Design, synthesis and inhibitory activity against human dihydroorotate dehydrogenase (hDHODH) of 1,3-benzoazole derivatives bearing amide units

Article abstract of DOI:10.1016/j.bmcl.2016.05.016

A series of 1,3-benzoazole derivatives possessing amide moieties were designed, synthesized and evaluated as inhibitors against human dihydroorotate dehydrogenase (hDHODH). Compounds A11, A14 and A26 exhibited good to excellent activities against hDHODH at the concentration of 10 μM. In particular, compound A14 displayed an IC₅₀ value of 0.178 μM with 2-fold preference over A771726. The result implied that a proper degree of steric size and electron density of the C-6 amide moiety was necessary to retain the inhibitory activity of the synthesized compounds.

Full text of DOI:10.1016/j.bmcl.2016.05.016

Accepted Manuscript
Design, synthesis and inhibitory activity against human dihydroorotate dehy-
drogenase (hDHODH) of 1,3-benzoazole derivatives bearing amide units
Jiling Li, Dang Wu, Xiaoyong Xu, Jin huang, Xusheng Shao, Zhong Li
PII:
S0960-894X(16)30501-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.05.016
BMCL 23875
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
15 January 2016
12 April 2016
4 May 2016
Please cite this article as: Li, J., Wu, D., Xu, X., huang, J., Shao, X., Li, Z., Design, synthesis and inhibitory activity
against human dihydroorotate dehydrogenase (hDHODH) of 1,3-benzoazole derivatives bearing amide units,
Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.05.016
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Design, synthesis and inhibitory activity
against human dihydroorotate
dehydrogenase (hDHODH) of 1,3-benzoazole
derivatives bearing amide units
Jiling Li, Dang Wu, Xiaoyong Xu, Jin Huang, Xusheng Shao* and Zhong Li*

Bioorganic & Medicinal Chemistry Letters
Design, synthesis and inhibitory activity against human dihydroorotate
dehydrogenase (hDHODH) of 1,3-benzoazole derivatives bearing amide units
Jiling Lia, Dang Wua, Xiaoyong Xua, Jin huanga, Xusheng Shaoa,  and Zhong Lia, b, 
aShanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
bShanghai Collaborative Innovation Center for Biomanufacturing Technology, 130 Meilong Road, Shanghai 200237, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of 1,3-benzoazole derivatives possessing amide moieties were designed, synthesized
and evaluated as inhibitors against human dihydroorotate dehydrogenase (hDHODH).
Compounds A11, A14 and A26 exhibited good to excellent activities against hDHODH at the
concentration of 10 μM. In particular, compound A14 displayed an IC50 value of 0.178 μM with
2-fold preference over A771726. The result implied that a proper degree of steric size and
electron density of the C-6 amide moiety was necessary to retain the inhibitory activity of the
synthesized compounds.
Keywords:
1,3-benzoazole
hDHODH
2009 Elsevier Ltd. All rights reserved.
Activity
Amide
Scaffold hopping
———
 Corresponding author. Tel.: +86-21-64253540; fax: +86-21-64252603; e-mail: lizhong@ecust.edu.cn.
 Corresponding author. Tel.: +86-21-64253967; fax: +86-21-64252603; e-mail: shaoxusheng@ecust.edu.cn

Dihydroorotate dehydrogenase (DHODH) is an enzyme
essential to the fourth and rate-limiting step in de novo
pyrimidine biosynthesis1 and it catalyzes the conversion of
dihydroorotate (DHO) to orotate concurrent (ORO) with the
reduction of ubiquinone.2,3 The significance of pyrimidine bases
for metabolism, cell and proliferation determines hDHODH as an
attractive chemotherapeutic target for the development of new
drug candidate in different biological and clinical applications for
cancer, arthritis and malaria.4,5
Figure 2. Molecular design of title compound
Leflunomide (1) and brequinar (2) are the most successful
examples of low-molecular weight inhibitors of hDHODH that
have been in clinical development (Figure 1).6,7 Leflunomide (1)
is the first hDHODH inhibitor8 that was approved for use in
human medicine in the treatment of rheumatoid arthritis9-11 and
turns out to be a pro-drug to the active metabolite A771726 (3),12-
As described in Scheme 1, under the action of 1-ethyl-3-(3-
dimethyllaminopropyl) carbodiimide hydrochloride and N-
hydroxybenzotriazole, the treatment of benzoic acids 4 with
appropriately substituted amines afforded the benzamide
derivatives 5 in 65 - 72% yields. The catalytic hydrogenation of
intermediates 5 with 15% Pd/C in methanol afforded the key
aniline derivatives 6 which were used for the further reaction
without purification. The methyl of 3-methoxybenzamide 8 was
removed in the presence of boron tribromide to yield the
resulting compound 9. A sample of aldehyde 12 was generated
by the reduction of pyrazole carboxylic acid 10 to 11 with
lithium aluminum hydride in dry tetrahydrofuran and then
oxidation of 11 to 12 with pyridinium chlorochromate. The
intermediate carboxylic acid 10 was synthesized according to the
methods reported in the literature. 34, 35
18
while brequinar (2) is an antitumor and immunosuppressive
agent
which
shows
immunosuppressive
activity.19-21
Unfortunately, severe side effects like leukocytopenia, mucositis
and abnormalities in liver enzymes have been observed during
clinical use of brequinar and lefluonomide.22,23 Consequently,
more efficient hDHODH inhibitors are needed as potential
prototypes for synthesis of new hDHODH inhibitors.
The application of heterocycles as amide bioisosteres is a
significant utility in drug design as the surrogates may lead to
compounds with enhanced pharmacokinetic and improved cell-
based potency properties.24 As an important biologically active
nucleus, 1,3-benzoazole is documented to exhibit widespread
potential pharmacological activities like anticancers, anti-
inflammatory agents, proton pump inhibitors, and etc.25-29
Simultaneously, 1,3-benzoazole has evolved as an effective
chemical isosteres of amide bonds and been widely used in
modern molecular design.30
Moreover, it is well documented that pyridine pyrazole is a
very important class of fused heterocycle due to its specific
physiological activity and the structural similarity with indole
and azaindole. N-aryl pyrazole analogs have shown potent
DHODH inhibitory activity with IC50 ranging from 13-100 nM.31-
33
Scheme 1. Synthesis of intermediates
The last synthetic route employed for the synthesis of the
target compounds A (C) was outlined in Scheme 2. The mixture
of aniline derivatives 6 and aldehyde 12 was heated at reflux
overnight in 1, 2-dichloroethane to get the title compound A. The
synthesis of compound B were similar to those of A3 in which 2-
hydroxy-3-nitrobenzoic acid was used as the starting material.
Surprisingly, the treatment of 9 and 12 generated the Schiff base
13 instead of the cyclization compound C under the same
condition. And the target compound C was finally obtained by
the oxidization of 13 with 3-dichloro-5,6-dicyano-1,4-
benzoquinone in dichloromethane in good yield.
Figure 1. Structures of representative inhibitors of hDHODH
Enlightened by all of the descriptions above, scaffold hopping
based on bioisosteres were proposed to identify new possible
chemotypes. The amide−aryl segment of the monocyclic series of
leflunomide (1) was replaced by a bicyclic1,3-benzoazole
scaffold and pyridylpyrazolyl was introduced into the
benzimidazole core structure to investigate whether there would
present some new beneficial pharmacokinetic properties (Figure
2). Herein, we described the molecular design, synthesis and
initial findings on inhibitory activities against hDHODH.

on the activity. Further investigations were performed to study
the effect of various substituted phenyls at R. Compounds A11
with o-methylphenyl and compound A14 with o-methoxy group
dramatically showed higher enzyme inhibitory activities than that
of the other mono-substituted counterparts, which implied that
inhibitory activity might be influenced by the size of the ortho-
substituent. Notably, the activity level of A14 rivaled that of
A771726, making it to be the most potent compound. Given the
potency observed with 2-methoxy analogs A14, additional
compounds featuring this element were explored. Nevertheless,
introducing electron-donating groups, electron-withdrawing
groups, or halogen atoms at different positions all showed
reduced potency compared to that A14. Among bis-substituted
compounds, the activity data revealed a clear preference for the
potency when substitution at C5-position was CF3 (A26) as
compared to that of OCH3 (A24), which indicated that an
electron-withdrawing group at the C5-position of 2-
methoxyphenyl contributed to the potency of synthesized
compounds. And, replacement of phenyl group at R position with
pyridine group to generate analogue A7 resulted in a sharp
decline of potency. The pyridine unit probably was an ineffective
group for activity. The biological activity data suggested that a
proper degree of electron density and steric size at R position was
necessary to retain the activity of the synthesized compounds,
which paved the way for further optimizations.
Scheme 2. The synthetic route for preparation of target compounds A, B
and C.
In order to explore SAR preliminarily, the compounds with
different groups at R position were synthesized and evaluated for
enzyme inhibition assay. And A771726 (teriflunomide/aubagio)
was used as a positive control. The inhibitory activities as well as
the IC50 of synthesized compounds were listed in Table 1. Firstly,
compounds (A1 - C) derived from alkyl groups exhibited lower
activity than the corresponding benzylated and phenylated
counterparts (A4 - A26) in following rank order: alkyl< benzyl<
phenyl. The results implied that the enzyme inhibition activity
might be influenced by the size or electronic effect of the
substituents. For comparison of benzoxazole and benzimidazole,
compound B and C displayed no obvious improvement over
analogue A3, suggesting that the skeleton had no apparent effect
Table 1. The inhibition activities against hDHODH of compounds A, B and C
Compound
R
Inhibition (%) at 10 μM
IC50 (μM)
Me
14.14
24.85
23.44
26.57
30.10
34.94
A1
A2
A3
B
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
Et
n-Pr
-
-
C
Benzyl
A4
4-CF3-benzyl
4-CH3-benzyl
32.04
22.74
A5
A6
n.t.
n.t.
n.t.
n.t.
n.t.
2-Picoline
27.58
43.04
43.99
A7
A8
A9
2,3,6-(F)3-benzyl
4-Cl-benzyl
4-OCH3-phenyl
36.06
A10
n.t.
2-CH3-phenyl
Phenyl
73.88
49.44
36.29
A11
A12
A13
0.676 ± 0.091
n.t.
4-CF3-phenyl
n.t.

2-OCH3-phenyl
60.51
A14
0.178 ± 0.065
2-F-phenyl
38.83
27.63
24.81
26.89
A15
A16
A17
A18
n.t.
n.t.
n.t.
n.t.
2-Br-phenyl
2-I-phenyl
2,4-(F)2-phenyl
2,4-(OCH3)2-phenyl
2,4-(CH3)2-phenyl
2,6-(OCH3)2-phenyl
1.303
48.76
0
A19
A20
A21
n.t.
n.t.
n.t.
3-CH3-phenyl
18.95
31.85
9.43
A22
A23
A24
n.t.
n.t.
2,3,4-(F)3-phenyl
2,5-(OCH3)2-phenyl
n.t.
n.t.
2-F-6-OCH3-phenyl
15.74
A25
A26
2-OCH3-5-CF3-phenyl
55.22
64.03
4.850 ± 0.230
0.356 ± 0.075
A771726
n.t.: not tested.
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In conclusion, a series of 1, 3-benzoazole derivatives
possessing C-6 amide units were designed, synthesized and
screened for their inhibitory activity against human
dihydroorotate dehydrogenase (hDHODH). Compounds A11,
A14 and A26 exhibited good to excellent activities against
hDHODH at the concentration of 10 μM. Even more remarkable,
compound A14 displayed the highest inhibition activity for
hDHODH with IC50 value of 0.178 μM, and was comparable to
that of A771726. The results indicated that the differences in
inhibitory activity might be due to variations in incorporation of
steric size and electrical property of the C-6 amide substitute
moieties. The relationships between structure and activity
obtained in this study could be beneficial for discovering new
hDHODH inhibitors and further chemo-biological optimization
on benzoheterocycle derivatives is well ongoing in our
laboratory.
Acknowledgments
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This work was financial supported by National Natural
Science Foundation of China (21472046, 21372079).
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