Discovery of 8-Membered Ring Sulfonamides as Inhibitors of Oncogenic Mutant Isocitrate Dehydrogenase 1

Article abstract of DOI:10.1021/acsmedchemlett.6b00264

Evidence suggests that specific mutations of isocitrate dehydrogenases 1 and 2 (IDH1/2) are critical for the initiation and maintenance of certain tumor types and that inhibiting these mutant enzymes with small molecules may be therapeutically beneficial. In order to discover mutant allele-selective IDH1 inhibitors with chemical features distinct from existing probes, we screened a collection of small molecules derived from diversity-oriented synthesis. The assay identified compounds that inhibit the IDH1-R132H mutant allele commonly found in glioma. Here, we report the discovery of a potent (IC₅₀ = 50 nM) series of IDH1-R132H inhibitors having 8-membered ring sulfonamides as exemplified by the compound BRD2879. The inhibitors suppress (R)-2-hydroxyglutarate production in cells without apparent toxicity. Although the solubility and pharmacokinetic properties of the specific inhibitor BRD2879 prevent its use in vivo, the scaffold presents a validated starting point for the synthesis of future IDH1-R132H inhibitors having improved pharmacological properties.

Full text of DOI:10.1021/acsmedchemlett.6b00264

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Letter
Discovery of 8-Membered Ring Sulfonamides as Inhibitors
of Oncogenic Mutant Isocitrate Dehydrogenase 1
Jason M. Law, Sebastian C. Stark, Ke Liu, Norah Eileen Liang, Mahmud M Hussain,
Matthias Leiendecker, Daisuke Ito, Oscar Verho, Andrew M. Stern, Stephen E.
Johnston, Yan-Ling Zhang, Gavin P. Dunn, Alykhan F. Shamji, and Stuart L. Schreiber
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00264 • Publication Date (Web): 18 Aug 2016
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Discovery of 8-Membered Ring Sulfonamides as Inhibitors of
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Jason M. Law,1,2 Sebastian C. Stark,1 Ke Liu,1 Norah E. Liang,1,2 Mahmud M. Hussain,1,2,3 Matthias
Leiendecker,1,2 Daisuke Ito,1 Oscar Verho,1,2 Andrew M. Stern,1 Stephen E. Johnston,1 Yan-Ling
Zhang,1 Gavin P. Dunn,4 Alykhan F. Shamji,*1 Stuart L. Schreiber*1,2,3
1Center for the Science of Therapeutics, Broad Institute, Cambridge, MA 02142. 2Department of Chemistry and
Chemical Biology, Harvard University, Cambridge, MA 02138. 3Howard Hughes Medical Institute, Cambridge, MA
02138. 4Department of Neurological Surgery, Washington Univeristy School of Medicine, St. Louis, MO 63110.
KEYWORDS: BRD2879, isocitrate dehydrogenase, 2-hydroxyglutarate, glioma, AML, cancer, diversity-oriented syn-
thesis, high-throughput screening, allele-selective probe, small-molecule probe
ABSTRACT: Evidence suggests that specific mutations of isocitrate dehydrogenases 1 and 2 (IDH1/2) are critical for the
initiation and maintenance of certain tumor types, and that inhibiting these mutant enzymes with small molecules may
be therapeutically beneficial. In order to discover mutant allele-selective IDH1 inhibitors with chemical features distinct
from existing probes, we screened a collection of small molecules derived from diversity-oriented synthesis. The assay
identified compounds that inhibit the IDH1-R132H mutant allele commonly found in glioma. Here, we report the discov-
ery of a potent (IC50 = 50 nM) series of IDH1-R132H inhibitors having 8-membered ring sulfonamides as exemplified by
the compound BRD2879. The inhibitors suppress (R)-2-hydroxyglutarate production in cells without apparent toxicity.
Although the solubility and pharmacokinetic properties of the specific inhibitor BRD2879 prevent its use in vivo, the scaf-
fold presents a validated starting point for the synthesis of future IDH1-R132H inhibitors having improved pharmacologi-
cal properties.
Systematic efforts to characterize the genomes of pa-
tient tumors are revealing the genomic alterations that
cause and maintain different cancers. Somatic mutations
in the genes encoding the isocitrate dehydrogenases IDH1
and IDH2 have been found in >70% of grade II-III gliomas
and secondary glioblastomas,1,2 ~17% of acute myeloid
leukemias (AML),3,4 ~56% of central and periosteal chon-
drosarcoma,5 and sporadically in other tumor types. Mu-
tations are nearly always heterozygous and occur fre-
quently at codons IDH1-R132, IDH2-R172, or IDH2-R140.
IDH enzymes normally catalyze the interconversion of
isocitrate and α-ketoglutarate (α-KG), but these muta-
tions unmask an otherwise cryptic NADPH-dependent
ketoreductase activity, allowing the enzyme to reduce α-
KG to (R)-2-hydroxyglutarate (R-2HG).3,6 As a result, R-
2HG levels are elevated >50-fold in samples from patients
with IDH mutations.3
are thought to cause the enhanced proliferation and im-
paired differentiation observed in IDH-mutant tumors.
The mutual exclusivity of IDH and TET2 mutations in
AML tumors7 and the ability of exogenous R-2HG to in-
duce leukemogenesis in blood cells11 further implicate R-
2HG as a critical mediator in how mutant IDH contrib-
utes to AML. In IDH1-mutant glioma models, hypermeth-
ylation of CTCF binding sites has been shown to cause
genomic insulator dysfunction leading to aberrant activa-
tion of PDGFRA, providing an explanation for how R-2HG
impacts cancer initiation in the brain.12
Chart 1. Structures of selected mutant IDH inhibitors
The pathogenesis of IDH-mutant tumors is thought to
center on the ability of R-2HG to act as an ‘oncometabo-
lite’. Due to its structural similarity to α-KG, R-2HG com-
petitively inhibits several α-KG-dependent dioxygenases
when present at the high concentrations observed in
IDH-mutant tumors. In particular, R-2HG impairs DNA
demethylation through inhibition of TET2,7 impairs his-
tone demethylation through inhibition of various lysine
demethylases,8,9 and modulates hypoxic stress response
through activation of EGLN1.10 These molecular changes
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aIDH1 inhibitor. bIDH2 inhibitor. cClinical compound.
need improvement to yield a suitable probe for in vivo
applications.
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The discovery of mutant allele-selective small-molecule
inhibitors of IDH1 and IDH2 has enabled the validation of
these enzymes as therapeutic targets. In particular, stud-
ies of AGI-5198, AGI-6780 and GSK321 in disease models
indicate that these inhibitors may shift cancer cells to-
wards a more mature and less proliferative state, suggest-
ing such compounds might form the basis of a future dif-
ferentiation therapy.13-16 Early results from clinical trials of
mutant-IDH inhibitors AG-12017 and AG-22118 in IDH-
mutant AML suggest that the compounds may be effec-
tive in this context.
To identify small molecules that inhibit IDH1-R132H,
we developed an in vitro assay measuring the enzyme’s
ketoreductase activity in 1536-well plates. Enzyme activity
was measured by detecting consumption of NADPH in a
diaphorase-coupled reaction with a fluorescence readout.
The screening buffer included 0.01% Tween 20 detergent
to minimize false positives due to compound aggregation.
IDH1 is reported to function with either Mn2+ or Mg2+ as a
cofactor, and we performed screens under both condi-
tions. Surprisingly, the screening results were markedly
different depending on the cofactor used (Figure S1). Alt-
hough enzyme turnover was substantially faster using the
IDH1-R132H-Mn2+ complex (Figure S2), the potency of
inhibitors in this model system proved to be a poor pre-
dictor of cellular activity (Table S1), and we prioritized the
Mg2+ complex for further study. The IDH1-R132H-Mg2+
complex was screened in duplicate against 89,093 com-
pounds from the Broad Institute’s DOS screening library.
Primary screening at 15 μM yielded 551 positives with ≥
60% inhibition in both replicates (hit rate 0.6%).
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We retested positives in 8-point dose in the primary
screening assay and in an orthogonal enzymatic assay
detecting NADPH by absorbance to confirm compound
activity and to mitigate detection-specific artifacts. We
then tested compounds for selectivity with respect to
wild-type IDH1. Wild-type IDH1 inhibition was measured
using an assay analogous to that used for the primary
screen, measuring the production of NADPH from NADP+
and isocitrate in a diaphorase-coupled reaction. Notably,
only 15 of the positives from this screen inhibited wild-
type IDH1 with an IC50 below 50 μM, and none of these
showed an IC50 below 20 µM. This allele-selectivity is con-
sistent with that seen for most previously published mu-
tant IDH1 inhibitors and is likely due to the substantial
differences in tertiary structure between wild-type and
R132H-mutant IDH1.24
Figure 1. BRD2879, but not its stereoisomers, potently in-
hibits IDH1-R132H in vitro. (a) Structure of BRD2879 (b) The
(2S, 5R, 6R) configuration shows >10x lower IC50 than its ste-
reoisomers. Values are geometric means of three independ-
ent experiments; typical CV is 35%. (c) NADPH-dependent
ketoreductase activity of IDH1-R132H after incubation with
compounds. BRD2879 inhibits IDH1-R132H with comparable
potency and Hill slope to previously disclosed AGI-5198. Val-
ues are mean ± SD of three independent experiments.
To prioritize the 103 confirmed hits for follow-up inves-
tigation, we examined preliminary structure–activity rela-
tionships (SAR) present in the screening data as well as
biological activity annotations in PubChem as a readout
of compound selectivity. The DOS screening library con-
sists of many groups of structural analogs for a given scaf-
fold, including nearly all stereoisomers of each com-
pound. This design enables the identification of series
that display SAR suggestive of a specific molecular inter-
action with the protein target. Here, we identified
BRD2879, an 8-membered sulfonamide containing 3 ste-
reocenters (2S, 5R, 6R) that has an IC50 10- to 1000-fold
lower than those of its stereoisomers (Figure 1A,B). This
stereochemistry–activity pattern was consistent among
structurally related compounds in the screening collec-
tion.
While the clinical progress of existing IDH inhibitors is
encouraging, the fact that cancers frequently develop re-
sistant alleles to targeted therapy motivates the need to
develop structurally and mechanistically diverse ‘next-in-
class’ compounds targeting IDH. The co-crystallization of
mutant IDH1 with probes such as GSK321, VVS and SYC-
435 has already uncovered distinct binding modes for
inhibitors of mutant IDH1 (Chart 1).16,19-21 To identify new
scaffolds for inhibition of IDH1-R132H, we screened com-
pounds derived from diversity-oriented synthesis (DOS).
The DOS collection contains molecules with increased sp3
content and myriad stereochemical features compared
with those found in standard commercial libraries,22,23 so
we hypothesized that our screen could reveal a probe
with unique binding characteristics. Here we report the
discovery of BRD2879, a potent and cell-active inhibitor
of IDH1-R132H with a markedly different structure from
previously reported probes. While BRD2879 exemplifies a
distinct class of IDH1 inhibitors, its physical properties
Scheme 1: Elaboration of the sulfonamide scaffolda
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fluorobenzene, TEA, XPhos-Pd-G3, MeCN, 70 °C, 14 h, 94%.
1
2
3
4
(d) DDQ, DCM, pH 7 buffer, rt, 5 h, 66%. (e) PPh3, DIAD,
phthalimide, THF, 0-23 °C, 18 h. (f) methylhydrazine, EtOH,
80 °C, 1 h, 36% (2 steps). (g) CH2O, Na(OAc)3BH, DCM, rt, 6
h, 51%.
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8
BRD2879 and other DOS-derived compounds have
been tested in a variety of screens conducted through the
NIH Molecular Libraries Program, and the data can be
analyzed for evidence of indiscriminate activity.25
BRD2879 was inactive in all 40 screening assays for which
results are available on PubChem, including a screen for
inhibitors of KDM4C, which like IDH1 uses α-KG as a co-
factor. Overall, the potency of BRD2879, its encouraging
preliminary SAR, and its lack of activity in other screens
led us to prioritize this scaffold for follow-up experiments.
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aReagents and conditions: (a) 1) TBSOTf, 2,6-lutidine,
DCM, rt, 1 h. 2) HF-pyr, THF, rt, 0.5 h. (b) cyclohexyl isocya-
nate, TEA, DCM, rt, 0.5 h, 86% (2 steps). (c) 1-ethynyl-4-
Table 1. Biochemical potency of BRD2879 analogsa
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aValues
are
geometric
means
of
at
least
3
independent
experiments;
typical
CV
is
35%.
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In order to confirm the activity of BRD2879 and explore
additional SAR of the scaffold, we resynthesized the probe
along with 36 analogs and tested their activity in an en-
zymatic assay. The core structure 1 was synthesized ac-
cording to the published procedure,26,27 and elaboration
to the final compounds was accomplished by N-capping
and Sonogashira reactions in either order, followed where
appropriate by functionalization of the primary alcohol
(exemplified in Scheme 1). A two-step Boc deprotection
avoiding strong acid was used to avoid concurrent remov-
al of the PMB group. All analogs were synthesized via this
route with the exception of the ‘reverse amide’ 36, which
required a modified synthesis of the structural core
(Scheme S1). The activity of resynthesized BRD2879 and
analogs was confirmed in dose by monitoring the kinetic
decrease in fluorescence of NADPH when incubated with
IDH1-R132H and α-KG (Figure 1C). In comparison to the
enzymatic assays used in screening, this direct fluores-
cence detection method allowed compound evaluation at
lower enzyme concentrations. The assay modification was
necessary to maintain [inhibitor] >> [enzyme] and to en-
sure that IC50 is a good approximation of binding affinity.
stereochemical SAR for BRD2879, we synthesized analogs
retaining the (2S, 5R, 6R) configuration. Activity is main-
tained when the R1 side chain is any of a variety of large
hydrophobic groups, with the 4-fluorophenyl substituent
in compound 3 showing a slight improvement over the 3-
fluorophenyl substituent in BRD2879. Compounds with
smaller or more polar substituents in this position show
reduced activity. Several analogs were synthesized with
the 3-pyridyl substituent in this position in order to im-
prove solubility, but this modification significantly re-
duced potency.
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Variations of the R2 substituent indicate a clear prefer-
ence for the cyclohexyl urea group, and decreasing the
size of the substituent leads to decreases in potency (5-8).
Aromatic or hydrophilic residues are poorly tolerated.
The urea linkage is essential, as replacement with an am-
ide in either orientation (13, 36) reduces activity, as does
N-methylation (16). These data suggest the urea may be a
site for specific interaction of the compounds with mu-
tant IDH1. Varying the primary alcohol did not signifi-
cantly alter potency in the small set of analogs tested (4,
37), suggesting an opportunity for future modification or
conjugation at this site.
To provide further evidence that BRD2879 inhibits
IDH1-R132H through direct binding of the protein, we
examined the effect of the compound on the melting
temperature of recombinant IDH1-R132H as measured by
differential scanning fluorimetry. The 3.7 °C melting point
shift observed in the presence of BRD2879 is consistent
with a direct binding interaction and is equivalent to the
shift seen in the presence of AGI-5198. We then per-
formed steady-state kinetics studies to determine the
mechanism of action of BRD2879, finding that the com-
pound is competitive with both the α-KG substrate and
the Mg2+ cofactor (Figure S3), and that binding of
BRD2879 and AGI-5198 is mutually exclusive (Figure S4).
Table 2. Key properties of BRD2879
a
In vitro enzyme inhibition, IC50
IDH1-R132H
0.05 μM
2.5 μM
IDH1-R132C
IDH1-WT
>20 μM
>20 μM
IDH2-R140Q
Thermal stabilizationb
IDH1-R132H
3.7 °C
1.4 °C
IDH1-R132C
Figure 2. BRD2879 is effective in HA1E-M cells. (a) BRD2879
treatment causes dose-dependent reduction in R-2HG se-
creted by cells, though at lower potency than AGI-5198. (b)
Cell viability, as measured by ATP levels, is maintained at
doses up to 10 µM but drops off at higher doses. Measure-
ments were taken after 72 hours of treatment. Values repre-
sent percentages normalized to DMSO-treated control sam-
ples, mean ± SD from three independent experiments, each
run in triplicate.
Cell-based R-2HG suppression,
c
EC50
HA1E-M-IDH1-R132H
Mechanism of inhibition
w.r.t. α-KG
0.3 μM
competitive
competitive
w.r.t. Mg2+
Solubility/Lipophilicity, pH 7.4
Thermodynamic solubility
LogD
5 μMd
5.19e
Our SAR studies focused on the alkyne (R1) and urea
(R2) side chains, as these are facile points of diversifica-
tion designed into the DOS library (Table 1). Recalling the
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ly sufficient to allow an efficacious dose in solution. We
f
Microsomal stability, t1/2
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3
4
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8
synthesized a small number of analogs in an attempt to
improve solubility of the probe, but these modifications
either reduced potency (25) or failed to improve solubility
as expected (4), indicating the need for further effort in
this area.
Mouse
0.7 min
0.7 min
Human
Plasma protein bindingd
Mouse
96.3%
99.5%
Although the high molecular weight, lipophilicity, and
low solubility of BRD2879 raise concerns that it may in-
hibit IDH1-R132H by nonspecific aggregation, we found
that its activity in vitro is unaffected by increasing con-
centrations of Tween 20 detergent (Figure S7). Further-
more, the low activity of BRD2879’s enantiomer suggests
that the compound’s activity may rely on specific interac-
tions with the target rather than simply its physical prop-
erties. The thermal stabilization of purified enzyme, lack
of activity against wild-type IDH1 and across many other
assays, and ability to suppress R-2HG production in cells
provide further evidence for this hypothesis. While
BRD2879 is of limited utility in its present form, explora-
tion of the SAR has revealed sites that are not critical for
compound potency and that may be modified to improve
solubility, selectivity, and susceptibility to metabolism.
BRD2879 represents a new structural class of mutant
IDH1 inhibitors that, with optimization, may prove useful
in the study of this enzyme and its role in cancer.
Human
aGeometric mean of at least three independent experi-
ments. bMean of 3 independent experiments, each with 7
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replicates. Mean of 3 independent experiments, each with 3
d
e
replicates. Mean of 3 replicates in one experiment. Single
experiment, includes 1% DMSO. fSingle experiment, calculat-
ed from a 6-point curve over 1 hour.
In order to confirm the activity of our inhibitors in a
more physiologically relevant context, we established a
cell-based model system for mutant IDH1 activity. Since it
has proved difficult to establish robust cell lines directly
from IDH-mutant tumors, we developed an engineered
model based on overexpression of IDH1-R132H in HA1E-M
cells (a HEK-293 derivative).28 These cells express high
levels of R-2HG, which is released into the growth media.
We determined the cellular activity of compounds by
measuring R-2HG present in conditioned media by liquid
chromatography-mass spectrometry (LC-MS) after 72
hours of compound treatment. After harvesting media, we
also determined viability of compound-treated cells by
observing cell morphology and measuring ATP levels. In
this model, AGI-5198 causes dose-dependent reduction in
R-2HG levels with an EC50 of 0.06 µM, consistent with
previous reports of its efficacy in cells.29 We found that
BRD2879 also reduced R-2HG levels, though at lower po-
tency (EC50 = 0.3 μM) as compared to its biochemical ac-
tivity (Figure 2A). Furthermore, the potency of BRD2879’s
stereoisomers in the cell-based assay correlated with their
potency in the enzymatic assay, providing evidence for an
IDH1-R132H-based mechanism-of-action in cells (Figure
S5).
ASSOCIATED CONTENT
Supporting Information
Supplementary figures, experimental details, compound
characterization, and abbreviations (PDF). The Supporting
Information is available free of charge on the ACS Publica-
tions website.
AUTHOR INFORMATION
Corresponding Author
*ashamji@broadinstitute.org, stuart_schreiber@harvard.edu
Funding Sources
To determine the toxicity of BRD2879, we measured in-
tracellular ATP levels and observed cell morphology of
our engineered HA1E-M cells and the AML-derived cell
lines U937 and THP1 after three days of compound treat-
ment (Figure 2B, Figure S6). By these measures, BRD2879
began to show inhibitory effects on cell viability at the 10
μM concentration required for near-complete suppression
of R-2HG production. U937 and THP1 are wild-type for
IDH1, and we do not expect IDH1-R132H inhibition to
affect the viability of engineered HA1E-M cells because
the cell line was already capable of growth and prolifera-
tion before the IDH1-R132H enzyme was introduced.
Thus, the observed toxicity is likely due to off-target ef-
fects and is possibly related to the compound’s poor solu-
bility.
This work was supported by the NCI’s Cancer Target Discov-
ery and Development grant (U01CA176152, awarded to S.L.S.)
S.L.S. is an Investigator at the Howard Hughes Medical Insti-
tute. M.M.H was supported by a Howard Hughes Medical
Institute Postdoctoral Fellowship. M.L. was supported by a
Kekulé fellowship (Fonds der Chemischen Industrie) and the
German Academic Exchange Service (DAAD). O.V. was sup-
ported by the Wenner-Gren Foundations and the Swedish
Chemical Society (Stiftelsen Bengt Lundqvists Minne).
ACKNOWLEDGMENT
We thank Dr. Jeremy R. Duvall, Dr. Ben Munoz, Dr. Zarko
Boskovic, Micah Maetani, and Shawn D. Nelson Jr. of the
Broad Institute for providing advice regarding compound
synthesis; and Dr. W. Frank An and Dr. Marshall Morn-
ingstar of the Broad Institute, Dr. Julie-Aurore Losman of
DFCI and Dr. Yongcheng Song of Baylor College of Medicine
for helpful discussions.
To assess further the suitability of BRD2879 for in vivo
use, we measured several relevant physical properties of
the probe (Table 2). The rapid degradation of the com-
pound by mouse and human liver microsomes indicates
optimization of the compound for metabolic stability will
be required before in vivo use is possible. Additionally, the
compound’s low solubility and high logD are liabilities
even in cell-based model systems, as the solubility is bare-
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Discovery of 8-Membered Ring Sulfonamides as Inhibitors of
Oncogenic Mutant Isocitrate Dehydrogenase 1
Jason M. Law,1,2 Sebastian C. Stark,1 Ke Liu,1 Norah E. Liang,1,2 Mahmud M. Hussain,1,2,3 Matthias
Leiendecker,1,2 Daisuke Ito,1 Oscar Verho,1,2 Andrew M. Stern,1 Stephen E. Johnston,1 Yan-Ling
Zhang,1 Gavin P. Dunn,4 Alykhan F. Shamji,*1 Stuart L. Schreiber*1,2,3
8
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