Optimization of 3-Pyrimidin-4-yl-oxazolidin-2-ones as Allosteric and Mutant Specific Inhibitors of IDH1

Article abstract of DOI:10.1021/acsmedchemlett.6b00334

High throughput screening and subsequent hit validation identified 4-isopropyl-3-(2-((1-phenylethyl)amino)pyrimidin-4-yl)oxazolidin-2-one as a potent inhibitor of IDH1^R132H. Synthesis of the four separate stereoisomers identified the (S,S)-diastereomer (IDH125, 1f) as the most potent isomer. This also showed reasonable cellular activity and excellent selectivity vs IDH1^wt. Initial structure-activity relationship exploration identified the key tolerances and potential for optimization. X-ray crystallography identified a functionally relevant allosteric binding site amenable to inhibitors, which can penetrate the blood-brain barrier, and aided rational optimization. Potency improvement and modulation of the physicochemical properties identified (S,S)-oxazolidinone IDH889 (5x) with good exposure and 2-HG inhibitory activity in a mutant IDH1 xenograft mouse model.

Full text of DOI:10.1021/acsmedchemlett.6b00334

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Letter
Optimization of 3-Pyrimidin-4-yl-oxazolidin-2-ones
as Allosteric and Mutant Specific Inhibitors of IDH1
Julian Roy Levell, Thomas Caferro, Gregg Chenail, Ina Dix, Julia Dooley, Brant Firestone, Pascal D Fortin,
John Giraldes, Ty Gould, Joseph D. Growney, Michael D Jones, Raviraj Kulathila, Fallon Lin, Gang Liu,
Arne Mueller, Simon van der Plas, Kelly Slocum, Troy Smith, Remi Terranova, B. Barry Touré, Viraj Tyagi,
Trixie Wagner, Xiaoling Xie, Ming Xu, Fan Yang, Liping X. Zhou, Raymond Pagliarini, and Young Shin Cho
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00334 • Publication Date (Web): 16 Dec 2016
Downloaded from http://pubs.acs.org on December 19, 2016
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ACS Medicinal Chemistry Letters
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Optimization of 3-Pyrimidin-4-yl-oxazolidin-2-ones as Allosteric and
Mutant Specific Inhibitors of IDH1
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Julian R. Levell, Thomas Caferro, Gregg Chenail, Ina Dix, Julia Dooley, Brant Firestone, Pascal D.
Fortin, John Giraldes, Ty Gould, Joseph D. Growney, Michael D. Jones, Raviraj Kulathila, Fallon Lin,
Gang Liu, Arne Mueller, Simon van der Plas, Kelly Slocum, Troy Smith, Remi Terranova,
B. Barry Touré, Viraj Tyagi, Trixie Wagner, Xiaoling Xie, Ming Xu, Fan S. Yang, Liping X. Zhou,
Raymond Pagliarini,* and Young Shin Cho*
Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, MA 02139
KEYWORDS mutant IDH1 inhibitor, allosteric inhibition, 2ꢀHG, preclinical inꢀvivo activity, 3ꢀpyrimidinꢀ4ꢀyloxazolidinꢀ2ꢀone, chiralityꢀ
defined potency
N
N
O
O
HN
N
N
HN
N
N
O
O
N
IDH125
IDHR132H IC50 0.22µM
IDH889
IDHR132H IC50 0.02µM
N
Cell 2-HG IC50 0.66µM
Cell 2-HG IC50 0.014µM
F
ABSTRACT:
High
throughput
screening
and
subsequent
hit
validation
identified
4ꢀisopropylꢀ3ꢀ(2ꢀ((1ꢀ
phenylethyl)amino)pyrimidinꢀ4ꢀyl)oxazolidinꢀ2ꢀone as a potent inhibitor of IDH1R132H. Synthesis of the four separate stereoisomers
identified the (S,S)ꢀdiastereomer (IDH125, 1f) as the most potent isomer. This also showed reasonable cellular activity and excelꢀ
lent selectivity vs. IDH1wt. Initial SAR exploration identified the key tolerances and potential for optimization. Xꢀray crystallogꢀ
raphy identified a functionally relevant allosteric binding site amenable to inhibitors which can penetrate the bloodꢀbrain barrier,
and aided rational optimization. Potency improvement and modulation of the physicoꢀchemical properties identified (S,S)ꢀ
oxazolidinone IDH889 (5x) with good exposure and 2ꢀHG inhibitory activity in a mutant IDH1 xenograft mouse model.
Hotspot heterozygous mutations in human cytoplasmic isoꢀ
citrate dehydrogenase 1 (IDH1) at Arg132 (R132*) have been
identified in multiple cancer types, including acute myeloid
leukemia (AML), glioma, chondrosarcoma and cholangiocarꢀ
cinoma.1 These mutations have been shown to confer a neoꢀ
morphic catalytic activity to produce high levels of intracelluꢀ
lar Rꢀ2ꢀhydroxyglutarate (2ꢀHG) and effect downstream epiꢀ
genetic markers on DNA and proteins.2,3 Catalytic inhibitors4ꢀ
10 of IDH1R132* have shown preclinical reduction of 2ꢀHG levꢀ
els in xenograft mouse models with mutant IDH1 tumors, and
have also shown preclinical tumor regression.4ꢀ7 Recent cliniꢀ
cal trials in AML patients with a specific inhibitor of IDH1
has shown clinical benefit, confirming the causal link between
this genetic mutation, the production of 2ꢀHG, and cancer.11
Efforts herein focused on the identification of compounds
which could potentially target all classes of mutantꢀIDH1 tuꢀ
mors, including those in the brain.
ic inhibitors with different mechanisms of action, which may
bind distal to this polar substrateꢀbinding site.
Figure 1: Sequence and sidechain representation of the substrateꢀ
binding site for IDH1R132H. Amino acids lining the pocket are
highlighted in yellow, mutation site R132H is shown in green.
High throughput screening was carried out with a NADPH
fluorescenceꢀbased biochemical assay using IDH1R132H hoꢀ
modimer protein, and orthogonal biochemical inhibition conꢀ
firmation using an LCMS readout of
2ꢀHG levels. Comꢀ
pounds 1a and 1b were identified as selective and functional
inhibitors of IDH1R132H from this screen. Both 1a and 1b were
screened as diastereomeric mixtures at the amine (Table 1,
Am), which necessitated the independent synthesis of the four
separate stereoisomers in order to determine the chiral preferꢀ
ence for ligand binding. Potency was found to be most strongꢀ
ly dependent upon the chirality at the amine center (Am), altꢀ
hough still significantly affected by the chirality at the oxazolꢀ
idinone (Ox) (Table 1). The preference for the Sꢀamine isomer
was maintained throughout the SAR on the series, but the efꢀ
The substrateꢀbinding site of mutant IDH1 is highly polar as
defined by the amino acids lining the pocket (Figure 1), in
addition to the activeꢀsite magnesium ion and NADPH cofacꢀ
tor. This suggests a low probability of being able to optimize a
compound for potent binding to this site whilst also fulfilling
the criteria most conducive to crossing the bloodꢀbrain barrier
(BBB).12 It was decided to explore the identification of catalytꢀ
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fect of Rꢀchirality at the oxazolidinone was sometimes only SAR by archive using IDH125 as the probe structure was unꢀ
marginal. As described elsewhere,13 compound 1f (IDH125)
was validated with multiple biophysical methods and this
compound enabled considerable understanding of the rationale
for selectivity and mechanism of action of this series. IDH125
was also confirmed as an essentially equipotent inhibitor of
both the homodimer IDH1R132H (IC50 0.22 µM) and IDH1R132C
(IC50 0.15 µM) proteins and slightly less potent for the heteroꢀ
dimer IDH1wtꢀIDH1R132H (IC50 0.97 µM) protein using
NADPH fluorescenceꢀbased biochemical assay.13 The cancerꢀ
specific mutation of IDH1 and lack of activity of IDH125
against wildꢀtype IDH1 up to 50 µM presented an opportunity
to develop an oncology drug with a high therapeutic index.
successful in identifying additional molecules in the Novartis
compound library with subꢀmicromolar activity, highlighting
that this substructure was somewhat unique. Initial analogs
were targeted to explore the tolerance for modification and/or
removal of different parts of the molecule (compounds 2a-d,
3a-f, and 4a-d in supporting information). Understanding of
this initial SAR was clarified by the determination of the Xꢀ
ray coꢀcrystal structure of IDH125 in the homodimer of
IDH1R132H, which showed IDH125 binding into an allosteric
site adjacent to the substrate binding site.13 Replacement of the
nitrogen at the 3ꢀposition of the pyrimidine with a carbon, and
either replacement of the 2ꢀamino with an oxygen or Nꢀ
alkylation all resulted in loss of activity due to lack of backꢀ
bone:ligand hydrogen bond donorꢀacceptor interactions to
Leu120. Small substituents such as methyl on the 4ꢀ and 5ꢀ
position of the pyrimidine were tolerated. The alphaꢀmethyl of
IDH125 binds into a small nook created by the backbone of
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Table 1. IDH1R132H biochemical activity of HTS hits and pure
resynthesized diastereomers.
N
O
Arg109, Glu110 and Ile128 as well as the sidechains of Ile130
,
HN
N
N
O
Ser278 and Met291 resulting in a good contact for a methyl
group but limited tolerance for larger groups in this region. An
unsubstituted oxazolidinone does not lead to the preferred
hydrophobic collapse conformation required for binding, but
there is tolerance for alternate oxazolidinone substituents beꢀ
yond isopropyl. Interestingly, the bound structure of IDH12513
closely overlays with the small molecule Xꢀray structure,
which suggests that hydrophobic collapse drives the molecule
to preorder into the active binding conformation. The nature of
the residues lining this allosteric pocket are also in stark conꢀ
trast to those lining the substrateꢀbinding site, and lend a more
hydrophobic character to the pocket (Figure 2). This pocket
has potential for potent binding of molecules which may also
be able to cross the BBB.
Am
1a-f
Chirality
oxazolidinone
(Ox)
Ox
at Biochem.
LCMS
Chirality at
amine (Am)
IC50 / µM
R:S = 1:1
S
0.8
8.5
19.7
4.2
4.6
1a
1b
1c
1d
1e
R:S = 1:1
R
R
R
S
R
S
R
1f
S
S
0.22
(IDH125)
Figure 2. Sequence and sidechain representation of the allosteric
pocket for IDH1R132H. Amino acids lining the pocket are highꢀ
lighted in yellow, mutation site R132H is shown in green
It was noted that IDH125 contains an aminopyrimidine core,
which is a common motif in ATPꢀcompetitive kinase inhibitor
scaffolds due to the structural similarity vs. the purine nucleus
of ATP.14 Therefore, initial thorough kinase profiling of
IDH125 in an internal kinase profiling panel, as well as an
external kinase profiling panel was carried out (supporting
information) IDH125 demonstrated virtually no inhibitory
activity across the entire kinome. This structural potential for
offꢀtarget activity was monitored with internal and external
kinase panels, but the lack of kinase activity was maintained
throughout the series.
Table 2. IDH1R132H biochemical and HCT116ꢀIDH1R132H cellular
activity of analogs exploring phenyl substitution
N
O
HN
N
N
O
2,4ꢀDichloropyrimidine underwent selective 4ꢀchloro substituꢀ
tion at room temperature, by reaction with the sodium salt of
(S)ꢀ4ꢀisopropylꢀoxazolidinꢀ2ꢀone (Scheme 1). Subsequent
displacement of the 2ꢀchloro at 140 °C with (S)ꢀ1ꢀ
phenylethanꢀ1ꢀamine in DMSO gave (S)ꢀ4ꢀisopropylꢀ3ꢀ(2ꢀ
(((S)ꢀ1ꢀphenylethyl)amino)pyrimidinꢀ4ꢀyl)oxazolidinꢀ2ꢀone
(IDH125, 1f). The other diastereomers were accessed using
the appropriate chiral building blocks.
R
5a-y
Biochem
LCMS
Cellular
IC50 / µM
R
IC50 / µM (2ꢀHG)
cyclohexyl
0.35
0.19
0.25
0.15
0.18
0.13
1.17
0.69
0.42
0.35
0.33
0.16
0.09
0.52
5a
5b
5c
5d
5e
5f
2ꢀfluorophenyl
3ꢀfluorophenyl
4ꢀfluorophenyl
3ꢀchlorophenyl
4ꢀchlorophenyl
Scheme 1. Synthetic scheme for 1f (IDH125)
O
NH2
HN
O
N
O
N
O
N
HN
N
N
O
Cl
N
N
Cl
N
Cl
NaH
O
DMSO
110 oC
72%
DMF
rt, 62%
3,4ꢀdichlorophenyl 0.097
2ꢀmethoxyphenyl 0.27
5g
5h
1f (IDH125)
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ACS Medicinal Chemistry Letters
uM at 100 mg/kg po) was deemed sufficient to explore
3ꢀmethoxyphenyl
4ꢀmethoxyphenyl
0.13
0.3
0.43
0.87
5i
1
2
3
4
5
6
7
8
IDH662 in an in vivo pharmacodynamic (PD) study monitorꢀ
ing compound effects on 2ꢀHG. However, at doses up to 600
mg/kg there was no significant alteration of 2ꢀHG levels in
xenograft tumor tissue in the HCT116ꢀIDH1R132H model (data
not shown). The estimated free compound concentration from
the measured plasma pharmacokinetics (PK) in this study was
below the cellular IC50 for mutant IDH1, which suggested that
the high plasma protein binding (>99%) limited productive
engagement of the target in vivo.
5k
3,4ꢀ
0.70
2.5
5m
dimethoxyphenyl
4ꢀcyanophenyl
1ꢀnaphthyl
2ꢀnaphthyl
pꢀbiphenyl
0.54
0.25
0.094
0.12
1.34
1.1
5n
5p
5q
5r
0.16
0.17
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It was decided to explore whether in vivo modulation of 2ꢀHG
inhibition was dependent on freeꢀfraction of drug in the plasꢀ
ma. The high protein binding was likely contributing to a lowꢀ
er in vivo clearance than predicted from the in vitro microsoꢀ
mal assays, suggesting that lower intrinsic clearance would be
needed in tandem with any increase of the free fraction in orꢀ
der to achieve high enough and effective exposures. In silico
and in vivo metabolic studies highlighted the benzylamine as a
key source of oxidative metabolic cleavage, so this was the
focus of the next iteration of targets.
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5s
4ꢀphenoxyphenyl
0.010
2.1
0.022
7.9
(IDH662)
5ꢀchloro pyrimidinꢀ
2ꢀyl
5t
0.081
0.2
5v
5w†
0.34
0.5
5x
Heterocyclic replacements of the phenyl group should lower
the oxidation potential of the benzylic carbon due to distribuꢀ
tion of the local electron density through to the conjugated,
and more electronegative, heteroatoms. Heterocycles would
also increase the local polarity, and this may be less well tolerꢀ
ated in the binding sites of cytochrome P450 proteins, leading
to reduced oxidative metabolism. This is supported by data
from Ioannidis et al, who have shown excellent in vitro and in
vivo PK properties with a structurally similar halopyrimidine
benzylamine for a series of Janus kinase 2 (JAK2) inhibitors.17
0.02
0.014
(IDH889)
† 1:1 mixture of diastereomers at amine sidechain
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The limited ability to improve the potency with simple modiꢀ
fications to the oxazolidinone, alphaꢀmethyl, or pyrimidine
regions of IDH125 necessitated exploration of modifications
to the phenyl group (Table 2). Saturation of the phenyl ring
and simple substitutions appeared to be tolerated around the
ring system (compounds 5a-m), although larger groups at the
ortho position were less tolerated than the meta or para posiꢀ
tions. In a similar fashion, 2ꢀnaphthyl 5p is also more potent
than 1ꢀnaphthyl 5q. In the paraꢀposition, the fluoro and chloro
groups appeared to give a slight boost in potency, but this was
lost with the larger methoxy group. This could have been due
to either a steric or electronic effect, but the electronꢀ
withdrawing pꢀcyano also showed a drop in potency, suggestꢀ
ing the loss of activity with electronꢀdonating pꢀmethoxy was
not a wholly electronic effect. Despite the drop in activity for
the pꢀmethoxy and pꢀcyanophenyl, there was not the precipiꢀ
tous drop usually seen for a steric clash with an inflexible part
of the protein. This suggested a low energy sidechain or backꢀ
bone movement of the protein in this region, which would
accommodate larger sidechains (i.e., for these intermediate
sized groups the energy penalty for protein reorganization was
not being compensated for by the interactions being made in
the newly formed pocket). This hypothesis is supported by the
potency seen with larger sidechains such as pꢀbiphenyl 5r and
pꢀphenoxyphenyl 5s (IDH662). Interestingly, IDH662 also
showed a significant boost in potency vs. the IDHwt protein
(IDHwt IC50 1.03 µM), suggesting that the allosteric pocket is
also accessible in the wt protein but is preferentially accessible
in the R132 mutated protein.13
The chemistry used to displace chloro from 2ꢀchloroꢀ4ꢀ
oxazolidinonyl pyrimidines with benzylamine derivatives was
not efficient with less nucleophilic heterocyclic amines such as
1ꢀ(5ꢀchloropyrimidinꢀ2ꢀyl)ethylamine. Therefore, the more
reactive 2,4ꢀdifluoropyrimidine was used to enable the syntheꢀ
sis
of
the
desired
products
via
2ꢀfluoroꢀ4ꢀ
oxazolidinonylpyrimidine.
The comparison of chloropyrimidine (5t) to chlorophenyl (5f)
shows a significant improvement in in vitro clearance in rat
microsomes (CLint 73 ꢁL/min.mg vs. CLint >900 ꢁL/min.mg),
but this results in a 16ꢀfold drop in biochemical activity and
50ꢀfold drop in cellular activity. A more moderate 8 to 10ꢀfold
drop in activity is observed for the phenoxypyrimidine analog
5v when compared to the analogous phenoxyphenyl IDH662.
However, <3ꢀfold drop in activity is seen for the phenylꢀ
pyrimidine analog (diastereomeric mixture 5w vs. biphenyl
5r). 5w also shows an improved clearance in rat microsomes
vs. pyrimidine 5t (5w CLint 24 ꢁL/min mg), whereas phenoxyꢀ
pyrimidine 5v was significantly higher (CLint 240 ꢁL/min mg).
Pyrimidine and paraꢀbiaryl motifs were maintained in subseꢀ
quent analogs, leading to identification of IDH889 [IDH1R132C
IC50 0.072 µM, IDH1WT IC50 1.38 µM].
IDH662 was profiled vs. a panel of in vitro assays and showed
an overall excellent selectivity for the mutant protein. IDH662
showed potent cellular inhibition of 2ꢀHG production in the
engineered HCT116ꢀIDH1R132H cell line (IC50 0.022 µM) and
inhibition of proliferation in the engineered MCF10A cellꢀline
(IC50 0.017 µM)13 (cell lines available from Horizon Discovꢀ
ery Group plc15ꢀ16). In comparison, IDH125 was considerably
weaker in the HCTꢀ116ꢀIDH1R132H cell line (IC50 0.66 µM).
Despite poor solubility (<5 µM at pH 6.8) and high in vitro
clearance across species, in vivo exposure in mice (AUC 1.1
µM.h, Cmax 0.39 µM at 10 mg/kg po, AUC 7.9 µM.h, Cmax 2.5
Xꢀray crystallography of IDH889 (PDB 5TQH) in homodimer
IDH1R132H is consistent with the structure of IDH125.13 The
aminopyrimidine core moiety interacts with the backbone of
Ile128 via a hydrogen bond donorꢀacceptor pair, the carbonyl of
the oxazolidinone forms a hydrogen bond interaction with
Leu120, and the alphaꢀmethyl fits into the methyl nook. The
molecule is in a hydrophobic collapse conformation with the
isopropyl in vanꢀderꢀWaals contact with the pyrimidine of the
amine sidechain. In addition, a hydrogen bond is observed
between the pyrimidine of the amine sidechain and Ser278, and
this specific interaction may be compensating for the expected
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increase in desolvation energy for a more polar ligand (vs. the ing. IDH889 also has excellent permeability and no efflux in
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3
4
biphenyl 5r).
the Cacoꢀ2 and human MDR1ꢀMDCK cell lines, supporting
the hypothesis that potent inhibition of mutant IDH1 function
by binding at the allosteric binding site is compatible with
brain penetration.
Figure 3. Xꢀray coꢀcrystal structure of IDH889 in IDH1R132H
.
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It was next tested whether the 2ꢀHG reduction was sufficient
to alter DNA methylation, a phenotype closely linked to IDH1
mutation and 2ꢀHG production.18ꢀ19 These studies were perꢀ
formed using Infinium HumanMethylation450 BeadChip arꢀ
rays (supporting information). Separately derived HCT116ꢀ
IDH1R132H and HCT116ꢀIDH1R132C cell line clones showed
consistent changes in both DNA hyperꢀ and hypomethylation
vs. their isogenic wildꢀtype counterpart cell line, that were
stable over time (Figure 5A). IDH889 caused timeꢀdependent
changes in these loci, with preferential hypomethylation of the
loci in mutant cells that were originally hypermethylated in
mutant vs. wildꢀtype cells. Importantly, IDH889 caused no
consistent changes in DNA methylation in the wildꢀtype conꢀ
trol cell line, highlighting that the DNA methylation changes
in the mutant clones were likely due to IDH1 mutation and 2ꢀ
HG production. To validate these changes in vivo, tumor samꢀ
ples collected at necropsy from the 25 mg/kg bid study (Figure
4B) were evaluated to compare global methylation status vs.
tumors obtained from untreated control animals (3 samꢀ
ples/group), as well as parental wildtype (WT) HCT116 tuꢀ
mors. Figure 5B shows that a large number of sites in the geꢀ
nome were either hypermethylated (red,) or hypomethylated
(green) in IDH1R132H+/ꢀ mutant HCT116 tumors vs. WT control
tumors. For the sites that were specifically altered in mutant
tumors, IDH889 treatment caused both the hypermethylated
and hypomethylated loci to trend towards reversion to their
WT status (figure 5C). The sites not altered by R132H mutaꢀ
tion (Figure 5B, grey) showed only minor changes as a group
upon IDH889 treatment (data not shown). Together, this data
suggests that the level of 2ꢀHG inhibition achieved in Figure
4B was sufficient to modulate mutant IDH1ꢀdependent DNA
methylation changes in vivo.
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IDH889 demonstrates significantly improved plasma exposure
in mice vs. IDH662 (AUC 3.6 µM.h, Cmax 1.7 µM at 10
mg/kg; AUC 55.5 µM.h, Cmax 14.2 µM at 100 mg/kg). This is
coupled with an improved solubility (39 µM at pH 6.8) and
measurable free fraction in mouse plasma (98% plasma proꢀ
tein binding), suggesting prolonged plasma concentration 20ꢀ
fold over the cellular IC50 could be achieved at a dose of 100
mg/kg.
In the HCT116ꢀIDH1R132H+/ꢀ xenograft model, IDH889 dosed
orally at 200 mg/kg inhibits new 2ꢀHG production but does
not eliminate 2ꢀHG from IDH1 mutant tissues. Thus, given the
high baseline tissue concentration of 2ꢀHG, several hours are
required to clear 2ꢀHG from the tissues. Peak reduction in
tumor 2ꢀHG levels after a single dose is observed 8ꢀ12 hours
post administration. 2ꢀHG levels begin to rebound as the free
concentration of IDH889 drops below the in vitro cellular IC50
(Figure 4A). At 48h post treatment, 2ꢀHG levels return to
baseline. Sustained 2ꢀHG inhibition greater than 24 hours was
achieved with BID (q12/12) administration of IDH889 for
four doses (Figure 4B).
In conclusion, a novel HTS hit was optimized for potency and
PK properties to generate a tool molecule (IDH889) suitable
for exploring the effect of inhibition of production of 2ꢀHG by
IDH1R132H in preclinical in vitro and in vivo cancer models.
IDH889 binds into an allosteric, inducedꢀfit pocket in
IDH1R132H, has good overall selectivity vs. the WT protein,
and inhibits both the IDH1R132H and IDH1R132C mutants, sugꢀ
gesting broad utility across the various known R132* mutaꢀ
tions. Oral dosing of IDH889 in a murine IDH1 mutant tumor
xenograft model shows robust reduction of tumor derived 2ꢀ
HG, a PD biomarker of mutantꢀIDH1R132* activity. In addition
to the potential treatment of AML, chondrosarcoma, cholangiꢀ
ocarcinoma and other forms of mutantꢀIDH1 driven cancers,
IDH889 demonstrates brain penetrant exposure. This suggests
potential utility in preclinical orthotopic tumor models, as well
as potential for the series to be optimizable for treating paꢀ
tients with IDH1ꢀmutant brain cancers.
Figure 4. A) Total and estimated free plasma IDH889 concentraꢀ
tion (mean + SD) and percent inhibition of baseline 2ꢀHG levels
(mean + SEM) in HCT116ꢀIDH1R132 xenograft tumor tissue folꢀ
lowing a single 200 mg/kg dose (dotted line is zero % inhibition,
dashed line represents cellular EC50); B) Percent inhibition (mean
± SEM) of 2ꢀHG levels in tumor tissue following four doses of
IDH889 at 25mg/kg po bid(q12/12).
ASSOCIATED CONTENT
Supporting Information
Synthetic procedures, analytical data, assay protocols, SAR of
compounds 2aꢀd, 3aꢀf and 4aꢀd, small molecule Xꢀray data of
IDH125 and 1e, kinase profiling of IDH125, assay protocols, PK
and PK/PD study protocols (PDF) is available free of charge on
the ACS Publications website.
In a rat PK study to assess brain:plasma ratio, IDH889
demonstrates favorable distribution to the brain (30 mg/kg po,
1.4 brain:blood ratio, AUCbrain 3117 nM.h, AUCblood 2222
nM.h), albeit with a lower free fraction due to slightly higher
rat brain protein binding (99.4%) vs rat plasma protein bindꢀ
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ACS Medicinal Chemistry Letters
AUTHOR INFORMATION
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Corresponding Authors
* Eꢀmail: raymond.pagliarini@novartis.com
* Eꢀmail: youngshin.cho@novartis.com
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Present Addresses
(J.R.L.) Constellation Pharmaceuticals, 215 First Street, Suite
200, Cambridge, MA 02142; (P.D.F.) and (B.T.) Relay Therapeuꢀ
tics, 215 First Street, Suite 300, Cambridge, MA 02142; (M.X.)
Dupont, 1007 Market St. Wilmington, DE 19898; (J.G.) National
Cancer Institute, 9609 Medical Center Dr., Rockville, MD 20850;
(L.X.Z.) Ipsen Bioscience, Inc., 650 East Kendall Street, Camꢀ
bridge, MA 02142, USA; (B.F.) Surface Oncology, 215 First
Street, Suite 400ꢀS, Cambridge, MA 02142
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Figure 5 : A) In vitro DNA methylation changes upon IDH889
treatment in the indicated HCT116 cell clones. Rows represent
individual sites in the genome, and columns within each cluster
represent time points (days 3, 7, 14, and 28). Green shading indiꢀ
cates hypomethylation and red shading indicates hypermethylaꢀ
tion, delta values as described in supporting information. B) Vioꢀ
lin plots showing in vivo DNA methylation changes in
IDH1R132H/+ mutant vs. wildꢀtype HCT116 xenograft tumors. Y
axis level of methylation for each site (1=methylated,
0=unmethylated: beta values, as described in supporting inforꢀ
mation), n=number of sites within each group, large dashed line
indicates median beta value, and smaller dashed lines indicate 75th
(top) and 25th (bottom) percentiles. C) violin plot as in B) for
IDH1R132H mutant tumors after treatment with IDH889 (tumors
from Figure 4B).
ACKNOWLEDGMENT
The authors would like to thank Thomas Smith, Kimberley Yue,
and Daniel Baird for screen optimization and execution; Kara
Herlihy, Tami Hood, and Suzanne Zhu for their help with the
development of the inꢀvitro activity assays; and Bill Sellers,
Markus Warmuth, Karin Briner, Juerg Zimmerman, Tim Ramsey
and Travis Stams for project support.
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Optimization of 3-Pyrimidin-4-yl-oxazolidin-2-ones as Allosteric and Mutant
Specific Inhibitors of IDH1
Julian R. Levell, Thomas Caferro, Gregg Chenail, Ina Dix, Julia Dooley, Brant Firestone, Pascal D.
Fortin, John Giraldes, Ty Gould, Joseph D. Growney, Michael D. Jones, Raviraj Kulathila, Fallon Lin,
Gang Liu, Arne Mueller, Simon van der Plas, Kelly Slocum, Troy Smith, Remi Terranova,
B. Barry Touré, Viraj Tyagi, Trixie Wagner, Xiaoling Xie, Ming Xu, Fan S. Yang, Liping X. Zhou,
Raymond Pagliarini,* and Young Shin Cho*
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Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, MA 02139
Graphical Abstract:
N
O
N
O
HN
N
N
O
HN
N
N
O
N
N
F
IDH125
IDH889
IDHR132H IC50 0.22 M
IDHR132H IC50 0.02 M
µ
µ
µ
µ
IC50 0.66 M
Cell 2-HG
IC50 0.014 M
Cell 2-HG
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