50=120 nM).
Given the above findings, we investigated conversion of the
C4-substituent of 1H-pyrrolo[2,3-b] pyridine-5-carboxamide
derivatives, as shown in Table 2. Similarly to N-methylcyclo-
hexylamine (11a), conversion to piperidine (11b) dramatically
decreased JAK inhibitory activity (JAK3, IC50=3200 nM), indi-
cating that the NH moiety at the C4-position was critical for
JAK inhibition. Therefore, a variety of secondary amine de-
rivatives were investigated. The size of the cyclohexyl ring of
14a was altered, and incorporation of 3-, 5-, and 7-membered
cycloalkyl rings was investigated. The JAK inhibitory activity
of these compounds increased with ring size, in order of cy-
clopropane (14b), cyclopentane (11c), and cycloheptane (11d).
Compared to 14 a, the cycloheptylamine analogue 11d showed
potent JAK inhibitory activity, particularly for JAK3 (JAK3,
JAK1, and JAK2 IC50=3.5, 25, and 13 nM, respectively), sug-
gesting the effect of hydrophobic interaction for JAK inhibi-
tion.
We next examined cleavage of the cyclohexyl ring of 14a,
and 3-pentylamine analogue 11e showed increased JAK3
inhibitory activity over 14a (IC50=7.7 n M). In contrast, replace-
ment of the cyclohexylamine with cyclohexylmethylamine
(11f) led to a slight decrease in JAK3 inhibitory activ-
ity (IC50=25 nM) compared to 14a, indicating that a branched
structure in the α-position of the C4-amino group was also
important for achieving good inhibitory activity. We also
investigated methylation of the cyclohexyl ring of 14a, and
2-methyl-cyclohexylamine analogue 14c had approximately
3-fold increased JAK3 inhibitory activity with equipotent
JAK1 and JAK2 inhibitory activity (JAK3, JAK1, and JAK2
IC50=5.1, 47, and 30 nM, respectively) compared to 14a. In the
Table 2. SARs of C4-Substitutent of 1H-Pyrrolo[2,3-b]pyridine-5-carboxamide Derivatives
Compd. R h JAK3
IC50
a) (nM)
h JAK1
IC50
a) (nM)
h JAK2
IC50
a) (nM)
Rat T cell
IC50
b) (nM)
14a 14 55 50 120
11b 3200 1900 2400 NTc)
14b 110 NTc)470 2600
11c 15 45 44 100
11d 3.5 25 13 63
11e 7.7 59 60 230
11f 25 290 71 300
14c 5.1 47 30 86
a) IC50 values are the average of duplicate experiments. b) Inhibitory effect on IL-2-stimulated T cell proliferation using rat spleen cells (n=2). c) NT=not tested.

346 Vol. 63, No. 5 (2015)
Chem. Pharm. Bull.
cellular assay, 11d and 14 c had increased inhibitory activity
(IC50=63 and 86 nM, respectively) compared to 14a, likely due
to an increase in JAK3 and JAK1 inhibitory activity. As for
JAK2 inhibitory activity, 14c had less potent than 11d. Given
its potential profile with regard to JAK inhibitory activity and
cellular potency, 14c was selected for further chemical modifi-
cation and biological evaluation.
As shown in Table 3, the effect of substituent on the cyclo-
hexyl ring moiety of 14 c was investigated. At the 2-position
of the ring, conversion to an ethyl (11g) or di-methyl group
(11h) maintained potent JAK3 inhibitory activity (IC50=5.2
and 3.7 nM, respectively) and increased cellular inhibitory
activity of IL-2-stimulated T cell proliferation (IC50=30 and
37 n M, respectively). We next assessed conversion of the sub-
stituted position of the methyl group. The 3-methylcyclohex-
ylamine analogue 11i showed potent JAK3 inhibitory activity
(IC50=3.0 nM), while the 4-methylcyclohexylamine analogue
11j had slightly decreased inhibitory activity (IC50=14 n M)
compared to 14 c. It was revealed that introduction of a sub-
stituted group to the cyclohexyl ring moiety was effective in
increasing JAK3 inhibition. Because a number of compounds
showed good in vitro pharmacological activity, their meta-
bolic stability was tested using rat liver microsomes prior to
in vivo evaluation. Results showed that 14c, 11g, 11h, and
11i had high intrinsic clearance (CLint) values of more than
1000 mL/min/kg.
In order to clarify the metabolic pathway of this series of
compounds, 14c was investigated to determine the profiles
of its metabolites in rat liver microsomes (Fig. 3). Findings
revealed that the scaffold moiety, 1H-pyrrolo[2,3-b] pyrid ine-5-
carboxamide, was hardly metabolized, with the methylcyclo-
hexyl ring mainly oxidized to afford hydroxylated compound
11k. The major metabolite 11k was synthesized and inves-
tigated for its biological profile, showing good JAK3 inhibi-
tory activity (IC50=9.7 n M) and significantly reduced values
for microsomal clearance (CLint=267 mL/min/kg) compared to
the parent compound 14c. The result indicated that decrease in
molecular lipophilicity by introducing polar functional group
was effective in improving metabolic stability (c Log P=1.0
and 2.8 for 11k and 14c, respectively).
Pharmacokinetic (PK) study of 14c and 11k was inves-
tigated in rats, and the parameters are shown in Table 4.
Table 3. SARs of Modification of Cyclohexyl Ring Moiety in C4-Substituent
Compd. R h JAK3
IC50
a) (nM)
h JAK1
IC50
a) (nM)
h JAK2
IC50
a) (nM)
Rat T cell
IC50
b) (nM)
Rat CLint
c)
(mL/min/kg) c Log Pd)
14c 5.1 47 30 86 >1000 2.8
11g 5.2 55 48 30 >1000 3.3
11h 3.7 54 26 37 >1000 3.1
11i 3.0 29 23 25 >1000 2.8
11j 14 50 38 97 NTe)2.8
11k 9.7 280 190 530 267 1.0
a) IC50 values are the average of duplicate experiments. b) Inhibitory effect on IL-2-stimulated T cell proliferation using rat spleen cells (n=2). c) In vitro metabolism with
rat liver microsomes in presence of NADPH-generating system (n=2). d) c Log P values are calculated using ACD/Labs Software, version 12.01. e) NT=not tested.

Vol. 63, No. 5 (2015) 347
Chem. Pharm. Bull.
In intravenous dosing, 14c showed high total clearance
(CLtot=62.4 mL/min/kg), comparable to hepatic blood flow in
rats, while, in oral dosing, 14c resulted in low exposure of
plasma concentration (Cmax=126 ng/mL and area under curve
from 0 to 24 h (AUC)0 –24=303 ng·h/mL) and low oral bioavail-
ability (F=11%). In a parallel artificial membrane permeability
ass ay ( PA M PA),2 9) 14c showed acceptable membrane perme-
ability (Pe=39×10−6 cm/s). Findings suggested that the poor
in vitro metabolic stability of 14c was reflected in the in vivo
PK profiles. In contrast, 11k showed over 5-fold improved oral
absorption (Cmax=1129 ng/mL and AUC0–24=1523 ng·h /mL),
possibly due to reduction of microsomal CYP-mediated drug
metabolism. Chemical modification of 1H-py rrolo[2,3-b]-
pyridine-5-carboxamide derivatives revealed that the hydro-
phobicity of the C4-substituent was critical for PK profile as
well as pharmacological activity.
Our optimization study on C4- and C5-substitutents of the
1H-pyrrolo[2,3-b] pyridine scaffold led to a large increase in
JAK3 inhibitory activity, and 14c achieved more than 200-fold
increase in activity over the initial compound 6. To validate
the effect of structural modification for JAK3 inhibitory activ-
ity, we analyzed docking calculations of 1H-pyrrolo[2,3-b]-
pyridine-5-carboxamide derivatives to human JAK3. Figure
4A shows the predicted binding mode of 14c to human JAK3.
This result shows that the 1H-pyrrolo[2,3-b] pyridine moiety
closely interacted with Glu903 and Leu905 in the hinge re-
gion, and the N1-hydrogen atom and N7-nitrogen atom served
as hydrogen donor and acceptor, respectively. In addition to
the hydrogen bond interaction, the proton of the C2-position
interacted with gate-keeper amino acid Met902, and the
aromaticity of the pyrrolopyridine ring contributed to CH–π
interaction with Val836 and Leu828. The 1H-pyrrolo[2,3-b]-
pyridine-5-carboxamide moiety was overlapped with the pyr-
rolopyrimidine of 1, while the C4-substituent occupied the
space in the hydrophobic cavity, corresponding to the amino-
piperidine moiety of 1.
With respect to C4- and C5-positions of 14 c, an intramolec-
ular hydrogen bond was observed between the NH proton of
the C4-amine and the carbonyl group of the C5-carboxamide.
The intramolecular interaction allowed the cycloalkyl group
at C4-position to direct efficiently to the hydrophobic cavity
of JAK3, which had a large space capable of accommodating
Table 4. Pharmacokinetic Parameters of 14c and 11k in Rats
Compd.
Intravenouslya)per osb)
AUC0–24
(ng· h/mL)
t1/2
(h)
Vdss
(L/kg)
CLtot
(mL/min/kg)
Cmax
(ng/mL)
tmax
(h)
AUC0–24
(ng· h/mL)
Fc)
(%)
14c 810 0.4 1.8 62.4 126 1.3 303 11
11k NTd)NTd)NTd)NTd)1129 1.5 1523 —
a) Dosed at 3 mg/kg (n=2). b) Dosed at 10 mg/kg (n=3). c) F=bioavailability. d) NT=not tested.
Fig. 5. Correlation between the Experimental Activity and the WaterMap Free Energy Liberation of Binding Site Waters for 1H-Pyrrolo[2,3-b]-
pyridine-5-carboxamide Derivatives in Tables 2 and 3

348 Vol. 63, No. 5 (2015)
Chem. Pharm. Bull.
bulky substituted groups. It was suggested that conformational
restriction by the intramolecular hydrogen bond contributed to
enhance affinity to the hydrophobic cavity, resulting in potent
JAK inhibitory activity of 14c compared to 6 and 11a. Fur-
ther, the carbamoyl group was located around the polar and
narrow hinge region, where a small f unctional group might
be tolerable. Substitution of the carbamoyl group seemed to
hinder access to the hinge region, which was disadvantageous
for binding to JAK3—findings consistent with SARs showing
that N-substituted carboxamide analogues (15a–c) had lower
JAK3 inhibitory activity than 14a. Given that both hydrogen
bond interaction with the hinge region and hydrophobic inter-
action with the cavity are essential for binding to JAK3, the
4-cycloalkylamino-1H-pyrrolo[2,3-b] pyridine-5-carboxamide
derivatives were favorably located in the binding pocket of
JAK 3.
In addition to docking calculations, to analyze SARs on
the C4-substitutent, we examined predicted binding energy
using the WaterMap program, a recently developed protocol
that combines molecular dynamics, solvent clustering, and
statistical thermodynamics to assess the enthalpy, entropy, and
free energy (ΔG) of water “hydration sites.”30, 31) The ΔG was
computed for the human JAK3 structure (PDB code: 3LXK),
and 11 unfavorable waters (ΔG=>2.0 kcal/mol) were detected
in the ligand binding pocket of human JAK3 (Fig. 4B).
Based on the docking calculations of our series of com-
pounds, the 1H-pyr rolo[2,3-b] pyridine-5-carboxamide scaf-
fold displaced 5 unfavorable water molecules (W1 to W5)
in the hinge region. In contrast, the aliphatic ring moiety of
the C4-substituent contributed to displacement of 3 unfavor-
able water molecules (W6 to W8) located in the hydrophobic
site. As shown in Fig. 5, weak correlation (R2=0.45) was
observed between the experimental inhibitory activity (pIC50)
and the WaterMap free energy liberation (ΔGpred). The ΔGpred
estimates the desolvation energy generated when a compound
displaces the waters in the binding pocket. In the hydropho-
bic site, 3 unfavorable water molecules (W6 to W8) were
displaced to enhance the inhibitory activity, associated with
an increase in spatial volume of the C4-substituent. This ob-
servation was consistent with SAR findings that enhancement
of the bulkiness of the C4-substituent led to an increase in
JAK3 inhibitory activity, and that substituted cyclohexylamine
analogues (14c and 11g) were more potent than unsubstituted
cyclohexylamine analogue 14a. The results of computational
analyses are useful for further optimization of the 1H-
pyr rolo[2,3-b] pyridine-5-carboxamide derivatives.
Conclusion
We synthesized 1H-pyrrolo[2,3-b] pyridine derivatives as
novel immunomodulators targeting JAK3 and evaluated the
biological profiles with respect to JAK inhibitory activity
and immunomodulating effects on T cell proliferation. The
results of a SAR study revealed that a carbamoyl group at the
C5-position and a substituted cycloalkylamino group at the
C4-position of the 1H-pyrrolo[2,3-b] pyridine scaffold played
an important role in increasing JAK3 inhibitory activity, and
14c was identified as a potent and moderately selective JAK3
inhibitor. Modification of the C4-substituent of 14c led to
increase in JAK3 inhibitory activity and cellular inhibitory
activity on IL-2-stimulated T cell proliferation (11g, 11h, and
11i). However, these compounds had high molecular lipo-
philicity and showed poor metabolic stability in liver micro-
somes. We clarified metabolic pathway of 14c to convert to
the hydroxylated compound 11k. Introduction of a hydroxy
group to the hydrophobic C4-substituent decreased molecular
lipophilicity, and thereby 11k showed a reduction in metabolic
clearance and subsequent improvement of oral absorption.
Docking calculations to JAK3 supported putative docking
modes of 1H-pyrrolo[2,3-b] pyridine-5-carboxamide deriva-
tives, indicating effective occupancy of the JAK3 binding
site to interact with the hinge region and hydrophobic cavity.
In addition, using the WaterMap program, the displacement
effect against unfavorable water molecules in the binding
pocket was correlated with JAK3 inhibitory activity. Com-
pared with the pyrrolopyrimidine derivatives such as 1 and 2,
C5-carbamoyl group of the 1H-pyr rolo[2,3-b] pyridine scaffold
allows for intramolecular hydrogen bond to maintain active
conformation in the JAK3 binding site. Given the findings,
the 1H-pyrrolo[2,3-b] pyridine-5-carboxamide derivatives are
promising as novel immunomodulators targeting JAK3 for the
treatment of immune diseases.
Experimental
1H-NMR spectra were recorded on a Br ucker Biospin
Avance400 or AV400M spectrometer. Chemical shifts are ex-
pressed in δ units using tetramethylsilane as an internal stan-
dard (NMR peak description: s=singlet; d=doublet; t=triplet;
q=quartet; m=multiplet; br=broad peak). Mass spectra (MS)
were recorded on a Hitachi LC/3DQMS M8000 or an Agilent
HP1100 LC/MCD spectrometer. High-resolution mass spec-
troscopy (HR-MS) spectra were recorded on a Waters LCT
Premier XE. Column chromatography was carried out using
silica gel 60N (Kanto Chemical, 63–210 µm) or HI-FLASH™
Column (Yamazen). The purity of all compounds screened
in biological assays was >95%, as judged by either or both
HPLC or elemental analyses. HPLC analysis was conducted
using a Hitachi LaChrom Elite system with a TOSOH TSK-
gel ODS-80TM column (150 mm×4.6 mm, 5 µm) at 40°C and
a 1.0 mL/min flow rate using acetonitrile and 0.01
M HClO4
aqueous solution as the eluent. Elemental analysis values were
recorded on an Elementar Vario EL III or YANACO MT-6
and were within 0.4% of the theoretical values Calcd for C,
H, and N. All animal experimental procedures were approved
by the Institutional Animal Care and Use Committee of As-
tellasPharma Inc. Further, the AstellasPharma Inc. Tsukuba
Research Center was awarded Accreditation Status by the
AAALAC International. All efforts were made to minimize
the number of animals used and to avoid suffering and dis-
tress.
4-Chloro-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-
pyrrolo [2,3-b]py ridine (4) To a solution of 4-chloro-1H-
pyr rolo[2,3-b] pyridine (3) (4.6 g, 30 mmol) in N,N-dimethylfor-
mamide (DMF) (46 mL), 60% NaH (1.4 g, 36 mmol) was added
portionwise at 0°C. The mixture was stir red at the same
temperature for 1 h, and then SEMCl (6.8 mL, 39 mmol) was
added. After stirring at the same temperature for additional
4 h, the mixture was poured into H2O (150 mL) and extracted
with ether (three times). The organic layer was then washed
with H2O, dried over MgSO4, and concentrated under reduced
pressure. The residue was purified by column chromatography
(n-hexane/ Et OAc=97/3 to 88/12) to give the title compound
(7.16 g, 84%). 1H-NMR (DMSO-d6) δ: −0.12 (9H, s), 0.80 (2H,

Vol. 63, No. 5 (2015) 349
Chem. Pharm. Bull.
t, J=1.6 Hz), 3.50 (2H, t, J=1.6 Hz), 5.64 (2H, s), 6.60 (1H, d,
J=3.7 Hz), 7.28 (1H, d, J=5.1 Hz), 7.77 (1H, d, J=3.7 Hz), 8.24
(1H, d, J=5.1 Hz). MS electrospray ionization (ESI) m/z: 283
(M+H)+
.
N-Cyclohexyl-N-methyl-1-{[2-(trimethylsilyl)ethoxy]-
methyl}-1H-pyrrolo[2,3-b]pyridin-4-amine (5) To a mix-
ture of 4 (350 mg, 1.24 mmol) and N-methylcyclohexylamine
(1.6 mL, 12.4 mmol) were added 2-(di-tert-butylphosphino)-
biphenyl (74 mg, 0.25 mmol), Cs2CO3 (403 mg, 1.24 mmol), and
Pd(OAc)2 (28 mg, 0.12 mmol). The mixture was stirred under
N2 gas atmosphere at 110°C for 2 h. After cooling to room
temperature, CHCl3 and MeOH were added for dilution. After
stirring at room temperature for 10 min, the mixture was fil-
trated through a pad of Celite®. The filtrate was concentrated
under reduced pressure, and the residue was purified by col-
umn chromatography (n-hexa ne/EtOAc=9/1 to 4/1) to give the
title compound (145 mg, 33%). 1H-NMR (DMSO-d6) δ: −0.08
(9H, s), 0.81 (2H, t, J=8.0 Hz), 1.09–1.23 (1H, m), 1.02–1.05
(1H, m), 1.31–1.45 (2H, m), 1.56–1.68 (3H, m), 1.71–1.87 (4H,
m), 2.95 (3H, s), 3.49 (2H, t, J=8.0 Hz), 3.90–3.99 (1H, m),
5.52 (1H, s), 6.28 (1H, d, J=5.6 Hz), 6.49 (1H, d, J=4.0 Hz),
7.31 (1H, d, J=4.0 Hz), 7.88 (1H, d, J=5.6 Hz). MS (ESI) m/z:
360 (M+H)+
.
N-Cyclohexyl-N-methyl-1H-py rrolo[2,3-b]pyridin-
4-amine (6) To a solution of 5 (140 mg, 0.39 mmol) in
CH2Cl2 (1.4 mL) was added TFA (1.5 mL, 19.6 mmol). The
mixture was stirred at room temperature for 2 h. After con-
centration under reduced pressure, the residue was dissolved
with CH2Cl2 (1.4 mL). To the solution were added 1 M NaOH
aqueous solution (1.6 mL, 1.56 mmol) and 1,2-diaminoethane
(78 µL, 1.17 mmol), and then the mixture was stirred at room
temperature for 16 h, extracted with CHCl3, and washed with
H2O. The organic layer was then dried over MgSO4 and con-
centrated under reduced pressure. The residue was purified
by column chromatography (CHCl3/MeOH=100/0 to 95/5) to
give the title compound (58 mg, 65%). 1H-NMR (DMSO-d6)
δ: 1.06–1.22 (1H, m), 1.29–1.44 (2H, m), 1.54–1.69 (3H, m),
1.69–1.87 (4H, m), 2.92 (3H, s), 3.91–4.02 (1H, m), 6.19 (1H, d,
J=5.8 Hz), 6.4 (1H, d, J=3.7 Hz), 7.13 (1H, d, J=3.6 Hz), 7.82
(1H, d, J=5.5 Hz), 11.25 (1H, s). MS (ESI) m/z: 230 (M+H)+
.
Anal. Calcd for C14H19N3· 0.1H 2O: C, 72.75; H, 8.37; N, 18.18.
Found: C, 72.96; H, 8.36; N, 18.2.
4-Chloro-1-(triisopropylsilyl)-1H-pyrrolo[2,3-b]pyridine
(7) To a solution of 3 (25 g, 164 mmol) in DMF (250 mL),
60% NaH (7.9 g, 197 mmol) was added portionwise at 5°C.
The mixture was stirred at the same temperature for 1 h, and
then TIPSCl (36 mL, 172 mmol) was added. After stirring
for additional 1 h, EtOAc and H2O were added to the mix-
ture. The organic layer was separated, washed with saturated
NaHCO3 aqueous solution and brine, dried over MgSO4, and
concentrated under reduced pressure. The residue was puri-
fied by column chromatography with n-hexane to give the title
compound (40 g, 79%). 1H-NMR (DMSO-d6) δ: 1.06 (18H, d,
J=7.5 Hz), 1.79–1.91 (3H, m), 6.68 (1H, d, J=3.5 Hz), 7.24 (1H,
d, J=5.2 Hz), 7.60 (1H, d, J=3.5 Hz), 8.20 (1H, d, J=5.2 Hz).
MS (ESI) m/z: 309 (M+H)+
.
Ethyl 4-Chloro-1H-pyrrolo[2, 3-b]pyridine-5-carboxylate
(8) To a solution of 7 (15 g, 48.6 mmol) in tetrahydrofuran
(THF) (150 mL), 1
M sec-BuLi in cyclohexane and n-hexane
(97.1 mL, 97.1 mmol) was added dropwise at −78°C under
Ar gas atmosphere. The mixture was stirred at the same
temperature for 1 h, and then ethyl chloroformate (9.29 mL,
97.1 mmol) was added at −78°C. After stirring at the same
temperature for additional 30 min, the mixture was quenched
with saturated NH4Cl aqueous solution and extracted with
EtOAc. The extract was washed with H2O and brine, dried
over MgSO4, and concentrated under reduced pressure. The
residue was dissolved in THF (120 mL), and 1
M TBAF in THF
(56 mL, 56 mmol) was added. The mixture was stirred at room
temperature for 1 h and then diluted with EtOAc, washed with
H2O, dried over MgSO4, and concentrated under reduced pres-
sure. The residue was triturated with IPE, and the precipitate
was filtrated to give the title compound (9.6 g, 88%). 1H-NMR
(DMSO-d6) δ: 1.36 (3H, t, J=7.1 Hz), 4.36 (2H, q, J=7.1 Hz),
6.64–6.67 (1H, m), 7.70–7.73 (1H, m), 8.71 (1H, s), 12.41 (1H,
br). MS (ESI) m/z: 223 (M−H)−.
4- Chloro-1H-pyrrolo[2,3 -b]pyridine-5-carboxylic Acid
(9) To a solution of 8 (10.5 g, 46.7 mmol) in EtOH (84 mL)
was added 1
M NaOH aqueous solution (140 mL, 140 mmol),
and the mixture was stirred at 60°C for 1.5 h. The mixture
was cooled to 4°C and acidified with 1
M HCl aqueous solu-
tion. The precipitate was filtrated and washed with H2O to
give the title compound (9.0 g, 98%). 1H-NMR (DMSO-d6) δ:
6.62–6.64 (1H, m), 7.67–7.70 (1H, m), 8.71 (1H, s), 12.32 (1H,
br s), 13.22 (1H, br s). MS (ESI) m/z: 195 (M−H)−.
4- Chloro-1H-pyrrolo[2,3 -b]pyridine-5-carboxamide (10)
To a solution of 9 (9.0 g, 45.8 mmol) in DMF (72 mL) was
added CDI (8.2 g, 50.4 mmol), and the mixture was stirred
at room temperature for 1 h. To the mixture was added 28%
NH4OH aqueous solution (10.0 mL, 164 mmol) dropwise at
4°C, and the mixture was stirred at room temperature for 1 h.
To the mixture was added EtOAc, and the precipitate was
filtrated to give the title compound (7.5 g, 84%). 1H-NMR
(DMSO-d6) δ: 6.57 (1H, d, J=3.6 Hz), 7.63 (1H, br s), 7.65 (1H,
d, J=3.6 Hz), 7.90 (1H, br s), 8.29 (1H, s), 12.12 (1H, br). MS
(ESI) m/z: 218 (M+Na)+
.
4-[Cyclohexyl(methyl)amino]-1H-pyrrolo[2,3 -b]pyridine-
5-carboxamide (11a) In the vessel of a microwave reac-
tor, 10 (25 mg, 0.128 mmol) and N-methylcyclohexylamine
(0.085 mL, 0.639 mmol) were suspended in n-BuOH (0.2 mL).
The vessel was then sealed, and the mixture was reacted at
150°C for 30 min under microwave irradiation. The mixture
was concentrated under reduced pressure, and the residue was
purified by column chromatography (CHCl3/MeOH=10 0/0
to 95/5) to give the title compound (9 mg, 26%). 1H-NMR
(DMSO-d6) δ: 0.97–1.37 (3H, m), 1.42–1.88 (7H, m), 2.91 (3H,
s), 3.49–3.66 (1H, m), 6.46–6.52 (1H, m), 7.24–7.33 (2H, m),
8.07 (1H, br), 8.21 (1H, s), 11.56 (1H, br s). MS (ESI) m/z: 273
(M+H)+
. Anal. Calcd for C15H20N4O·0.5H2O: C, 64.03; H,
7.52; N, 19.91. Found: C, 63.98; H, 7.52; N, 20.15.
4- (Piperidin-1-yl)-1H-pyrrolo[2 ,3 -b]pyridine-5-carbox-
amide (11b) The title compound was prepared from 10 and
piperidine in accordance with the procedure for preparing
11a in 38% yield. 1H-NMR (DMSO-d6) δ: 1.57–1.73 (6H,
m), 3.27–3.42 (4H, m), 6.56–6.59 (1H, m), 7.27–7.36 (2H, m),
7.85–7.93 (1H, m), 8.18 (1H, s), 11.56 (1H, br). MS (ESI) m/z:
245 ( M+H)+
. Anal. Calcd for C13H16N4O: C, 63.91; H, 6.60; N,
22.93. Found: C, 63.81; H, 6.62; N, 22.81.
4-(Cyclopentylamino)-1H-pyrrolo[2, 3-b]pyridine- 5-car-
boxamide (11c) The title compound was prepared from 10
and cyclopentylamine in accordance with the procedure for
preparing 11a in 29% yield. 1H-NMR (DMSO-d6) δ: 1.43–1.78

350 Vol. 63, No. 5 (2015)
Chem. Pharm. Bull.
(6H, m), 1.90–2.10 (2H, m), 4.35–4.50 (1H, m), 6.57–6.63
(1H, m), 7.00 (1H, br), 7.08–7.14 (1H, m), 7.64 (1H, br), 8.34
(1H, s), 9.61–9.70 (1H, m), 11.43 (1H, br s). MS (ESI) m/z: 245
(M+H)+
. Anal. Calcd for C13H16N4O · 0.1H 2O: C, 63.45; H,
6.64; N, 22.77. Found: C, 63.64; H, 6.64; N, 22.62.
4-[(Cyclohexylmethyl)amino]-1H-pyrrolo[2, 3-b]pyridine-
5-carboxamide (11f) The title compound was prepared
from 10 and cyclohexanemethylamine in accordance with
the procedure for preparing 11a in 39% yield. 1H-NMR
(DMSO-d6) δ: 0.92–1.34 (5H, m), 1.50–1.88 (6H, m), 3.45
(2H, dd, J=6.0, 12.0 Hz), 6.54– 6.60 (1H, m), 6.98 (1H, br),
7.08–7.10 (1H, m), 7.65 (1H, br), 8.34 (1H, s), 9.61–9.66 (1H,
m), 11.43 (1H, br s). MS (ESI) m/z: 273 (M+H)+
. Anal. Calcd
for C15H20N4O · 0.1H 2O: C, 65.72; H, 7.43; N, 20.44. Found: C,
65.83; H, 7.38; N, 20.29.
4-{[(1S,2R)-2-(Hydroxymethyl)cyclohexyl]amino}-1H-
pyrrolo[2,3-b]pyridine-5-carboxamide (11k) The title
compound was prepared from 10 and [(1R,2S)-2-amino-
cyclohexyl] methanol hydrochloride in accordance with the
procedure for preparing 11a in 41% yield. 1H-NMR (DMSO-
d6) δ: 1.13–2.00 (9H, m), 3.19–3.56 (2H, m), 4.51–4.64 (1H,
m), 6.78– 6.84 (1H, m), 7.29–7.36 (1H, m), 7.69 (1H, br), 8.38
(1H, br), 8.53 (1H, s), 10.96–11.05 (1H, m), 12.51 (1H, br s).
MS (ESI) m/z: 289 (M+H)+
. HR-MS m/z: 289.1666 (M+H)+
(Calcd for C15H18N4O: 288.1586).
4-{[(1S,2R)-2-Ethylcyclohexyl]amino}-1H-py rrolo[2,3-b]-
pyridine-5-carboxamide (11g) In the vessel of a micro-
wave reactor, to a solution of 10 (100 mg, 0.511 mmol) in
N-methylpyrrolidone (NMP) (1 mL) were added (1S,2R)-2-eth-
ylcyclohexanamine hydrochloride (167 mg, 1.02 mmol) and
N,N-diisopropylethylamine (DIPEA) (0.27 mL, 1.53 mmol).
The vessel was sealed, and the mixture was reacted at 160°C
for 1.5 h under microwave irradiation. After cooling to room
temperature, H2O was added, and the mixture was extracted
with CHCl3. The organic layer was separated, washed with
H2O, dried over MgSO4, and concentrated under reduced pres-
sure. The residue was purified by column chromatography
(CHCl3/MeOH=100/0 to 90/10) to give the title compound
(58 mg, 40%). 1H-NMR (DMSO-d6) δ: 0.78 (3H, t, J=7.2 Hz),
1.21–1.68 (10H, m), 1.82–1.89 (1H, m), 4.29–4.32 (1H, m),
6.51–6.53 (1H, m), 7.00 (1H, br), 7.08–7.11 (1H, m), 7.67 (1H,
br), 8.35 (1H, s), 9.87–9.92 (1H, m), 11.43 (1H, br s). MS (ESI)
m/z: 287 (M+H)+
. Anal. Calcd for C16H22N4O ·0.3H2O: C,
65.86; H, 7.81; N, 19.20. Found: C, 65.65; H, 7.43; N, 19.25.
4-(Cycloheptylamino)-1H-pyrrolo[2, 3-b]pyridine-5-car-
boxamide (11d) The title compound was prepared from 10
and cycloheptylamine in accordance with the procedure for
preparing 11g in 57% yield. 1H-NMR (DMSO-d6) δ: 1.4 6–1.71
(10H, m), 1.89–2.10 (2H, m), 4.07–4.23 (1H, m), 6.50–6.54
(1H, m), 7.00 (1H, br), 7.08–7.13 (1H, m), 7.62 (1H, br), 8.31
(1H, s), 9.67 (1H, d, J=8.1 Hz), 11.43 (1H, br s). MS (ESI) m/z:
273 (M+H)+
. Anal. Calcd for C15H20N4O·0.3H2O: C, 64.86; H,
7.48; N, 20.17. Found: C, 64.82; H, 7.38; N, 20.10.
4-(Pentan-3-ylamino)-1H-pyrrolo[2,3 -b]pyr idine-5 -car-
boxamide (11e) The title compound was prepared from 10
and 3-aminopentane in accordance with the procedure for
preparing 11g in 24% yield. 1H-NMR (DMSO-d6) δ: 0.92 (6H,
t, J=7.6 Hz), 1.48–1.69 (4H, m), 3.87–3.97 (1H, m), 6.50–6.53
(1H, m), 6.93 (1H, br), 7.08–7.12 (1H, m), 7.69 (1H, br), 8.35
(1H, s), 9.55 (1H, d, J=8.8 Hz), 11.42 (1H, br s). MS (ESI) m/z:
247 ( M+H)+
. Anal. Calcd for C13H18 N4O: C, 63.39; H, 7.37; N,
22.75. Found: C, 63.22; H, 7.24; N, 22.65.
4-[(2,2-Dimethylcyclohexyl)amino]-1H-py rrolo[2,3-b]-
pyridine-5-carboxamide (11h) The title compound was
prepared from 10 and 2,2-dimethylcyclohexan-1-amine hydro-
chloride in accordance with the procedure for preparing 11g
in 66% yield. 1H-NMR (DMSO-d6) δ: 0.94 (3H, s), 1.00 (3H,
s), 1.30–1.54 (6H, m), 1.63–1.71 (1H, m), 1.83–1.91 (1H, m),
3.69–3.77 (1H, m), 6.47–6.50 (1H, m), 6.95 (1H, br), 7.08–7.12
(1H, m), 7.69 (1H, br), 8.34 (1H, s), 9.83 (1H, d, J=8.8 Hz),
11.42 (1H, br s). MS (ESI) m/z: 287 (M+H)+
. HR-MS m/z:
28 7.1867 (M+H)+ (Calcd for C16H22N4O, 286.1794).
4-[(3-Methylcyclohexyl)amino]-1H-pyrrolo[2,3 -b]pyr i-
dine-5 -carboxamide (11i) The title compound was pre-
pared from 10 and 3-methylcyclohexylamine in accordance
with the procedure for preparing 11g in 77% yield. 1H-NMR
(DMSO-d6) δ: 0.87–0.92 (3H, m), 0.97–1.81 (8H, m), 2.02–2.12
(1H, m), 3.82–3.92 (0.4H, m), 4.33–4.39 (0.6H, m), 6.47–6.55
(1H, m), 6.98 (1H, br), 7.10–7.16 (1H, m), 7.76 (1H, br), 8.35
(0.4H, s), 8.36 (0.6H, s), 9.60 (0.4H, d, J=7.6 Hz), 10.01 (0.6H,
d, J=8.4 Hz), 11.49 (1H, br s). MS (ESI) m/z: 273 (M+H)+
.
HR-MS m/z: 273.1719 (M+H)+ (Calcd for C15H20N4O,
272.1637).
4-[(4-Methylcyclohexyl)amino]-1H-pyrrolo[2,3 -b]pyri-
dine-5 -carboxamide (11j) The title compound was prepared
from 10 and 4-methylcyclohexylamine in accordance with the
procedure for preparing 11g in 95% yield. 1H-NMR (DMSO-
d6) δ: 0.88–0.94 (3H, m), 1.09–1.31 (3H, m), 1.37–1.58 (2H,
m), 1.62–1.81 (3H, m), 2.03–2.11 (1H, m), 3.76–3.86 (0.4H,
m), 4.21–4.29 (0.6H, m), 6.46–6.53 (1H, m), 6.97 (1H, br),
7.09–7.14 (1H, m), 7.74 (1H, br), 8.34 (0.4H, s), 8.36 (0.6H,
s), 9.55 (0.4H, d, J=8.0 Hz), 9.97 (0.6H, d, J=8.0 Hz), 11.43
(1H, br s). MS (ESI) m/z: 273 (M+H)+
. HR-MS m/z: 273.1714
(M+H)+ (Calcd for C15H20N4O, 272.1637).
Ethyl 4 -{[(1S,2R)-2-Methylcyclohexyl]amino}-1H-pyrrolo
-
[2, 3-b]pyridine-5-carboxylate (12c) In the vessel of a
microwave reactor, 8 (258 mg, 1.15 mmol) and (1S,2R)-2-
methylcyclohexanamine hydrochloride (292 mg, 1.95 mmol)
were suspended in n-BuOH (1.03 mL), and DIPEA (0.70 mL,
4.02 mmol) was added. The vessel was then sealed, and the
mixture was reacted at 160°C for 2 h under microwave ir-
radiation. The mixture was concentrated under reduced pres-
sure, and the residue was purified by column chromatogra-
phy (n-hexane/ Et OAc=3/1 to 1/1) to give the title compound
(185 mg, 53%). 1H-NMR (DMSO-d6) δ: 0.91 (3H, d, J=6.9 Hz),
1.32 (3H, t, J=7.1 Hz), 1.35–2.16 (9H, m), 4.23–4.34 (3H, m),
6.59 (1H, d, J=3.5 Hz), 7.17 (1H, d, J=3.5 Hz), 8.68 (1H, s),
9.02–9.06 (1H, m), 11.66 (1H, br). MS (ESI) m/z: 302 (M+H)+
.
Ethyl 4-(Cyclohexylamino)-1H-pyrrolo[2, 3- b]pyridine-5-
carboxylate (12a) The title compound was prepared from 8
and cyclohexylamine in accordance with the procedure for
preparing 12c in 56% yield. 1H-NMR (DMSO-d6) δ: 1.32 (3H,
t, J=7.1 Hz), 1.33–1.77 (8H, m), 1.99–2.08 (2H, m), 3.95–4.08
(1H, m), 4.26 (2H, q, J=7.1 Hz), 6.55 (1H, d, J=3.5 Hz), 7.18
(1H, d, J=3.5 Hz), 8.54 (1H, s), 8.84–8.88 (1H, m), 11.67 (1H,
br s). MS (ESI) m/z: 288 (M+H)+
.
Ethyl 4-(Cyclopropylamino)-1H-pyrrolo[2, 3- b]pyridine-
5-carboxylate (12b) The title compound was prepared from
8 and cyclopropylamine in accordance with the procedure
for preparing 12c in 100% yield. 1H-NMR (DMSO-d6) δ:
0.57–0.72 (2H, m), 0.87–1.04 (2H, m), 1.31 (3H, t, J=7.1 Hz),
3.00–3.15 (1H, m), 4.25 (2H, q, J=7.1 Hz), 6.97–7.07 (1H, m),

Vol. 63, No. 5 (2015) 351
Chem. Pharm. Bull.
7.12–7.23 (1H, m), 8.52 (1H, s), 8.76 (1H, d, J=2 .1 Hz), 11.68
(1H, s). MS (ESI) m/z: 246 (M+H)+
.
4-{[(1S,2R)-2-Methylcyclohexyl]amino}-1H-py rrolo[2,3-
b]pyridine-5 -carboxylic Acid (13c) To a solution of 12c
(150 mg, 0.50 mmol) in EtOH (1.5 mL) was added 2
M NaOH
aqueous solution (1.12 mL, 2.24 mmol), and the mixture was
stirred under reflux for 20 h. After cooling to room tempera-
ture, the mixture was acidified with 1
M HCl aqueous solution
(pH=4–5) and extracted with a mixture solution of CHCl3
and MeOH (4 : 1). The organic layer was separated, dried over
MgSO4, and concentrated under reduced pressure to give
the title compound (175 mg, >100%), which was used in the
next reaction without further purification. 1H-NMR (DMSO-
d6) δ: 0.93 (3H, d, J=6.9 Hz), 1.23–2.01 (9H, m), 4.38–4.40
(1H, m), 6.88– 6.89 (1H, m), 7.37–7.40 (1H, m), 8.64 (1H, s),
10.20–10.24 (1H, m), 12.76 (1H, br s), 13.80 (1H, br). MS (ESI)
m/z: 274 (M+H)+
.
4-(Cyclohexylamino)-1H-pyrrolo[2,3-b]pyridine-5-car-
boxylic Acid (13a) The title compound was prepared from
12a in accordance with the procedure for preparing 13c
in 73% yield. 1H-NMR (DMSO-d6) δ: 1.14–2.16 (10H, m),
4.03–4.22 (1H, m), 6.77 (1H, d, J=6.8 Hz), 7.33–7.39 (1H, m),
8.59 (1H, s), 9.80–9.90 (1H, m), 12.48 (1H, br s), 13.83 (1H, br).
MS (ESI) m/z: 260 (M+H)+
4-(Cyclopropylamino)-1H-pyrrolo[2, 3- b]pyridine-5- car-
boxylic Acid (13b) The title compound was prepared from
12b in accordance with the procedure for preparing 13c
in 88% yield. 1H-NMR (DMSO-d6) δ: 0.56–0.71 (2H, m),
0.86–1.02 (2H, m), 2.96–3.14 (1H, m), 6.96–7.05 (1H, m),
7.10–7.19 (1H, m), 8.49 (1H, s), 8.89–9.06 (1H, m), 11.61 (1H,
s), 12.38 (1H, br). MS (ESI) m/z: 218 (M+H)+
.
4-{[(1S,2R)-2-Methylcyclohexyl]amino}-1H-py rrolo[2,3-
b]pyridine-5-carboxamide (14c) To a solution of 13c
(170 mg, 0.622 mmol) in DMF (1.7 mL) were added HOBt
(126 mg, 0.933 mmol) and EDC (145 mg, 0.933 mmol) at room
temperature, and the mixture was stirred at 60°C for 1 h.
After cooling to room temperature, 28% NH4OH aqueous
solution (0.17 mL, 1.24 mmol) was added, and the mixture was
stirred at the same temperature for 1 h. After adding H2O, the
mixture was extracted with a solution of CHCl3 and MeOH
(4 : 1). The organic layer was separated and washed with H2O,
and the extract was dried over MgSO4 and concentrated under
reduced pressure. The residue was purified by column chro-
matography (CHCl3/MeOH=100/0 to 90/10) to give the title
compound (102 mg, 60%). 1H-NMR (DMSO-d6) δ: 0.90 (3H,
d, J=6.8 Hz), 1.20–1.98 (9H, m), 4.11–4.22 (1H, m), 6.46–6.55
(1H, m), 6.99 (1H, br), 7.07–7.13 (1H, m), 7.64 (1H, br), 8.36
(1H, s), 9.85–9.90 (1H, m), 11.43 (1H, br). MS (ESI) m/z: 273
(M+H)+
. Anal. Calcd for C15H20N4O: C, 66.15; H, 7.40; N,
20.57. Found: C, 65.81; H, 7.42; N, 20.31.
4-(Cyclohexylamino)-1H-pyrrolo[2,3-b]pyridine-5-car-
boxamide (14a) The title compound was prepared from 13a
in accordance with the procedure for preparing 14c in 60%
yield. 1H-NMR (DMSO-d6) δ: 1.14–2.01 (10H, m), 3.91–4.01
(1H, m), 6.48– 6.54 (1H, m), 7.03 (1H, br), 7.10–7.13 (1H, m),
7.70 (1H, br), 8.34 (1H, s), 9.64–9.68 (1H, m), 11.43 (1H, br s).
MS (ESI) m/z: 259 (M+H)+
. Anal. Calcd for C14H18N4O: C,
65.09; H, 7.02; N, 21.69. Found: C, 65.08; H, 7.12; N, 21.40.
4-(Cyclopropylamino)-1H-pyrrolo[2, 3- b]pyridine-5- car-
boxamide (14b) The title compound was prepared from 13b
in accordance with the procedure for preparing 14c in 74%
yield. 1H-NMR (DMSO-d6) δ: 0.49–0.66 (2H, m), 0.80–1.00
(2H, m), 2.90–3.09 (1H, m), 6.90–7.02 (1H, m), 7.03 (1H, br),
7.04–7.18 (1H, m), 7.73 (1H, br), 8.35 (1H, s), 9.58 (1H, d,
J=2.1 Hz), 11.45 (1H, s). MS (ESI) m/z: 217 (M+H)+
. Anal.
Calcd for C11H12N4O·0.05H2O · 0 .1CH 3OH: C, 60.75; H, 5.70;
N, 25.53. Found: C, 60.97; H, 5.62; N, 25.11.
4-(Cyclohexylamino)-N-methyl-1H-pyrrolo[2, 3-b]pyri-
dine-5 -carboxamide (15a) To a solution of 13a (25 mg,
0.096 mmol) in DMF (0.375 mL) were added HOBt (19.5 mg,
0.145 mmol), EDC (22.5 mg, 0.145 mmol), and MeNH2·HCl
(9.8 mg, 0.145 mmol), and the mixture was stirred at 55°C
for 1 h. To the solution were added H2O and EtOAc, and the
mixture was extracted with EtOAc. The extract was then
washed with H2O, dried over MgSO4, and concentrated under
reduced pressure. The residue was purified by column chro-
matography (CHCl3/MeOH=100/0 to 90/10) to give the title
compound (5 mg, 19%). 1H-NMR (DMSO-d6) δ: 1.18–1.81 (8H,
m), 1.91–2.05 (2H, m) 2.73 (3H, d, J=4.4 Hz), 3.84–4.01 (1H,
m), 6.47– 6.50 (1H, m), 7.11–7.14 (1H, m), 8.14–8.24 (1H, m),
8.27 (1H, s), 9.36–9.45 (1H, m), 11.42 (1H, br s). MS (ESI) m/z:
273 (M+H)+
. Anal. Calcd for C15H20N4O·0.5H2O: C, 64.03; H,
7.52; N, 19.91. Found: C, 63.91; H, 7.57; N, 20.07.
N-Cyclohexyl-4-(cyclohexylamino)-1H-py rrolo[2,3-b]-
pyridine-5-carboxamide (15b) The title compound was
prepared from 13a and cyclohexylamine in accordance with
the procedure for preparing 15a in 34% yield. 1H-NMR
(DMSO-d6) δ: 1.05–2.01 (20H, m), 3.63–3.82 (1H, m),
3.84–4.01 (1H, m), 6.48–6.49 (1H, m), 7.11–7.13 (1H, m),
7.96 (1H, d, J=7.7 Hz), 8.31 (1H, s), 9.32 (1H, d, J=8.0 Hz),
11.44 (1H, br s). MS (ESI) m/z: 341 (M+H)+
. Anal. Calcd for
C20H28N4O· 0.2H2O: C, 69.82; H, 8.32; N, 16.28. Found: C,
69.82; H, 8.35; N, 16.06.
4-(Cyclohexylamino)-N-phenyl-1H-pyrrolo[2,3-b]pyri-
dine-5 -carboxamide (15c) The title compound was prepared
from 13a and aniline in accordance with the procedure for
preparing 15a in 37% yield. 1H-NMR (DMSO-d6) δ: 1.15 –1.81
(8H, m), 1.92–2.09 (2H, m), 3.91–4.11 (1H, m), 6.59–6.60 (1H,
m), 7.04–7.44 (4H, m), 7.68 (2H, d, J=7.9 Hz), 8.50 (1H, s),
9.10–9.21 (1H, m), 10.13 (1H, br s), 11.77 (1H, br s). MS (ESI)
m/z: 335 (M+H)+
. Anal. Calcd for C20H22N4O·0.2H2O: C,
71.07; H, 6.68; N, 16.58. Found: C, 70.98; H, 6.62; N, 16.60.
Docking Calculation Docking calculations were per-
formed on the crystal structure of tofacitinib (1) bound to
JAK3 (PDB code: 3LXK2 8)). The protein-ligand complex was
prepared with the Protein Preparation Wizard in Maestro (ver-
sion 9.3; Schrödinger, LLC, New York, NY, 2012), with impref
applying the appropriate side-chain protonation states, refine-
ment, and structure minimization. Docking grids were gener-
ated and defined based on the centroid of tofacinitib in the
ATP binding site incorporating hydrogen bond constraints to
the hinge region and hydrophobic region constraints. Ligands
were prepared using LigPrep (version 2.5; Schrödinger, LLC,
New York, NY, 2012) and Confgen (version 2.3; Schrödinger,
LLC, New York, NY, 2012), and the energy-minimized con-
formation of each ligand was used to input molecules into
docking calculations. Ligand receptor docking was carried out
using XP mode in Glide (version 5.8; Schrödinger, LLC, New
York, NY, 2012). The top-scoring pose, as assessed by GlideS-
core, was employed for discussions.
Wat erMap A WaterMap (version 1.4; Schrödinger, LLC,
New York, NY, 2012) calculation was conducted on the crystal

352 Vol. 63, No. 5 (2015)
Chem. Pharm. Bull.
structure of tofacitinib bound to JAK3 (PDB code: 3LXK) via
the structure preparation method described above. WaterMap
was run in the default mode using the tofacitinib structure to
define the binding site but removing the structure in the MD
simulation. ΔGpred of binding and ligand strain energies were
calculated using the ab initio form of the displaced-solvent
functional as described by Abel et al.30)
Human JAK Assay The human JAK1, JAK2, and
JAK3 kinase-domains were purchased from Carna Biosci-
ences, Inc. (Kobe, Japan), and the assay for kinase inhibitory
activity32) was performed using a streptavidin-coated 96-well
plate. A final 50-µL reaction mixture contained 15 mM Tris–
HCl (pH 7.5), 0.01% Tween 20, 2 mM dithiothreitol (DTT),
10 m M MgCl2, 250 nM Biotin-Lyn-Substrate-2 (Biotin-XEQED
EPEGD YFEWL EPE, X=ε-Acp; Peptide Institute, Inc.,
Osaka, Japan) and ATP. The final concentrations of ATP
were 200, 10, and 8 µM for JAK1, JAK2, and JAK3, respec-
tively. Test compounds were dissolved in dimethyl sulfoxide
(DMSO). The reaction was initiated by adding the kinase
domain (JAK1: 60 ng/mL, JAK2: 20 ng/mL, JAK3: 16 ng/
mL), followed by incubation at room temperature for 1 h. Ki-
nase activity was measured as the rate of phosphorylation of
Biotin-Lyn-Substrate-2 using horseradish peroxidase (HRP)-
conjugated anti-phosphotyrosine antibody (HRP-PY-20; Santa
Cruz Biotechnology, Inc., Santa Cruz, CA, U.S.A.) using
a phosphotyrosine-specific enzyme-linked immunosorbent
assay (ELISA). The experiments were performed in duplicate
for test compounds, except for 1, and the IC50 value of each
experiment was calculated using linear regression analysis.
Assays for 1 were performed in four experiments, and the IC50
values were calculated using Sigmoid-Emax non-linear regres-
sion analysis.
Rat T Cell Proliferation Spleen cells from male Lewis
rats (Charles River Japan, Inc., Kanagawa, Japan) were sus-
pended in RPMI1640 (Sigma, St. Louis, MO, U.S.A.) supple-
mented with 10% fetal bovine serum, 100 U/mL penicillin,
100 µg/mL streptomycin, and 50 µM 2-mercaptoethanol at a
density of 1.5×106 cells/mL. Rat splenocytes were cultured
with 1 µg/mL concanavalin A (Sigma) for 24 h at 37°C in 5%
CO2. After being washed, 4×10 4 splenocytes were incubated
with 3 ng/mL IL-2 (BD Biosciences, San Diego, CA, U.S.A.)
and test compounds at designated concentrations in 96-well
tissue culture plates. After incubation for 3 d, alamarBlue®
(Life Technologies, Carlsbad, CA, U.S.A.) was added to each
of the test wells, followed by incubation for 6 h. The fluores-
cence intensity was measured at an excitation wavelength of
545 nm and an emission wavelength of 590 nm. The experi-
ments were performed in duplicate for test compounds, and
the IC50 value of each experiment was calculated using linear
regression analysis.
In Vitro Liver Microsomal Stability To estimate stabil-
ity against rat hepatic CYPs, test compound (0.2 or 1.0 µM)
was incubated with male SD rat liver microsomes (0.2 mg pro-
tein/mL) in the presence of reduced nicotinamide adenine di-
nucleotide phosphate (NADPH) (1 mM) and ethylenediamine-
tetraacetic acid (EDTA) (0.1 mM) in phosphate buffer (100 mM)
at 37°C. The percentage compound remaining was determined
via LC/MS/MS.
Pharmacokinetic Study The pharmacokinetic character-
ization was conducted in male SD rats. Compound 14c was
administrated at 10 mg/kg orally and 3 mg/kg intravenously in
50% polyethylene glycol. Compound 11k was administrated at
10 mg/kg orally in 50% propylene glycol. Blood samples were
taken at multiple points up to 24 h after a single administra-
tion of 14c and 11k. Concentrations of the unchanged com-
pound in plasma were determined using LC/MS/MS.
Acknowledgment We deeply thank Dr. Mitsuru Ohkubo
for his useful advice in this study, and Ms. Misato Ito, Ms.
Masako Kuno, Dr. Noboru Chida, Dr. Masamichi Inami, Dr.
Hidetsugu Murai, and Mr. Keitaro Kadono for performing the
biological experiments. We also thank Dr. Tor u Asano for his
helpful support in preparation of this manuscript. The authors
are grateful to the staff of the Division of Analytical Science
Laboratories for elemental analysis and spectral measure-
ments.
Conflict of Interest All authors were employees of Astel-
las Pharma Inc. when this study was conducted and have no
further conflicts of interest to declare.
Supplementary Materials The online version of this ar-
ticle contains supplementary materials.
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