Quinoxalinylurea derivatives as a novel class of JSP-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.01.094

A series of quinoxalinylurea-based inhibitors are synthesized and shown to be the novel and potent inhibitors against Jnk Stimulatory Phosphatase-1 (JSP-1), which is a special member of dual-specificity protein phosphatase (DSP) family. Biological assay a

Full text of DOI:10.1016/j.bmcl.2007.01.094

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2118–2122
Quinoxalinylurea derivatives as a novel class of JSP-1 inhibitors
Li Zhang,a,ꢀ Beiying Qiu,b,ꢀ Bing Xiong,a Xin Li,a Jingya Li,b
Xin Wang,a Jia Lib,* and Jingkang Shena,*
aState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
555 Zuchongzhi Road, Shanghai 201203, PR China
bThe National Center for Drug Screening, 189 Guoshoujing Road, Shanghai 201203, PR China
Received 13 November 2006; revised 26 January 2007; accepted 30 January 2007
Available online 2 February 2007
Abstract—A series of quinoxalinylurea-based inhibitors are synthesized and shown to be the novel and potent inhibitors against Jnk
Stimulatory Phosphatase-1 (JSP-1), which is a special member of dual-specificity protein phosphatase (DSP) family. Biological assay
and computational modeling studies showed the compounds were reversible and noncompetitive inhibitors of JSP-1. JSP-1 inhib-
itors may be useful for the treatment of inflammatory, vascular, neurodegenerative, metabolic, and oncological diseases in humans
associated with dysfunctional Jnk signaling.
Ó 2007 Elsevier Ltd. All rights reserved.
JSP-1 (Jnk Stimulatory Phosphatase-1),1 also referred to
as VHX (VHR-related MKPX)2 or JKAP (Jnk path-
way-associated phosphatase),3 is a special member of
the dual-specificity protein phosphatases (DSPs),4 which
belong to the protein tyrosine phosphatases (PTPs),5 a
super-family of enzymes with similarly conserved motif
HCXXGXXR and the same tertiary structure. PTPs
dephosphorylate proteins with phosphate on serine,
threonine, and/or tyrosine, and play important regula-
tive roles in cellular signal transduction. In recent years,
interest in PTPs as potential drug targets for several
serious diseases such as cancers, autoimmune diseases,
and diabetes has rapidly increased.6,7 While some mem-
bers of DSPs like VHR (human VH1-related) and
MKP1 are negative regulators of mitogen activated
protein kinases (MAPK), JSP-1 is a positive regulator
for the Jun NH2-terminal kinase (Jnk) pathway. The
study of Belmont’s group3 indicated that JSP-1 is
necessary for optimal Jnk activation. However, JSP-1
does not exert its effects directly on Jnk, it appears to work
upstream of Jnk itself, by activating MKK41 and MKK73
kinases, which phosphorylate and activate Jnk.
apoptosis.8 Dysfunctional Jnk signaling is associated
with inflammatory, vascular, neurodegenerative, meta-
bolic, and oncological diseases in humans.9 Therefore,
effect of JSP-1 on the Jnk signal pathway makes it worth
studying as a potential novel therapeutic target.
To date, reports regarding small molecule JSP-1 inhibi-
tors are few.10 Through a high-throughput screening of
our sample collection, a quinoxalinylurea-based small
molecule compound A1 (Fig. 1) was discovered and
showed inhibiting activity toward JSP-1 with the 50%
inhibitory concentration (IC50) of 12.01 0.15 lM in
an in vitro biological assay.
Quinoxalines are an important class of nitrogen-con-
taining heterocycles with antibacterial activity,11,12
angiotensin II receptor,13,14 and AMPA receptor antag-
onist activity.15,16 In this paper, a series of quinoxali-
O
O
H
N
N
N
N
O
The Jnk pathway plays broad roles in cellular response
to various forms of stresses, growth stimulation, and
O
N
Keywords: JSP-1; Inhibitor; Quinoxalinylurea.
A1 IC50 = 12.01 0.15µM
*
Corresponding authors. Tel./fax: +86 21 50806896 (J.S.); e-mail
addresses: jli@mail.shcnc.ac.cn; jkshen@mail.shcnc.ac.cn
These authors contributed equally to this work.
Figure 1. Inhibitor of JSP-1 discovered by
screening.
a high-throughput
ꢀ
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.094

L. Zhang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2118–2122
2119
nylurea-based small molecules are reported as novel and
potent JSP-1 inhibitors.
the corresponding 6-aminoquinoxaline derivatives 8,
which were treated with triphosgene to provide crude
2,3-disubstituted-6-isocyanato-quinoxalines derivatives
1321 to be used in the next reaction without further puri-
fication. On the other hand, piperidine-3-carboxylic
acids 9 were treated with (Boc)2O to give Boc-protected
piperidine-3-carboxylic acids 10, which were coupled
with various amines in the presence of 1-ethyl-3-(3-dim-
ethylaminopropyl)carbodiimide hydrochloride (EDC)
and 1-hydroxy benzotriazole (HOBt) to afford Boc-pro-
tected piperidine-3-amides 11. After removing Boc-pro-
tecting group, the corresponding piperidine-3-amide
hydrochlorides 12 were produced. Finally, the designed
compounds were obtained by the reaction of 13 with
piperidine-3-amides 12.
With the goal to investigate the structure–activity rela-
tionship (SAR) of these quinoxalinylurea-based small
molecules, a library with 28 of these analogs were
designed and synthesized. Keeping the N-6-quinoxali-
nyl-1,3-piperidinedicarboxamide moiety, aryl and alter-
native lipophilic groups such as phenyl, 4-methylphenyl,
4-fluorophenyl, 4-bromophenyl, and methoxyl were
employed as replacements for the furyl groups in the 2
and 3 positions on the quinoxaline ring, and cyclic
primary and secondary amines such as pyrrolidine, piper-
idine, cyclopentylamine, and cyclohexylamine were used
to displace diethylamide of A1 in an effort to investigate
whether these groups were essential for retaining the
activity in this series.
All synthesized compounds were screened for their abil-
ity to inhibit JSP-1 in an in vitro enzymatic assay.22
A
The synthetic routes of these compounds described in
this study are outlined in Scheme 1. In general, 2,3-di-
substituted 6-nitro-quinoxaline 3 was obtained by the
condensation of 1,2-dicarbonyl compound 1 with 4-ni-
tro-o-phenylenediamine 2.17,18 2,3-dimethoxy-substitut-
ed analog of 6-nitro-quinoxaline 7 was synthesized by
the condensation of diethyl oxalate 4 with 4-nitro-o-
phenylenediamine 2, followed by the chlorination with
POCl3, and then the substitution with CH3ONa.19,20
The catalytic hydrogenation of 6-nitro-quinoxaline 3
and 7 in the presence of 10% Pd/C in EtOH afforded
part of biological results with representative structures
are listed in Table 1, and the other compounds whose
IC50 exceed 40 lM were not described in detail. The re-
sults showed that substitution at positions 2 and 3 on
the quinoxaline ring plays an important role in the
inhibitory activity. Keeping the furyl groups at positions
2 and 3, compounds (A2–A4) showed potent inhibition
against JSP-1. Replacements of the furyl groups in posi-
tions 2 and 3 of the quinoxaline ring with phenyl, or
substituted phenyl groups resulted in decreased potency
(A5–A12), while replacement with the lipophilic methoxyl
O
N
O
N
R1
R1
R1
NH2
N
N
O
N
N
a
H2N
H2N
b
O
O
R1
+
R1
R1
O
8
6
3
1
2
O
O
N
O
N
O
O
N
Cl
Cl
N
HO
HO
N
N
d
c
H2N
H2N
O
O
O
+
O
N
O
5
2
4
O
N
e
CH3O
CH3O
N
N
O
7
R2
R2
N
f
g
h
HN
OH
N
N
OH
N
HN
HCl
R3
R3
boc
boc
O
O
O
O
9
12
11
10
H
R1
NCO
N
N
R1
R1
N
NH2
R1
R1
N
N
O
N
N
i
j
R1
O
N
N
R2
R3
8
13
A
Scheme 1. Reagents and conditions: (a) EtOH, reflux, 24 h (90–96%); (b) H2, 10% Pd/C, EtOH, rt, 3 h (90–96%); (c) reflux, 24 h (89%); (d) POCl3,
reflux, 3.5 h (66%); (e) CH3ONa, CH3OH, rt, 3 h (91%); (f) (Boc)2O (98%); (g) HNR2R3, EDC, HOBt, CH2Cl2, rt (95%); (h) HCl in dioxane (90–
95%); (i) triphosgene, DIPEA, CH2Cl2, rt; (j) 12, DIPEA, CH2Cl2, rt.

2120
L. Zhang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2118–2122
Table 1. Activities of quinoxalinylurea derivatives
H
R1
R1
N
O
N
N
N
O
N
R2
R3
Compound R1
NR2R3
JSP-1 IC50 (lM)a
A1
A2
2-Furyl
2-Furyl
2-Furyl
2-Furyl
Phenyl
Phenyl
Diethylamine
Piperidine
12.01 0.15
8.39 1.50
A3
A4
Cyclopentylamine 3.02 0.18
Cyclohexylamine 2.25 0.24
A5
A6
Diethylamine
Piperidine
>40
>40
>40
A7
A8
4-Methylphenyl Diethylamine
4-Methylphenyl Cyclopentylamine >40
A9
4-Fluorophenyl Diethylamine
4-Fluorophenyl Cyclohexylamine
4-Bromophenyl Diethylamine
4-Bromophenyl Pyrrolidine
>40
>40
A10
A11
A12
A13
A14
>40
>40
Methoxy
Methoxy
Piperidine >100
Cyclohexylamine >100
a Data are means of three independent experiments.
group was not tolerated (A13–A14). In this series, com-
pound A4 showed the most potent inhibition of JSP-1.
Detailed enzymatic kinetics studies revealed that com-
pound A4 is a reversible and noncompetitive inhibitor
of JSP-1 (Figs. 2 and 3),22,23 suggesting that this com-
pound may utilize an allosteric mechanism to inhibit
JSP-1.
Figure 3. Noncompetitive inhibitors of JSP-1.
As mentioned by Wiesmann et al.,24 when an inhibitor is
bound to the allosteric modulation pocket of protein
tyrosine phosphatase-1B (PTP1B), it could lock the
PTP1B at the inactive conformation. This could be com-
pared to our case with JSP-1. Although three-dimen-
tional structure of JSP-1 was solved by Yakota et al.,
(PDB access code 1WRM),25 we are aware that JSP-1
in the crystal structure is remaining in an active confor-
mation. Through structure and sequence alignments, the
catalytic domain of the inactive conformation of MAPK
phosphatase Pyst1 (PDB access code 1MKP) was uti-
lized as a structure template to model the inactive con-
formation of JSP-1. The allosteric binding site was
indicated by the superimposed structure of PTP1B
(PDB access code 1T48), which contained helix a3 (res-
idues 65–80) and helix a6 (residues 137–152) in JSP-1.
The advanced docking software AUTODOCK3.0 was
used to dock the A4 into this binding pocket
(Fig. 4).26 As suggested by the docking study, there
are several hydrophobic residues close to the piperi-
dine-3-carboxamide and this binding interaction provid-
ed evidence that the large side chain group on the
piperidine-3-carboxamide may improve inhibiting JSP-
1 activity.
To corroborate the hypothesis coming from the docking
study, a series of 2,3-difuryl quinoxalinyl ureas incorpo-
rating various bulky alkyl amines shown in Table 2 were
designed for the second round. Difuryl substitutes at
positions 2 and 3 of the quinoxaline ring were retained
and alternative substitutes on the piperidine carboxam-
ide were explored. The compounds A15–A23 were pre-
pared by the same method as described above. As
listed in Table 2, the results of the in vitro enzymatic as-
say showed that activity was influenced by the substitu-
ents at the piperidine-3-carboxamide. Replacement of
the diethyl group with small alkyl groups resulted in
reducing JSP-1 inhibitory activity (A15, A19–A20).
Figure 2. Reversible inhibitors of JSP-1.

L. Zhang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2118–2122
2121
Figure 4. (A) View of allosteric site in presence of compound A4. (B) View of allosteric site in presence of compound A17.
Table 2. Activities of quinoxalinylurea derivatives
Acknowledgment
This work was financially supported by the National
Natural Science Foundation of China (Grants
30230400 and 30572255).
H
N
R1
R1
N
O
N
N
O
N
R2
R3
References and notes
Compound R1
NR2R3
JSP-1 IC50 (lM)a
1. Shen, Y.; Luche, R.; Wei, B.; Gordon, M. L.; Diltz, C. D.;
Tonks, N. K. Proc. Natl. Acad. Sci. U.S.A. 2001, 98,
13613.
2. Alonso, A.; Merlo, J. J.; Na, S. Q.; Kholod, N.;
Jaroszewski, L.; Kharitonenkov, A.; Williams, S.; Godzik,
A.; Posada, J. D.; Mustelin, T. J. Biol. Chem. 2002, 277,
5524.
3. Chen, A. J.; Zhou, G. S.; Juan, T.; Colicos, S. M.;
Cannon, J. P.; Cabriera-Hansen, M.; Meyer, C. F.;
Jurecic, R.; Copeland, N. G.; Gilbert, D. J.; Jenkins, N.
A.; Fletcher, F.; Tan, T. H.; Belmont, J. W. J. Biol. Chem.
2002, 277, 36592.
A15
A16
A17
A18
A19
A20
A21
A22
A23
2-Furyl Dimethylamine >40
2-Furyl N-Methyl-butylamine 5.55 0.56
2-Furyl N-Ethyl-butylamine
2-Furyl Dipropylamine
2-Furyl Ethylamine
2.35 0.65
2.50 0.06
>40
2-Furyl Isopropylamine
2-Furyl tert-Butylamine
2-Furyl Isobutylamine
2-Furyl n-Butylamine
>40
8.05 0.97
8.36 0.18
2.79 0.25
a Data are means of three independent experiments.
4. Alonso, A.; Rojas, A.; Godzik, A.; Mustelin, T.. In Top.
Curr. Genetics; Arino, J., Alexander, D. R., Eds.; Spring-
er-Verlag: Berlin, Heidelberg, 2004; Vol. 5, p 333.
5. Alonso, A.; Sasin, J.; Bottini, N.; Friedberg, I.; Friedberg,
I.; Osterman, A.; Godzik, A.; Hunter, T.; Dixon, J.;
Mustelin, T. Cell 2004, 117, 699.
6. van Huijsduijnen, R. H.; Bombrun, A.; Swinnen, D. Drug
Discovery Today 2002, 7, 1013.
7. Hoffman, B. T.; Nelson, M. R.; Burdick, K.; Baxter, S. M.
Curr. Pharm. Des. 2004, 10, 1161.
8. Davis, R. J. Cell 2000, 103, 239.
Replacing with long chain or sterically hindered alkyl
groups, the compounds A16–A18 and A21–A23 showed
significant activity comparable with A2–A4. Further-
more, enzymatic kinetics studies on compound A17 also
indicated that it is a reversible and noncompetitive
inhibitor of JSP-1 (Figs. 2 and 3).
9. Manning, A. M.; Davis, R. J. Nat. Rev. Drug Disc. 2003,
2, 554.
10. Cutshall, N. S.; O’Day, C.; Prezhdo, M. Bioorg. Med.
Chem. Lett. 2005, 15, 3374.
11. Makino, K.; Sakata, G.; Morimoto, K.; Ochiai, Y.
Heterocycles 1985, 23, 2025.
12. Siddiqui, A. A.; Khan, S. A.; Bhatt, S. Asian J. Chem.
2002, 14, 1109.
13. Kim, K. S.; Qian, L. G.; Dickinson, K. E. J.; Delaney, C.
L.; Bird, J. E.; Waldron, T. L.; Moreland, S. Bioorg. Med.
Chem. Lett. 1993, 3, 2667.
14. Kim, K. S.; Qian, L. G.; Bird, J. E.; Dickinson, K. E. J.;
Moreland, S.; Schaeffer, T. R.; Waldron, T. L.; Delaney,
C. L.; Weller, H. N.; Miller, A. V. J. Med. Chem. 1993, 36,
2335.
In summary, we have synthesized and investigated preli-
minary SAR for a novel series of quinoxalinylurea
derivatives with JSP-1 inhibitory activity based on the
compound A1 obtained through high-throughput
screening. The result of in vitro biological experiment
showed that these compounds were noncompetitive
and reversible inhibitors of JSP-1. Through computa-
tional modeling, an allosteric site in JSP-1 was found,
and based on the docking study we provided a hypoth-
esis that a hydrophobic subpocket is close-by and can be
utilized to improve the binding affinity. The biological
assay performed on the second round compounds con-
firmed that bulky alkyl groups appended to the piperi-
dine-3-carboxamide would benefit the affinity to JSP-1.
The further study is going on.
15. Takano, Y.; Shiga, F.; Asano, J.; Ando, N.; Uchiki, H.;
Anraku, T. Bioorg. Med. Chem. Lett. 2003, 13, 3521.

2122
L. Zhang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2118–2122
16. Catarzi, D.; Colotta, V.; Varano, F.; Calabri, F. R.;
Filacchioni, G.; Galli, A.; Costagli, C.; Carla, V. J. Med.
Chem. 2004, 47, 262.
17. Alamino, R. J. U.S. Patent 4,540,693, 1985; Chem. Abstr.
1986, 104, 88601.
18. Abdel-Jalil, R. J.; Al-Qawasmeh, R. A.; Voelter, W.;
Heeg, P.; El-Abadelah, M. M.; Sabri, S. S. J. Heterocycl.
Chem. 2000, 37, 1273.
19. Katoh, A.; Takahashi, M.; Ohkanda, J. Chem. Lett. 1996,
369.
inhibition) versus inhibitor concentration [I] by using the
following equation% Inhibition = 100/{1 + (IC50/[I])k},
where k is the Hill coefficient. For mechanism studies,
data were analyzed using a nonlinear regression fit
according to classical Michaelis–Menten kinetics models.
23. The reversibility of inhibition is easily determined by
measuring the recovery of enzymatic activity after dialysis
of the enzyme–inhibitor complex against assays. To test for
reversibility, excessive compounds A4 and A17 were incu-
bated with 100 nM enzyme. Then the mixture was dialyzed
against the assay buffer and the activity of the enzyme was
tested in different times. If the inhibitor was reversible,
enzymatic activity could recover to some extent; and if the
inhibitor was irreversible, enzymatic activity could not
recover at any time. Figure 2 shows the activity of JSP-1 was
recovered to some extent after dialysis, compared with the
mixture without dialysis. It verified that compounds A4 and
A17 were reversible JSP-1 inhibitors.
24. Wiesmann, C.; Barr, K. J.; Kung, J.; Zhu, J.; Erlanson, D.
A.; Shen, W.; Fahr, B. J.; Zhong, M.; Taylor, L.; Randal,
M.; McDowell, R. S.; Hansen, S. K. Nat. Struct. Mol.
Biol. 2004, 11, 730.
25. Yokota, T.; Nara, Y.; Kashima, A.; Matsubara, K.;
Misawa, S.; Kato, R.; Sugio, S. Proteins 2007, 66, 272.
26. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.;
Hart, W. E.; Belew, R. K.; Olson, A. J. J. Comput. Chem.
1998, 19, 1639.
20. Cheeseman, G. W. H. J. Chem. Soc. 1962, 1170.
21. Cotarca, L.; Delogu, P.; Nardelli, A.; Sunjic, V. Synthesis
1996, 553.
22. The JSP-1 activity was determined at room temperature
by monitoring the hydrolysis of 3-o-methylfluorescein
phosphate (OMFP). The studies have shown that OMFP
is an ideal substrate for dual-specificity phosphatases as
they evoke faster kinetics for hydrolysis of OMFP
compared with pNPP. In a typical 100 lL assay mixture
containing 50 mM BisÆTris, pH 6.5, 1 mM EDTA, 1 mM
DTT, 10 lM OMFP, and 30 nM purified recombinant
GST-fusion human JSP-1, the enzyme activity was con-
tinuously monitored with excitation 485 nm/emission
535 nm filter set for 3 min and the initial rate of the
hydrolysis was determined using the linear region of the
enzymatic reaction kinetic curve. IC50 was calculated from
the nonlinear curve fitting of percent inhibition (%