Discovery of a novel class of pyridine derivatives that selectively inhibits mutant isocitrate dehydrogenase 2

Article abstract of DOI:10.1111/cbdd.13139

This paper presents synthesis and structure–activity relationship of pyridine derivatives as inhibitors of mutant isocitrate dehydrogenase 2 (IDH2). A series of 2,4,6-trisubstituted pyridine derivatives have been prepared and evaluated in vitro. Among these compounds, 14n exhibited excellent inhibition activity with the IC₅₀ of 54.6?nm, which is approximately onefold improvement compared to drug candidate AG-221 (Enasidenib) that is in Phase III trial. Exquisite selectivity of 14n for IDH2 R140Q mutant isoform was demonstrated by the poor activity against the wild-type IDH1 and IDH2.

Full text of DOI:10.1111/cbdd.13139

MR. GUOYI YAN (Orcid ID : 0000-0002-3953-6190)
PROFESSOR YUANWEI CHEN (Orcid ID : 0000-0001-5405-7828)
Article type
: Research Article
Discovery of a novel class of pyridine derivatives that
selectively inhibits mutant Isocitrate dehydrogenase 2
Fangying Wang 1, †, Zhuolin Li 1, †, Tao Zhang 1, Guoyi Yan 1, Mingxing Hu 1, Lifeng Zhao 3,
1. 2, *
*, Yinglan Zhao 1, *, Yuanwei Chen
1 State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of
Biotherapy, West China Hospital, West China Medical School of Sichuan University,
Chengdu 610041, China.
2 Hinova Pharmaceuticals Inc., Suite 402, Building B, #5 South KeYuan Road, Chengdu,
610041, China.
3 Chengdu University, Sichuan Industrial Institute of Antibiotics, Chengdu 610052, China.
† These authors contributed equally to this work.
Running Title: pyridine derivatives as mutant IDH2 inhibitors
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13139
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Correspondence
Lifeng Zhao: lifengzhao@scu.edu.cn (L-f Zhao)
Yinglan Zhao: zhaoyinglan@scu.edu.cn (Y-l Zhao)
Yuanwei Chen: ywchen@scu.edu.cn (Y-w Chen)
Phone: 86-28-85164063; Fax: 86-28-85164060.
Keywords: IDH; IDH2 R140Q mutation; pyridine derivatives; enzyme-based assay; SAR
study.
ABSTRACT
1. Current paper presents synthesis and Structure-Activity Relationship of pyridine
derivatives as inhibitors of mutant isocitrate dehydrogenase 2 (IDH2). A series of
2,4,6-trisubsitituted pyridine derivatives have been prepared and evaluated in vitro.
Among these compounds, 14n exhibited excellent inhibition activity with the IC50 of 54.6
nM, which is approximately 1-fold improvement compared to drug candidate AG-221
(Enasidenib) that is in Phase III trial. Exquisite selectivity of 14n for IDH2 R140Q
mutant isoform was demonstrated by the poor activity against the wild-type IDH1 and
IDH2.
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Introduction
Metabolic enzymes are important targets for cancer research and anti-cancer drug
discovery.[1, 2] Isocitrate dehydrogenase (IDH) is one of the metabolic enzymes most closely
associated with cancer risk and clinical outcome. In the tricarboxylic acid cycle, IDH
catalyzes the oxidative decarboxylation of isocitric acid (ICT) to α-ketoglutaric acid (α-KG)
using Mg2+ and NADP+ (or NAD+) as cofactors.[3] There are three IDH isozymes in humans,
with IDH1 located in cytoplasm and IDH2 and 3 located in mitochondria.[4, 5] Point mutations
affecting IDH1 Arg132 (R132) and IDH2 Arg140 or Arg172 (R140 or R172) are observed in
a wide range of cancers including low-grade gliomas, secondary glioblastomas (GBM),
melanoma, angioimmunoblastic T-cell lymphomas, myeloproliferative neoplasms,
myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML).[6-10] Moreover,
clinical data implicates the IDH mutation is an early event in cancer initiation and
development.[11, 12]
Cancer-associated point mutations in IDH1 and IDH2 result in loss-of-function for the
oxidative decarboxylation of isocitrate and confer a neomorphic activity which allows
reduction of α-KG to the oncometabolite R-2-hydroxyglutarate (2-HG).[13] High
concentrations of 2-HG have been shown to competitively inhibit α-KG dependent
dioxygenases, including histone and DNA demethylases.[14, 15] Several studies have
demonstrated that 2-HG producing IDH mutants can cause hypermethylation of histone and
DNA which may contribute to tumorigenesis.[16, 17] Taken together, these findings provide
proof-of-concept that mutant IDH1/2 may act as an oncogene and a compelling drug target
for new therapies.
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A majority of representative small-molecule inhibitors of mutant IDH1 that are under
investigation are showed in Figure 1A.[18-25] These compounds have reduced 2-HG at cellular
level and showed on-target activity in cancer cells. On the other hand, it has been reported
that the mutant IDH2 plays an dominant role in the occurrence and development of
hematologic malignancies, as well as Grade II/III gliomas and GBM,[11, 12] with the IDH2
R140Q mutation being the most frequent (75%–80%) and the IDH2 R172K mutation being
the second (20%).[26] Despite the high mutation rate of IDH2, there are only 2 inhibitors of
mutant IDH2 reported in literature (Figure 1B): a preclinical candidate AGI-6780[27] and a
Phase III compound AG-221.[28] The later compound showed drug-related adverse effects
which included differentiation syndrome, leukocytosis and nausea.[29] New inhibitors of
mutant IDH2 with improved activity and safety profile are needed. Thus, we devote our effort
to develop small molecules that can selectively inhibit IDH2 R140Q mutant.
The structure of AG-221 has 4 key moieties: the triazine central core and three
substituents: a nitrogen linked heterocycle A, another nitrogen linked alkyl R group, and an
aromatic moiety B (Figure 2). In addition to this, the heterocycle A and B were capped with
a trifluoromethyl, respectively. We explored similar lay out with pyridine as the central core.
Herein, we reported the synthesis, biological evaluation, and structural–activity relationship
of novel IDH2 R140Q mutant inhibitors based on 2,4,6-trisubsitituted pyridine scaffold in the
study.
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2. Experimental section
2.1 Chemistry
The general syntheses of these compounds are summarized in schemes 1, 2 and 3.
Compounds 14a–14p and 17a-17d were prepared in sequential steps as described in scheme
1. As depicted in scheme 1, 2-chlorin-4-iodopyridine (11) was chosen as substrate. When
using isopropylamine as amine component, it was found that the Buchwald-Hartwig coupling
progressed exclusively in 4-position of 11 in 78% yield due to the higher reactivity of the
iodine.[30] In another route, a competing nucleophilic substitution proved to be progressed in
positions 2 and 4 of the reactant 11. The potent electron-withdrawing chlorine group of the
pyridine facilitated selective substitution at 2-position of 11. [31] Intermediate 13, achieved
through the Suzuki coupling reaction, was reacted with various arylamines and
heteroarylamines to afford 14a-14p. The intermediate 15, obtained through the Nucleophilic
substitution reaction, was reacted with different heteroarylamines to afford 16a–16d.
Compounds 17a-17d were prepared through the Suzuki coupling. Applying similar reactions,
compounds 19a-19i, 21a-21e and 24a-24b were prepared according to schemes 2 and 3.
2.2 Enzyme-based assay
Compounds were evaluated their abilities to inhibit the IDH2 R140Q mutant in an
enzyme-based assay. The results were expressed in inhibition% at 0.5 µM, 2 µM and IC50.
The inhibition% at 0.5 µM or 2 µM presented the relative response (%) of the tested
compounds compared to DMSO, while the IC50 values represented the concentration of the
tested compounds for 50% IDH2 R140Q mutant inhibition.
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3. Results and discussion
The bioactivity results of compounds 14 series is showed in Table 1. The readily
obtained compound 14a was firstly synthesized. In view of the poor activity of the compound
14a and the structure characteristic of AG-221, we decide to introduce a potent
electron-withdraw group trifluoromethyl on the ortho-, meta- and para- position of the
benzene ring, respectively. Gratifyingly, compound 14c is preferable to 14b and 14d with an
IC50 value of 287 nM, while compound 14c still has a large discrepancy with the positive
control AG-221 of which the IC50 value was 86.3 nM. To investigate the structure activity
relationship more clearly, we replace trifluoromethyl with halogens (14e, 14f) and the
electron-donating groups (14g, 14h). In comparison to compound 14c, other groups on
aromatic ring were detrimental to the activity. When group A was a pyridine ring, we
suspected the position of nitrogen atom may have an impact on activity. Although there is no
vital difference among them (14i, 14j, 14k), the replacement of pyridine to benzene have a
slight improvement. Encouraged by this subtle change, we introduce some meta-substitution
trifluoromethylpyridine. Evaluation of the substituents revealed that replacement of the
phenyl group with heteroaryl group is much more effective in the presence of the
meta-substitution trifluoromethyl. Interestingly, 14n showed an IC50 value of 54.6 nM, which
exhibited approximate 1-fold improvement than AG-221. The second most active compound
is 14l, with an IC50 value of 190 nM. Compounds substituted with other six-membered
heteroaryl ring such as 14p showed moderate activity.
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Next, we tried to investigate the influence of the position of nitrogen atom in the
pyridine nucleus (17a-17d). However, all compounds exhibited reduced potency compared to
14l-14o.
On the basis of the favorable activity of the compounds 14n and 14l, we then continued
to explore the SAR of heteroaryl B. Replacing the benzene ring in compound 14l with
3-Trifluoromethylphenyl ring (19b) improved bioactivity almost 3-fold, while analog (19a)
offered a moderate improvement in binding affinity. Simultaneously, halogens (19d, 19e) and
the electron-donating groups (19f, 19g) were used to replace the trifluoromethyl.
Satisfactorily, 3-Chlorophenyl ring 19e restored the bioactivity to 59.7 nM, while the use of
substituted pyridine group (19h, 19i) reduced even lost the activity. Evaluation of Ar4
substituents revealed that ortho-substitution was most favored (21a) for the aromatic ring,
while replacement of the phenyl group with cyclohexene afforded only micromolar potency
analogs. Furthermore, 3,5-Bis(trifluoromethyl)benzene ring 21d showed reduced potency.
Finally, we tested several alkyl and aromatic amines with the aim of improving potency
further. However, isopropylamine is preferable to the aminomethylcyclopropane and
benzylamine (24a, 24b).
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 R132H mutant, wild-type IDH1 and wild-type IDH2. As shown
in Table 2, 14n showed modest activity against the IDH1 R132H mutant and its exquisite
selectivity for IDH2 R140Q mutant subtype was demonstrated by the poor activity against
the wild-type IDH1 and the wild-type IDH2.
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To examine the possible binding modes of 14n, docking analysis was performed using
Autodock 4.0.[32] The crystal structure of IDH2 R140Q mutant (PDB 4JA8)[27] was used. Two
hydrogen bonds interaction between 14n and IDH2 R140Q mutant were identified. The
fluorine of the trifluoromethyl group formed a hydrogen bond with Gln316, while the NH of
the isopropylamine group formed additional hydrogen bond with the carbonyl of Gln316. The
benzene moiety formed hydrophobic interactions with the side chains of Trp164, Val297,
Trp306, Leu320 and Leu298 (Figure 3). In comparison with AG-221, the binding mode of
14n was similar based on the reported crystal structure of AG-221 and IDH2 R140Q
mutant.[33] The hydrogen bonds between inhibitors and Gln316 and the hydrophobic
interactions with the side chains of Trp164, Val297, Leu320 and Leu298 contributed to the
high inhibitory potency of the two inhibitors. However, that no hydrogen bond was formed
between 14b and Gln316 is a reason for 14b with no activity.
4. Conclusion
In summary, we have synthesized a series of 2,4,6-trisubsitituted pyridine derivatives as
IDH2 R140Q mutant inhibitors. Compounds 14n, 19b and 19e were found to have potent
inhibition activity, which were preferable to the positive control AG-221. 14n, with an IC50
value of 54.6 nM, exhibits very weak activity against wild-type IDH1 and IDH2 and shows a
high selectivity of >550-fold. Taken together, our findings in the present study provide
valuable information on novel structures, which hold a great potential for further
development and optimization.
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Acknowledgments
The authors greatly appreciate the financial support from National Natural Science
Foundation of China (grant 81472418 and 81672951).
Conflict of Interest
The authors have declared no conflict of interest.
Supplementary Materials
Synthetic details, characterization data for all compounds and biological procedure
reported in this manuscript are described in Method S1. NMR Spectra Data are showed in
Figure S1.
Methods S1. Synthetic procedure.
Figure S1. NMR Spectra Data of Synthetic Compounds.
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Caption
Figure 1. Examples of IDH1 mutant inhibitors (A) and IDH2 R140Q mutant inhibitors (B)
Figure 2. Four important parts of the Phase III compound AG-221 and the scaffold of
2,4,6-trisubsitituted pyridine derivatives
Scheme 1. Synthesis of Compounds 14a-14p and 17a-17d
Reagents and conditions: (I) Isopropylamine, Cs2CO3, Pd(OAc)2, BINAP, Toluene, 100
℃, 12 hr, yeild 78%; (II) Pd(dba)2, S-Phos, K3PO4, Phenylboronic acid, Toluene, 110℃, 18
hr, yeild 46%; (III) Cs2CO3, Pd(OAc)2, BINAP, Ar1-NH2, Dioxane, 80℃, 24 hr, yeild
64%-91%; (IV) Isopropylamine, DIEA, NMP, 150℃, 24 hr, yeild 51%; (V) Pd2(dba)3,
X-Phos, Cs2CO3, Ar2-NH2, Dioxane, 50℃, 24 h, yeild 71%-93%; (VI) Pd(PPh3)4, Na2CO3,
Phenylboronic acid, Dioxane/H2O = 3/1, 90℃, 24h, yeild 83%-95%.
Scheme 2. Synthesis of Compounds 19a-19i and 21a-21e
Reagents and conditions: (I) Cs2CO3, Pd(OAc)2, BINAP,
2-Amino-6-(trifluoromethyl)pyridine, Dioxane, 80℃, 24h, yeild 88%; (II) Pd(PPh3)4,
Na2CO3, Ar3-B(OH)2, Dioxane/H2O = 3/1, 90℃, 24h, yeild 68%-86%. (III) Cs2CO3,
Pd(OAc)2, BINAP, 2-Amino-4-(trifluoromethyl)pyridine, Dioxane, 80℃, 24hr, yeild 77%;
(IV) Pd(PPh3)4, Na2CO3, Ar4-B(OH)2, Dioxane/H2O = 3/1, 90℃, 24 hr, yeild 66%-81%.
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Scheme 3. Synthesis of Compounds 24a-24b
Reagents and conditions: (I) Cs2CO3, Pd(OAc)2, BINAP, R1-NH2, Toluene, 100℃, 12
hr, yeild 55%-62%; (II) Cs2CO3, Pd(OAc)2, BINAP, 2-Amino-4-(trifluoromethyl)pyridine,
Dioxane, 80℃, 24 hr, yeild 56%-67%; (III) Pd(PPh3)4, Na2CO3, Phenylboronic acid,
Dioxane/H2O = 3/1, 90℃, 24 hr, yeild 62%-67%.
Table 1. In vitro IDH2 R140Q mutant inhibitory activities
a, bEnzyme inhibition% compared to DMSO controls at 0.5 µM or 2 µM. Each
compound was tested in triplicate; the data are presented as the mean ± SD. cIC50 values for
enzymatic inhibition of IDH2 R140Q mutant protein. The IC50 values were tested in
triplicate; the data are presented as the mean ± SD.
Table 2. Selectivity profiling of potent 2,4,6-trisubsitituted pyridine derivatives
aThe IC50 values for R140Q, R132H, IDHwt and IDH2wt homodimers are the mean of at
least three determinations performed as described in the Supporting Information, the data are
presented as the mean ± SD.
Figure 3. Proposed binding mode of compound 14n in IDH2 R140Q mutant model
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