Benzimidazole Derivatives as Potent JAK1-Selective Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.5b01263

The Janus kinase (JAK) family comprises four members (JAK1, JAK2, JAK3, and Tyk2) that play a key role in mediating cytokine receptor signaling. JAK inhibition thus modulates cytokine-mediated effects. In particular, selective inhibition of JAK1 or JAK3 may provide an efficient therapeutic agent for the treatment of inflammatory diseases, with minimized side effects. In this study, as part of our continued efforts to develop a selective JAK1 inhibitor, a series of 1,2-disubstituted benzimidazole-5-carboxamide derivatives was prepared and their inhibitory activities against all four JAK isozymes were evaluated. A clear structure-activity relationship was observed with respect to JAK1 selectivity; this highlighted the importance of hydrogen bond donors at both N¹ and R₂ positions located within a specific distance from the benzimidazole core. One of the synthesized compounds, 1-(2-aminoethyl)-2-(piperidin-4-yl)-1H-benzo[d]imidazole-5-carboxamide (5c), showed remarkable JAK1 selectivity (63-fold vs JAK2, 25-fold vs JAK3, and 74-fold vs Tyk2). Molecular docking revealed that the 2-aminoethyl and piperidin-4-yl substituents of 5c function as probes to differentiate the ATP-binding site of JAK1 from that of JAK2, resulting in preferential JAK1 binding. A kinase panel assay confirmed the JAK1 selectivity of 5c, which showed no appreciable inhibitory activity against 26 other protein kinases at 10 μM.

Full text of DOI:10.1021/acs.jmedchem.5b01263

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Brief Article
Benzimidazole Derivatives as Potent JAK1-Selective Inhibitors
Mi Kyoung Kim, Heerim Shin, Kwang-su Park, Hyungmi Kim, Jiseon
Park, Kangjeon Kim, Joonwoo Nam, Hyunah Choo, and Youhoon Chong
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01263 • Publication Date (Web): 09 Sep 2015
Downloaded from http://pubs.acs.org on September 11, 2015
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Benzimidazole Derivatives as Potent JAK1-Selective Inhibi-
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Mi Kyoung Kim,† Heerim Shin,† Kwang-su Park,† Hyungmi Kim,† Jiseon Park,∥ Kangjeon Kim,∥
Joonwoo Nam,∥ Hyunah Choo,*,‡,§ Youhoon Chong*,†
†Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Hwayang-
dong, Gwangjin-gu, Seoul 05029, Korea
∥R&D center, Jeil Pharmaceutical Co., Ltd. 7 Cheongganggachangro, Baegam-myun, Cheoin-gu, Yongin-city,
Kyunggi-Do 17172, Korea
‡Center for Neuro-Medicine, Korea Institute of Science and Technology, 5, Hwarang-ro 14-gil, Seoungbuk-gu, Seoul
02792, Korea
§Department of Biological Chemistry, University of Science and Technology, 217 Gajeong-ro, Youseong-gu, Daejeon
34113, Korea.
KEYWORDS. Benzimidazole, Aminoalkyl, Piperidin-4-yl, Janus kinase 1, Selectivity.
ABSTRACT: The Janus kinase (JAK) family comprises four members (JAK1, JAK2, JAK3, and Tyk2) that play a key role in
mediating cytokine receptor signaling. JAK inhibition thus modulates cytokine-mediated effects. In particular, selective
inhibition of JAK1 or JAK3 may provide an efficient therapeutic agent for the treatment of inflammatory diseases, with
minimized side effects. In this study, as part of our continued efforts to develop a selective JAK1 inhibitor, a series of 1,2-
disubstituted benzimidazole-5-carboxamide derivatives was prepared and their inhibitory activities against all four JAK
isozymes were evaluated. A clear structure-activity relationship was observed with respect to JAK1 selectivity; this high-
lighted the importance of hydrogen bond donors at both N1 and R2 positions located within a specific distance from the
benzimidazole core. One of the synthesized compounds, 1-(2-aminoethyl)-2-(piperidin-4-yl)-1H-benzo[d]imidazole-5-
carboxamide (5c), showed remarkable JAK1 selectivity (63-fold vs JAK2, 25-fold vs JAK3, and 74-fold vs Tyk2). Molecular
docking revealed that the 2-aminoethyl and piperidin-4-yl substituents of 5c function as probes to differentiate the ATP-
binding site of JAK1 from that of JAK2, resulting in preferential JAK1 binding. A kinase panel assay confirmed the JAK1
selectivity of 5c, which showed no appreciable inhibitory activity against 26 other protein kinases at 10 μM.
tors (IL-3, IL-5, granulocyte macrophage colony-
stimulating factor, erythropoietin, and thrombopoietin)
and functions as a homodimer to play an essential role in
red blood cell formation4. As a result, JAK2-knockout
mice develop anemia5, while a unique gain-of-function
JAK2 point mutation (V617F) results in myeloproliferative
disorders (MPD) in humans6. Taken together, studies of
cytokine-mediated signaling have revealed pathologic
roles of specific cytokines in inflammatory diseases and
MPD, which can be abrogated by inhibition of the associ-
ated JAK isozymes7. Consequently, enormous efforts have
been devoted to the development of JAK inhibitors, which
culminated in the recent approval of two JAK inhibitors,
ruxolitinib8 and tofacitinib9, for the treatment of MPD
and rheumatoid arthritis (RA), respectively (Chart 1); rux-
olitinib is a JAK1/JAK2-selective inhibitor with reduced
potency against JAK38, while tofacitinib is a pan-JAK in-
hibitor that inhibits JAK1, JAK2, and JAK3 to an approxi-
mately equal extent9. However, it is still unclear whether
INTRODUCTION
The cytoplasmic domains of cytokine receptors that me-
diate the intracellular signaling of type I/II cytokines are
associated with specific Janus protein tyrosine kinases
(JAK1, JAK2, JAK3, and Tyk2). For example, interleukins
(IL) responsible for adaptive immune functions (IL-2, IL-
4, IL-7, IL-9, IL-15, and IL-21) specifically bind to cytokine
receptors that contain a common gamma chain (γc,
CD132) and are associated with intracellular JAK3 and
JAK1.1 JAK3 is characterized by its exclusive association
with γc, and loss-of-function mutation of JAK3 results in
complete abrogation of signaling by γc-cytokines; this
causes severe combined immunodeficiency2. In contrast,
JAK1 is widely expressed and, by pairing with JAK2 and/or
Tyk2, is involved in a broad range of cytokine signaling to
affect several proinflammatory cytokines associated with
the innate immune response (γc, gp130, type I/II interfer-
on, IL-6, IL-10 subfamily)3. JAK2 is exclusively associated
with the receptors for various cytokines and growth fac-
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Page 2 of 8
Chart 1. Structures of the JAK inhibitors
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2
3
4
5
6
7
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9
CF3
NH
O
O
S
N
CN
S
O
O
NC
O
N
NC
N
N
N
H
O
N
N
N
N
N
N
N
N
N
O
N
N
N
N
NH
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N
N
N
N
N
H
N
N
H
H
H
Filgotinib
Decernotinib
Tofacitinib
Ruxolitinib
Baricitinib
CN
O
CN
N
N
F
N N
F
OH
HO
O
HN
N
N
N
N
N
F3C
N
H2N
O
N
H
N
N
H
N
N
N
NH
H
H
INCB-039110
Peficitinib
Imidazopyrrolo-
pyridine 31
Pyridone 6
Table 1. JAK1-selectivity of known inhibitors in enzyme assay
IC50 (nM)
Selectivitya
Compds
JAK1
6
JAK2
JAK3
>400
2.5b
810
-
Tyk2
53
11
JAK1/JAK2
JAK1/JAK3
JAK1/Tyk2
Baricitinib
Decernotinib
Filgotinib
6
13
25
-
1
1
>67
0.2
81
9
1
11
10
-
116
-
3
12
-
INCB-039110c
>20
>200
0.2
-
Peficitinib
3.9
-
1.9e
5.0
-
0.71
-
280e
4.8
-
12e
1
1
ABT-494
74d
-
Imidazopyrrolopyridine-31
-
36
147
6
aSelectivity in enzyme assay = (IC50 against JAK2, JAK3, or Tyk2) / (IC50 against JAK1). Ki value. Under development for on-
b
c
cology indications. dSelectivity in cellular assays. eInhibition constants (Ki’s)
inhibition of multiple JAK isozymes is a prerequisite for
the clinical efficacy of JAK inhibitors, or if an isozyme-
selective inhibitor would show improved efficacy and
safety profiles. In this context, it is worth noting that,
even though the clinical efficacy of tofacitinib in patients
with RA has been attributed to the suppression of JAK3
activity8,10, a dominant role of JAK1 over JAK3 in RA has
been strongly proposed11. On the other hand, a recent
report by Pfizer stated that inhibition of either JAK1 or
JAK3 was enough to abrogate cytokine signaling12. Ac-
cordingly, there is a compelling need for the development
of selective JAK1 or JAK3 inhibitors. Moreover, from a
therapeutic point of view, inhibition of JAK2 should be
avoided because the induction of anemia, a dose-limiting
side effect of tofacitinib, has been ascribed to the con-
comitant inhibition of JAK213.
RA (Table 1). Among those, filgotinib16, INCB-03911017,
ABT-49419 and imidazopyrrolopyridine-31 are reported
20
to show JAK1-selectivity while decernotinib15 is the best
characterized selective JAK3 inhibitor. In particular, ABT-
494, a highly selective JAK1 inhibitor (74-fold vs. JAK2),
turned out to be effective in RA patients without unfavor-
able effects on erythropoietin signaling and peripheral NK
cell counts.19 Thus, it is anticipated that the clinical stud-
ies with JAK1 or JAK3 selective inhibitors will confirm a
relationship between the isoform selectivity and side-
effect profiles. In this context, further investigation to
identify novel scaffolds of JAK1-selective inhibitors is re-
quired.
Recently, our research group reported that 3-alkynolyl-
indazole-7-carboxamide derivatives with a linear tether,
3-alkynol (dashed circle, Chart 2), which serves as an iso-
zyme-specific probe group, showed selective inhibition of
JAK1 over other JAK isozymes21. Based on this observation,
the present study developed compounds with a novel
Currently, several JAK inhibitors with different isoform
specificity profiles14-20 are in the clinical or preclinical
stage for the treatment of autoimmune diseases including
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Journal of Medicinal Chemistry
11.3 μM, respectively) and JAK3 (IC50 = 1.5 and 1.8 μM, re-
benzimidazole-5-carboxamide scaffold and an aminoalkyl
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3
4
5
6
7
8
functionality (dashed circle, Chart 2). In addition, to op-
timize selectivity and potency for the target enzyme, the
benzimidazole core structure was further substituted with
an alkyl substituent at the 2-position. Here, we describe
the synthesis and biological evaluation of a series of these
1-aminoalkyl-2-alkyl-1H-benzo[d]imidazole-5-
spectively). Surprisingly, however, shortening the 3-
aminopropyl chain of 4a by one carbon potentiated the
JAK1-inhibitory activity of the resulting benzimidazole
derivative 5a by more than 60-fold (IC50 = 0.1 μM) without
altering inhibition of other JAK isozymes. As a result, the
2-aminoethyl-substituted benzimidazole derivative 5a
showed high JAK1 selectivity over other JAK isozymes (29-
fold vs JAK2, 15-fold vs JAK3, and 31-fold vs Tyk2). Based
on this observation, the N1 substituent was fixed as a 2-
aminoethyl group and a series of benzimidazole deriva-
tives with various R2 substituents was prepared (5b–5o).
Within the series, the R2-alkyl derivatives (5a–5d) showed
a clear structure-activity relationship whereby an amino
group located within a specific distance from the benzim-
idazole core played a key role in maintaining potent JAK1-
inhibitory activity. Thus, compounds with 2-aminoethyl
(5a) or piperidin-4-yl (5c) functionalities at R2 showed
potent inhibitory activity against JAK1, with IC50 values of
0.1 and 0.05 μM, respectively, while derivatives with a
longer R2-alkylamino group [5b, R2 = (CH2)3NH2] or with
an R2-hydrocarbon substituent [5d, R2 = cyclohexyl] were
significantly less potent. On the other hand, the R2-alkyl
substituents did not affect the inhibitory activities of the
corresponding benzimidazole derivatives (5a–5d) against
other JAK isozymes (JAK2, JAK3, and Tyk2). Taken to-
gether, the R2-dependence of the inhibitory activities of
these benzimidazole derivatives was specific to JAK1, and
compound 5c, with a piperidin-4-yl group at R2, showed
remarkable JAK1 selectivity (63-fold vs JAK2, 25-fold vs
JAK3, and 74-fold vs Tyk2). On the other hand, among the
benzimidazole derivatives with an aromatic substituent at
R2 (5e–5o), only 5k (R2 = 4-OH-Ph) with a hydroxyl group
at the 4-position of the aromatic group showed JAK1-
selective inhibitory activity (11-fold vs JAK2, 20-fold vs
JAK3, and 26-fold vs Tyk2). Based on these observations,
common structural features within the set of JAK1-
selective inhibitors (5a, 5c, and 5k) could be identified; an
alkyl chain bearing a hydrogen bond donor (-NH or -OH)
is substituted at the 1-position of the benzimidazole core.
carboxamide derivatives, resulting in the identification of
a potent JAK1 inhibitor with remarkable JAK1 selectivity.
9
Chart 2. Design of the title compound
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RESULTS AND DISCUSSION
The
1-aminoalkyl-2-alkyl-1H-benzo[d]imidazole-5-
carboxamides (4 and 5), were prepared from commercial-
ly available 4-fluoro-3-nitrobenzoic acid (1) in 5 steps
(Scheme
dimethylaminopropyl)carbodiimide] coupling of 1 with 1-
hydroxy-1H-benzotriazole (HOBT)-ammonium salt
1).
First,
EDC
[1-ethyl-3-(3-
(NH3·HOBT) in a 4:1 mixture of CH3CN and N,N-
dimethylformamide (DMF) yielded the corresponding
benzamide, which underwent nucleophilic aromatic sub-
stitution by 1,3-diaminopropane or ethylenediamine fol-
lowed by N-Boc protection, to give the corresponding 4-
alkylamino-3-nitrobenzamide (2a or 2b), with 75% or 79%
yield. The nitro functionality of 2a or 2b was then reduced
to give the key intermediate, 4-alkylamino-3-
aminobenzamide (3a or 3b), with 67% or 91% yield. These
diaminobenzamides were then cyclized to the corre-
sponding benzimidazole-5-carboxamides by treatment
with the appropriate aldehydes in the presence of sodium
hydrogen sulfite (NaHSO3)22 with 10–67% yields. Finally,
deprotection of the Boc groups provided the desired
compounds (4 and 5).
In a separate experiment, the inhibitory activities of 5c
against the JAK isozymes were measured in the presence
of 1 mM ATP (Table 3), instead of at the Km12,24, in order to
mimic the physiological level of ATP. As expected12,24, the
observed IC50 values for 5c were 114–205-fold higher in the
presence of 1 mM ATP than those obtained at the Km val-
ues (Table 2); these data shifts were smallest for JAK1,
which has the highest Km for ATP. As a result, the JAK1
selectivity of 5c became more prominent under these
physiologically relevant assay conditions.
Z′-LYTE™ Kinase Assay Kits (Invitrogen) were used to
determine the in vitro inhibitory activity of the benzimid-
azole derivatives against the JAK isozymes; the Tyr 6 pep-
tide was used to analyze JAK1 and JAK3 effects and the
Tyr 3 peptide was used for Tyk2. Pyridone-6 (Chart 1)23, a
pan-JAK inhibitor which is used for quality control of the
assay kits, was used as a positive control. Assays were per-
formed at the ATP Km and the IC50 values of the synthe-
sized compounds against each JAK isozyme, as well as
their JAK1-selectivities, are summarized in Table 2. The
benzimidazole derivatives with a 3-aminopropyl group
substituted at the N1 position (4a and 4b) showed only
moderate inhibitory activity against JAK1 (IC50 = 6.1 and
The JAK1 selectivity of this benzimidazole derivative was
then investigated using a molecular docking study. The
most potent JAK1-selective inhibitor, 5c, was docked into
the ATP-binding site of JAK1 (PDB ID = 4EHZ)20 or JAK2
(PDB ID = 4F09)20 using the flexible ligand docking soft-
ware, Glide, incorporated into the Schrödinger molecular
modeling software suite; the best docking poses are
shown in Figure 1. The ATP-binding sites of JAK1 and
JAK2 are differentiated by two amino acids, one in the
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Scheme 1. Synthesis of the benzimidazole derivatives (4 and 5).
Page 4 of 8
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Table 2. Inhibitory activities of the benzimidazole derivatives for the indicated enzymes
IC50 (μM)a
Selectivityb
Compds
JAK1
6.1±0.8
11.3±0.8
0.1±0.01
2.2±0.3
0.05±0.01
4.3±0.3
11.6±1.2
18.2±0.7
2.9±0.2
3.7±0.2
3.1±0.1
JAK2
JAK3
1.5±0.1
1.8±0.3
1.5±0.3
1.1±0.1
Tyk2
JAK1/JAK2
JAK1/JAK3
0.2
0.2
15
JAK1/Tyk2
4a
2.2±0.3
20.5±1.2
2.9±1.3
1.6±0.2
3.15±0.2
4.1±0.2
2.8±0.3
2.7±0.3
2.6±0.3
2.6±0.1
5.9±0.5
4.8±0.2
1.3±0.2
2.5±0.2
13.5±0.9
4.7±0.6
1.6±0.2
0.001
6.4±0.9
2.7±0.1
3.1±0.2
2.5±0.2
3.7±0.3
3.3±0.2
4.2±0.2
7.5±0.2
2.5±0.1
3.5±0.3
2.7±0.2
3.2±0.3
3.2±0.1
3.7±0.1
5.8±0.2
4.3±0.5
2.8±0.3
0.005
0.4
2
1
4b
0.2
31
5a
29
0.7
63
1
5b
0.5
25
1
5c
1.26±0.1
2.5±0.1
14.1±0.6
2.1±0.3
1.9±0.3
2.3±0.1
1.9±0.2
2.1±0.2
2.4±0.1
2.9±0.3
10.5±0.4
1.9±0.2
2.5±0.1
0.01
74
0.8
0.4
0.4
0.9
0.9
0.9
0.3
26
1
5d
0.6
1
5e
0.2
0.1
0.9
0.7
2
5f
0.1
5g
0.7
0.6
0.6
0.2
20
5h
5i
5j
9.3±0.5
0.1±0.0
3.1±0.3
32.4±1.4
8.1±0.4
3.4±0.1
0.004
0.5
11
5k
5l
0.8
0.4
0.6
0.5
2
0.9
0.3
0.2
0.7
5m
0.2
0.5
0.8
0.2
5n
5o
Pyridone-6c
0.2
a
The ATP concentration (Km) was 87 μM for JAK1, 35 μM for JAK2, 16 μM for JAK3, and 25 μM for Tyk2. The means ± SD of
three independent experiments are shown. bSelectivity = (IC50 against JAK2, JAK3, or Tyk2) / (IC50 against JAK1). cPan-JAK inhibi-
tor23 used as a positive control in this study.
αD-helix (Glu966 in JAK1, Asp939 in JAK2) and the other
in the glycine-rich loop (His885 in JAK1, Asn859 in JAK2)
(Figure 1). Comparison of these two pairs of amino acid
residues (Glu966 vs Asp939, His885 vs Asn859) revealed
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Table 3. Inhibitory activity of 5c against JAK isozymes in the presence of 1 mM ATP.
1
2
3
4
5
6
IC50 (μM)a
Selectivityb
Compds
JAK1
JAK2
437.3±3.1
1.4±0.3
JAK3
Tyk2
759.5±5.6
1.7±0.3
JAK1/JAK2
JAK1/JAK3
JAK1/Tyk2
5c
5.7±0.8
0.6±0.1
232.5±7.5
0.9±0.2
76
2
41
1
133
3
Pyridone-6
7
8
aThe means ± SD of three independent experiments. bSelectivity = (IC50 against JAK2, JAK3, or Tyk2) / (IC50 against JAK1).
9
that they had different side chain lengths. Thus, amino
acids with longer side chains, Glu966 (JAK1) and Asn859
(JAK2), protrude into the ATP-binding site, which results
in constriction of ligand binding in the “upper” portion of
JAK1 (Glu966–Leu881, 5.2 Å, Figure 1a) and the “lower”
portion of JAK2 (Arg980–Asn859, 4.1 Å, Figure 1b).
flected by the docking scores (G-score; -8.95 for JAK1 and
-7.75 for JAK2). Taken together, this molecular docking
information clearly showed that the 2-aminoethyl and
piperidin-4-yl moieties (attached to the benzimidazole
core of 5c) differentiate the ATP-binding sites of JAK1 and
JAK2; 5c preferentially binds to the ATP-binding site of
JAK1 by positioning the bulkier piperidin-4-yl substituent
in the open space and forming multiple favorable contacts
with the enzyme.The commercial KinaseProfiler™ Service
(Eurofins Scientific, Inc.) was utilized to evaluate the se-
lectivity of compound 5c against a panel of 27 kinases
(Figure 2). KinaseProfiler™ assay protocols measure the
percent inhibition of phosphorylation of a peptide sub-
strate in the presence of fixed concentrations of ATP that
were close to the Km for each kinase (Figure 2). Except
JAK1, no appreciable inhibition of these protein kinases
was observed in the presence of 10 μM 5c, with most
maintaining >65% of their control activity.
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Figure 1. Docked structures of 5c to (a) JAK1 (PDB ID: 4EHZ)
and (b) JAK2 (PDB ID: 4F09). Dotted lines show hydrogen
bonds. Hydrophobic contacts between 5c and the ATP-
binding sites are shown as dashed circles. Block arrows indi-
cate the widths of the ATP-binding sites.
Interestingly, the best docking poses of 5c to JAK1 and
JAK2 showed that the more sterically demanding piperi-
din-4-yl moiety of 5c was positioned away from the con-
stricted area. The binding mode of 5c to JAK1 is character-
ized by positioning the piperidin-4-yl group at the “lower”
open side of the ATP-binding pocket (Figure 1a), forming
a hydrogen bond with Asn1008 and multiple favorable
contacts with Arg1007, Gly882, Val889, and Asp1021. In
addition, the smaller and charged 2-aminoethyl group is
placed near the polar side chain of Glu966. On the other
hand, upon binding to JAK2, the benzimidazole moiety of
5c rotates about the aryl-carbonyl bond (~190.4o) to locate
the 2-aminoethyl and piperidin-4-yl substituents at the
opposite side of the ATP-binding pocket (Figure 1b); the
piperidin-4-yl group, at the upper side of the ATP-binding
pocket, is located between Asp939 and Leu855, while the
2-aminoethyl functionality forms a hydrogen bond to
Asn981 at the bottom of the ATP-binding site. Overall,
the binding mode of 5c to JAK2 is less favorable because
of the lack of hydrophobic contacts around the 2-
aminoethyl substituent, as well as the positioning of the
nonpolar hydrocarbons in the piperidin-4-yl ring between
the polar side chain of Asp939 and the backbone carbonyl
group of Leu855. The preference of 5c for JAK1 was re-
Figure 2. Kinase panel assays were performed in the pres-
ence of 5c (10 μM) and ATP (Km concentration) using Eu-
rofins KinaseProfiler™ service. The y-axis represents percent-
age of remaining kinase activity (% Activity).
However, due to high polarity, the JAK1-selective inhibi-
tor 5c showed low PAMPA permeability (Pe = 0.1 x 10-6
cm/s), lack of activity in cell-based assay25 (Supporting
Information) and poor oral bioavailability (0.7%) in rat.
On the other hand, 5c demonstrated generally favorable
pharmacokinetic properties: following intravenous (i.v.)
administration to rats, 5c exhibited moderate volumes of
distribution (Vss, 0.5 l/kg) and low systemic plasma clear-
ance (CL, 0.4 l/hr/kg) resulting in plasma half-life (t1/2) of
9.1 h (Table S1, Supporting Information). In light of the
unsatisfactory pharmacokinetic properties, the JAK1-
selective 1,2-disubstituted benzimidazole-5-carboxamide
scaffold needs further optimization.
CONCLUSIONS
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Since each JAK isozyme is employed in various cytokine which was purified by column chromatography (SiO2,
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pathways regulating immune function, specific inhibition
of a single JAK isozyme can provide a beneficial therapeu-
tic option in patients with immunologic disorders such as
RA. Pfizer’s pan-JAK inhibitor, tofacitinib, was recently
approved for the treatment of RA, and its clinical efficacy
has been attributed to the suppression of JAK3 activity.
However, since both JAK1 and JAK3 are associated with γc
cytokine receptors, it is unclear whether the clinical effi-
cacy of tofacitinib results from its concomitant inhibition
of JAK3 and JAK1, or selective inhibition of one JAK iso-
zyme. The development of selective JAK1 or JAK3 inhibi-
tors, and investigation of their effects, is therefore re-
quired in order to address this issue. For this purpose, we
set out to develop JAK1-selective inhibitors, based on our
previous observations that a linear alkyl tether can func-
tion as a probe to differentiate the ATP-binding site of
JAK1 from those of the other JAK isozymes. We synthe-
sized a series of 1,2-disubstituted benzimidazole deriva-
tives and evaluated their inhibitory activities against all
four JAK isozymes. These benzimidazole derivatives
showed clear structure-activity relationships that con-
firmed the importance of hydrogen bond donors at both
N1 and R2 positions for JAK1 selectivity; a 2-aminoethyl
group and an alkyl chain bearing a hydrogen bond donor
were favored at N1 and R2, respectively. Compound 5c,
with a piperidin-4-yl group at R2, showed unprecedented
JAK1 selectivity (63-fold vs JAK2, 25-fold vs JAK3, and 74-
fold vs Tyk2). A molecular docking study revealed that 5c
preferentially bound to the ATP-binding site of JAK1 by
positioning the bulkier piperidin-4-yl substituent at the
open space and by forming multiple favorable contacts
with the enzyme. A kinase panel assay also showed that
compound 5c (10 μM) did not inhibit a panel of 26 other
protein kinases. The JAK1-selective benzimidazole deriva-
tives identified in this study thus have the potential to
elucidate the biological consequences of specific JAK1
inhibition, specifically in terms of therapeutic interven-
tion for RA.
CH2Cl2:MeOH:NH4OH:water = 80:20:1:1) to give the ben-
zoimidazole-derivative as a dark yellow powder. This
benzoimidazole-derivative was then dissolved in MeOH
(3 ml) and treated with 6 N HCl (0.1 ml). The reaction
mixture was stirred at room temperature for 8 h and then
concentrated under reduced pressure. The crude product
was purified by column chromatography (SiO2,
CH2Cl2:MeOH:NH4OH:water = 60:35:5:5) to give 4a (11 mg,
60% yield) as a yellow powder.
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ASSOCIATED CONTENT
Supporting Information
General experimental procedures, 1H and 13C nuclear magnet-
ic resonance (NMR) spectra for new compounds, and meth-
ods for the molecular docking study. This material is availa-
ble free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
* For H. C.: phone, +82-2-958-5157; E-mail, hchoo@kist.re.kr.
* For Y. C.: phone, +82-2-2049-6100; Fax: +82-2-454-8217, E-
mail, chongy@konkuk.ac.kr.
Author Contributions
All authors have given approval to the final version of the
manuscript.
ACKNOWLEDGMENT
This research was supported by a Korean Health Technology
R&D project grant, Ministry of Health & Welfare, Republic of
Korea (A111698), by the Basic Science Research Program
through the National Research Foundation of Korea (NRF)
funded
by
the
Ministry
of
Education
(NRF-
2013R1A1A2006455), and by a grant from the Priority Re-
search Centers Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Educa-
tion, Science and Technology (2009-0093824). Additional
funding was provided by the Korea Institute of Science and
Technology (KIST) Institutional Program (2E25580, 2E25473,
and 2E25240).
EXPERIMENTAL SECTION
ABBREVIATIONS
General procedure for the preparation of 3-amino-4-
alkylaminobenzamide (3a and 3b). Palladium on char-
coal (10% w/w) was added to a solution of 2 (1.4 mmol) in
MeOH (15 ml) and charged with hydrogen (balloon, 1
atm). After stirring for 8 h at room temperature, solvent
was removed under reduced pressure to give a crude
product which was purified by column chromatography
(SiO2, CH2Cl2:MeOH:NH4OH:water = 60:35:5:5) to give 3.
JAK, Janus kinase; IL, interleukin; MPD, myeloproliferative
disorders; RA, rheumatoid arthritis; EDC, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide; HOBT, 1-hydroxy-1H-
benzotriazole; DMF, N,N-dimethylformamide; Boc, tert-
butoxycarbonyl; EtOAc, ethyl acetate; MeOH, methanol;
PAMPA, parallel artificial membrane permeability assay.
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