Synthesis and evaluation of novel 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives as potent and orally efficacious immunomodulators targeting JAK3

Article abstract of DOI:10.1016/j.bmc.2015.05.034

Janus kinases (JAKs) regulate various inflammatory and immune responses and are targets for the treatment of inflammatory and immune diseases. As a novel class of immunomodulators targeting JAK3, 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives are promising candidates for treating such diseases. In chemical modification of lead compound 2, the substitution of a cycloalkyl ring for an N-cyanopyridylpiperidine in C4-position was effective for increasing JAK3 inhibitory activity. In addition, modulation of physical properties such as molecular lipophilicity and basicity was important for reducing human ether-a-go-go-related gene (hERG) inhibitory activity. Our optimization study gave compound 31, which exhibited potent JAK3 inhibitory activity as well as weak hERG inhibitory activity. In cellular assay, 31 exhibited potent immunomodulating effect on IL-2-stimulated T cell proliferation. In a pharmacokinetic study, good metabolic stability and oral bioavailability of 31 were achieved in rats, dogs, and monkeys. Further, 31 prolonged graft survival in an in vivo rat heterotopic cardiac transplant model.

Full text of DOI:10.1016/j.bmc.2015.05.034

Accepted Manuscript
Synthesis and evaluation of novel 1H-pyrrolo[2,3-b]pyridine-5-carboxamide
derivatives as potent and orally efficacious immunomodulators targeting JAK3
Yutaka Nakajima, Takayuki Inoue, Kazuo Nakai, Koichiro Mukoyoshi, Hisao
Hamaguchi, Keiko Hatanaka, Hiroshi Sasaki, Akira Tanaka, Fumie Takahashi,
Shigeki Kunikawa, Hiroyuki Usuda, Ayako Moritomo, Yasuyuki Higashi,
Masamichi Inami, Shohei Shirakami
PII:
S0968-0896(15)00434-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.05.034
BMC 12330
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
2 April 2015
14 May 2015
15 May 2015
Please cite this article as: Nakajima, Y., Inoue, T., Nakai, K., Mukoyoshi, K., Hamaguchi, H., Hatanaka, K., Sasaki,
H., Tanaka, A., Takahashi, F., Kunikawa, S., Usuda, H., Moritomo, A., Higashi, Y., Inami, M., Shirakami, S.,
Synthesis and evaluation of novel 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives as potent and orally
efficacious immunomodulators targeting JAK3, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/
1
0.1016/j.bmc.2015.05.034
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Synthesis and evaluation of novel 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives as
potent and orally efficacious immunomodulators targeting JAK3
Yutaka Nakajima,* Takayuki Inoue, Kazuo Nakai, Koichiro Mukoyoshi, Hisao Hamaguchi,
Keiko Hatanaka, Hiroshi Sasaki, Akira Tanaka, Fumie Takahashi, Shigeki Kunikawa, Hiroyuki
Usuda, Ayako Moritomo, Yasuyuki Higashi, Masamichi Inami, Shohei Shirakami*
Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585,
Japan
*Corresponding author:
Tel: +81 (0)29-829-6185
Fax: +81 (0)29 852 5387
E-mail: yutaka.nakajima@astellas.com (Y. Nakajima), shohei.shirakami@astellas.com (S.
Shirakami).
1

Abstract
Janus kinases (JAKs) regulate various inflammatory and immune responses and are targets for
the treatment of inflammatory and immune diseases. As a novel class of immunomodulators
targeting JAK3, 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives are promising candidates
for treating such diseases. In chemical modification of lead compound 2, the substitution of a
cycloalkyl ring for an N-cyanopyridylpiperidine in C4-position was effective for increasing
JAK3 inhibitory activity. In addition, modulation of physical properties such as molecular
lipophilicity and basicity was important for reducing human ether-a-go-go-related gene (hERG)
inhibitory activity. Our optimization study gave compound 31, which exhibited potent JAK3
inhibitory activity as well as weak hERG inhibitory activity. In cellular assay, 31 exhibited
potent immunomodulating effect on IL-2-stimulated T cell proliferation. In a pharmacokinetic
study, good metabolic stability and oral bioavailability of 31 were achieved in rats, dogs, and
monkeys. Further, 31 prolonged graft survival in an in vivo rat heterotopic cardiac transplant
model.
Keywords: JAK (Janus kinase), JAK3 inhibitor, immunomodulator, transplant rejection, docking
calculation, hERG (human ether-a-go-go-related gene)
2

1
. Introduction
Janus kinases (JAKs) are cytoplasmic protein tyrosine kinases involved in a variety of
signal transduction pathways mediated by cytokines, such as interleukins (ILs), growth factors,
1
-5
and interferons (IFNs). Cytokine receptors associated with JAKs are stimulated by the binding
of cytokines, and JAKs are activated to induce phosphorylation of receptors. Activated JAKs
subsequently phosphorylate signal transducers and activators of transcription proteins (STATs)
in the cytoplasm, thereby inducing the dimerization of STATs. These dimer complexes of
STATs are then transferred to the nucleus and activate transcription to regulate gene expression.
JAKs consist of JAK1, JAK2, JAK3, and TYK2, which each mediate various
1
inflammatory and immune responses in their corresponding cytokine receptors. Expression of
-5
JAK3 is restricted to hematopoietic cells, while other JAKs are ubiquitously expressed in whole
6
body. JAK3 is linked to the common γc subunit of the cytokine receptors for IL-2, IL-4, IL-7,
,7
1
IL-9, IL-15, and IL-21 and regulates cytokine-induced cell development. Genetic analysis of
-5
severe combined immunodeficiency patients revealed genetic mutations of JAK3 and decreased
8
JAK3 protein expression. JAK3-knockout mice also exhibit immunodeficiency, with markedly
,9
1
0,11
reduced numbers of functional T cells with no other remarkable phenotype
Like JAK3,
JAK1 is associated with the IL-2 receptor and collaboratively mediates IL-2 signal cascade with
1
-5
JAK3 to regulate T cell proliferation. In addition, JAK1 plays important roles in the signaling
of IL-6 and IFN-γ, which is related to inflammatory responses. JAK2 mediates the signaling of
hematopoietic growth factors such as erythropoietin, granulocyte colony-stimulating factor, and
thrombopoietin and regulates the differentiation and proliferation of erythrocytes, neutrophils,
1
-5
and thrombocytes.
3

Due to the contribution to inflammatory and immune responses, JAK inhibitors have been
examined for utility in treating inflammatory and immune disorders, such as rheumatoid arthritis
1
2-16
(
RA), psoriasis, inflammatory bowel disease, and organ transplant rejection.
Tofacitinib
(
compound 1, Figure 1), which has been approved for treating RA as a pan-JAK inhibitor,
1
7-19
exhibits in vivo efficacy in rat and monkey transplant models.
In organ transplantation, T
cells recognize the allografts as antigens, become activated, and produce IL-2 to promote their
2
0
differentiation and proliferation. T cell-mediated immunity therefore impairs allograft survival
and results in transplant rejection. Current immunosuppressive therapeutic regimens consisting
of calcineurin inhibitors (CNIs), such as tacrolimus and cyclosporin A, are highly effective in the
prevention of acute transplant rejection. CNIs play an important role in inhibition of IL-2
production and the following T cell activation. In spite of potent efficacy, the long-term
application of CNIs would be associated with side effects such as nephrotoxicity and
2
1-23
neurotoxicity.
JAK3 inhibition may be a novel and safe immunomodulating treatment for
organ transplantation, due to the effect on IL-2-dependent T cell proliferation and limited JAK3
2
4,25
expression on lymphoid cells.
In our laboratory, a series of 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives was
found to exhibit potent and moderately selective JAK3 inhibitory activity, and compound 2
2
Figure 2) was identified as a lead compound. The hydrophobic cycloalkyl ring at the C4-
6
(
position of these derivatives was important for JAK3 inhibitory activity, due to the interaction
with the hydrophobic cavity of JAK3. However, a number of compounds suffered from poor
metabolic stability in liver microsomes. To increase JAK3 inhibitory activity and improve
pharmacokinetic (PK) profile, further modification of the C4-substituent was attempted. As
shown in Figure 2, we investigated the conversion of the cycloalkyl ring to a piperidine ring to
4

modulate molecular lipophilicity and thereby improve metabolic stability, and N-substitution of
the piperidine is considered to facilitate novel interaction with JAK3 at the back of the
hydrophobic cavity.
In addition to the above, the issue of human ether-a-go-go-related gene (hERG) channel
inhibition was also examined for these compounds. The hERG channel is a member of the
voltage-gated potassium ion channels, and its inhibition is related to QT interval prolongation,
2
which is a risk factor of cardiotoxicity in clinical stage. hERG blockers such as terfenadine (3),
7
cisapride (4), and MK-499 (5) contain characteristic basic amine and hydrophobic groups
(
Figure 3), and the docking mode of these compounds in the hERG pore domain is suggested by
an in silico molecular modeling study, indicating a cation-π interaction with Tyr652 and π-
2
8,29
stacking interaction with Tyr652 or Phe656.
Based on the reports of hERG blockers,
molecular lipophilicity and basicity are related to hERG blockade, and we modified the physical
properties of our test compounds to avoid the unintentional hERG inhibitory activity.
Here, we describe the structure-activity relationship (SAR) study of 4-(piperidin-4-
ylamino)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives with a focus on JAK3 inhibition
and decreased hERG inhibition. In addition, the efficacy of 31 was investigated in an in vivo rat
3
0
heterotopic cardiac transplant model.
5

2
. Chemistry
Scheme 1 shows the synthesis of 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives.
2
Compound 6 was prepared as previously described and treated with several amines under
6
microwave irradiation to give the desired compounds 7a-d.
Scheme 2 shows the synthesis of a series of 4-aminopiperidine analogues. In a similar
manner to Scheme 1, 6 was reacted with N-Boc-4-aminopiperidine to give compound 8, and
following deprotection of Boc group under an acidic condition gave intermediate 9. 9 underwent
acetylation and mesylation by treatment with Ac
Condensation of 9 with cyanoacetic acid under treatment with HOBt and EDC gave 10c.
Reductive alkylation of 9 with 3-cyanobenzaldehyde using NaBH(OAc) gave 10d.
Scheme 3 shows the synthesis of N-arylpiperidine compounds. Treatment of 9 with 4-
fluorobenzonitrile and K CO gave N-cyanophenyl analogue 11a. For N-pyridyl analogues,
2
O to give 10a and with MsCl to give 10b.
3
2
3
reaction with 2-chloropyridines containing an electron-withdrawing group such as a cyano,
trifluoromethyl, and ester group gave 11b-d. N-Cyanopyrazinyl analogue 11e and N-
cyanopyridazinyl analogue 11f were synthesized in a similar manner with corresponding aryl
chlorides. Treatment of 11d with ammonia in a sealed tube gave carboxamide 12a. Reduction of
4
the ester group of 11d with LiAlH gave alcohol 12b.
Scheme 4 shows the synthesis of the 3-methylpiperidine analogue 19. Commercially
available 1-benzyl-3-methylpiperidin-4-one (13) was reacted with (2,4-dimethoxybenzyl)amine
under reductive amination by using NaBH(OAc)
3
to give a racemic mixture of cis-4-amino-3-
O gave 15 and successive treatment with
methylpiperidine 14. Treatment of 14 with (CF CO)
3
2
TFA followed by alkaline resulted in deprotection of the 2,4-dimethoxybenzyl group and
trifluoroacetyl group to give amine 16. Reaction of 6 and 16 under microwave irradiation gave
6

1
7. Deprotection of the benzyl group by Pd-catalyzed hydrogenation gave 18, and target
compound 19 was obtained following nucleophilic reaction with 2-chloro-5-cyanopyridine.
Scheme 5 shows the synthesis of chiral 3-fluoropiperidine analogues 31 and 35.
Commercially available N-Boc-4-piperidone (20) was treated with trimethylsilyl chloride
®
TMSCl) to give silyl enol ether 21, and subsequent fluorination with Selectfluor gave 3-fluoro-
(
4
-oxopiperidine 22. Reductive amination of 22 treated with benzylamine and NaBH(OAc)
3
provided a racemic mixture of cis-4-(benzylamino)-3-fluoropiperidine, which was separated by
3
1,32
chiral column chromatography to give (3S,4R)-isomer 23 and (3R,4S)-isomer 24.
Deprotection of the benzyl group of 23 and 24 by Pd-catalyzed hydrogenation gave 25 and 26,
respectively. Nucleophilic substitution of ethyl 4-chloro-1H-pyrrolo[2,3-b]pyridine-5-
2
6
carboxylate (27) with 25 was conducted to give 28. The ester group of 28 was hydrolyzed to
give 29, and successive condensation with aqueous ammonia gave carboxamide 30. The Boc
group of 30 was deprotected by TFA, and then treated with 2-chloro-5-cyanopyridine to give 31.
The enantiomer 35 was prepared in a manner similar to that for 31 by using 26 instead of 25.
7

3
. Results and discussion
Synthesized compounds were evaluated for human JAK3 inhibitory activity, and IC50
values were calculated. JAK1 inhibitory activity was evaluated in addition to JAK3 inhibitory
activity, as JAK3 and JAK1 collaboratively regulate the IL-2 signaling pathway. JAK2 inhibitory
activity was also investigated, as the inhibition of JAK2 may be related to adverse hematopoietic
3
3
effects such as anemia. The IL-2 signaling pathway is important for T cell-mediated immune
response, and selected compounds were investigated for inhibition of IL-2-stimulated T cell
proliferation using rat spleen cells to evaluate the cellular effect via the inhibition of JAK3 and
JAK1. An in vivo rat heterotopic cardiac transplant model was investigated using ACI rats as
cardiac donors and Lewis rats as cardiac recipients, and median survival times (MST) of grafts
3
0
were analyzed.
Lead compound 2 inhibited JAK3 (IC50 = 5.1 nM) and T cell proliferation (IC50 = 86 nM).
However, the microsomal intrinsic clearance (CLint) value of 2 was high (>1000 mL/min/kg) due
26
to oxidative metabolism of the hydrophobic C4-substituent. Therefore, to avoid drug
metabolism, aminopiperidine was introduced to the C4-position. We suspected that conversion to
a heterocyclic ring might improve metabolic stability.
Table 1 shows results of SAR of aminopiperidine derivatives. Regarding JAK3 inhibitory
activity, N-methylpiperidine analogue 7a exhibited an 18-fold decreased activity (IC50 = 90 nM)
compared to 2. The C4-substituent of the 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives
2
6
contributed to hydrophobic interaction with JAK3. To enhance affinity for JAK3 in the back
pocket of the hydrophobic region, N-substitution of the piperidine of 7a was attempted.
Introduction of an acetyl group (10a) and mesyl group (10b) to the piperidine led to weak JAK3
inhibitory activity (IC50 = 340 and 150 nM, respectively), suggesting that a small polar
functional group was unfavorable for binding to JAK3. Introduction of a benzyl group was then
8

examined, with the N-benzyl piperidine analogue 7b exhibiting a 4-fold increase in JAK3
inhibitory activity (IC50 = 23 nM) compared to 7a.
To investigate spatial tolerance around the piperidine moiety, the direction of the benzyl
group was modified. Given that the 3-aminopiperidine analogues 7c and 7d exhibited weak
JAK3 inhibitory activity (IC50 = 240 and 130 nM, respectively), the 4-aminopiperidine core was
selected for further modification. To increase JAK3 inhibitory activity , the incorporation of a
cyano group into the C4-substituent was investigated due to the interaction with JAK3 in the
3
back of the binding pocket reported for known JAK inhibitors. As expected, N-cyanoacetyl
4
analogue 10c exhibited a 7-fold increase in JAK3 inhibitory activity (IC50 = 46 nM) compared to
1
0a. In contrast, introduction of a cyano group to the phenyl ring of 7b resulted in moderate
JAK3 inhibitory activity (10d; IC50 = 25 nM).
To restrict conformation of the N-substituent and direct the cyanophenyl group towards the
back of the binding pocket of JAK3, the methylene linker of 10d was modified, and the N-
cyanophenyl analogue 11a largely increased JAK3 inhibitory activity with an IC50 value of 3.4
nM. Substitution of the phenyl ring of 11a to a pyridine was then investigated, with the N-
cyanopyridyl analogue 11b exhibiting a further increase in JAK3 inhibition (IC50 = 1.3 nM). The
effect of functional groups other than a cyano group on the pyridine ring was then investigated.
However, introduction of a trifluoromethyl (11c), carbamoyl (12a), and hydroxymethyl group
(
12b) all resulted in decreased JAK3 inhibitory activity (IC50 = 25, 5.2, and 9.2 nM, respectively).
The series of N-arylpiperidine derivatives (11a−c, and 12a,b) exhibited moderately selective
JAK3 inhibition, with about 10-fold decrease in the potency of JAK1 and JAK2 inhibition (Table
1
). In an in vitro test of metabolic stability using rat liver microsomes, 11a and 11b exhibited
relatively low CLint values (262 and 204 mL/min/kg, respectively) compared to lead compound 2,
9

although the molecular lipophilicity of 11a and 11b was similar or higher than that of 2 (CLogP
of 2, 11a, and 11b = 2.8, 3.2, and 2.8, respectively). Conversion of cyclohexyl ring to piperidine
seemed to reduce drug metabolism on C4-substituent moiety, and the 4-aminopiperidine
derivatives were preferable for improving metabolic stability. In a functional cellular assay, 11b
exhibited potent inhibition of IL-2-stimulated T cell proliferation, with an IC50 value of 27 nM
(
Table 3). Given these results, we identified 11b as a new lead compound. However, 11b
exhibited moderate inhibition of the hERG channel, with an IC50 value of 13 µM (Table 2).
Table 2 shows the results of modification of the pyridine and piperidine moiety of 11b. To
28,29
decrease hERG inhibitory activity, reduced molecular lipophilicity and basicity is important.
We attempted conversion of the pyridine to other hetero aromatic rings, and the pyrazine
analogue 11e and pyridazine analogue 11f maintained potent JAK3 inhibitory activity (IC50 = 3.6
and 1.2 nM, respectively) with decreased molecular lipophilicity (CLogP = 2.4 and 1.6,
respectively). Possibly due to the decreased lipophilicity and the reduced electron density of the
aromatic ring, 11e and 11f substantially decreased hERG inhibitory activity with IC50 values of
more than 100 µM. Modification of the piperidine moiety of 11b was then investigated.
Consistent with a previous finding that a methyl group of cyclohexyl ring of 2 contributed to
2
6
increased JAK3 inhibitory activity, introduction of a methyl group to the piperidine of 11b
resulted in potent JAK3 inhibitory activity (19; IC50 = 0.78 nM). However, the 3-
methylpiperidine analogue 19 retained moderate hERG inhibitory activity (IC50 = 29 µM),
associated with high molecular lipophilicity (CLogP = 3.4).
Therefore, instead of a methyl group, introduction of a fluoro group to the 3-position of the
piperidine was investigated (31 and 35). Introduction of fluorine atom to piperidine reduces pKa
value of the nitrogen atom (see Supplementary data). We considered that the 3-fluoropiperidine
10

analogues 31 and 35 resulted in not only a slight decrease in lipophilicity (CLogP = 2.7) but also
decrease in the basicity of piperidine nitrogen due to the electron-withdrawing effect of fluorine
3
5,36
atom.
In stereochemistry of the 3-fluoropiperidine-4-amine, the (3R, 4S)-isomer 35 was
equipotent to 11b for JAK3 inhibitory activity (IC50 = 1.7 nM), while the (3S, 4R)-isomer 31
exhibited a 4-fold increase in the activity (IC50 = 0.30 nM). With respect to hERG inhibition, 31
and 35 showed markedly decreased hERG inhibition (IC50 >100 µM), possibly due to decreased
molecular lipophilicity and basicity. In addition, 31 maintained about 10-fold JAK3 selectivity
toward JAK1 and JAK2. Further, 31 showed weak inhibitory activity toward 32 representative
kinases in kinase panel assay (see Supplementary data).
In the cellular assay of IL-2-stimulated T cell proliferation, less potent inhibition was
exerted by 11e (IC50 = 77 nM) and 11f (IC50 = 140 nM) compared to 11b despite retaining good
JAK3 inhibitory activity (Table 3). To investigate the difference between enzymatic and cellular
inhibition, the membrane permeability of these compounds was evaluated in a parallel artificial
e
membrane permeability assay (PAMPA). In contrast to the high effective permeability (P ) value
-
6
of 11b (P
e
e
= 26 × 10 cm/sec), both 11e and 11f exhibited low membrane permeability (P =
-
6
-6
0.2 × 10 and <0.2 × 10 cm/sec, respectively), suggesting weak pharmacological effect on
<
cellular assay. In contrast, 31 exhibited potent inhibition of T cell proliferation (IC50 = 11 nM)
-6
e
due to excellent JAK3 inhibitory activity and acceptable membrane permeability (P = 9 × 10
cm/sec). Given these findings, 31 was identified as a potent immunomodulator without risk of
hERG-related cardiac disorder.
The SAR study of 4-(piperidin-4-ylamino)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide
derivatives revealed that the cyanopyridine and fluoro group of 31 contributed to potent JAK3
inhibitory activity. To verify the effect of the C4-substituent on JAK3 inhibition, docking
11

calculation of 31 was conducted as shown in Figure 4. Docking calculations were performed for
3
4
the crystal structure of 1 bound to JAK3 (PDB code: 3LXK ) using the XP mode in Glide
version 5.8; Schrödinger, LLC, New York, NY, 2012). The predicted binding mode of 31
toward human JAK3 was similar to our previously reported series of 1H-pyrrolo[2,3-b]pyridine-
(
2
-carboxamide derivatives. The 1H-pyrrolo[2,3-b]pyridine moiety occupied the ATP-binding
6
5
site to form hydrogen bond interactions with Glu903 and Leu905, and the N1-hydrogen atom
served as a hydrogen donor and N7-nitrogen atom as an acceptor. In addition, the proton of the
C2-position interacted with the gatekeeper amino acid Met902, and the 1H-pyrrolo[2,3-
b]pyridine ring formed CH-π interactions with Val836 and Leu828. Characteristic intramolecular
interaction between carbonyl group of C5-carboxamide and proton of C4-amine was observed,
and due to the intramolecular hydrogen bond, the fluoropiperidine moiety was retained in the
hydrophobic region, with the fluoro group located around the rim of the pocket for the
hydrophobic interaction. Further, the cyanopyridine moiety extended to the back of the binding
pocket. The pyridine ring was located near the acidic side chain of Asp967, suggesting that ionic
interactions contributed to binding affinity to JAK3. In the binding pocket, the cyano group
interacted with side chain of Arg953, and the hydrogen bond interaction was considered
important for potent JAK3 inhibitory activity.
As shown in Table 4, in in vitro metabolic stability test using liver microsomes, 31 showed
low metabolic clearance values in not only humans but also other species, such as rats, dogs, and
monkeys. Table 5 shows PK parameters of 31 after oral (po) and intravenous (iv) administration.
In rats, 31 exhibited low total clearance (CLtot = 5.3 mL/min/kg), with favorable oral absorption
and good oral bioavailability (F = 71%). PK profiles of 31 were similar in dogs (F = 49%) and
monkeys (F = 64%), with good oral bioavailability in these species as well.
12

3
1 was also evaluated for in vivo efficacy to prevent allograft rejection in a rat heterotopic
3
0
cardiac transplantation model (Table 6). In this model, untreated allografts were ultimately
rejected with a graft survival period of five days after transplantation. Following oral
administration, 31 exhibited dose-dependent and effective prevention of allograft rejection to
prolong the graft survival time compared to the vehicle (MST = 11.5 and 22 days at 10 and 20
mg/kg, respectively). These results suggest that 31 may be useful for the prevention of organ
transplant rejection.
13

4
. Conclusion
We synthesized
4-(piperidin-4-ylamino)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide
derivatives and evaluated for the JAK inhibitory activity and immunomodulating effect on IL-2-
stimulated T cell proliferation. Incorporation of a cyanopyridine to the piperidine nitrogen in C4-
position led to potent JAK3 inhibitory activity (11b). The novel C4-substituent was favorable for
interaction with JAK3 binding pocket. However, 11b also exhibited moderate inhibition of
hERG channels. Although conversion of the pyridine of 11b to a pyrazine (11e) and pyridazine
(
11f) effectively reduced hERG inhibitory activity, these compounds exerted less potent cellular
inhibition due to low membrane permeability. We therefore introduced a fluoro group that
reduced the basicity of the piperidine nitrogen and in turn hERG inhibition (31). In addition, the
fluoro group contributed to further increase in JAK3 inhibitory activity, and 31 exhibited potent
inhibition of T cell proliferation. The in vivo efficacy of the novel and potent JAK3 inhibitor 31
was also investigated in an organ transplant model. The pharmacokinetic profile of 31 was
favorable with good metabolic stability and oral bioavailability in rats, dogs, and monkeys. In a
rat heterotopic cardiac transplant model, 31 effectively prevented allograft rejection and
prolonged graft survival. These findings suggest that 31 may be a candidate immunomodulator
for the prevention of organ transplant rejection.
14

5
5
. Experimental
.1. Chemistry
1
H NMR spectra were recorded on a Bruker Biospin AV400 or AV400M spectrometer.
Chemical shifts are expressed in δ units using tetramethylsilane as an internal standard (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 spectroscopy (HRMS) spectra were recorded on a
Waters LCT Premier XE. Column chromatography was conducted using silica gel 60N (Kanto
TM
Chemical, 63-210 µm) or a HI-FLASH Column (Yamazen). The purity of all compounds
screened in biological assays was >95%, as determined by HPLC or elemental analyses, or a
combination of the two. 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
4
mL/min flow rate using acetonitrile and 0.01 M HClO 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 calculated for C, H, and N.
5
.1.1. 4-[(1-Benzylpiperidin-4-yl)amino]-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (7b)
In the vessel of a microwave reactor, 4-amino-1-benzylpiperidin (973 mg, 5.11 mmol) and
3
Et N (0.71 mL, 5.11 mmol) were added to a solution of 4-chloro-1H-pyrrolo[2,3-b]pyridine-5-
carboxamide (6) (200 mg, 1.02 mmol) in NMP (2 mL). The vessel was sealed, and the mixture
was reacted at 180 °C for 1.5 h under microwave irradiation. After cooling to room temperature,
2
the mixture was diluted with EtOAc and then washed with H O. The organic layer was separated,
dried over MgSO , and concentrated under reduced pressure. The residue was purified by NH
4
15

silica gel column chromatography (CHCl
3
/MeOH = 100/0 to 95/5) to give the title compound
344 mg, 96%). H NMR (400 MHz, DMSO-d ): δ 1.46–1.57 (2H, m), 1.95–2.04 (2H, m), 2.21–
.31 (2H, m), 2.64–2.75 (2H, m), 3.33 (2H, s), 3.92–4.02 (1H, m), 6.47–6.50 (1H, m), 7.00 (1H,
1
(
6
2
br), 7.11–7.14 (1H, m), 7.22–7.28 (1H, m), 7.30–7.35 (4H, m), 7.77 (1H, br), 8.36 (1H, s), 9.71
+
1H, d, J = 8.0 Hz), 11.46 (1H, brs). MS (ESI) m/z: 350 (M + H) . Anal. calcd for
(
20 23 5 2
C H N O·1.1H O: C, 65.06; H, 6.88; N, 18.97. Found: C, 65.25; H, 6.91; N, 18.64.
5
.1.2. 4-[(1-Methylpiperidin-4-yl)amino]-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (7a)
The title compound was prepared from 6 and 4-amino-1-methylpiperidine in accordance
1
with the procedure for preparing 7b in 32% yield. H NMR (400 MHz, DMSO-d
6
): δ 1.45–1.57
(
2H, m), 1.94–2.02 (2H, m), 2.14–2.24 (5H, m), 2.59–2.70 (2H, m), 3.88–3.99 (1H, m), 6.47–
6
.50 (1H, m), 6.97 (1H, br), 7.11–7.14 (1H, m), 7.73 (1H, br), 8.35 (1H, s), 9.68 (1H, d, J = 8.0
+
Hz), 11.45 (1H, brs). MS (ESI) m/z: 274 (M + H) . Anal. calcd for C14
H
19
N
5
O·0.3MeOH: C,
6
0.70; H, 7.20; N, 24.75. Found: C, 60.78; H, 7.01; N, 24.63.
5
.1.3.
4-{[(3R)-1-Benzylpiperidin-3-yl]amino}-1H-pyrrolo[2,3-b]pyridine-5-carboxamide
(
7c)
The title compound was prepared from 6 and (R)-1-benzyl-3-aminopiperidine in
1
accordance with the procedure for preparing 7b in 22% yield. H NMR (400 MHz, DMSO-d
6
) δ
1
.44–1.61 (2H, m), 1.66–1.87 (2H, m), 2.17–2.55 (3H, m), 2.66–2.77 (1H, m), 3.44–3.56 (2H,
m), 4.06–4.16 (1H, m), 6.39–6.42 (1H, m), 6.98 (1H, br), 7.03–7.06 (1H, m), 7.18–7.24 (1H, m),
.27–7.32 (2H, m), 7.40 (2H, d, J = 7.2 Hz), 7.63 (1H, br), 8.33 (1H, s), 9.73 (1H, d, J = 8.0 Hz),
7
16

+
1.38 (1H, brs). MS (ESI) m/z: 350 (M + H) . Anal. calcd for C20
1
6
H
23
N
5
O·0.4H
2
O: C, 67.36; H,
.73; N, 19.64. Found: C, 67.54; H, 6.55; N, 19.42.
5
.1.4.
4-{[(3S)-1-Benzylpiperidin-3-yl]amino}-1H-pyrrolo[2,3-b]pyridine-5-carboxamide
(
7d)
The title compound was prepared from 6 and (S)-1-benzyl-3-aminopiperidine in
1
accordance with the procedure for preparing 7b in 34% yield. H NMR (400 MHz, DMSO-d
6
) δ
1
.44–1.61 (2H, m), 1.66–1.87 (2H, m), 2.17–2.55 (3H, m), 2.66–2.77 (1H, m), 3.44–3.56 (2H,
m), 4.06–4.16 (1H, m), 6.39–6.42 (1H, m), 6.98 (1H, br), 7.03–7.06 (1H, m), 7.18–7.24 (1H, m),
7
1
6
.27–7.32 (2H, m), 7.40 (2H, d, J = 7.2 Hz), 7.63 (1H, br), 8.33 (1H, s), 9.73 (1H, d, J = 8.0 Hz),
+
1.38 (1H, brs). MS (ESI) m/z: 350 (M + H) . Anal. calcd for C20
H
23
N
5
O·0.3H
2
O: C, 67.70; H,
.70; N, 19.74. Found: C, 67.95; H, 6.65; N, 19.52.
5
.1.5.
tert-Butyl
4-[(5-carbamoyl-1H-pyrrolo[2,3-b]pyridin-4-yl)amino]piperidine-1-
carboxylate (8)
The title compound was prepared from 6 and N-Boc-4-aminopiperidine in accordance with
1
the procedure for preparing 7b in 60% yield. H NMR (400 MHz, DMSO-d
6
) δ 1.31–1.45 (11H,
m), 1.95–2.03 (2H, m), 3.04–3.24 (2H, m), 3.73–3.83 (2H, m), 4.10–4.20 (1H, m), 6.57 (1H, d, J
=
3.2 Hz), 7.01 (1H, br), 7.15 (1H, d, J = 3.2 Hz), 7.79 (1H, br), 8.37 (1H, s), 9.72 (1H, d, J = 8.0
+
Hz), 11.50 (1H, brs). MS (ESI) m/z: 360 (M + H) .
5
.1.6. 4-(Piperidin-4-ylamino)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide dihydrochloride
(
9)
17

To a solution of 8 (0.97 g, 2.70 mmol) in EtOH (14.6 mL), 4 M HCl in dioxane (10.1 mL,
4
0.5 mmol) was added, and the mixture was stirred at room temperature overnight. After
concentration under reduced pressure, IPE was added to the residue. The precipitate was filtrated
and washed with IPE to give the title compound (0.97 g, >100%), which was used in the next
1
reaction without further purification. H NMR (400 MHz, DMSO-d
6
) δ 1.75–1.87 (2H, m), 2.17–
2
7
1
.25 (2H, m), 3.16–3.33 (4H, m), 4.40–4.50 (1H, m), 6.97–7.01 (1H, m), 7.39–7.42 (1H, m),
.77 (1H, br), 8.48 (1H, br), 8.61 (1H, s), 9.09–9.32 (2H, m), 10.78–10.84 (1H, m), 12.75 (1H, s),
+
4.82 (1H, br). MS (ESI) m/z: 260 (M + H) .
5
.1.7. 4-[(1-Acetylpiperidin-4-yl)amino]-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (10a)
To a solution of 9 (70 mg, 0.211 mmol) in THF (1.4 mL), Ac
DIPEA (0.22 mL, 1.26 mmol) were added, and the mixture was stirred at room temperature for
8 h and then diluted with CHCl . To the mixture, saturated NaHCO aqueous solution was
added, and the organic layer was separated, dried over MgSO , and concentrated under reduced
pressure. The residue was purified by column chromatography (CHCl /MeOH = 100/0 to 90/10)
to give the title compound (45 mg, 71%). H NMR (400 MHz, DMSO-d ) δ 1.27–1.37 (1H, m),
.41–1.51 (1H, m), 1.95–2.07 (5H, m), 3.02–3.10 (1H, m), 3.24–3.29 (1H, m), 3.69–3.76 (1H,
m), 4.03–4.11 (1H, m), 4.17–4.26 (1H, m), 6.57–6.60 (1H, m), 6.99 (1H, br), 7.13–7.17 (1H, m),
2
O (25 µL, 0.263 mmol) and
1
3
3
4
3
1
6
1
7
.74 (1H, br), 8.37 (1H, s), 9.72 (1H, d, J = 8.4 Hz), 11.48 (1H, brs). MS (ESI) m/z: 302 (M +
+
H) . Anal. calcd for C15
19 5 2 2
H N O ·0.3H O: C, 58.73; H, 6.44; N, 22.83. Found: C, 58.86; H, 6.47;
N, 22.45.
18

5
.1.8.
carboxamide (10b)
To a solution of 9 (40 mg, 0.154 mmol) and Et
4-{[1-(Methylsulfonyl)piperidin-4-yl]amino}-1H-pyrrolo[2,3-b]pyridine-5-
3
N (0.065 mL, 0.463 mmol) in DMF (0.8
mL), MsCl (0.014 mL, 0.185 mmol) was added dropwise under ice cooling, and the mixture was
then stirred at the same condition for 2 h. The precipitate was filtrated, and then dissolved in a
small amount of MeOH. IPE was added to the solution, and the precipitate was filtrated and
1
washed with IPE to give the title compound (22 mg, 42%). H NMR (400 MHz, DMSO-d
6
) δ
1
4
.49-1.59 (2H, m), 2.06–2.15 (2H, m), 2.90 (3H, s), 3.06–3.14 (2H, m), 3.44–3.51 (2H, m),
.08–4.18 (1H, m), 6.56–6.58 (1H, m), 7.03 (1H, brs), 7.15–7.18 (1H, m), 7.80 (1H, brs), 8.61
+
1H, s), 9.73 (1H, d, J = 8.0 Hz), 11.52 (1H, s). MS (ESI) m/z: 338 (M + H) . Anal. calcd for
(
14 19 5 3 2
C H N O S·0.8H O: C, 47.80; H, 5.90; N, 19.91. Found: C, 48.20; H, 5.53; N, 19.53.
5
.1.9. 4-{[1-(Cyanoacetyl)piperidin-4-yl]amino}-1H-pyrrolo[2,3-b]pyridine-5-carboxamide
(
10c)
To a solution of 9 (50 mg, 0.150 mmol), cyanoacetic acid (25.6 mg, 0.301 mmol), and Et N
3
(
0.042 mL, 0.301 mmol) in DMF (1 mL), HOBt (46.1 mg, 0.301 mmol) and EDC (46.7 mg,
.301 mmol) were added. The mixture was stirred at room temperature overnight, diluted with
CHCl , and then washed with H O. The organic layer was separated, and the aqueous layer was
0
3
2
extracted with CHCl . Combined organic layer was dried over MgSO and concentrated under
3
4
reduced pressure. The residue was purified by column chromatography (CH
2
2
Cl /MeOH = 10/1)
1
to give the title compound (20 mg, 41%). H NMR (400 MHz, DMSO-d
6
) δ 1.29–1.40 (1H, m),
1
.46–1.58 (1H, m), 1.98–2.07 (2H, m), 3.09–3.18 (1H, m), 3.27–3.37 (1H, m), 3.56–3.64 (1H,
m), 4.03–4.13 (3H, m), 4.19–4.29 (1H, m), 6.60–6.62 (1H, m), 7.04 (1H, brs), 7.15–7.18 (1H, m),
19

7
.81 (1H, brs), 8.38 (1H, s), 9.74 (1H, d, J = 8.0 Hz), 11.52 (1H, brs). MS (ESI) m/z: 327 (M +
+
+
18 4
H) . HRMS (M + H) calcd for C15H N O: 326.1491. Found: 327.1578.
5
.1.10.
carboxamide (10d)
To a solution of 9 (100 mg, 0.301 mmol) in CH
mg, 0.602 mmol), NaBH(OAc) (128 mg, 0.602 mmol), and Et
added successively. The mixture was stirred at room temperature overnight, and saturated
NaHCO aqueous solution was then added. The organic layer was separated, dried over MgSO4,
4-{[1-(3-Cyanobenzyl)piperidin-4-yl]amino}-1H-pyrrolo[2,3-b]pyridine-5-
2
Cl
2
(1 mL), 3-cyanobenzaldehyde (78.9
N (0.084 mL, 0.602 mmol) were
3
3
3
and concentrated under reduced pressure. The residue was purified by column chromatography
1
/MeOH = 100/0 to 90/10) to give the title compound (45 mg, 40%). H NMR (400 MHz,
(
CHCl
3
DMSO-d
6
) δ 1.49–1.59 (2H, m), 1.97–2.05 (2H, m), 2.24–2.35 (2H, m), 2.65–2.75 (2H, m), 3.57
(
2H, s), 3.94–4.04 (1H, m), 6.48–6.51 (1H, m), 6.98 (1H, brs), 7.12–7.13 (1H, m), 7.55 (1H, t, J
=
7.6 Hz), 7.67–7.77 (4H, m), 8.36 (1H, s), 9.71 (1H, d, J = 8.0 Hz), 11.46 (1H, s). MS (ESI)
+
m/z: 375 (M + H) . Anal. calcd for C21
H
22
N
6
O·0.3H
2
O: C, 66.40; H, 6.00; N, 22.12. Found: C,
6
6.51; H, 5.90; N, 21.73.
5
.1.11.
4-{[1-(4-Cyanophenyl)piperidin-4-yl]amino}-1H-pyrrolo[2,3-b]pyridine-5-
carboxamide (11a)
To a solution of 9 (100 mg, 0.301 mmol) in DMSO (10 mL), K
mmol) and 4-fluorobenzonitrile (54.7 mg, 0.451 mmol) were added, and the mixture was stirred
at 80 °C. After cooling to room temperature, CHCl was added, and the mixture was washed
with H O, dried over MgSO , and concentrated under reduced pressure. The residue was purified
by column chromatography (CHCl /MeOH = 100/0 to 90/10) to give the title compound (22 mg,
2
3
CO (125 mg, 0.903
3
2
4
3
20

1
1%). H NMR (400 MHz, DMSO-d
2
6
) δ 1.46–1.57 (2H, m), 2.05–2.13 (2H, m), 3.24–3.35 (2H,
m), 3.76–3.83 (2H, m), 4.22–4.32 (1H, m), 6.62 (1H, dd, J = 2.0, 3.6 Hz), 7.01 (1H, brs), 7.06
(
2H, d, J = 9.2 Hz), 7.15–7.18 (1H, m), 7.57 (2H, d, J = 9.2 Hz), 7.79 (1H, brs), 8.37 (1H, s),
+
.74 (1H, d, J = 8.4 Hz), 11.50 (1H, s). MS (ESI) m/z: 361 (M + H) . Anal. calcd for
9
20 20 6 2
C H N O·0.5H O·0.5MeOH: C, 63.88; H, 6.01; N, 21.80. Found: C, 63.99; H, 5.83; N, 21.81.
5
.1.12.
carboxamide (11b)
To a solution of 9 (75 mg, 0.226 mmol) in 1,3-dimethyl-2-imidazolidinone (DMI) (0.75
mL), Et N (0.13 mL, 0.903 mmol) and 2-chloro-5-cyanopyridine (62.6 mg, 0.451 mmol) were
added, and the mixture was then stirred at 160 °C for 1 h. After cooling to room temperature,
EtOAc was added, and the mixture was washed with H O, dried over MgSO , and concentrated
under reduced pressure. The residue was purified by column chromatography (CHCl /MeOH =
) δ 1.41–
4-{[1-(5-Cyanopyridin-2-yl)piperidin-4-yl]amino}-1H-pyrrolo[2,3-b]pyridine-5-
3
2
4
3
1
1
1
6
9
00/0 to 95/5) to give the title compound (46 mg, 56%). H NMR (400 MHz, DMSO-d
6
.52 (2H, m), 2.05–2.14 (2H, m), 3.40–3.49 (2H, m), 4.18–4.37 (3H, m), 6.62–6.35 (1H, m),
.99 (1H, d, J = 9.2 Hz), 7.01 (1H, br), 7.16–7.19 (1H, m), 7.81 (1H, br), 7.85 (1H, dd, J = 2.4,
.2 Hz), 8.38 (1H, s), 8.49 (1H, d, J = 2.0 Hz), 9.76 (1H, d, J = 8.0 Hz), 11.51 (1H, brs). MS
+
ESI) m/z: 362 (M + H) . Anal. calcd for C H N O·0.8H
(
2
O: C, 60.72; H, 5.52; N, 26.09. Found:
19
19
7
C, 60.82; H, 5.96; N, 26.03.
5
.1.13.
4-({1-[5-(Trifluoromethyl)pyridin-2-yl]piperidin-4-yl}amino)-1H-pyrrolo[2,3-
b]pyridine-5-carboxamide (11c)
The title compound was prepared from 9 and 2-chloro-5-(trifluoromethyl)pyridine in
1
accordance with the procedure for preparing 11b in 26% yield. H NMR (400 MHz, DMSO-d
6
) δ
21

1
.42–1.52 (2H, m), 2.05–2.13 (2H, m), 3.38–3.47 (2H, m), 4.16–4.24 (2H, m), 4.27–4.36 (1H,
m), 6.62–6.65 (1H, m), 7.01 (1H, d, J = 9.2 Hz), 7.02 (1H, br), 7.16–7.19 (1H, m), 7.79 (1H, dd,
J = 2.4, 9.2 Hz), 7.82 (1H, br), 8.37 (1H, s), 8.41–8.43 (1H, m), 9.76 (1H, d, J = 8.0 Hz), 11.51
+
1H, brs). MS (ESI) m/z: 405 (M + H) . Anal. calcd for C H F N O·0.3H
(
2
O: C, 55.69; H, 4.82;
1
9
19
3
6
N, 20.51. Found: C, 55.99; H, 4.93; N, 20.16.
5
.1.14.
Methyl
6-{4-[(5-carbamoyl-1H-pyrrolo[2,3-b]pyridin-4-yl)amino]piperidin-1-
yl}nicotinate (11d)
The title compound was prepared from 9 and methyl 6-chloronicotinate in accordance with
1
the procedure for preparing 11b in 65% yield. H NMR (400 MHz, DMSO-d
6
) δ 1.42–1.52 (2H,
m), 2.06–2.14 (2H, m), 3.40–3.48 (2H, m), 3.79 (3H, s), 4.18–4.36 (3H, m), 6.62–6.65 (1H, m),
6
9
.93 (1H, d, J = 9.2 Hz), 7.00 (1H, br), 7.16–7.18 (1H, m), 7.79 (1H, br), 7.95 (1H, dd, J = 2.4,
.2 Hz), 8.37 (1H, s), 8.66 (1H, d, J = 2.0 Hz), 9.75 (1H, d, J = 8.0 Hz), 11.51 (1H, brs). MS
+
ESI) m/z: 395 (M + H) . Anal. calcd for C H N O ·0.8H
(
2
O: C, 58.76; H, 5.82; N, 20.56. Found:
20
22
6
3
C, 58.62; H, 5.43; N, 20.39.
5
.1.15.
carboxamide (11e)
The title compound was prepared from 9 and 5-chloropyrazine-2-carbonitrile in
4-{[1-(5-Cyanopyrazin-2-yl)piperidin-4-yl]amino}-1H-pyrrolo[2,3-b]pyridine-5-
1
accordance with the procedure for preparing 11b in 22% yield. H NMR (400 MHz, DMSO-d
6
) δ
1
.47–1.57 (2H, m), 2.09–2.17 (2H, m), 3.47–3.55 (2H, m), 4.27–4.39 (3H, m), 6.63–6.66 (1H,
m), 7.02 (1H, br), 7.17–7.19 (1H, m), 7.79 (1H, br), 8.38 (1H, s), 8.48 (1H, d, J = 1.6 Hz), 8.56
+
1H, d, J = 1.6 Hz), 9.75 (1H, d, J = 8.0 Hz), 11.52 (1H, brs). MS (ESI) m/z: 363 (M + H) . Anal.
(
22

calcd for C18
N, 29.35.
18 8 2
H N O·0.5H O·0.2MeOH: C, 57.86; H, 5.28; N, 29.66. Found: C, 57.90; H, 5.12;
5
.1.16.
carboxamide (11f)
The title compound was prepared from 9 and 6-chloropyridazine-3-carbonitrile in
4-{[1-(6-Cyanopyridazin-3-yl)piperidin-4-yl]amino}-1H-pyrrolo[2,3-b]pyridine-5-
1
accordance with the procedure for preparing 11b in 50% yield. H NMR (400 MHz, DMSO-d
6
) δ
1
.47–1.57 (2H, m), 2.10–2.18 (2H, m), 3.50–3.58 (2H, m), 4.28–4.40 (3H, m), 6.64–6.67 (1H,
m), 7.01 (1H, br), 7.17–7.20 (1H, m), 7.41 (1H, d, J = 9.6 Hz), 7.79 (1H, br), 7.86 (1H, d, J = 9.6
+
Hz), 8.38 (1H, s), 9.76 (1H, d, J = 8.0 Hz), 11.52 (1H, brs). MS (ESI) m/z: 385 (M + Na) . Anal.
18 8 2
calcd for C18H N O·0.9H O: C, 57.10; H, 5.27; N, 29.60. Found: C, 57.37; H, 4.91; N, 29.26.
5
.1.17. 4-{[1-(5-Carbamoylpyridin-2-yl)piperidin-4-yl]amino}-1H-pyrrolo[2,3-b]pyridine-5-
carboxamide (12a)
A mixture of 11d (100 mg, 0.254 mmol) and 2 M NH
0 °C in a sealed tube. After cooling to room temperature, the mixture was concentrated under
3
in MeOH (1 mL) was stirred at
9
reduced pressure. The residue was purified by column chromatography (CHCl /MeOH = 10/1) to
3
1
give the title compound (19 mg, 20%). H NMR (400 MHz, DMSO-d
6
) δ 1.41–1.52 (2H, m),
2
.04–2.13 (2H, m), 3.31–3.43 (2H, m), 4.14–4.22 (2H, m), 4.25–4.34 (1H, m), 6.62–6.62 (1H,
m), 6.89 (1H, d, J = 9.2 Hz), 7.00 (1H, brs), 7.13 (1H, brs), 7.16-7.18 (1H, m), 7.72 (1H, brs),
7
.75 (1H, brs), 7.96 (1H, dd, J = 2.4, 9.2 Hz), 8.37 (1H, s), 8.62 (1H, d, J = 2.4 Hz), 9.76 (1H, d,
+
J = 8.4 Hz), 11.51 (1H, brs). MS (ESI) m/z: 380 (M + H) . Anal. calcd for
19 21 7 2 2
C H N O ·0.3H O·MeOH: C, 57.62; H, 6.19; N, 23.52. Found: C, 57.54; H, 6.19; N, 23.40.
23

5
.1.18.
b]pyridine-5-carboxamide (12b)
LiAlH (9.6 mg, 0.254 mmol) was added to a solution of 11d (100 mg, 0.254 mmol) in
THF (1 mL) at 0 °C under N gas atmosphere, and the mixture was stirred at the same
temperature. After adding H O and 15% NaOH aqueous solution, the mixture was stirred at
room temperature, and the precipitate was removed by filtration. The filtrate was concentrated
4-({1-[5-(Hydroxymethyl)pyridin-2-yl]piperidin-4-yl}amino)-1H-pyrrolo[2,3-
4
2
2
under reduced pressure. The residue was purified by column chromatography (CHCl
3
/MeOH =
1
0/1) to give the title compound (74 mg, 80%). H NMR (400 MHz, DMSO-d
1
6
) δ 1.41–152 (2H,
m), 2.02–2.11 (2H, m), 3.23–3.33 (2H, m), 4.01–4.08 (2H, m), 4.20–4.29 (1H, m), 4.35 (2H, d, J
5.6 Hz), 4.98 (1H, t, J = 5.6 Hz), 6.60–6.62 (1H, m), 6.86 (1H, d, J = 8.8 Hz), 6.99 (1H, brs),
=
7
.15–7.17 (1H, m), 7.49 (1H, dd, J = 2.4, 8.8 Hz), 7.77 (1H, brs), 8.05 (1H, d, J = 2.4 Hz), 8.37
+
1H, s), 9.73 (1H, d, J = 8.0 Hz), 11.50 (1H, brs). MS (ESI) m/z: 367 (M + H) . Anal. calcd for
(
19 22 6 2 2
C H N O ·0.6H O·0.1MeOH: C, 60.30; H, 6.25; N, 22.09. Found: C, 60.35; H, 5.96; N, 21.76.
5
.1.19. cis-1-Benzyl-N-(2,4-dimethoxybenzyl)-3-methylpiperidin-4-amine (14)
To a solution of 1-benzyl-3-methylpiperidin-4-one (13) (5.0 g, 24.6 mmol) and (2,4-
dimethoxybenzyl)amine (3.7 mL, 24.6 mmol) in DCE (75 mL), NaBH(OAc)
mmol) was added at room temperature. After stirring for 2 h at the same temperature, 1 M NaOH
aqueous solution (10 mL) was added to the mixture. The mixture was extracted with CHCl , and
the organic layer was separated, dried over MgSO , and concentrated under reduced pressure.
The residue was purified by column chromatography (CHCl /MeOH = 100/0 to 90/10) to give
the title compound (3.7 g, 42%). H NMR (400 MHz, DMSO-d ) δ 0.90 (3H, d, J = 6.8 Hz),
3
(10.4 g, 49.2
3
4
3
1
6
24

1
.41–1.58 (2H, m), 1.83–1.92 (1H, m), 1.97–2.08 (2H, m), 2.38–2.52 (2H, m), 2.58–2.67 (1H,
m), 3.24–3.46 (3H, m), 3.53–3.57 (2H, m), 3.73 (3H, s), 3.75 (3H, s), 6.45 (1H, dd, J = 2.4, 8.0
Hz), 6.51 (1H, d, J = 2.4 Hz), 7.16 (1H, d, J = 8.0 Hz), 7.19–7.24 (1H, m), 7.26–7.32 (4H, m).
+
MS (ESI) m/z: 355 (M + H) .
5
.1.20.
cis-N-(1-Benzyl-3-methylpiperidin-4-yl)-N-(2,4-dimethoxybenzyl)-2,2,2-
trifluoroacetamide (15)
3
To a solution of 14 (3.7 g, 10.4 mmol) in DCE (74 mL), were added Et N (5.8 mL, 41.8
mmol) and (CF
temperature for 2 h. After adding H
washed with H O, dried over MgSO
3
CO)
2
O (2.2 mL, 15.7 mmol) at 4 °C, and the mixture was stirred at the same
O, the mixture was extracted with CHCl . The extract was
, and concentrated under reduced pressure. The residue was
2
3
2
4
purified by column chromatography (n-hexane/EtOAc = 5/1 to 4/1) to give the title compound
1
4.1 g, 87%). H NMR (400 MHz, DMSO-d
(
6
) δ 1.04 (3H, d, J = 6.8 Hz), 1.09–1.19 (1H, m),
1
.32–1.51 (1H, m), 1.87–2.17 (3H, m), 2.21–2.31 (1H, m), 2.57–2.65 (1H, m), 2.73–2.85 (1H,
m), 3.42–3.50 (1H, m), 3.74 (3H, s), 3.81 (3H, s), 3.93–4.17 (1H, m), 4.31–4.70 (2H, m), 6.44–
6
4
.54 (1H, m), 6.86–6.92 (1H, m), 6.89 (1H, d, J = 8.4 Hz), 7.19–7.33 (5H, m). MS (ESI) m/z:
+
51 (M + H) .
5
.1.21. cis-1-Benzyl-3-methylpiperidin-4-amine (16)
TFA (16.8 mL) was added to 15 (4.1 g, 9.10 mmol) at room temperature, and the mixture
was stirred at 50 °C for 24 h. After concentration under reduced pressure, the residue was
dissolved with CHCl and neutralized with saturated NaHCO aqueous solution. The organic
layer was separated, dried over MgSO , and concentrated under reduced pressure. After
3
3
4
25

trituration and washing with IPE, the residue was dissolved with MeOH (81 mL). After adding 1
M NaOH aqueous solution (27 mL, 27.0 mmol), the mixture was stirred under reflux for 2 h.
After concentration under reduced pressure, the residue was extracted with CHCl
3
. The extract
was washed with saturated NaHCO
3
aqueous solution, dried over MgSO , and concentrated
4
1
under reduced pressure to give the title compound (1.4 g, 75%). H NMR (400 MHz, DMSO-d
6
)
δ 0.84 (3H, d, J = 7.0 Hz), 1.14–1.40 (1H, m), 1.41–1.58 (2H, m), 1.65–1.74 (1H, m), 2.10–2.31
(
(
3H, m), 2.40–2.53 (1H, m), 2.73–2.77 (1H, m), 3.20–3.45 (3H, m), 7.20–7.33 (5H, m). MS
+
ESI) m/z: 205 (M + H) .
5
.1.22.
carboxamide (17)
In the vessel of a microwave reactor, to a solution of 6 (240 mg, 1.23 mmol) in NMP (1.2
mL), 16 (501 mg, 2.45 mmol) and Et N (0.51 mL, 3.68 mmol) were added. The vessel was
sealed, and the mixture was reacted at 180 °C for 1.5 h under microwave irradiation. After
cis-4-[(1-Benzyl-3-methylpiperidin-4-yl)amino]-1H-pyrrolo[2,3-b]pyridine-5-
3
cooling to room temperature, EtOAc and H
2
O were added to the mixture, and the precipitate was
1
filtrated and washed with EtOAc to give the title compound (270 mg, 61%). H NMR (400 MHz,
DMSO-d
6
) δ 0.92 (3H, d, J = 6.8 Hz), 1.72–1.85 (2H, m), 2.05–2.13 (1H, m), 2.21–2.52 (4H, m),
3
7
1
.41–3.51 (2H, m), 4.14–4.21 (1H, m), 6.50–6.52 (1H, m), 6.99 (1H, br), 7.10–7.12 (1H, m),
.22–7.27 (1H, m), 7.31–7.33 (4H, m), 7.77 (1H, br), 8.37 (1H, s), 9.91 (1H, d, J = 8.4 Hz),
+
1.45 (1H, brs). MS (ESI) m/z: 364 (M + H) .
5
.1.23.
cis-4-[(3-Methylpiperidin-4-yl)amino]-1H-pyrrolo[2,3-b]pyridine-5-carboxamide
(
18)
26

To a solution of 17 (120 mg, 0.33 mmol) in EtOH (2.5 mL), 20% Pd(OH)
2
(50% wet) (150
mg) was added, and the mixture was stirred under H
2
gas atmosphere at 50 °C for 2 h. The
®
catalyst was filtrated through a pad of Celite and the filtrate was concentrated under reduced
1
pressure to give the title compound (75 mg, 83%). H NMR (400 MHz, DMSO-d
6
) δ 0.89 (3H, d,
J = 6.8 Hz), 1.60–1.74 (2H, m), 1.94–2.03 (1H, m), 2.61–2.70 (2H, m), 2.73–2.81 (2H, m), 4.15–
4
9
.24 (1H, m), 6.50–6.52 (1H, m), 6.97 (1H, br), 7.09–7.12 (1H, m), 7.76 (1H, br), 8.36 (1H, s),
+
.89 (1H, d, J = 8.4 Hz), 11.44 (1H, brs). MS (ESI) m/z: 274 (M + H) .
5
.1.24.
cis-4-{[1-(5-Cyanopyridin-2-yl)-3-methylpiperidin-4-yl]amino}-1H-pyrrolo[2,3-
b]pyridine-5-carboxamide (19)
To a solution of 18 (30 mg, 0.11 mmol) in DMI (0.75 mL), 2-chloro-5-cyanopyridine (30
mg, 0.22 mmol) and Et
60 °C for 2 h. After cooling to room temperature, CHCl
The organic layer was separated, washed with H O, dried over MgSO
reduced pressure. The residue was purified by column chromatography (CHCl
3
N (0.061 mL, 0.439 mmol) were added, and the mixture was stirred at
and H O were added to the mixture.
, and concentrated under
/MeOH = 100/0
to 90/10) to give the title compound (28 mg, 68%). H NMR (400 MHz, DMSO-d ) δ 0.90 (3H,
d, J = 6.8 Hz), 1.74–1.89 (2H, m), 2.13–2.21 (1H, m), 3.58–3.66 (1H, m), 3.71–3.79 (2H, m),
.83–3.91 (1H, m), 4.36–4.44 (1H, m), 6.60–6.62 (1H, m), 7.00 (1H, d, J = 9.2 Hz), 7.02 (1H,
1
3
2
2
4
3
1
6
3
br), 7.15–7.17 (1H, m), 7.80 (1H, br), 7.83 (1H, dd, J = 2.4, 9.2 Hz), 8.40 (1H, s), 8.47 (1H, d, J
+
2.4 Hz), 10.02 (1H, d, J = 8.4 Hz), 11.51 (1H, brs). MS (ESI) m/z: 376 (M + H) . HRMS (M +
=
+
H) calcd for C20
H
21
N
7
O: 375.1808. Found: 376.1887.
5
.1.25. tert-Butyl 4-[(trimethylsilyl)oxy]-3,6-dihydropyridine-1(2H)-carboxylate (21)
27

Under N
DMF (635 mL), TMSCl (193 mL, 1.53 mol) and Et
mixture was stirred at 90 °C for 21 h. After cooling to room temperature and dilution with n-
hexane (600 mL), the mixture was added to a mixture of n-hexane (1.9 L) and 5.5% NaHCO
aqueous solution (2.5 L). The organic layer was separated, washed with H O and brine, dried
, and concentrated under reduced pressure to give the title compound (343 g, 99%).
2
gas atmosphere, to a solution of N-Boc-4-piperidone (20) (254 g, 1.27 mol) in
3
N (444 mL, 3.19 mol) were added, and the
3
2
over MgSO
4
1
H NMR (400 MHz, CDCl
3
) δ 0.20 (9H, s), 1.47 (9H, s), 2.07–2.14 (2H, m), 3.49–3.55 (2H, m),
+
.84–3.90 (2H, m), 4.77–4.82 (1H, m). MS (ESI) m/z: 294 (M + Na) .
3
5
.1.26. tert-Butyl 3-fluoro-4-oxopiperidine-1-carboxylate (22)
®
To a solution of 21 (342 g, 1.26 mol) in CH
was added under ice cooling, and the mixture was stirred at the same condition for 50 min. After
concentration under reduced pressure, EtOAc and 5.5% NaHCO aqueous solution (1.9 L) were
added to the residue. The organic layer was separated, and the aqueous layer was extracted with
EtOAc. The combined organic layer was washed with brine, dried over Na SO , and
3
CN (6.8 L), Selectfluor (440 g, 1.12 mol)
3
2
4
concentrated under reduced pressure. The residue was triturated with a mixture of n-hexane (450
mL) and EtOAc (50 mL) for 1 h, and the precipitate was filtrated and washed with n-hexane to
1
give the title compound (274 g, 75%). H NMR (400 MHz, DMSO-d
6
) δ 1.38 (9H, s), 1.47–1.54
(
1H, m), 1.61–1.71 (1H, m), 3.67–3.82 (1H, m), 3.91-4.03 (1H, m), 4.08–4.25 (1H, m), 5.93 (1H,
-
brs), 5.97–5.99 (1H, m). MS (ESI) m/z: 216 (M - H) .
5
5
.1.27. tert-Butyl (3S,4R)-4-(benzylamino)-3-fluoropiperidine-1-carboxylate (23)
.1.28. tert-Butyl (3R,4S)-4-(benzylamino)-3-fluoropiperidine-1-carboxylate (24)
28

Under N
mol) were added to a solution of 22 (366 g, 1.69 mol) in DCE (5.5 L) under ice cooling, and the
mixture was stirred at room temperature for 2.5 h. After adding 20% K CO aqueous solution (3
L), the mixture was stirred for 10 min. The organic layer was separated, washed with H O and
brine, dried over MgSO , and concentrated under reduced pressure. The residue was purified by
column chromatography (n-hexane/EtOAc = 85/15 to 70/30, then CHCl /MeOH = 98/2 to 94/6)
to give a racemic mixture of 23 and 24 (312 g, 60%). H NMR (400 MHz, DMSO-d ) δ 1.32–
.49 (12H, m), 1.64–1.71 (1H, m), 2.01–2.10 (1H, m), 2.54–3.09 (3H, m), 3.84–3.98 (1H, m),
2 3
gas atmosphere, benzylamine (193 mL, 1.77 mol) and NaBH(OAc) (536 g, 2.53
2
3
2
4
3
1
6
1
4
.06–4.22 (1H, m), 4.68–4.88 (1H, m), 7.19–7.24 (1H, m), 7.28–7.36 (4H). MS (ESI) m/z: 309
+
M + H) . The racemic mixture (75 g) was purified by chiral column chromatography
(
(
2
CHIRALPAK AD-H, n-hexane/EtOH/Et NH = 90/10/0.1) to give 23 (32.5 g) and 24 (31.1 g),
respectively. The optical purity of 23 and 24 was determined by HPLC analysis with
CHIRALPAK AD-H (0.46 cm i.d. × 25 cm) eluted with n-hexane/EtOH/Et NH (90/10/0.1, flow
rate: 1.0 mL/min). 23: >98% ee, t = 8.14 min. 24: >98% ee, t = 6.46 min. The absolute
2
R
R
stereochemistry was determined by X-ray analysis of the derivative synthesized from 23 (data
not shown).
5
.1.29. tert-Butyl (3S,4R)-4-amino-3-fluoropiperidine-1-carboxylate (25)
Under N gas atmosphere, HCO NH (24.1 g, 382 mmol) and H O (29.5 mL) were added
2
2
4
2
to a solution of 23 (29.5 g, 95.7 mmol) in EtOH (295 mL), and the mixture was stirred at room
temperature for 5 min. After adding 10% Pd-C (50% wet) (10.2 g), the mixture was stirred under
reflux for 1 h. After cooling to room temperature, the Pd-catalyst was filtrated through a pad of
®
Celite . The filtrate was concentrated under reduced pressure, and the residue was purified by
29