Design and synthesis of novel 2-arylbenzimidazoles as selective mutant isocitrate dehydrogenase 2 R140Q inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.127070

A series of novel 2-arylbenzimidazoles have been designed, synthesized and evaluated for their inhibitory activity against IDH2 R140Q mutant. The preliminary results indicated that four compounds 7b, 7c, 7m and 7r displayed the potent inhibitory activity against IDH2 R140Q mutant. Among them, compound 7c showed the highest inhibitory activity, with the IC₅₀ value of 0.26 μM, which was more active than positive control enasidenib. The exquisite selectivity of 7c for IDH2 R140Q mutant isoform was demonstrated by the poor activity against the IDH1 R132C mutant, IDH1 R132H mutant, wild-type IDH1, IDH2 R172K mutant and the wild-type IDH2.

Full text of DOI:10.1016/j.bmcl.2020.127070

Journal Pre-proofs
Design and synthesis of novel 2-arylbenzimidazoles as selective mutant isoci-
trate dehydrogenase 2 R140Q inhibitors
Zhenyu Li, Xingkang Wu, Lejiao Jia, Jun Li, Rui Zhang, Hui Tang, Zhiying Li,
Huagang Bu, Chengwu Shen
PII:
S0960-894X(20)30148-7
DOI:
https://doi.org/10.1016/j.bmcl.2020.127070
BMCL 127070
Reference:
Bioorganic & Medicinal Chemistry Letters
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21 October 2019
26 February 2020
28 February 2020
Please cite this article as: Li, Z., Wu, X., Jia, L., Li, J., Zhang, R., Tang, H., Li, Z., Bu, H., Shen, C., Design and
synthesis of novel 2-arylbenzimidazoles as selective mutant isocitrate dehydrogenase 2 R140Q inhibitors,
Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127070
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Design and synthesis of novel 2-arylbenzimidazoles as selective
mutant isocitrate dehydrogenase 2 R140Q inhibitors
Zhenyu Lia, Xingkang Wub, Lejiao Jiac, Jun Lia, Rui Zhanga, Hui Tanga, Zhiying Lid, Huagang
Bud, and Chengwu Shena,

aDepartment of Pharmacy, Shandong Provincial Hospital Affiliated to Shandong University, Jinan
250021, P. R. China
bModern Research Center for Traditional Chinese Medicine, Shanxi University, Taiyuan, Shanxi,
030006, P. R. China
cDepartment of Pharmacy, Shandong University Qilu Hospital, No. 107 West Wenhua Road, Jinan
250012, Shandong, P. R. China
dKey Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences,
Shandong University, No. 44 West Wenhua Road, Jinan 250012, Shandong, P. R. China
Abstract
A series of novel 2-arylbenzimidazoles have been designed, synthesized and evaluated
for their inhibitory activity against IDH2 R140Q mutant. The preliminary results
indicated that four compounds 7b, 7c, 7m and 7r displayed the potent inhibitory
activity against IDH2 R140Q mutant. Among them, compound 7c showed the highest
inhibitory activity, with the IC50 value of 0.26 μM, which was more active than positive
control enasidenib. The exquisite selectivity of 7c for IDH2 R140Q mutant isoform
was demonstrated by the poor activity against the IDH1 R132C mutant, IDH1 R132H
mutant, wild-type IDH1, IDH2 R172K mutant and the wild-type IDH2.
Keywords: 2-Arylbenzimidazoles; Synthesis; IDH2 R140Q; Inhibitors
*Corresponding author. Tel./Fax: +86 0531 68778252; e-mail: scw810@126.com
1

Isocitrate dehydrogenase (IDH) is one of the metabolic enzymes most closely
associated with cancer risk and clinical outcome.1 In the tricarboxylic acid cycle, IDH
catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) using
Mg2+ and NADP+ (or NAD+) as cofactors, participating in various molecular processes
including histone and DNA modifcations.2-6 Heterozygous point mutations in the active
site arginine residues of IDH1 (R132) and IDH2 (R140 and R172) are observed in
cancers including low-grade gliomas, secondary glioblastomas, angioimmunoblastic T-
cell lymphomas, acute myeloid leukemia (AML) and so on.7-11 Approximately 10-40%
of patients with AML carry mutations in the IDH1/2 gene, making the two proteins
promising therapeutic targets in AML.4, 12 In AML patients with IDH2 mutations, R140
mutations occur in approximately 80% of patients as compared to R172 mutations that
occur in only 20%.13
IDH2 mutations confer a gain-of-function and lead to neomorphic enzymatic
activity of the mutated IDH enzyme.14 Instead of catalyzing the conversion of isocitrate
to α-KG, R172 and R140 mutant IDH2 both catalyze the conversion of α-KG to 2-
hydroxyglutarate (2-HG) (Fig. 1).15 Supra-normal levels of intracellular 2-HG lead to
hypermethylation of target genes and a block in cellular differentiation.16 Currently, the
IDH2 R140Q inhibitor enasidenib has been approved by FDA for the clinical treatment
of IDH2 mutation-positive AML (Fig. 2), which opens up a new avenue for the therapy
of AML patients. Therefore, it is essential to develop novel IDH2 R140Q inhibitors
with improved potency and exquisite selectivity.
Fig. 1 Reactions catalyzed by wild-type and mutant IDH2.
2

Fig. 2 Chemical structure of Enasidenib approved by the FDA for AML treatment.
Treatment with AGI-6780 induced differentiation of TF-1 erythroleukemia and
primary human AML cells in vitro. These data provide proof-of-concept that inhibitors
targeting mutant IDH2 R140Q could have potential applications as a differentiation
therapy for cancer.16 However, the selectivity of AGI-6780 for inhibiting IDH2 R140Q
is not ideal. Due to the cocrystal structure of IDH2 R140Q/AGI-6780 reported,16 it is
possible to develop novel IDH2 R140Q inhibitors through structure-based drug design.
In this study, we take AGI-6780 as a lead compound, and scaffold hopping strategy was
used to design a novel series of 2-arylbenzimidazoles as IDH2 R140Q mutant inhibitors
(Fig. 3). In the designed compounds, various groups were introduced to investigate the
structure-activity relationship (SAR).
Fig. 3 Design strategy of novel 2-arylbenzimidazoles IDH2 R140Q inhibitors.
Chlorosulfonylation of commercially available 2-nitrobromobenzene (1) to obtain
intermediate (2), followed by reaction with cyclopropylamine afforded the
cyclopropylsulfonamide (3). Palladium catalyzed cross-coupling of the aryl bromide
with 3-thiopheneboronic acid yielded the biaryl sulfonamide (4). Reduction of the aryl-
3

nitro group with iron yielded the aniline (5), which was reacted with di-2-pyridyl
thionocarbonate to afford the isothiocyanate (6). The title compounds 7a-t were
prepared by the condensation of the isothiocyanate 6 with various substituted o-
phenylenediamines, respectively (Scheme 1). The spectral data for all the synthesized
compounds are provided in the supporting information.
Scheme 1 Reagents: (i) chlorosulfonic acid, 120°C ; (ii) cyclopropylamine, DIEPA,
dichloromethane, room temperature; (iii) 3-thiopheneboronic acid, Pd2Cl2(dppf), cesium carbonate,
dioxane, 100°C ; (iv) ammonium chloride, iron powder, methanol, H2O, 80°C, 5 h; (v) di-2-pyridyl
thionocarbonate, acetonitrile, overnight, 40 ºC; (vi) substituted o-phenylenediamine, room
temperature.
Setting AGI-5198, AGI-6780 and enasidenib as the positive controls, all the
synthesized compounds were evaluated for their abilities to inhibit the IDH2 R140Q
mutant in an enzyme-based assay. The results were expressed in IC50, which
represented the concentration of the tested compounds for 50% IDH2 R140Q mutant
inhibition. The selectivity of the derivatives with IC50 values less than 1 μM were then
tested for their inhibitory activity against IDH1 R132C mutant, IDH1 R132H mutant,
wild-type IDH1, IDH2 R172K mutant and the wild-type IDH2.
Table 1 In vitro IDH2 R140Q mutant inhibitory activities
4

Compd
R
H
IC50 (μM)
5.41±0.31
0.73±0.09
0.26±0.04
3.63±0.51
11.89±4.57
2.02±0.13
46.94±5.09
81.00±15.14
97.87±18.02
14.09±4.68
>100
7a
2-Cl
7b
2-Br
7c
2-NO2
3-F
7d
7e
3-Br
7f
3-OMe
3-NO2
3-CN
7g
7h
7i
3-CF3
7j
3-COOMe
3-COOEt
2,4-2Me
2,4-2F
3,4-2Me
3,4-2F
3,4-2Cl
3,4-2Br
3-F,4-Cl
3-CF3,4-Cl
7k
>100
7l
0.96±0.07
4.94±0.30
1.61±0.09
13.80±2.14
2.72±0.16
0.65±0.09
10.35±2.03
4.34±0.25
0.06±0.02
7m
7n
7o
7p
7q
7r
7s
7t
AGI-6780
Enasidenib
0.31±0.09
In general, from the obtained data, it was observed that some of the 2-
arylbenzimidazoles, 7b, 7c, 7m and 7r displayed remarkable IDH2 R140Q mutant
inhibitory activity. Particularly, the compound 7c showed the highest inhibitory activity
against IDH2 R140Q mutant, with the IC50 value of 0.26 μM, which was more active
than positive control enasidenib.
The SARs analysis showed that the nature of the substituents greatly influenced
the IDH2 R140Q mutant inhibitory activity of these compounds. In the monosubstituted
series, the inductive effects of substituents can significantly reflect active sequence of
the derivatives. It followed the rules: the weaker the negative inductive effect is, the
5

stronger the activity is, e.g. 7c (2-Br) > 7b (2-F) and 7f (3-Br) > 7e (3-Cl) > 7j (3-CF3) >
7h (3-NO2). Moreover, the halogen disubstituted series also followed the rules, e.g. 7r
(3,4-2Br) > 7q (3,4-2Cl) > 7p (3,4-2F).
In the disubstituted series, when substituents were electron-donating methyl groups,
they were more active than electron-withdrawing fluoro-substituted derivatives, e.g.
7m (2,4-2Me) > 7n (2,4-2F) and 7o (3,4-2Me) > 7p (3,4-2F). However, electron-
donating methyl-substituted derivatives were less active than weak electron-
withdrawing bromo-substituted derivatives, e.g. 7r (3,4-2Br) > 7o (3,4-2Me),
illustrating that the induction effect of substituents greatly affects the activity of
compounds.
Table 2 Selectivity profiling of potent 2-arylbenzimidazoles
IDH1
WT
IDH1
IDH1
IDH2
WT
IDH2
IDH2
R132C
R132H
R140Q
R172K
Compd
IC50 (μM)
>100
IC50 (μM) IC50 (μM) IC50 (μM) IC50 (μM) IC50 (μM)
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.73±0.09
0.26±0.04
0.96±0.07
0.65±0.09
>100
78.8
>100
7b
7c
>100
>100
>100
>100
51.8
7m
>100
>100
>100
>100
7r
>100
0.39±0.07
>100
0.17±0.01
>100
>100
AGI-5198
AGI-6780
Enasidenib
0.06±0.02
0.31±0.09
>100
10.39±1.61
1.08±0.23
>100
>100
>100
As the SAR investigation revealed functional group modifications that provided
potent inhibitors in the IDH2 R140Q mutant enzymatic assay, we selected a focused
set of analogs for evaluation against IDH1 R132C mutant, IDH1 R132H mutant, wild-
type IDH1, IDH2 R172K mutant and wild-type IDH2. As shown in Table 2, 7b, 7c, 7m
and 7r showed exquisite selectivity for IDH2 R140Q mutant subtype and was
demonstrated by the poor activity against the IDH1 R132C mutant, IDH1 R132H
mutant, wild-type IDH1, IDH2 R172K mutant and the wild-type IDH2.
To examine the possible binding mode between 7c and IDH2 R140Q, docking
analysis was performed using Autodock vina 1.1.217 and the results were shown in Fig.
4. The maximum binding affinity between 7c and the IDH2 R140Q was predicted to be
-6.8 kcal/mol. In comparison with AG-6780, the binding mode of 7c was similarly
based on the reported crystal structure of AG-6780 (Fig. 4A). The 3-thienyl group of
6

the compound 7c located at the hydrophobic pocket, surrounded by the residues B/Val-
297, B/Trp-306 and B/Ile-319, while cyclopropane ring of the compound 7c stretched
into another hydrophobic pocket that consisted of A/Leu-160, A/Trp-164, A/Val-297,
A/Leu-298 and A/Leu-320, forming a strong hydrophobic binding (Fig. 4B). Detailed
analysis showed that the benzimidazole scaffold of the 7c formed anion-π interactions
with the residues A/Asp-312 and B/Asp-312, respectively. Importantly, the four key
hydrogen bond interactions was observed between the 7c and the residues A/Gln-316
(bond lengths: 2.0 and 3.5 Å) and B/Gln-316 (bond lengths: 2.8 and 3.2 Å), which was
the main interaction between the 7c and the IDH2 R140Q (Fig. 4B). All these
interactions helped 7c to anchor in the binding site of IDH2 R140Q. In summary, the
above molecular simulations give us rational explanation of the interactions between
the 7c and the IDH2 R140Q, which provided valuable information for the development
of the IDH2 R140Q inhibitors.
Fig. 4 Molecular docking simulation of the interactions between 7c and IDH2 R140Q. (A)
Compound 7c was docked into the binding site of IDH2 R140Q with AGI-6780 overlapped (total
view). (B) Detailed view of the binding mode between 7c and IDH2 R140Q. IDH2 R140Q was
represented with cartoon (chain A was colored with green, and chain B was colored with cyan), and
the representative binding residues were shown in lines; 7c and AGI-6780 were represented with
rose red and violet sticks, respectively. The hydrogen bonds were shown as yellow dotted lines.
In conclusion, a series of novel 2-arylbenzimidazoles were designed, synthesized
and biologically evaluated for their inhibitory activity against IDH2 R140Q mutant.
Compounds 7b, 7c, 7m and 7r showed exquisite selectivity for IDH2 R140Q mutant
7

subtype and demonstrated poor activity against the IDH1 R132C mutant, IDH1 R132H
mutant, wild-type IDH1, IDH2 R172K mutant and the wild-type IDH2. Particularly,
the compound 7c showed the highest inhibitory activity against IDH2 R140Q mutant,
with the IC50 value of 0.26 μM. Taken together, our findings in the present study provide
valuable information on novel structures, which presents a most promising lead for
further investigation.
Acknowledgments
This work was supported by the National Natural Science Foundation of China project
(No. 81502921), Programs of Science and Technology of Higher Education of Shanxi
Province (No. 2019L0094) and Career Development Funds of Shanxi University (No.
226545031).
Conflict of interest
The authors declare no conflict of interest.
8

References
1. Fangrui W, Hong J, Baisong Z, et al. Journal of medicinal chemistry 2015; 58:6899.
2. Xu X, Zhao J, Xu Z, et al. Journal of Biological Chemistry 2004; 279:33946.
3. Chen J, Yang J, Sun X, et al. Scientific Reports 2017; 7:16458.
4. Medeiros B C, Fathi A T, DiNardo C D, et al. Leukemia 2017; 31:272.
5. Waitkus M S, Diplas B H, Yan H. Neuro-oncology 2016; 18:16.
6. Sim H-W, Nejad R, Zhang W, et al. Journal of Clinical Oncology 2017; 35:15_suppl:2037.
7. De Carli E, Wang X, Puget S. The New England Journal of Medicine 2009; 360:2248.
8. Dang L, Jin S, Su S M. Trends in Molecular Medicine 2010; 16:387.
9. Cairns R, Iqbal J, Lemonnier F, et al. Blood 2012; 119:1901.
10. Kao H-W, Liang D-C, Wu J-H, et al. Neoplasia 2014; 16:481.
11. Lin C C, Hou H A, Chou W C, et al. American Journal of Hematology 2014; 89:137.
12. DiNardo C D, Ravandi F, Agresta S, et al. American Journal of Hematology 2015; 90:732.
13. Stein, M. E. Best Practice & Research Clinical Haematology 2015; 28:112.
14. Green C L, Evans C M, Lu Z, et al. Blood 2011; 118:409.
15. Losman J-A, Kaelin W G. Genes & Development 2013; 27:836.
16. Wang F, Travins J, Delabarre B, et al. Science 2013; 340:622.
17. Trott O, Olson A J. Journal of Computational Chemistry 2010; 31:455.
9

Highlights
●Synthesis of novel IDH2 R140Q inhibitors with benzimidazole scaffold via six steps
reaction.
●Selectivity of 7c for IDH2 R140Q mutant was the best among the twenty compounds.
●7c might be a lead compound deserved further structural optimization.
10

Graphical abstract
The exquisite selectivity of 7c for IDH2 R140Q mutant isoform was demonstrated by
the poor activity against the IDH1 R132C mutant, IDH1 R132H mutant, wild-type
IDH1, IDH2 R172K mutant and the wild-type IDH2.
11