Novel JAK1-selective benzimidazole inhibitors with enhanced membrane permeability

Article abstract of DOI:10.1016/j.bmcl.2016.05.078

The previously identified Janus kinase 1 (JAK1)-selective inhibitor, 1-(2-aminoethyl)-2-(piperidin-4-yl)-1H-benzo[d]imidazole-5-carboxamide (2), suffered from low cell permeability, which resulted in poor pharmacokinetic properties. In this study, by introducing less polar hydrogen bond donors at N¹(a hydroxyalkyl or a methylaminoalkyl group) and C2 (a cyclohexanol group) positions, a series of novel benzimidazole derivatives were prepared, which exhibited selective JAK1 inhibitory activity (IC₅₀against JAK1?=?0.08–0.15?μM; JAK1-selectivity?=?26–40 fold vs JAK2, 12–23 fold vs JAK3, and 38–54 fold vs Tyk2) along with significantly increased lipophilicity (3.3–15.8 times) as well as membrane permeability (6.3–12 times).

Full text of DOI:10.1016/j.bmcl.2016.05.078

Accepted Manuscript
Novel JAK1-Selective Benzimidazole Inhibitors with Enhanced Membrane
Permeability
Hyungmi Kim, Mi Kyoung Kim, Hyunah Choo, Youhoon Chong
PII:
S0960-894X(16)30589-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.05.078
BMCL 23937
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 April 2016
23 May 2016
27 May 2016
Please cite this article as: Kim, H., Kim, M.K., Choo, H., Chong, Y., Novel JAK1-Selective Benzimidazole Inhibitors
with Enhanced Membrane Permeability, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/
10.1016/j.bmcl.2016.05.078
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Submitted to Bioorganic & Medicinal Chemistry Letters
Novel JAK1-Selective Benzimidazole Inhibitors with Enhanced Membrane
Permeability
Hyungmi Kim,a,† Mi Kyoung Kim,a,† Hyunah Choob,c and Youhoon Chonga,*
aDepartment of Integrative Bioscience and Biotechnology, Bio/Molecular Informatics Center,
b
Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul 143-701, Korea; Center for Neuro-
Medicine, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seoungbuk-gu,
c
Seoul 136-791, Korea; Department of Biological Chemistry, Korea University of Science
and Technology, Youseong-gu, Daejeon 305-350, Korea
†These two authors contributed equally on this work.
*Corresponding author. E-mail: chongy@konkuk.ac.kr, Tel: +82-2-2049-6100
Graphical Abstract
JAK Inhibition (IC50, μM)
Property
JAK1
0.05
0.08
JAK2
3.2
JAK3
1.3
Tyk2
3.7
LogP
LogPe
-5.12
-4.12
2
-0.37
0.69
3a
3.2
1.8
4.3
Keywords: Janus kinase, Rheumatoid arthritis, Benzimidazole, Selective inhibition
- 1 -

ABSTRACT
The previously identified Janus kinase 1 (JAK1)-selective inhibitor, 1-(2-aminoethyl)-2-
(piperidin-4-yl)-1H-benzo[d]imidazole-5-carboxamide (2), suffered from low cell
permeability, which resulted in poor pharmacokinetic properties. In this study, by introducing
less polar hydrogen bond donors at N1 (a hydroxyalkyl or a methylaminoalkyl group) and C2
(a cyclohexanol group) positions, a series of novel benzimidazole derivatives were prepared,
which exhibited selective JAK1 inhibitory activity (IC50 against JAK1 = 0.08 ~ 0.15 μM;
JAK1-selectivity = 26 ~ 40 fold vs JAK2, 12 ~ 23 fold vs JAK3, and 38 ~ 54 fold vs Tyk2)
along with significantly increased lipophilicity (3.3 ~ 15.8 times) as well as membrane
permeability (6.3 ~ 12 times).
- 2 -

Cytokine receptors are specifically coupled to various Janus protein tyrosine kinases (JAK1,
JAK2, JAK3 and Tyk2). Upon binding to cytokines, the cytokine receptors transmit
information related to immune function,1 inflammation,2 or hematopoiesis3-5 by way of JAK-
STAT (signal transducer and activator of transcription) pathway. In particular, cytokines such
as interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21 bind to the cytokine receptors with γc
chain which is exclusively associated with JAK3 and JAK1.2 Therefore, both JAK3 and
JAK1 have been targeted for therapeutic intervention of immunologic disorders such as
rheumatoid arthritis (RA).2 Xeljanz (Tofacitinib, CP-690,550) was the first-in-class JAK
inhibitor approved for the treatment of RA.6-7 However, because Xeljanz was revealed as a
potent pan-JAK inhibitor with low isozyme-selectivity,8 debate continues about whether
highly isozyme-specific JAK inhibitors are necessarily the best approach for the control of
RA with an optimized risk/benefit ratio. Another unresolved point is whether the signal
transduction through γc-containing cytokine receptors is governed by JAK3 or JAK1.9-11 Thus,
in order to put an end to this controversy and to facilitate JAK inhibitor-based anti-RA drug
discovery, identification of novel JAK3-selective12-13 or JAK1-selective14-15 inhibitors is
urgently required.
Over the past few years, we have been in active pursuit of the JAK1-selective inhibitors
based on the initial observation that a linear tether (dashed box, 1, Fig. 1) attached to a hinge-
binding motif (circle, 1, Fig. 1) of an ATP-competitive kinase inhibitor 3-alkynolyl-indazole-
7-carboxamide derivative (1, Fig. 1) serves as an isozyme-specific probe group to provide
selective inhibition of JAK1 over JAK2.16 Various scaffolds with a linear tether have been
prepared,
and
recently
we
identified
1-(2-aminoethyl)-2-(piperidin-4-yl)-1H-
benzo[d]imidazole-5-carboxamide (2, Fig. 1) as a potent JAK1-selective inhibitor (IC50
against JAK1 = 0.05 μM; 63-fold vs JAK2, 25-fold vs JAK3, and 74-fold vs Tyk2).17
- 3 -

However, due to the lack of cell permeability conferred by the polar hydrogen bond donors
(aminoethyl and piperidinyl groups) at N1 and C2 positions, compound 2 showed poor
pharmacokinetic properties.17 In this study, by introducing less polar hydrogen bond donors at
N1 (a hydroxyalkyl or a methylaminoalkyl group) and C2 (a cyclohexanol group) positions of
the benzimidazole core (3a ~ 3g, Fig. 1), we attempted to discover novel membrane-
permeable benzimidazole derivatives with potent and selective JAK1 inhibitory activity.
Figure 1. Design of novel benzimidazole derivatives (3a ~ 3g) based on the previously
identified JAK1-selective inhibitors (1 and 2)
The title compounds (3a ~ 3g) were synthesized from commercially available 4-fluoro-3-
nitrobenzoic
acid
(4)
in
5
steps
(Scheme
1).17
EDC
[1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide] coupling of 4 with 1-hydroxy-1H-benzotriazole (HOBt)-
- 4 -

ammonium salt (NH3·HOBt) in a 1:4 mixture of N,N-dimethylformamide (DMF) and
acetonitrile (CH3CN) provided 4-fluoro-3-nitrobenzamide (97% yield), which underwent
nucleophilic aromatic substitution with various amines to give 4-amino-3-nitrobenzamides 5a
~ 5e in 74% ~ 96% yields. After reduction, 5a ~ 5e were converted to the corresponding 3,4-
diaminobenzamides 6a ~ 6e in 52% ~ 61% yields. Formation of the benzimidazole core
structure was accomplished by condensation of 6a ~ 6e with trans-4-((tert-
butyldimethylsilyl)oxy)cyclohexanecarbaldehyde or tert-butyl 4-formylpiperidine-1-
carboxylate in the presence of sodium metabisulfite (Na2S2O5)18. The protecting groups in the
resulting benzimidazole derivatives were then removed by treatment with HCl or TFA to
provide a series of the benzimidazole derivatives with a C2-cyclohexanol substituent (3a ~
3e) or with a C2-piperidine substituent (3f ~ 3g) in 16% ~ 30% yields.
- 5 -

Scheme 1. Synthesis of the benzimidazole derivatives (3a ~ 3g)
In vitro inhibitory activity of the benzimidazole derivatives (3a ~ 3g) on the JAK isozymes
was determined using Z′-LYTE™ Kinase Assay Kit-Tyr 6 Peptide (JAK1, JAK2 and JAK3)
- 6 -

and Tyr 3 Peptide (Tyk2) (Invitrogen). Pyridone-6,19 a pan-JAK inhibitor which is used for
quality control of the assay kit, was used as a positive control. Assays were performed at the
ATP Km, and the IC50 values of 3a ~ 3g against each JAK isozyme as well as their JAK1-
selectivities over other JAK isozymes are summarized in Table 1.
Table 1. Inhibitory activity (IC50) of the benzimidazole derivatives against JAK isozymes
IC50 (μM)a
JAK2
JAK1 Selectivityb
vs
vs
vs
JAK1
JAK3
Tyk2
JAK2 JAK3 Tyk2
0.08±0.01
7.0±0.2
3.2±0.2
29.7±0.9
12.1±0.3
3.9±0.2
18.5±0.9
3.1±0.3
1.8±0.1
20.6±1.2
8.5±0.2
1.9±0.1
12.4±0.5
1.5±0.1
4.3±0.5
42.2±2.8
25.7±1.5
5.6±0.2
30.7±1.4
4.1±0.4
40
4
23
3
54
6
3a
3b
3c
3d
3e
3f
2.0±0.1
6
4
13
38
6
0.15±0.08
5.3±0.3
26
4
12
2
0.09±0.02
34
16
46
6
5.4±0.9
27.3±3.4
3.2±0.2
14.3±1.2
1.3±0.1
31.0±3.8
3.7±0.3
5
3
3g
2c
0.05±0.01
63
25
74
aEach experiment was repeated at least three times
bSelectivity = (IC50 against JAK2 or JAK3) / (IC50 against JAK1)
cReference 19
The benzimidazole derivatives synthesized in this study (3a ~ 3g) showed modest to potent
inhibition against a series of JAK isozymes. In particular, 3a, 3d and 3f showed potent (IC50
against JAK1 = 0.08 ~ 0.15 μM) and selective (26 ~ 40 fold vs JAK2, 12 ~ 23 fold vs JAK3,
and 38 ~ 54 fold vs Tyk2) inhibitory activity against JAK1 (Table 1). As demonstrated in our
previous work17, structural comparison of these compounds with others revealed that 3a, 3d
and 3f share common structural elements for selective inhibition of JAK1; hydrogen bond
donors are substituted at the N1 position via an ethylene linker. In contrast, one-carbon
- 7 -

homologues with a N1-propyl side chain (3b, 3e and 3g) showed only moderate inhibitory
activity (IC50 against JAK1 = 5.3 ~ 7.0 μM) with low JAK1 selectivity (4 ~ 5 fold vs JAK2, 2
~ 3 fold vs JAK3, and 6 fold vs Tyk2). Furthermore, introduction of a methyl group to the N1-
ethylene linker was detrimental for JAK1 inhibition, which resulted in significant decrease in
inhibitory activity as well as JAK1 selectivity of 3c (IC50 against JAK1 = 2.0 μM; 6-fold vs
JAK2, 4-fold vs JAK3, and 13-fold vs Tyk2). Unlike substitution at the N1-ethylene moiety,
methylation at the terminal amino group did not affect the inhibitory activity of the
benzimidazole derivatives, and 3f with a N1-2-(methylamino)ethyl substituent showed similar
inhibitory activity as well as JAK1 selectivity (IC50 against JAK1 = 0.09 μM; 34-fold vs
JAK2, 16-fold vs JAK3, and 46-fold vs Tyk2) compared with 3a (IC50 against JAK1 = 0.08
μM; 40-fold vs JAK2, 23-fold vs JAK3, and 54-fold vs Tyk2). On the other hand, 4-
hydroxycyclohexyl (3a ~ 3e) or 4-piperidinyl (3f and 3g) group substituted at the C2 position
did not affect the inhibitory activity of the resulting benzimidazole derivatives; 3d (IC50
against JAK1 = 0.15 μM; 26-fold vs JAK2, 12-fold vs JAK3, and 38-fold vs Tyk2) and 3f
(IC50 against JAK1 = 0.09 μM; 34-fold vs JAK2, 16-fold vs JAK3, and 46-fold vs Tyk2)
showed similar inhibitory activity and JAK1 selectivity.
The logP value of a compound, the logarithm of its partition coefficient between n-octanol
and water, is a well-established measure of the compound's lipophilicity. The logP values of
the benzimidazole derivatives with potent and selective inhibitory activity against JAK1 (3a,
3d and 3f) were thus calculated (Table 2) and, as anticipated, they showed significantly
increased partition coefficient (P) values (3.3 ~ 15.8 times) compared with 2 (Fig. 1).
The novel JAK1-selective inhibitors 3a, 3d and 3f were also evaluated for their ability to pass
through an artificial membrane. The parallel artificial membrane permeability assay
(PAMPA), a gold standard in vitro method for determining drug permeability, was
- 8 -

performed20 by using the pre-coated plate which includes polystyrene filter plate with
polyvinylidene fluoride membrane. The PAMPA data summarized in Table 2 show that, in
terms of passive permeability (effective permeability coefficients, Pe), the benzimidazole
derivatives 3a, 3d and 3f were more permeable than 2 by 10.0, 12.0 and 6.30 times,
respectively.
Table 2. Lipophilicity (LogP)a and permeability across artificial membranesb of JAK1-
selective inhibitors (2) and benzimidazole derivatives (3a~3g)
Compd.
LogP
3a
0.69
3d
0.83
3f
2
0.15
-0.37
Log Pe (cm/s)
-4.12±0.02
-4.04±0.04
-4.32±0.06
-5.12±0.06
aLogP was calculated by ChemDraw Ultra 12.0
bAssay was performed in triplicate.
In summary, current attempt to improve membrane permeability of the JAK1-selective
inhibitor 2 includes introduction of less polar hydrogen bond donors at N1 and C2 positions of
the benzimidazole core. In particular, changing the polar amino groups at N1 and C2 positions
of 2 by methylation and/or substitution with a 4-hydroxycyclohexyl group resulted in
significant increase in lipophilicity (3.3 ~ 15.8 times) as well as membrane permeability (6.3
~ 12 times) of the resulting benzimidazole derivatives (3a, 3d and 3f) without substantial loss
of inhibitory activity against JAK1 (IC50 against JAK1 = 0.08 ~ 0.15 μM; JAK1-selectivity =
26 ~ 40 fold vs JAK2, 12 ~ 23 fold vs JAK3, and 38 ~ 54 fold vs Tyk2). A comprehensive in
vitro and in vivo assessment of the biological activity of 3a, 3d and 3f are underway to
confirm their therapeutic potential against RA.
Acknowledgments
This paper was supported by Konkuk University in 2015.
- 9 -

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