4-Amino-pyrrolopyridine-5-carboxamide: A novel scaffold for JAK1-selective inhibitors

Article abstract of DOI:10.1248/cpb.c13-00807

Despite a high level of interest in selective Janus kinase 1 (JAK1) inhibitors and their potential for the treatment of inflammatory diseases such as rheumatoid arthritis (RA), only a few such inhibitors have been reported to date. In this study, a novel 4-amino-1H-pyrrolo[2,3-b]pyridine-5-carboxamide scaffold was designed through structural modification of the potent JAK1-selective inhibitor, C2-methyl imidazopyrrolopyridine. Among the series studied, the 4-(2-aminoethyl)amino-pyrrolopyridine derivative, 2j, exhibited a significant 24.7-fold JAK1/JAK2 selectivity along with reasonable inhibitory activity against JAK1 (IC₅₀=2.2 μM). The noticeable JAK1-selectivity of 2j was then tackled through a molecular docking study, which showed that the aminoethyl functionality of 2j is well positioned to discriminate the subtle but significant difference in the size of the ligand binding sites between JAK1 and JAK2.

Full text of DOI:10.1248/cpb.c13-00807

March 2014
Communication to the Editor
Chem. Pharm. Bull. 62(3) 217–220 (2014)
217
4
-Amino-pyrrolopyridine-5-
cific for JAK3 as initially thought, but it is rather a potent
8)
pan-JAK inhibitor, suggesting that the clinical efficacy of the
compound against RA is unlikely to be driven by the selec-
tive inhibition of JAK3. In addition, accumulated evidence
carboxamide: A Novel Scaffold for
JAK1-Selective Inhibitors
has revealed that in signal transduction through γ -containing
#
#
c
Heerim Shin, Mi Kyoung Kim, and
Youhoon Chong*
cytokine receptors, JAK1 plays a dominant role, while JAK3
exhibits only a secondary functional activity that merely en-
9
–13)
Department of Bioscience and Biotechnology, Bio/Molecular hances the effect of JAK1.
Accordingly, JAK1 inhibition
Informatics Center, Konkuk University; Hwayang-dong, Gwangjin- has become a relevant treatment option for RA. For successful
gu, Seoul 143–701, Korea.
Received October 14, 2013; accepted December 24, 2013
treatment of RA, JAK1 inhibitor has also been anticipated to
have a high JAK1-over-JAK2 selectivity because the therapy-
related anemia and thrombocytopenia observed with JAK2
inhibitors are particularly dangerous for patients receiving im-
Despite a high level of interest in selective Janus kinase
1
(JAK1) inhibitors and their potential for the treatment of
14)
munomodulating therapy.
inflammatory diseases such as rheumatoid arthritis (RA),
only a few such inhibitors have been reported to date. In
Despite a high level of interest in selective JAK inhibi-
tors and their potential for immune-modulating therapies,
this study,
-carboxamide scaffold was designed through structural
modification of the potent JAK1-selective inhibitor, C2-
methyl imidazopyrrolopyridine. Among the series studied, the 1) shows potent and selective inhibitory activity against
a
novel 4-amino-1H-pyrrolo[2,3-b]pyridine-
15,16)
only a few JAK1 inhibitors have been reported to date.
Among them, C2-methyl imidazopyrrolopyridine (1, Fig.
5
16)
4
-(2-aminoethyl)amino-pyrrolopyridine derivative, 2j, exhib- JAK1, i.e., K of 10nM, and JAK1 selectivity of 20, which
i
ited a significant 24.7-fold JAK1/JAK2 selectivity along with guided us to design a novel JAK1-selective inhibitor based
reasonable inhibitory activity against JAK1 (IC ꢀ2.2µM).
5
0
on a pyrrolopyridine scaffold. 4-Amino-1H-pyrrolo[2,3-b]-
pyridine-5-carboxamide (2, Fig. 1), thus designed, has amino
and carboxamide functionalities at the 4- and 5-positions of
the pyrropyridine scaffold, respectively, which are anticipated
to mimic the imidazole moiety of 1 through the formation of
an intramolecular hydrogen bond. A short and modular syn-
thesis of the structural variants for an extensive study on the
structure–activity relationship is an additional advantage of
exploring 4-amino-pyrrolopyridine-5-carboxamides as a scaf-
The noticeable JAK1-selectivity of 2j was then tackled
through a molecular docking study, which showed that the
aminoethyl functionality of 2j is well positioned to discrimi-
nate the subtle but significant difference in the size of the
ligand binding sites between JAK1 and JAK2.
Key words Janus kinase 1 (JAK1); pyrrolopyridine; rheuma-
toid arthritis
The Janus kinase/signal transducers and activators of tran- fold for the design of a novel JAK1-selective inhibitor.
scription (JAK/STAT) pathway, a major signaling cascade in
The synthesis of 4-amino-pyrrolopyridine-5-carboxamide
response to inflammatory and proliferative signals, consists of derivatives 2a through k was accomplished starting from
the Janus protein tyrosine kinase family (JAK3, JAK2, JAK1, the commercially available 7-azaindole (Chart 1). In a three-
and TYK2) and STAT family of transcription factors. Upon step sequence, 7-azaindole was selectively chlorinated at the
17)
ligand-receptor binding, the JAKs get activated, resulting 4-position to give 3 in 84% yield, which was then treated
in the phosphorylation, dimerization, and nuclear transloca- with triisopropylsilyl chloride (TIPSCl) to provide N1-protect-
18)
tion of the downstream STAT proteins, which regulate the ed 4-chloropyrrolopyridine 4 in 76% yield. The ortho-lithi-
transcription of STAT-dependent genes. Depending on the ation of 4, followed by trapping with methyl chloroformate,
19)
specific cytokines coupled with JAKs, ligand-receptor binding furnished the key intermediate 5 in 65% yield. Nucleophilic
2
0)
culminates in an intracellular response such as an immune aromatic substitution of 5 with various amines, followed
1)
2)
3,4)
function, inflammation, or hematopoiesis.
For instance,
the gamma common (γ ) family of cytokines, i.e., interleu-
c
kin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21, is exclusively
5
)
associated with JAK1 and JAK3, and a loss-of-function
mutation in the γ chain or JAK3 results in severe combined
c
immunodeficiency (SCID). As a result, abrogation of signaling
by γ -cytokines through the inhibition of γ -linked JAK3 has
c
c
long been regarded as an attractive target for the treatment of
2
,6)
immunologic disorders such as rheumatoid arthritis (RA).
Pfizer’s Xeljanz (Tofacitinib, CP-690550), the first-in-class
7
)
JAK inhibitor approved for the treatment of RA, was initially
6
)
reported to be a selective JAK3 inhibitor, spurring many
pharmaceutical companies to launch JAK3 medicinal chemis-
try programs.
However, it has become clear that tofacitinib is not as spe-
The authors declare no conflict of interest.
Fig. 1. Structure of C-2 Methyl Imidazopyrrolopyridine (1) and 4-Amino-
1H-pyrrolo[2,3-b]pyridine-5-carboxamide (2)
#
These authors contributed equally to this work.
*
To whom correspondence should be addressed. e-mail: chongy@konkuk.ac.kr
© 2014 The Pharmaceutical Society of Japan

2
18
Vol. 62, No. 3
Reagent and conditions: (a) mCPBA, DME/heptane; (b) POCl , 85°C; (c) NaOH, H O, pH 10; (d) NaH, TlPSCl, THF, 80°C; (e) sec-BuLi, THF, 30min, −78°C; (f) methyl
3
2
chloroformate, THF; (g) RNH , DIPEA, i-PrOH; (h) NaOH, EtOH; (i) EDC, NH Cl, HOBt, DIPEA, CH CN/DMF.
2
4
3
Chart 1. Syntheses of the Title Compounds, 2a–k
21)
by hydrolysis and amidation, gave the desired products 2a three compounds with promising JAK isozyme selectivity pro-
through k in combined yields of 23 to 61%. files were selected (2i–k) and their IC50 values were evaluated
The in vitro inhibitory activity of 4-amino-pyrrolopyridine- (Table 2).
-carboxamide derivatives 2a through k on the JAK isozymes As anticipated, the 4-hydroxyalkylamino- (2i) and the
5
was determined using Z′-LYTE™ Kinase Assay Kit-Tyr 6 4-aminoalkylamino- (2j,k) pyrrolopyridine derivatives exhib-
Peptide (JAK1 through JAK3) and Tyr 3 Peptide (Tyk2) (In- ited significant JAK1/JAK2 selectivity of 20.5–24.7, along with
vitrogen) according to the manufacturer’s instruction. The reasonable inhibitory activity against JAK1 (IC =2.2–2.6µM).
5
0
pan-JAK inhibitor, “pyridine 6,” was used as a positive con- Also noteworthy is the JAK1/Tyk2 selectivity of 9.8–11.3
2
2)
trol, and the percentage of inhibition of each JAK isozyme observed with this class of compound. In addition, the high
obtained using 10µM of the title compounds is summarized in membrane permeability of the most promising compound 2j
Table 1.
measured in a parallel artificial membrane permeability assay
2
3,24)
Depending on the amino functionality attached at the 4-po- (PAMPA, log P =−3.21, Table 2)
sition of the pyrrolopyridine scaffold, the title compounds opment of the 4-aminoalkylamino-pyrrolopyridine derivatives
warrants further devel-
e
can be divided into three groups: arylalkyl (2a–d), alkyl as potential JAK1-selective inhibitors.
(2e–h), and hydroxyalkyl/aminoalkyl amine (2i–k) deriva-
The noticeable JAK1 selectivity of this particular series of
tives. Among these series, the alkylamine derivatives, 2e–k, pyrrolopyridine derivatives was then tackled through a mo-
inhibited JAK1 kinase activity more efficiently than did the lecular docking study. The compound 2j was therefore docked
16)
arylalkylamine-substituted derivatives, 2a–d. Interestingly, into the ATP-binding site of JAK1 (PDB ID=4EHZ) and
16)
the JAK1 inhibitory activity was significantly decreased with JAK2 (PDB ID=4F09) by using the flexible ligand docking
increased steric bulk to give compounds with unsubstituted software Glide incorporated into the Schrödinger molecular
and short amino functionality, 2a and e, which were the most modeling software suite. Although the ligand-binding sites
active in each series. In particular, pyrrolopyridine derivatives of JAK1 and JAK2 share almost the same features, the best
with short alkylamine substituents such as 2e and f showed docking poses of 2j for the two enzymes showed subtle, but
the most potent inhibitory activity against JAK1 with 65.4% significant, differences (Fig. 2).
and 58.2% inhibition at 10µM, respectively (Table 1).
A comparison of the ligand-binding sites of JAK1 and
Based on the inhibitory activity at 10µM concentration, JAK2 revealed that the distance between the hinge motif,

March 2014
219
i.e., Leu959/Glu957 for JAK1 and Leu932/Glu930 for JAK2, the hydroxyalkyl and aminoalkyl substituents endow a 4-amino-
and the bottom of the ligand-binding site, i.e., Arg1007/ pyrrolopyridine-5-carboxamide scaffold with JAK1 selectiv-
Asn1008 for JAK1 and Arg980/Asn981 for JAK2, is about ity over JAK2, an observation supported through a molecular
0.3–0.6Å longer for JAK2 (the block arrow shown in Fig. 2). docking study. Based on the results, an extensive study on the
The docking pose of 2j to JAK1 showed that the aminoethyl structure–activity relationship of pyrrolopyridine derivatives
functionality of 2j is well positioned for discriminating the as JAK1-selective inhibitors is ongoing, and the results will be
different sizes of the ligand-binding sites of JAK1 and JAK2. reported elsewhere.
Specifically, compound 2j fits snuggly in the ligand-binding
site of JAK1 to form two hydrogen bonds with backbone and
Acknowledgments This research was supported by a
side-chain carbonyl groups of Arg1007 and Asn1008, respec- Grant from the Korean Health Technology R&D project, Min-
tively (Fig. 2). In contrast, owing to the larger binding site of istry of Health & Welfare, Republic of Korea (A111698), by
JAK2, compound 2j binds at a slightly higher region of the the Priority Research Centers Program through the National
ligand-binding site (double-headed arrow shown in Fig. 2), Research Foundation of Korea (NRF) funded by the Ministry
resulting in the formation of only one hydrogen bond with the of Education, Science and Technology (2009-0093824), and by
backbone carbonyl group of Arg980 (Fig. 2), and lowering the a Grant from Next Generation Bio-Green 21 PJ007982 (Korea
docking score (G-score=−8.22) compared to that of JAK1 (G- Rural Development Administration).
score=−8.45).
Overall, the title compounds showed significantly decreased
1
6)
activity compared with the C-2 methyl imidazopyrrolopyridine
1, Fig. 1). Presumably, this loss of activity might have resulted
from the competition for hinge motif binding between the
-amino-5-carboxamide functionalities and the pyrrolopyridine
(
4
scaffold. Nevertheless, this proof-of-concept study revealed that
Table 1. % Inhibition of the JAK Isozymes by 10µM of the Title Com-
pounds
a)
%
Inhibition
Compound
JAK1
JAK2
JAK3
Tyk2
2
2
2
2
2
2
2
2
2
2
2
a
b
c
d
e
f
50.3± 3.1
41.4± 2.6
37.6± 2.4
27.1± 1.9
65.4± 3.7
58.2± 2.1
45.2± 1.8
40.7± 2.2
49.4± 2.4
52.6± 3.2
49.8± 3.6
4.3
15.7± 1.8
15.9± 1.2
21.1± 0.8
21.2± 0.8
28.8± 2.1
30.7± 2.3
24.4± 1.8
29.4± 2.3
12.6± 1.1
10.4± 0.8
15.2± 1.6
1.3
43.3± 2.1
46.8± 3.4
41.9± 2.0
48.3± 1.5
54.5± 1.9
42.6± 2.0
38.5± 1.2
45.5± 2.1
30.5± 1.3
32.5± 1.7
42.6± 1.4
12.6
32.8± 1.6
13.5± 0.8
13.9± 1.2
35.7± 1.7
18.4± 1.3
21.5± 1.5
20.1± 1.0
31.6± 1.9
11.5± 0.9
15.7± 1.3
10.8± 1.0
5.1
g
h
i
j
Fig. 2. Superposition of the Docking Modes of 2j to JAK1 (Atoms in
Atom-Type Color, Ribbons in Pink) and JAK2 (Atoms and Ribbons in
Green)
k
b)
Pyridine 6
nM)
c)
c)
c)
c)
(
(3.3)
(2.5)
(10.9)
(3.6)
Hydrogen bonds are shown as dotted lines, and the size of the ligand binding site
is denoted as a block arrow. The double-headed arrow indicates an upward shift of
a) Each experiment was repeated at least three times. b) IC values (nM) of the
pan-JAK inhibitor, ‘pyridine 6.’ c) IC50 values reported in ref. 22.
50
22)
2j upon binding to JAK2. (Color images were converted into gray scale.)
Table 2. JAK Isozyme Selectivity and PAMPA Permeability of Three Selected Compounds (2i–k)
Selectivity
a)
IC50 (µM)
logP (PAMPA)
e
Compound
b)
(cm/s)
JAK1
JAK2
JAK3
Tyk2
JAK1/JAK2
JAK1/Tyk2
c)
2
2
2
i
2.4
2.2
2.6
49.2
54.4
59.4
1.9
1.7
2.0
27.1
24.1
25.6
20.5
24.7
22.8
11.3
11.0
9.8
ND
j
−3.21± 0.02
c)
k
ND
a) Selectivity=IC against JAK2 (or Tyk2)/IC50 against JAK1. b) P (effective permeability) was determined at 25µM concentration of the test compound after incubation
5
0
e
for 1h. c) Not determined.

2
20
Vol. 62, No. 3
J. M., Grosveld G., Ihle J. N., Cell, 93, 385–395 (1998).
2) Smolen J. S., Beaulieu A., Rubbert-Roth A., Ramos-Remus C.,
Rovensky J., Alecock E., Woodworth T., Alten R., OPTION Investi-
gators, Lancet, 371, 987–997 (2008).
3) Haan C., Rolvering C., Raulf F., Kapp M., Druckes P., Thoma G.,
Behrmann I., Zerwes H. G., Chem. Biol., 18, 314–323 (2011).
4) Verstovsek S., Clin. Cancer Res., 16, 1988–1996 (2010).
5) Kulagowski J. J., Blair W., Bull R. J., Chang C., Deshmukh G.,
Dyke H. J., Eigenbrot C., Ghilardi N., Gibbons P., Harrison T. K.,
Hewitt P. R., Liimatta M., Hurley C. A., Johnson A., Johnson T.,
Kenny J. R., Kohli P. B., Maxey R. J., Mendonca R., Mortara K.,
Murray J., Narukulla R., Shia S., Steffek M., Ubhayakar S., Ultsch
M., van Abbema A., Ward S. I., Waszkowycz B., Zak M., J. Med.
Chem., 55, 5901–5921 (2012).
6) Zak M., Mendonca R., Balazs M., Barrett K., Bergeron P., Blair W.
S., Chang C., Deshmukh G., DeVoss J., Dragovich P. S., Eigenbrot
C., Ghilardi N., Gibbons P., Gradl S., Hamman C., Hanan E. J.,
Harstad E., Hewitt P. R., Hurley C. A., Jin T., Johnson A., Johnson
T., Kenny J. R., Koehler M. F. T., Bir Kohli P., Kulagowski J. J.,
Labadie S., Liao Z., Liimatta M., Lin Z., Lupardus P. J., Maxey R.
J., Murray J. M., Pulk R., Rodriguez M., Savage S., Shia S., Steffek
M., Ubhayakar S., Ultsch M., van Abbema A., Ward S. I., Xiao L.,
Xiao Y., J. Med. Chem., 55, 6176–6193 (2012).
References
1
1)
Pesu M., Laurence A., Kishore N., Zwillich S. H., Chan G., O’Shea
J. J., Immunol. Rev., 223, 132–142 (2008).
O’Shea J. J., Pesu M., Borie D. C., Changelian P. S., Nat. Rev. Drug
Discov., 3, 555–564 (2004).
2)
1
3
)
Clark S. C., Kamen R., Science, 236, 1229–1237 (1987).
1
1
4) Metcalf D., Nature (London), 339, 27–30 (1989).
5
)
)
Rochman Y., Spolski R., Leonard W. J., Nat. Rev. Immunol., 9,
4
80–490 (2009).
6
Changelian P. S., Flanagan M. E., Ball D. J., Kent C. R., Magnuson
K. S., Martin W. H., Rizzuti B. J., Sawyer P. S., Perry B. D., Bris-
sette W. H., McCurdy S. P., Kudlacz E. M., Conklyn M. J., Elliott
E. A., Koslov E. R., Fisher M. B., Strelevitz T. J., Yoon K., Whipple
D. A., Sun J., Munchhof M. J., Doty J. L., Casavant J. M., Blumen-
kopf T. A., Hines M., Brown M. F., Lillie B. M., Subramanyam
C., Shang-Poa C., Milici A. J., Beckius G. E., Moyer J. D., Su C.,
Woodworth T. G., Gaweco A. S., Beals C. R., Littman B. H., Fisher
D. A., Smith J. F., Zagouras P., Magna H. A., Saltarelli M. J., John-
son K. S., Nelms L. F., Des Etages S. G., Hayes L. S., Kawabata T.
T., Finco-Kent D., Baker D. L., Larson M., Si M. S., Paniagua R.,
Higgins J., Holm B., Reitz B., Zhou Y. J., Morris R. E., O’Shea J. J.,
Borie D. C., Science, 302, 875–878 (2003).
van der Heijde D., Tanaka Y., Fleischmann R., Keystone E., Kremer
J., Zerbini C., Cardiel M. H., Cohen S., Nash P., Song Y. W., Teg-
zová D., Wyman B. T., Gruben D., Benda B., Wallenstein G., Krish-
naswami S., Zwillich S. H., Bradley J. D., Connell C. A., ORAL
Scan Investigators, Arthritis Rheum., 65, 559–570 (2013).
Meyer D. M., Jesson M. I., Li X., Elrick M. M., Funckes-Shippy C.
L., Warner J. D., Gross C. J., Dowty M. E., Ramaiah S. K., Hirsch
J. L., Sabbye M. J., Barks J. L., Kishore N., Morris D. L., J. In-
flamm., 7, 41 (2010).
1
7
)
1
7) Yo M., Shoji H., Pyuji O., Joel R. W., Yingfu L., Feryan A., Yusuke
N., WO 002772A1 (2011).
1
8) Heinrich T., Seenisamy J., Emmanuvel L., Kulkarni S. S., Bomke J.,
Rohdich F., Greiner H., Esdar C., Krier M., Grädler U., Musil D., J.
Med. Chem., 56, 1160–1170 (2013).
8)
1
9) L’Heureux A., Thibault C., Ruel R., Tetrahedron Lett., 45, 2317–
319 (2004).
0) Bi Y., Yu G., Rotella D. P., Macor J. E., WO Patent 20489A2 (2002).
1) Ishichi Y., Yamada M., Kamei T., Fujimori I., Nakada Y., Yukawa
2
2
2
9)
Rodig S. J., Meraz M. A., White J. M., Lampe P. A., Riley J. K.,
Arthur C. D., King K. L., Sheehan K. C., Yin L., Pennica D., John-
son E. M. Jr., Schreiber R. D., Cell, 93, 373–383 (1998).
T., Sakauchi N., Ohba Y., Tsukamoto T., WO 046882A1 (2012).
2) Thompson J. E., Cubbon R. M., Cummings R. T., Wicker L. S.,
Frankshun R., Cunningham B. R., Cameron P. M., Meinke P. T.,
Liverton N., Weng Y., DeMartino J. A., Bioorg. Med. Chem. Lett.,
2
10) Shimoda K., Kato K., Aoki K., Matsuda T., Miyamoto A., Shiba-
mori M., Yamashita M., Numata A., Takase K., Kobayashi S.,
Shibata S., Asano Y., Gondo H., Sekiguchi K., Nakayama K.,
Nakayama T., Okamura T., Okamura S., Niho Y., Nakayama K., Im-
munity, 13, 561–571 (2000).
1
2, 1219–1223 (2002).
3) Kansy M., Senner F., Gubernator K., J. Med. Chem., 41, 1007–1010
1998).
2
2
(
4) Cho S. Y., Kim M. K., Park K.-S., Choo H., Chong Y., Bioorg. Med.
Chem., 21, 1671–1679 (2013).
11) Parganas E., Wang D., Stravopodis D., Topham D. J., Marine J. C.,
Teglund S., Vanin E. F., Bodner S., Colamonici O. R., van Deursen