Radiolabeled inhibitors as probes for imaging mutant IDH1 expression in gliomas: Synthesis and preliminary evaluation of labeled butyl-phenyl sulfonamide analogs

Article abstract of DOI:10.1016/j.ejmech.2016.04.066

Introduction: Malignant gliomas frequently harbor mutations in the isocitrate dehydrogenase 1 (IDH1) gene. Studies suggest that IDH mutation contributes to tumor pathogenesis through mechanisms that are mediated by the neomorphic metabolite of the mutant

Full text of DOI:10.1016/j.ejmech.2016.04.066

European Journal of Medicinal Chemistry 119 (2016) 218e230
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Radiolabeled inhibitors as probes for imaging mutant IDH1 expression
in gliomas: Synthesis and preliminary evaluation of labeled
butyl-phenyl sulfonamide analogs
,
Satish K. Chitneni a *, Zachary J. Reitman b, David M. Gooden c, Hai Yan b,
Michael R. Zalutsky a
a Department of Radiology, Duke University Medical Center, Durham, NC 27710, USA
b Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA
c Small Molecule Synthesis Facility, Duke University, Durham, NC 27708, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Introduction: Malignant gliomas frequently harbor mutations in the isocitrate dehydrogenase 1 (IDH1)
gene. Studies suggest that IDH mutation contributes to tumor pathogenesis through mechanisms that are
mediated by the neomorphic metabolite of the mutant IDH1 enzyme, 2-hydroxyglutarate (2-HG). The
aim of this work was to synthesize and evaluate radiolabeled compounds that bind to the mutant IDH1
enzyme with the goal of enabling noninvasive imaging of mutant IDH1 expression in gliomas by positron
emission tomography (PET).
Received 22 February 2016
Received in revised form
25 April 2016
Accepted 26 April 2016
Available online 28 April 2016
Methods: A small library of nonradioactive analogs were designed and synthesized based on the
chemical structure of reported butyl-phenyl sulfonamide inhibitors of mutant IDH1. Enzyme inhibition
assays were conducted using purified mutant IDH1 enzyme, IDH1-R132H, to determine the IC50 and the
maximal inhibitory efficiency of the synthesized compounds. Selected compounds, 1 and 4, were labeled
with radioiodine (125I) and/or 18F using bromo- and phenol precursors, respectively. In vivo behavior of
the labeled inhibitors was studied by conducting tissue distribution studies with [125I]1 in normal mice.
Cell uptake studies were conducted using an isogenic astrocytoma cell line that carried a native IDH1-
R132H mutation to evaluate the potential uptake of the labeled inhibitors in IDH1-mutated tumor cells.
Results: Enzyme inhibition assays showed good inhibitory potency for compounds that have iodine or a
fluoroethoxy substituent at the ortho position of the phenyl ring in compounds 1 and 4 with IC50 values
Keywords:
IDH mutation
Glioma
Mutant IDH1 inhibitor
PET imaging
Fluorine-18
Radioiodine
of 1.7 mM and 2.3 mM, respectively. Compounds 1 and 4 inhibited mutant IDH1 activity and decreased the
production of 2-HG in an IDH1-mutated astrocytoma cell line. Radiolabeling of 1 and 4 was achieved
with an average radiochemical yield of 56.6 20.1% for [125I]1 (n ¼ 4) and 67.5 6.6% for [18F]4 (n ¼ 3).
[
125I]1 exhibited favorable biodistribution characteristics in normal mice, with rapid clearance from the
blood and elimination via the hepatobiliary system by 4 h after injection. The uptake of [125I]1 in tumor
cells positive for IDH1-R132H was significantly higher compared to isogenic WT-IDH1 controls, with a
maximal uptake ratio of 1.67 at 3 h post injection. Co-incubation of the labeled inhibitors with the
corresponding nonradioactive analogs, and decreasing the normal concentrations of FBS (10%) in the
incubation media substantially increased the uptake of the labeled inhibitors in both the IDH1-mutant
and WT-IDH1 tumor cell lines, suggesting significant non-specific binding of the synthesized labeled
butyl-phenyl sulfonamide inhibitors.
Conclusions: These data demonstrate the feasibility of developing radiolabeled probes for the mutant
IDH1 enzyme based on enzyme inhibitors. Further optimization of the labeled inhibitors by modifying
the chemical structure to decrease the lipophilicity and to increase potency may yield compounds with
improved characteristics as probes for imaging mutant IDH1 expression in tumors.
© 2016 Elsevier Masson SAS. All rights reserved.
* Corresponding author. Department of Radiology, Duke University Medical
Center, Box 3808, Durham, NC 27710, USA.
E-mail address: satish.chitneni@duke.edu (S.K. Chitneni).
http://dx.doi.org/10.1016/j.ejmech.2016.04.066
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

S.K. Chitneni et al. / European Journal of Medicinal Chemistry 119 (2016) 218e230
219
1. Introduction
2. Results and discussion
Isocitrate dehydrogenase 1 encodes for the IDH1 enzyme, which
plays important roles in cellular metabolism, energy production
and maintenance of normal redox status in cells. IDH1 is localized
in the cell cytoplasm and peroxisomes where it catalyzes the con-
2.1. Chemistry
Butyl-phenyl sulfonamide inhibitors of mutant IDH were among
the first compounds shown to inhibit mutant IDH1 activity in vitro.
version of isocitrate to
a
-ketoglutarate (
a
-KG) and simultaneously
From
this
chemical
class,
N-(4-butylphenyl)-3-(4-(2-
reduces NADP to NADPH [1].
a
-Ketoglutarate serves as a key in-
methoxyphenyl)piperazine-1-carbonyl)-4-
termediate in the tricarboxylic acid cycle (TCA) in cells, and as
substrate for several important dioxygenase enzymes. NADPH is a
key cofactor in the cholesterol and lipid synthesis pathways and
also contributes to cell defense against oxidative damage induced
by reactive oxygen species (ROS) [2]. Recent genome-wide muta-
tion analysis studies revealed that gliomas frequently harbor mu-
tations in IDH1 and, to a lesser extent, the homologous IDH2 genes
[3,4]. IDH1 mutations are present in over 75% of WHO grade 2 and
grade 3 gliomas and in secondary gliomas that develop from lower-
grade tumors [2,4,5]. IDH mutations are generally associated with
better prognosis when compared to glioma patients with wild-type
IDH (WT-IDH) tumors of the same histological type [4,6]. The
median overall survival of patients with IDH-mutated glioblastoma
and anaplastic astrocytoma is approximately 2e3 times longer than
for patients whose tumors have WT-IDH1 [4], strongly suggesting
the prognostic significance of IDH1 and IDH2 mutations in glioma
[7].
methylbenzenesulfonamide A (Fig. 1) was chosen as the reference
compound, which has an IC50 value of <1 mM against the mutant
IDH1-R132H enzyme [17]. A series of nonradioactive iodo- and/or
fluorinated analogs of A were synthesized for structureeactivity
relationship studies and to identify potent inhibitors for radio-
labeling (Fig. 2). The basic synthesis route involved the reaction of
commercially
available
5-(N-(4-butylphenyl)sulfamoyl)-2-
methylbenzoic acid with appropriate phenyl piperazine derivative
in the presence of the coupling reagents EDC and HOBt in DMF
(Scheme 1) [17]. For the iodophenyl analogs 1 and 2, the piperazine
derivatives 1-(2-iodophenyl)piperazine and 1-(4-iodophenyl)
piperazine were synthesized following a literature report [17], and
were subsequently coupled to the benzoic acid derivative in 62%
yield for 1 and 71% yield for 2. The fluoroethyl analogs 4 and 5 were
synthesized by introduction of a fluoroethoxy function at the ortho-
or para-position of the phenyl ring in the phenyl piperazine moiety.
For these reactions, fluoroethoxyphenyl piperazine derivatives
were synthesized by using the corresponding phenol piperazine via
a 3-step synthesis route [18]. First, the secondary amine function in
the piperazine ring was protected with a Boc-group in quantitative
yields. Fluoroethylation of the boc-protected 2- or 4-hydroxypheyl
piperazine was achieved by using fluoroethyl bromide (FEtBr) in
about 75% yield. Removal of the boc protective groups with a
mixture of TFA and CH2Cl2 (50/50, v/v) yielded the fluoroethox-
yphenyl piperazine derivatives in quantitative yields. The synthe-
sized 2- and 4-fluoroethoxyphenyl piperazine analogs were then
reacted with the methylbenzoic acid derivative to obtain the fluo-
roethoxy analogs 4 and 5 in 40e47% yield (Scheme 1). For com-
pound 3, the iodine atom was introduced in place of the methyl
group in the methylbenzoic acid derivative in a two-step synthesis
(9) and was then coupled to 1-(-2-methoxyphenyl)piperazine in
59% yield (Scheme 2).
Scheme 3 shows the synthesis route for the fluoroalkyl de-
rivatives 6 and 7. Introduction of the fluorine atom into the side
chain in these compounds was achieved by means of a fluoropropyl
(6) or a fluorobutyl (7) function. For these syntheses, the fluoroalkyl
aniline derivatives 12a and 12b were synthesized first, and were
subsequently reacted with 5-(chlorosulfonyl)-2-methylbenzoic
acid to obtain 13a or 13b. The synthesis of 4-(3-fluoropropyl)ani-
line 12a was achieved using commercially available 3-(4-
nitrophenyl)propionic acid (structure not shown) where the car-
boxylic acid function was first reduced to an alcohol using borane in
THF to get 10a. Treatment of the alcohol derivatives 10a and 10b
Studies suggest that IDH mutations also play significant roles in
tumorigenesis. IDH mutation is an early genetic event in the for-
mation of gliomas, and contributes to tumor pathogenesis through
multiple mechanisms that are mediated by the neomorphic activity
of the mutated IDH1 enzyme [5,8,9]. IDH mutation results in a loss
of normal catalytic activity - conversion of isocitrate to
the IDH enzyme but also imparts a new gain-of-function that en-
ables the mutant IDH to convert -KG to 2-hydroxyglutarate (2-HG)
a-KG - for
a
in a stereospecific manner (D-2-HG) [10]. Consequently, tumors
that are positive for IDH1 mutation have ~100-fold higher levels of
D-2-HG compared to tumors with normal IDH1 (WT-IDH1).
Excessive D-2-HG in IDH1-mutated cells competitively inhibits
several key cellular dioxygenases that are normally dependent on
a
-KG as a co-factor for their enzyme activity [11]. This competitive
inhibition is presumed to be due to the structural similarity of D-2-
HG with the -KG, and results in the impairment of the activity of
multiple cellular dioxygenases leading to stabilization and activa-
tion of the transcription factor hypoxia-inducible factor-1 (HIF-
), epigenetic dysregulation leading to histone methylation and
a
a
1
a
DNA hypermethylation, and impairment of normal collagen
maturation [12e14]. IDH1 mutations also directly impact the
cellular metabolism of small biochemicals such as amino acids,
glutathione derivatives and TCA cycle intermediates, suggesting the
occurrence of widespread metabolic changes in IDH1-mutated
tumor cells [15,16].
In view of these diverse roles and the impact of IDH1 mutations
on cellular metabolism and epigenetics, there is a strong rationale
for development of noninvasive imaging methods (e.g. PET) to
evaluate mutant IDH1 expression in gliomas. Such a method could
enable determination of the IDH1 mutation status in glioma pa-
tients noninvasively, predict prognosis and assist in the design and
implementation of novel therapeutics for patients with malignant
brain tumors as well as other cancers that carry these mutations
[2,5]. In this work, we have synthesized and evaluated radiolabeled
compounds that have inhibitory activity against the most
commonly occurring IDH1 mutation subtype, IDH1-R132H [17].
Selected compounds were labeled with radioiodine and/or
fluorine-18, and in vitro studies and preliminary in vivo studies
were conducted using the labeled inhibitors in order to assess their
suitability as probes for mutant IDH1.
Fig. 1. Chemical structure of the reference mutant IDH1 inhibitor A.

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IC50
(μM)
Inhibition
Compound R1
R2
R3
R4
Efficiency (%)
102.6 ± 10.6
106.7 ± 11.6
4.1
A
1
2
3
4
5
6
7
OCH3
H
H
I
CH3
CH3
CH3
I
CH3
CH3
CH3
CH3
CH3
CH3
F
2.2
1.7
n.a.
n.t.
2.3*
1.2
2.7
2.4
I
H
OCH3
OCH2CH2F
H
H
H
n.t
CH3
106.6 ± 18.3*
26.4 ± 13.6
98.7 ± 18.0
81.3 ± 17.1
OCH2CH2F CH3
OCH3
OCH3
H
H
CH3
CH3
CH2F
n.a. - not active; n.t. - not tested
*mean value derived from three separate experiments (e.g. see Fig. 3)
Fig. 2. Chemical structures, inhibitory potency and the maximal inhibition values (%) for the synthesized mutant IDH1-R132H inhibitors and the reference analog A. Inhibitory
efficiency is presented as mean SD (n ¼ 3e4) for each of the compounds.
Scheme 1. General synthesis route for preparation of the ortho- and para-substituted inhibitors 1, 2, 4 and 5.
with XTalFluor-M in the presence of DBU as the promotor yielded
the fluoroalkyl-nitrobenzene analogs 11a and 11b in 41% and 53%
yield, respectively (Scheme 3). Reduction of the nitro group to an
amino function by Pd/C-catalyzed hydrogenation followed by sul-
famoylation of the anilines with 5-(chlorosulfonyl)-2-
methylbenzoic afforded the fluoroalkylphenyl sulfonamide in-
termediates 13a and 13b in 69e74% yield. Compounds 13a and 13b
were then coupled to 1-(2-methoxyphenyl)piperazine to obtain the
final compounds 6 and 7 in 85% and 78% yield, respectively. The
synthesis of the radiolabeling precursors 14 and 15 was achieved as
(piperazin-1-yl)phenol as starting materials and with a chemical
yield of 77% and 54%, respectively. The synthesized compounds
were evaluated by mass spectrometry for identity confirmation and
characterized by NMR spectroscopy. The purity of the synthesized
nonradioactive analogs and the radiolabeling precursors was veri-
fied by analytical HPLC, and was found to be between 94% and 98%.
2.2. Inhibitory activity of the nonradioactive analogs against the
mutant IDH1
described for
1
using 1-(2-bromophenyl)piperazine and 2-
The inhibitory activity of the nonradioactive iodo- and fluoro

S.K. Chitneni et al. / European Journal of Medicinal Chemistry 119 (2016) 218e230
221
Scheme 2. Synthesis scheme for the iodo analog 3.
Scheme 3. Synthesis scheme for the fluoropropyl- and fluorobutyl phenyl sulfonamide analogs 6 and 7.
analogs was evaluated by incubating serially-diluted (2-fold) so-
lutions of the compounds with purified IDH1-R132H enzyme in
multiwell plates. Fig. 2 shows the half-maximal inhibitory con-
centration (IC50) values and the maximal enzyme inhibition ach-
ieved for the synthesized compounds in the inhibition assays. Fig. 3
shows the inhibitory curves derived from these experiments for the
reference methoxy analog A and for some of the unlabeled in-
The inhibitory efficiency of 1 and 4 in terms of maximal enzyme
inhibition also was similar to that for the reference methoxy analog,
all showing near-complete inhibition (100%) of mutant IDH1 ac-
tivity in the tested concentration range (0.19e392
mM). On the
other hand, substitution of an iodo- or fluoroethoxy function at the
para position of the phenyl ring resulted in a substantial decrease in
potency for compounds 2 and 5 against mutant IDH1. While the
para-fluoroethoxy derivative 5 displayed partial inhibitory activity
hibitors synthesized in this work, at 0.19e392 mM concentrations.
The results showed that the substitution of the ortho-methoxy
group in the reference compound A with an iodine atom or a flu-
oroethoxy function did not affect inhibitory potency for com-
pounds 1 and 4 against the mutant IDH1. The IC50 of 1 and 4 against
with an IC50 of 1.2 mM but a maximum enzyme inhibition of only
~30%, the para-iodo analog 2 did not show any significant activity
against mutant IDH1, suggesting that the structural modification at
the para-position was not well-tolerated for the binding affinity.
Introduction of a fluorine atom in place of hydrogen or the terminal
methyl group in the butyl side chain also resulted in good retention
the mutant IDH1 in these experiments was 1.7 and 2.3
mM,
respectively, as compared to 2.2 M for the reference Compound A.
m

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Fig. 3. Determination of inhibitory potency for the nonradioactive analogs using purified IDH1-R132H enzyme. The IC50 values are shown in parenthesis for the corresponding
compounds. The para-fluoroethoxy analog 5 displayed a partial inhibitory response with a mean inhibition efficiency of 26.4%.
of inhibitory activity for compounds 6 and 7, with IC50 values of
2.7 M and 2.4 M, and an inhibitory efficiency of 99% and 81%,
respectively.
We next evaluated the ability of the nonradioactive compounds
to bind to and inhibit mutant IDH1 activity in tumor cells that are
positive for the IDH1-R132H mutation. For these experiments, an
astrocytoma cell line with a native IDH1-R132H mutation was used
[19] with an isogenic astrocytoma cell line with WT-IDH1 serving
that were present inside the cells prior to their incubation with
the inhibitors are exported into the supernatant with time and
partially contributed to the concentrations observed at 8 h after
incubation with the test compounds. These results are consistent
with recent studies that reported a 2-HG inhibitory half-life (50%
inhibition) of about 5 h for a structurally unrelated mutant IDH
inhibitor [20], and also suggest that incubation times of up to 48 h
may be required to achieve complete inhibition of 2-HG production
by mutant IDH1 inhibitors in cell based assays [20,21]. Neverthe-
less, these results confirm the ability of the unlabeled inhibitors 1
and 4 to inhibit the mutant IDH1 enzyme in tumor cells that are
positive for an IDH1 mutation.
m
m
as a control. Cells were treated with the inhibitors 1 or 4 (50 mM),
and the 2-HG concentrations in cell supernatants were analyzed by
LC/MS at 4 and 8 h after incubation [14,19]. Comparison of 2-HG
levels in vehicle-treated cell lines showed that the concentration
of 2-HG in IDH1-mutated astrocytoma cells was about 30-fold
higher than that in isogenic wild-type IDH1 control cells (WT-
IDH1), confirming the expression of mutant IDH1 protein and its
neomorphic activity in the IDH1-mutated astrocytoma cell line
(Fig. 4). Addition of the nonradioactive inhibitor 1 or 4 to the cul-
ture media decreased 2-HG concentration in mutant IDH1 cell su-
pernatants by 43% for compound 1 and by 50% for compound 4 at
8 h post-incubation. It is likely that the residual amounts of 2-HG
2.3. Radiochemistry
Radioiodination of 1 ([125I]1) was achieved through a Cu(I)-
catalyzed Br-to-*I exchange reaction in acidic and reducing condi-
tions using gentisic acid as depicted in Scheme 4 [22]. After label-
ing, the crude reaction mixture was purified by RP-HPLC, and the
HPLC peak corresponding to the [125I]1 was further purified by a C18
Sep-Pak cartridge in order to remove acetonitrile and TFA from the
product before being used for biological experiments. The average
radiochemical yield for [125I]1 was 56.6 20.1% (n ¼ 4). 18F labeling
of the fluoroethoxy analog 4 was achieved by heating the phenol
precursor 14 with the secondary labeling agent [18F]fluoroethyl
bromide ([18F]FEtBr) in the presence of cesium carbonate in DMF
(Scheme 5) [23]. The decay corrected radiochemical yield for [18F]
FEtBr synthesis was 48.7 10.8 (n ¼ 4), and [18F]4 was obtained
with an average 18F-fluoroethylation efficiency of 67.5
6.6%
(n ¼ 3). In one synthesis, 1.25 GBq of [18F]4 was obtained (0.65 GBq
at the end of synthesis) starting from 4 GBq of [18F]fluoride, rep-
resenting an overall radiochemical yield of about 31% relative to
initial [18F]fluoride activity. The total synthesis time including HPLC
purification was 109 5 min (n ¼ 3) for [18F]4. The radiochemical
purity was >98% for both labeled compounds. Identity confirma-
tion was achieved by co-elution with the corresponding nonra-
dioactive analogs by analytical HPLC (Fig. 5).
2.4. Tissue distribution studies
Tissue distribution studies were conducted using [125I]1 in order
to evaluate the normal tissue distribution characteristics of the
labeled compound and to assess its suitability for development as
Fig. 4. Inhibition of the mutant IDH1 activity (2-HG production) by the nonradioactive
analogs 1 and 4 (50
mM) at 8 h after incubation in IDH1 mutated astrocytoma cells. The
2-HG levels in the WT-IDH1 control cells are also shown in the Figure for comparison.

S.K. Chitneni et al. / European Journal of Medicinal Chemistry 119 (2016) 218e230
223
Scheme 4. Radiosynthesis of [125I]1 using the bromo precursor 14.
Scheme 5. Radiosynthesis of [18F]4 using [18F]fluoroethyl bromide and the phenol precursor 15.
Fig. 5. Identity conformation of the HPLC-purified radiolabeled inhibitors [125I]1 (A) and [18F]4 (B) by co-elution with the corresponding nonradioactive analog on an analytical
HPLC.

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S.K. Chitneni et al. / European Journal of Medicinal Chemistry 119 (2016) 218e230
an imaging agent for mutant IDH1. Table 1 shows the % ID values
and Fig. 6 shows the % ID/g data for [125I]1 in BALB/C mice from
5 min to 4 h post injection. The biodistribution data is consistent
with general expectations for a lipophilic small molecule radio-
tracer, with rapid clearance from the blood and elimination via the
hepatobiliary system. At 5 min p.i., about 12% ID was present in the
blood, with the activity cleared rapidly with time to about 0.13% ID
at 4 h. Except for liver, the activity concentration in the vital organs
was <0.2% ID/g at 4 h, demonstrating good clearance of radioac-
tivity from normal tissues by 4 h. The excretion of the labeled
compound thorough kidneys and into urine was minimal with only
3.50
1.08%ID present in kidneys at 5 min, and a maximum of
2.84% ID excreted into the urine (1 h). About 30% ID was present in
liver at 5 min, with the activity declining with time, most likely
reflecting clearance into the intestines. At 2 h, approximately 60%
ID was detected in the hepatobiliary system, suggesting this as the
major metabolic route for elimination of the labeled inhibitor. The
activity concentration (%ID/g) in the thyroid gland at 5 min was
Fig. 6. Tissue distribution data for the radioiodinated inhibitor [125I]1 in BALB/C mice
at 5 mine4 h after injection. Data is shown as %ID/g values for different organs (n ¼ 5
animals/time point).
1.69
0.93 but decreased to 0.42
0.16 by 4 h after injection,
indicative of the stability of [125I]1 towards dehalogenation in vivo.
The uptake of [125I]1 in the brain was 0.39 0.13%ID at 5 min, and
remained very low at later time points (ꢀ0.07%ID/g at 1e4 h).
Preliminary evaluation of [18F]l4 also revealed similar results when
2.5. Cell uptake studies
10 mM 4 was used for co-incubation experiments. Taken together,
these data suggest that the labeled inhibitors undergo significant
non-specific binding with increasing concentrations of unlabeled
inhibitors and/or with decreasing concentrations of FBS in the in-
cubation media. In part, this non-specific binding can be attributed
to the lipophilicity of the labeled inhibitors which have a calculated
LogP (cLogP) of 5.94 and 5.15 for the iodo- and fluoroethyl de-
rivatives 1 and 4, respectively, as compared to 4.93 for the reference
methoxy analog A. The cLogP of the fluoroalkyl analogs 6 and 7 is
somewhat lower, 3.78 and 4.22, respectively, suggesting that
radiolabeled analogs of these two compounds may show less non-
specific binding compared to that observed for [125I]1. In addition to
the affinity or the interaction of the labeled molecule with the
mutant IDH1 protein, lipophilicity also plays a critical role in the
uptake and specific retention of the labeled inhibitors in mutant
IDH1 tumor cells. Several PET radiotracers have proven useful for
imaging intracellular targets in human gliomas despite their hy-
drophilic (e.g. [18F]FMISO for hypoxia imaging; cLogP: ꢁ0.52) or
very lipophilic (e.g. [11C](R)-PK11195 for mitochondrial translocator
protein (TSPO) imaging; CLogP: 4.58) [24,25] characteristics, sug-
gesting practical evaluation of all candidate radiotracers in relevant
biological models (in vitro and in vivo) before a lead compound is
identified for new imaging targets such as mutant IDH1. While high
lipophilicity may increase the risk of non-specific binding of small
molecule compounds to proteins, studies suggest that efficient cell
membrane permeability requires a LogP of >0.8 for compounds
that are undergoing passive diffusion to enter cells [26]. Using the
information obtained from the present study, work is in progress to
synthesize and evaluate labeled compounds from additional
chemical classes that have been shown to have higher potency than
the butyl-phenyl sulfonamide inhibitors and also inhibited the
neomorphic activity of mutant IDH1 in tumor models in vivo
[21,27].
The uptake of the labeled inhibitors in mutant IDH expressing
cells was evaluated using the same astrocytoma cell line used for
the 2-HG inhibition assays by incubating the cells with HPLC-
purified [125I]1 for 15 mine4 h. At 15 min after incubation, about
0.5% of the added activity was present in the mutant IDH1 cell line,
and increased to 0.80 0.04% at 3 h (P < 0.05). Comparison of the
uptake of [125I]1 in the mutant-IDH1 and WT-IDH1 cell lines
revealed an average uptake ratio of 1.66 at 2 h (P < 0.05) and 1.67 at
3 h (P < 0.05) after incubation (Fig. 7A). Based on these data, a 3-h
incubation was chosen for further studies evaluating the effect of
increasing concentrations of unlabeled 1 (0.1e100 mM) on cell up-
take of [125I]1 and the binding specificity in mutant IDH1 tumor
cells. In these experiments, co-incubation with cold compound
resulted in
concentration-dependent manner in both the mutant-IDH1 and
WT-IDH1 cell lines (P < 0.05 except for 0.1 M; Fig. 7B). The
maximal increase in [125I]1 uptake was observed at 10
M 1 where
the uptake was 53-fold higher than that for no-carrier-added [125I]
1, and the uptake decreased at higher concentrations (50 M and
100 M). Similarly, decreasing the concentration of the FBS from
a substantial increase in [ a
125I]1 uptake in
m
m
m
m
10% in the normal incubation media to 0.1% also resulted in
increased uptake of [125I]1 in both the cell lines (Fig. 7C).
Table 1
Biodistribution of 125I-labeled 1 in BALB/C mice. Data is presented as percent
injected dose (%ID) and as mean SD for five animals for each time point.
Organ
5 min
1 h
2 h
6.57 1.77
4 h
3.41 0.62
Liver
Spleen
Lungs
29.43 5.60
0.36 0.12
1.69 0.83
1.11 0.39
3.50 1.08
0.03 0.01
0.40 0.12
6.37 5.80
0.32 0.18
11.83 1.99
0.15 0.05
13.79 4.85
3.92 1.45
3.31 0.74
13.30 0.69
0.05 0.01
0.17 0.02
0.12 0.02
0.88 0.14
0.01 0.01
0.65 0.18
38.13 1.49
0.04 0.03
0.71 0.27
0.03 0.01
8.21 0.77
1.21 0.14
2.90 1.07
0.03 0.01
0.07 0.03
0.04 0.01
0.43 0.15
0.02 0.03
0.63 0.82
53.24 7.84
0.05 0.05
0.29 0.09
0.02 0.01
3.91 0.87
0.60 0.29
1.25 0.53
0.01 0.01
0.03 0.01
0.01 0.00
0.16 0.03
0.01 0.01
1.22 2.12
41.40 6.02
0.06 0.03
0.13 0.01
0.01 0.00
1.57 0.34
0.22 0.14
0.56 0.09
Heart
Kidneys
Bladder
Stomach
Intestines
Thyroid
Blood
Brain
Muscle
Bone
2.6. Conclusions
A series of nonradioactive mutant IDH1 inhibitors were syn-
thesized based on a butyl-phenyl sulfonamide chemical scaffold.
Enzyme inhibition assays using purified IDH1-R132H led to the
identification of 1 and 4 as lead compounds for radiolabeling, with
Skin
IC50 of about 2
mM. Radiolabeling of compounds 1 and 4 was

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225
mutant IDH1 expressing tumor cells with radionuclides suitable for
PET imaging.
3. Experimental section
3.1. Reagents and general methods
The solvents and regents used in this work were purchased from
VWR International, LLC, or from SigmaeAldrich, and were used as
supplied. The reference compound N-(4-butylphenyl)-3-(4-(2-
methoxyphenyl)piperazine-1-carbonyl)-4-
methylbenzenesulfonamide (A) was purchased from Enamine LLC,
New Jersey. Melting points were determined using a Fisher-Johns
Melting Point Apparatus. Radioiodine was obtained from Perki-
nElmer as [125I]NaI solution in 0.1 N NaOH. [18F]Fluoride was sup-
plied by PETNET Solutions (RaleigheDurham, NC), and was
delivered on a Sep-Pak Light Accell plus QMA anion exchange
cartridge (Waters). Purification and quality control analysis of the
radioiodinated compound [125I]1 was performed on Beckman Gold
HPLC system equipped with gradient solvent system, variable
wavelength UV detector set at 254 nm, and a radiometric detector.
Data were acquired and analyzed using the software (32 Karat®)
provided by the vendor. [18F]4 was purified with a HPLC system that
consisted of an Eldex Model 1SMP pump and a Knauer Model K-
2501 UVeVIS detector (254 nm). Effluent from the UVeVIS detector
was passed through a Carroll & Ramsey Associates Model 105S
radiation detector. Chromatograms were recorded and analyzed
using PeakSimple software (SRI Instruments). Quality control
analysis of [18F]4 was performed on Beckman Gold HPLC system
similar to that used for [125I]1. LogP values were calculated for the
final compounds based on their chemical structure using ACD/
ChemSketch software (www.acdlabs.com).
3.2. Chemistry
3.2.1. N-(4-Butylphenyl)-3-(4-(2-iodophenyl)piperazine-1-
carbonyl)-4-methylbenzenesulfonamide (1)
To a stirred solution of 1-(2-iodophenyl)piperazine (0.065 g,
0.23 mmol) in dimethylformamide (8 mL) was added 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC; 0.048 g, 0.26 mmol),
N,N-diisopropylethylamine (0.18 mL, 0.78 mmol) at 0 ꢂC. After
5 min, hydroxybenzotriazole (HOBt; 0.040 g, 0.26 mmol) and 5-(N-
(4-butylphenyl)sulfamoyl)-2-methylbenzoic
acid
(0.091
g,
0.23 mmol) were added and the resulting mixture was stirred at
room temperature (RT) overnight. The crude mixture was diluted
with water (80 mL) and extracted with ethyl acetate (3 ꢃ 50 mL).
The combined organic layers were washed with water followed by
brine, and filtered over sodium sulfite. The filtrate was concen-
trated under reduced pressure, and subjected to purification using
silica gel eluted with gradient mixtures of ethyl acetate (EtOAc) and
hexanes (0 / 50%) to obtain 1 as an off-white solid (0.087 g, 62%
Fig. 7. Uptake of [125I]1 in astrocytoma cells that are positive for an IDH1-R132H
mutation, and in WT-IDH1 cells. (A) Percent added activity taken up in mutant IDH1
and WT-IDH1 astrocytoma cells at 0.25e4 h after incubation. (B) Uptake of [125I]1 in
the presence of increasing concentrations of the unlabeled 1 in the incubation media
yield), mp: 83e84 ꢂC. 1H NMR (CDCl3, 400 MHz):
d 7.86 (d,
J ¼ 7.6 Hz, 1H), 7.61 (m, 2H), 7.36e7.28 (m, 2H), 7.03e6.95 (m, 5H),
6.85 (t, J ¼ 7.6 Hz, 1H), 4.06 (m, 2H), 3.27 (m, 2H), 3.05 (m, 2H), 2.79
(m, 2H), 2.47 (t, J ¼ 7.2 Hz, 2H), 2.37 (s, 3H), 1.49 (m, 2H), 1.27 (m,
(0.1e100 mM, 3 h). (C) Effect of varying concentrations of FBS (%) in the incubation
media on [125I]1 uptake at 3 h after incubation.
2H) 0.87 (t, J ¼ 7.2 Hz, 3H). 13C NMR (CDCl3, 125 MHz)
d 168.25,
152.40, 140.45, 140.06, 137.12, 136.51, 133.81, 131.15, 129.34, 129.12,
127.63, 126.12, 124.97, 122.50, 121.17, 98.29, 52.74, 52.02, 47.28,
42.09, 34.91, 33.39, 28.00, 22.24, 19.26, 13.85. LC-MS (DART): m/z
calcd. for C28H33IN3O3S ([M þ H]þ): 618.1287; observed: 618.1282.
achieved using 125I and [18F]fluoroethyl bromide, respectively, in
good radiochemical yields and purity. Biodistribution studies with
[
125I]1 showed favorable normal tissue distribution characteristics;
however, cell uptake studies using mutant IDH1 and WT-IDH1 tu-
mor cell lines revealed high nonspecific binding for the labeled
inhibitors. Further optimization of these compounds by decreasing
the lipophilicity and increasing the inhibitory potency may yield
compounds with more favorable uptake characteristics for labeling
3.2.2. N-(4-Butylphenyl)-3-(4-(4-iodophenyl)piperazine-1-
carbonyl)-4-methylbenzenesulfonamide (2)
To a stirred solution of 1-(4-iodophenyl)piperazine (0.40 g,
1.4 mmol) in dimethylformamide (40 mL) at 0 ꢂC was added EDC

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(0.30 g, 1.58 mmol) and N,N-diisopropylethylamine (1.0 mL,
4.3 mmol). After 5 min, HOBt (0.25 g, 1.58 mmol) and 5-(N-(4-
washed with water (2 ꢃ 200 mL) and dried. The solid residue was
dissolved in EtOAc (400 mL) and the resulting amber solution was
washed with brine (3 ꢃ 400 mL) then dried (Na2SO4). The drying
agent was removed by filtration and the filtrate was concentrated
to dryness under reduced pressure to afford the crude product
(11 g, 63%) as a light orange solid. The crude product was used in the
next step without further purification.
butylphenyl)sulfamoyl)-2-methylbenzoic
acid
(0.091
g,
0.23 mmol) were added and the mixture was allowed to warm to
RT, and stirred for 36 h. The reaction mixture was then diluted with
water and extracted with ethyl acetate (3 ꢃ 100 mL). The combined
organic layers were washed with brine, dried with sodium sulfite
and filtered. The filtrate was concentrated under reduced pressure,
and the residue was purified using silica gel via flash column
chromatography eluted with 45% EtOAc in hexanes. The fractions
containing the title compound 2 were combined and evaporated to
give the product as an off-white solid (0.62 g, 71% yield), mp:
3.2.6. 5-(N-(4-Butylphenyl)sulfamoyl)-2-iodobenzoic acid (9)
Solid 5-(chloromethylsulfonyl)-2-iodobenzenecarboxylic acid 8
(5.0 g, 11 mmol, 1.1 eq.) was added in one portion to a solution of 4-
butylaniline (1.6 mL, 10 mmol) in pyridine (40 mL). The reaction
was allowed to warm to RT as the cooling bath melted. Stirring was
continued overnight (15 h) after which time overnight analysis of
the reaction by TLC (50% EtOAc in hexanes) indicated complete
consumption of the starting aniline. The reaction mixture was
diluted with CH2Cl2 (50 mL) and washed with 1 N HCl (50 mL). The
aqueous wash was extracted with CH2Cl2 (3 ꢃ 50 mL). The organic
extracts were combined. Silica gel (~2 g) was added and the
mixture was concentrated to dryness under reduced pressure. Flash
column chromatography (RediSepRf SiO2 (40 g), 100% CH2Cl2 / 5%
MeOH in CH2Cl2) gave the product as a light brown brittle glass
(0.713 g, 16%). Analysis of the material by LCMS showed a single
peak at multiple wavelengths (210 nm, 221 nm, 254 nm and
280 nm). The area under those peaks corresponded to the expected
mass-to-charge ratio (m/z ¼ 476 [(M þ H)þ] and 474 [(MꢁH)ꢁ]) and
the material was used without further characterization.
154e156 ꢂC. 1H NMR (CDCl3, 400 MHz):
d 7.60e7.53 (m, 3H), 7.29
(m, 1H), 7.03 (d, J ¼ 8.4, 2H), 6.95 (d, J ¼ 8.4, 2H), 6.65 (m, 3H), 3.92
(m, 2H), 3.21 (m, 4H), 2.97 (m, 2H), 2.51 (t, J ¼ 7.6 Hz, 2H), 2.35 (s,
3H) 1.52 (m, 2H), 1.28 (m, 2H), 0.87 (t, J ¼ 7.2 Hz, 3H). 13C NMR
(CDCl3, 125 MHz)
d 168.07, 150.25, 140.38, 140.06, 137.92, 137.12,
136.14, 133.82, 131.18, 129.09, 127.73, 124.87, 122.46, 118.71, 82.75,
49.46, 49.02, 46.48, 41.43, 34.93, 33.41, 22.24, 19.23, 13.85. LC-MS
(DART): m/z calcd. for
observed: 618.1294.
C
28H33IN3O3S ([M
þ
H]þ): 618.1287;
3.2.3. N-(4-Butylphenyl)-3-(4-(2-(2-fluoroethoxy)phenyl)
piperazine-1-carbonyl)-4-methylbenzenesulfonamide (4)
Compound 4 was prepared a described for 1, but using 1-(2-(2-
fluoroethoxy)phenyl)piperazine (0.31 g, 1.38 mmol) and 5-(N-(4-
butylphenyl)sulfamoyl)-2-methylbenzoic acid (0.5 g, 1.4 mmol) as
starting materials to get 0.36 g of 4 as a white solid (47% yield), mp:
152e153 ꢂC. 1H NMR (CDCl3, 400 MHz):
d
7.63 (s, 1H), 7.58 (d,
3.2.7. N-(4-Butylphenyl)-4-iodo-3-(4-(2-methoxyphenyl)
J ¼ 7.6 Hz, 1H), 7.28 (d, J ¼ 8.0 Hz, 1H), 7.03e6.87 (m, 8H), 6.64 (s,
1H), 4.77 (m, J ¼ 47.2 Hz, 2H), 4.25 (m, J ¼ 28.0 Hz, 2H), 3.97 (m, 2H),
3.27 (m, 2H), 3.15 (m, 2H), 2.92 (m, 2H), 2.48 (t, J ¼ 7.2 Hz, 2H), 2.36
(s, 3H), 1.50 (m, 2H), 1.28 (m, 2H), 0.88 (t, J ¼ 7.2 Hz, 3H). 13C NMR
piperazine-1-carbonyl)benzenesulfonamide (3)
N,N-Diisopropylethylamine (1.1 mL, 6.3 mmol, 4 eq.) was added
in one portion at room temperature to a (near) solution of the acid 9
(0.713 g, 1.6 mmol), HOBt (0.37 g, 2.3 mmol, 1.5 eq.), EDC (0.45 g,
2.3 mmol, 1.5 eq.) and 1-(-2-methoxyphenyl)piperazine (0.9 g,
3.9 mmol, 2.5 eq.) in anhydrous CH2Cl2 (25 mL). After 1 h, analysis
of the reaction mixture by LCMS indicated complete consumption
of starting acid. Silica gel (~3 g) was added and the mixture was
concentrated under reduced pressure. Flash column chromatog-
raphy (RediSepRf SiO2 (40 g), 100% CH2Cl2 / 5% MeOH in CH2Cl2)
gave the amide 3 as brownish-orange solid (0.6 g, 59%), mp:
(CDCl3, 125 MHz)
d 168.14, 150.98, 141.06, 140.36, 139.91, 137.15,
136.55, 133.84, 131.02, 129.06, 127.58, 124.85, 123.45, 122.55, 122.11,
118.80, 113.60, 82.51, 81.15, 67.72, 67.57, 51.06, 50.36, 47.11, 41.96,
34.86, 33.37, 22.20, 19.20, 13.82. HRMS (DART): m/z calcd. for
C
30H37FN3O4S ([M þ H]þ): 554.2483; observed: 554.2481.
3.2.4. N-(4-Butylphenyl)-3-(4-(4-(2-fluoroethoxy)phenyl)
piperazine-1-carbonyl)-4-methylbenzenesulfonamide (5)
87e89 ꢂC. 1H NMR (CDCl3, 400 MHz):
d
7.86 (d, J ¼ 8.4 Hz, 1H), 7.62
Compound 5 was synthesized as described for 1, but using 1-(4-
(2-fluoroethoxy)phenyl)piperazine (0.14 g, 0.62 mmol) as the
starting material. Purification of the crude mixture by flash column
chromatography eluted with 0 / 60% EtOAc in hexanes provided
the product 5 as an off-white solid in 40% yield (0.12 g), mp:
(d, J ¼ 2.4 Hz, 1H), 7.29 (dd, J ¼ 2.4 H, 8.4 Hz, 1H), 7.03 (m, 3H),
6.96e6.86 (m, 5H), 6.80 (s, 1H), 3.96 (m, 2H), 3.86 (s, 3H), 3.31 (m,
1H), 3.17 (m, 2H), 3.08 (m, 2H), 2.89 (m, 1H), 2.49 (t, J ¼ 7.2 Hz, 2H),
1.51 (m, J ¼ 7.2 Hz, 2H), 1.29 (m, J ¼ 7.2 Hz, 2H), 0.88 (t, J ¼ 7.2 Hz,
3H). 13C NMR (CDCl3, 125 MHz)
d 167.89, 152.15, 149.56, 142.81,
207e208 ꢂC. 1H NMR (CDCl3, 400 MHz):
d
7.61e7.58 (m, 2H), 7.27
140.70, 140.32, 140.05, 139.85, 136.16, 133.52, 129.19, 128.36, 126.44,
125.50, 123.68, 122.77, 121.03, 118.47, 111.33, 98.36, 55.39, 50.69,
50.16, 47.16, 42.09, 34.92, 33.38, 22.23, 13.84. ESI-MS: m/z calcd for
(m, 1H), 7.04e6.87 (m, 8H), 4.74 (m, J ¼ 47.6 Hz, 2H), 4.17 (m,
J ¼ 28.0 Hz, J ¼ 4.0 Hz, 2H), 3.93 (m, 2H), 3.21 (m, 2H), 3.11 (m, 4H),
2.87 (m, 2H), 2.50 (t, J ¼ 7.6 Hz, 2H), 2.35 (s, 3H), 1.29 (m, 2H), 0.88
C
28H33IN3O4S [(M þ H)þ]: 634.1231; observed: 634.1227.
(t, J ¼ 7.2 Hz, 3H). 13C NMR (CDCl3, 125 MHz)
d 168.04, 153.23,
145.44, 140.36, 140.04, 137.11, 136.31, 133.86, 131.14, 129.09, 127.66,
124.88, 122.50, 118.89, 115.49, 82.66, 81.30, 67.67, 67.50, 51.23,
50.78, 46.87, 41.75, 34.92, 33.40, 22.23, 19.23, 13.83. LC-MS (DART):
m/z calcd. for C30H37FN3O4S ([M þ H]þ): 554.2489; observed:
554.2489.
3.2.8. 3-(4-Nitrophenyl)propan-1-ol (10a)
3-(4-Nitrophenyl)propionic acid (5.12 g, 26.20 mmol) in anhy-
drous THF (15 mL) was cooled in a NaCl bath, and a solution of BH3
in THF (33 mL, 33 mmol, 1.3 eq.) was added dropwise over 5 min.
Upon complete addition, the cooling bath was removed and the
mixture was stirred for 2 h after which time analysis of the reaction
by TLC (2% MeOH in CH2Cl2) indicated complete consumption of
starting material. Silica gel (~8 g) was added and the mixture was
concentrated to dryness under reduced pressure. Flash column
chromatography (RediSep® Rf SiO2 (80 g), 100% CH2Cl2 / 5% MeOH
in CH2Cl2) gave the product as an orange oil (4.26 g, 90%). Analysis
of the material by LCMS showed a single peak at multiple wave-
lengths (210 nm, 221 nm, 254 nm and 280 nm). The area under
those peaks corresponded to the expected mass-to-charge ratio (m/
3.2.5. 5-(Chlorosulfonyl)-2-iodobenzoic acid (8)
Chlorosulfonic acid (50 mL, 752 mmol) was cooled in an ice-
NaCl bath under an atmosphere of argon. To this was added o-
iodobenzoic acid (12.4 g, 50 mmol) in small portions over 5 min.
The cooling bath was removed and the solution was allowed to
warm to room temperature then heated overnight to 115 ꢂC (oil
bath temperature). The reaction mixture was then cooled to RT and
poured slowly over ice (~1 L). The resulting precipitate was filtered,

S.K. Chitneni et al. / European Journal of Medicinal Chemistry 119 (2016) 218e230
227
z ¼ 182 [(M þ H)þ]) and the material was used without further
3.2.12. N-(4-(3-Fluoropropyl)phenyl)-3-(4-(2-methoxyphenyl)
piperazine-1-carbonyl)-4-methylbenzenesulfonamide (6)
characterization.
N,N-Diisopropylethylamine (2.2 mL, 12.5 mmol, 4 eq.) was
added in one portion at RT to a (near) solution of the benzoic acid
derivative 13a (1.10 g, 3.13 mmol), HOBt (0.73 g, 4.70 mmol, 1.5 eq.),
EDC (0.9 g, 4.7 mmol, 1.5 eq.) and 1-(-2-methoxyphenyl)piperazine
(1.78 g, 7.80 mmol, 2.5 eq.) in anhydrous CH2Cl2 (50 mL). After 1 h,
analysis of the reaction mixture by LCMS indicated complete con-
sumption of the starting material 13a. Silica gel (~3 g) was added
and the mixture was concentrated under reduced pressure. Flash
column chromatography (RediSep® Rf SiO2 (40 g), 100%
CH2Cl2 / 50% EtOAc in CH2Cl2) gave the amide compound 6 as an
off-white solid (1.40 g, 85%), mp: 198e199 ꢂC. 1H NMR (CDCl3,
3.2.9. 1-(3-Fluoropropyl)-4-nitrobenzene (11a)
A solution of 3-(4-nitrophenyl)propan-1-ol 10 (2.5 g,13.8 mmol)
in anhydrous CH2Cl2 (47 mL) was cooled in a dry ice/acetone bath.
The solution was treated sequentially with 1,8-Diazabicyclo[5.4.0]
undec-7-ene (DBU, 3.2 mL, 21 mmol, 1.5 eq.) and XtalFluor-M® (5 g,
21 mmol, 1.5 eq.). The mixture was stirred in the cold bath for
30 min then allowed to warm to room temperature overnight. The
following morning, analysis of the reaction mixture by TLC (10%
EtOAc in hexanes) indicated complete consumption of starting
material. The reaction was quenched with saturated aqueous
NaHCO3 (50 mL) and the mixture was stirred for 30 min. Following
phase separation, the aqueous phase was extracted with CH2Cl2
(2 ꢃ 25 mL). The combined organic extracts were dried (MgSO4).
The drying agent was removed by filtration. Silica gel (~5 g) was
added and the filtrate was concentrated to dryness under reduced
pressure. Flash column chromatography (RediSep® Rf SiO2 (80 g),
100% hexanes / 10% EtOAc in hexanes) gave the product as a clear
yellow oil (1.04 g, 41%). Analysis of the material by LCMS showed a
single peak at multiple wavelengths (210 nm, 221 nm, 254 nm and
280 nm). The area under those peaks corresponded to the expected
mass-to-charge ratio (m/z ¼ 184 [(M þ H)þ]) and the material was
used without further characterization.
400 MHz):
d
7.66 (s, 1H), 7.59 (d, J ¼ 8.0 Hz, 1H), 7.28 (m, 3H),
7.05e6.89 (m, 8H), 4.45 (t, J ¼ 5.6 Hz, 1H), 4.33 (t, J ¼ 5.6 Hz, 1H),
3.96 (m, 2H), 3.87 (s, 3H), 3.29 (t, J ¼ 4.8 Hz, 2H), 3.11 (m, 2H), 2.90
(m, 2H), 2.36 (s, 3H), 1.95 (m, 1H), 1.91 (m, 1H), 1.66 (s, 1H). 13C NMR
(CDCl3, 125 MHz)
d 168.11, 152.15, 140.29, 139.98, 138.39, 137.14,
136.55, 134.41, 131.06, 129.18, 127.55, 124.88, 123.68, 122.43, 121.03,
118.45, 111.35, 83.51, 82.20, 55.38, 51.03, 50.47, 47.10, 41.94, 31.84,
31.68, 30.59, 30.56, 19.21. ESI-MS: m/z calcd. for C28H33FN3O4S
[(M þ H)þ]: 526.2170; observed: 526.2169.
3.2.13. N-(4-(4-Fluorobutyl)phenyl)-3-(4-(2-methoxyphenyl)
piperazine-1-carbonyl)-4-methylbenzenesulfonamide (7)
N,N-Diisopropylethylamine (1.8 mL, 10.5 mmol, 4 eq.) was
added in one portion at RT to a (near) solution of the acid 13b (1.0 g,
2.62 mmol), HOBt (0.61 g, 3.90 mmol, 1.5 eq.), EDC (0.75 g,
3.90 mmol, 1.5 eq.) and 1-(-2-methoxyphenyl)piperazine (1.25 g,
6.60 mmol, 2.5 eq.) in anhydrous CH2Cl2 (50 mL). After 1 h, analysis
of the reaction mixture by LCMS indicated complete consumption
of the starting acid. Silica gel (~3 g) was added and the mixture was
concentrated under reduced pressure. Flash column chromatog-
raphy (RediSepRf SiO2 (40 g), 100% CH2Cl2 / 50% EtOAc in CH2Cl2)
gave the amide 7 as an off white solid (1.1 g, 78%), mp: 160e161 ꢂC.
3.2.10. 4-(3-Fluoropropyl)aniline (12a)
A solution of the nitro compound 11 (1.04 g, 5.7 mmol) and 10%
Pd/C (0.25 g) in MeOH (15 mL) was stirred overnight under a
balloon of H2 after which time, analysis of the reaction mixture by
TLC (10% EtOAc in hexanes) indicated complete consumption of
starting material. The mixture was filtered through a pad of Celite.
The pad was washed with MeOH (50 mL). Silica gel (~2 g) was
added and the filtrate was concentrated to dryness under reduced
pressure. Flash column chromatography (RediSep® Rf SiO2 (40 g),
100% hexanes / 100% EtOAc in hexanes) gave the product as a
clear amber liquid (0.62 g, 71%). Analysis of the material by LCMS
showed a single peak at multiple wavelengths (210 nm, 221 nm,
254 nm and 280 nm). The area under those peaks corresponded to
the expected mass-to-charge ratio (m/z ¼ 154 [(M þ H)þ]) and the
material was used without further characterization.
1H NMR (CDCl3, 400 MHz)
d
7.62 (s, 1H), 7.58 (d, J ¼ 8.0 Hz, 1H), 7.25
(m, 3H), 7.05e6.86 (m, 8H), 4.45 (t, J ¼ 5.6 Hz,1H), 4.33 (t, J ¼ 5.6 Hz,
1H), 3.97 (m, 2H), 3.85 (s, 3H), 3.26 (t, J ¼ 4.8 Hz, 2H), 3.10 (m, 2H),
2.87 (m, 2H), 2.34 (s, 3H), 1.66e1.62 (m, 5H). 13C NMR (CDCl3,
125 MHz)
d 168.11, 152.16, 140.31, 139.97, 139.34, 137.13, 136.53,
134.21, 131.04, 129.07, 127.56, 124.87, 123.67, 122.45, 121.02, 118.46,
111.34, 84.43, 83.12, 55.38, 51.01, 50.50, 47.10, 41.94, 34.64, 29.86,
29.70, 26.78, 26.75, 19.21. ESI-MS: m/z calcd for C29H35FN3O4S
[(M þ H)þ]: 540.2327; observed: 540.2329.
3.2.11. 5-(N-(4-(3-Fluoropropyl)phenyl)sulfamoyl)-2-
methylbenzoic acid (13a)
Solid
5-(chloromethylsulfonyl)-2-methylbenzenecarboxylic
acid (1.04 g, 4.43 mmol, 1.1 eq.) was added in one portion to an
ice-bath cooled solution of 4-(3-fluoropropyl)aniline 12a (0.617 g,
4.03 mmol) in pyridine (15 mL). The reaction was allowed to warm
to room temperature as the cooling bath melted. Stirring was
continued overnight (15 h) after which time analysis of the reaction
by TLC (50% EtOAc in hexanes) indicated complete consumption of
the starting aniline. The reaction mixture was diluted with CH2Cl2
(50 mL) and washed with 1 N HCl (50 mL). The aqueous wash was
extracted with CH2Cl2 (3 ꢃ 50 mL). The organic extracts were
combined. Silica gel (~2 g) was added and the mixture was
concentrated to dryness under reduced pressure. Flash column
chromatography (RediSep® Rf SiO2 (40 g), 100% hexanes / 100%
EtOAc in hexanes) gave the product as an off-white solid (1.10 g,
74%). Analysis of the material by LCMS showed a single peak at
multiple wavelengths (210 nm, 221 nm, 254 nm and 280 nm). The
area under those peaks corresponded to the expected mass-to-
charge ratio (m/z ¼ 368 [(M þ H)þ] and 366 [(MꢁH)ꢁ]) and the
material was used without further characterization.
3.2.14. 3-(4-(2-Bromophenyl)piperazine-1-carbonyl)-N-(4-
butylphenyl)-4-methylbenzenesulfonamide (14)
This was synthesized as described for compound 1 using 1-(2-
bromophenyl)piperazine (0.17 g, 0.70 mmol) and 5-(N-(4-
butylphenyl)sulfamoyl)-2-methylbenzoic acid (0.25 g, 0.70 mmol)
as starting materials. Purification of the crude material by flash
column chromatography (0 / 50% EtOAc in hexanes) yielded the
bromo precursor 14 as an off-white solid (0.31 g, 77%), mp:
86e87 ꢂC. 1H NMR (CDCl3, 400 MHz):
d 7.61e7.57 (m, 2H), 7.31e7.21
(m, 2H), 7.1e6.92 (m, 6H), 6.62 (s, 1H), 3.98 (m, 2H), 3.25 (m, 2H),
3.09 (m, 2H), 2.84 (m, 2H), 2.47 (t, J ¼ 8.0 Hz, 2H) 2.37 (s, 3H), 1.51
(m, 2H), 1.28 (m, 2H), 0.87 (t, J ¼ 7.2 Hz, 3H). 13C NMR (CDCl3,
125 MHz)
d 168.25, 149.63, 140.40, 139.99, 137.10, 136.45, 133.85,
131.14, 129.09, 128.39, 127.64, 125.10, 124.96, 122.48, 121.11, 119.98,
52.06, 51.37, 47.16, 41.99, 34.90, 33.38, 22.23, 19.24, 13.83. LC-MS
(DART): m/z calcd. for C28H33BrN3O3S [(M þ H)þ]: 572.1406;
observed: 572.1398.

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S.K. Chitneni et al. / European Journal of Medicinal Chemistry 119 (2016) 218e230
3.2.15. N-(4-Butylphenyl)-3-(4-(2-hydroxyphenyl)piperazine-1-
carbonyl)-4-methylbenzenesulfonamide (15)
HPLC system that consisted of an XTerra® C18 column (5
mm,
4.6 ꢃ 250 mm) eluted with gradient mixtures of acetonitrile (A) and
water (B) containing 0.1% TFA in both the solvents. The % of
acetonitrile was kept constant at 5% for the first 5 min and was then
increased to 90% over 30 min at a flow rate of 1 mL/min. The UV
absorbance was measured at 254 nm. [18F]4 eluted with a retention
time of 27.2 min compared to 27.0 min for the cold analog 4.
This was synthesized as described for compound 1, but starting
from 2-(piperazin-1-yl)phenol (0.15 g, 0.84 mmol) and 5-(N-(4-
butylphenyl)sulfamoyl)-2-methylbenzoic acid (0.3 g, 0.84 mmol).
Purification of the crude mixture by flash column chromatography
(0 / 50% EtOAc in hexanes) gave the phenol precursor 15 as an off-
white solid (0.23 g, 54%), mp: 158e160 ꢂC. 1H NMR (CDCl3,
400 MHz):
d
7.63e7.57 (m, 2H) 7.31 (d, J ¼ 8.4 Hz, 1H) 7.17e6.87 (m,
3.4. Mutant IDH1 inhibition assays
8H), 6.63 (s, 1H) 3.95 (m, 2H), 3.23 (m, 2H), 2.94 (m, 2H), 2.68 (m,
2H), 2.46 (t, J ¼ 7.6 Hz, 2H), 2.38 (s, 3H), 1.49 (m, 2H), 1.27 (m, 2H),
The inhibitory efficiency of the synthesized nonradioactive an-
alogs against mutant IDH1 was evaluated using purified IDH1-
R132H enzyme in standard 384-well plates. Recombinant IDH1-
R132H protein was derived as described previously [10] by
expression with a C-terminal 6ꢃHis Tag in E. coli followed by pu-
rification using a Ni-NTA spin column (Qiagen, Hilden, Germany).
For inhibition assays, serially-diluted solutions of nonradioactive
0.87 (t, J ¼ 7.2 Hz, 3H). 13C NMR (CDCl3, 125 MHz)
d 168.22, 151.09,
140.44, 140.01, 137.95, 137.12, 136.24, 133.80, 131.21, 129.11, 127.73,
126.94, 124.93, 122.43, 121.29, 120.28, 114.54, 52.68, 52.22, 47.37,
42.25, 34.84, 33.37, 22.20, 19.25, 13.81. LC-MS: m/z calcd. for
C
28H34N3O4S ([M þ H]þ): 508.2270; observed: 508.2269.
3.3. Radiochemistry
test compounds in DMSO (1
loaded with 25 L of substrate mixture consisting of
rate (2 mM) and NADPH (8 M). After centrifugation of the plate at
300g for 3 min, a diluted solution of IDH1-R132H enzyme (25 L;
7.5 ng/ L) was added to the reaction mixture. After a second
centrifugation, the mixture was incubated at RT for 50 min fol-
lowed by the addition of developer mixture (25 L) containing
resazurin (30 M) and diaphorase enzyme (36 g/mL). The mixture
was incubated for 5 min, and the fluorescence intensity in reaction
mixtures was measured by reading the plates on a POLARstar Op-
tima plate reader (BMG Labtech, Germany) using 544 nm and
590 nm as excitation and emission wavelengths, respectively. It
should be noted that the fluorescence reaction is enabled by the
conversion of resazurin to a highly fluorescent resorufin by the
diaphorase enzyme, and the signal intensity is proportional to the
unconsumed NADPH present in the reaction mixture. Efficient in-
hibition of the mutant IDH1 by the test compounds results in a high
fluorescence signal due to the presence of high NADPH in the re-
action mixture, whereas low fluorescence in wells is an indicative
of uninhibited reactions.
m
L) were added to wells that were pre-
m
a
-ketogluta-
3.3.1. N-(4-Butylphenyl)-3-(4-(2-[125I]iodophenyl)piperazine-1-
carbonyl)-4-methylbenzenesulfonamide ([125I]1)
m
m
To a solution of the bromo precursor 14 in ethanol (0.2 mg in
m
20
10 mg/mL solution of gentisic acid (ethanol/H2O, 75/25, v/v) and
18 L of 0.013 M CuSO4 (H2O). The reaction mixture was purged
with argon for 5 min followed by the addition of [125I]NaI (3e13
L,
mL; 0.35 mmol) in a 0.2 mL Reacti-Vial™ were added 80 mL of a
m
m
m
m
m
46e145 MBq). The reaction vial was tightly closed using a screw cap
with septum (PTFE/silicone disc) and was heated at 140 ꢂC for 1 h in
an oil bath. The mixture was cooled to RT, and was purified using
RP-HPLC on an XTerra® C18 column (5
m
m, 4.6 mm ꢃ 250 mm;
Waters) eluted with 60% acetonitrile in water with 0.1% TFA, at a
flow rate of 1 mL/min. The HPLC peak corresponding to [125I]1 was
collected (tR ¼ 26e28 min), diluted to 10 mL with water and loaded
onto a pre-conditioned Sep-Pak® SPE cartridge (C18, Waters). After
rinsing the cartridge with water (5 mL), the labeled product was
eluted with ethanol and 50-mL fractions were collected. Most of the
activity was eluted in fractions 3e6, which were used for biological
experiments. The identity confirmation of the purified [125I]1 was
achieved by co-injection with the unlabeled analog 1 on the HPLC
system as described for the purification.
3.5. Functional inhibition assay
The ability of compounds 1 and 4 to inhibit the neomorphic
activity of the mutant, IDH1-R132H, was evaluated using isogenic
anaplastic astrocytoma cell lines carrying a native IDH1-R132H
mutation or WT-IDH1. The IDH1-R132H-mutated astrocytoma cell
line was a subclone derived from a WHO grade III anaplastic as-
trocytoma; this line was described previously [19]. The isogenic
WT-IDH1 cell line was a sister subclone derived at the same time
from the same astrocytoma tumor cells. The WT-IDH1 cell line had
lost the IDH1-R132H allele during cell line passage and retained
only the IDH1-WT allele. Sanger sequencing was used to confirm
that only the wild type IDH1 sequence and no mutant IDH1-R132H
sequence was retained in the WT-IDH1 cell line. Likewise, Sanger
sequencing was used to confirm that the WT-IDH1 cell line retained
all other known genetic alterations found in the original tumor and
in the isogenic IDH1-R132H cell line, including TP53 (TP53
p.G245V) and ATRX (ATRX p.R781X) mutations. The AmpFlSTR®
Identifiler® Direct PCR Amplification Kit (Applied Biosystems) was
used to confirm that the D2S1338 locus at chromosome 2q35 near
the IDH1 locus was reduced to one marker for the WT-IDH1 cell
line, consistent with loss of heterozygosity at the IDH1 locus for the
WT-IDH1 subline.
3.3.2. N-(4-Butylphenyl)-3-(4-(2-(2-[18F]fluoroethoxy)phenyl)
piperazine-1-carbonyl)-4-methylbenzenesulfonamide ([18F]4)
[
18F]Fluoride, obtained on a QMA® cartridge, was eluted into a
conical reaction vial with a Kryptofix (37.2 mg/mL) and K2CO3
(3.3 mg/mL) solution in acetonitrile/water (0.75 mL, 95/5, v/v).
Removal of solvents and further drying of the activity was achieved
by heating the reaction vial at 100 ꢂC and by azeotropic distillation
with acetonitrile (3 ꢃ 0.75 mL) under a stream of N2. The dried
activity was allowed to cool to RT, and a solution of 2-bromoethyl
triflate in O-dichlorobenzene was added to the reaction vessel
(5 m
L in 0.7 mL). The mixture was then heated at 120 ꢂC while
bubbling with N2, and the resulting [18F]fluoroethyl bromide ([18F]
FEtBr) along with the N2 sweep gas was passed through a C18 Sep-
Pak before collecting the activity in a conical reaction vial that
contained the phenol precursor 15 (0.5 mg) and Cs2CO3 in DMF
(0.25 mL). Upon completion of the distillation and/or collecting
sufficient activity (~20 min), the reaction vial was tightly closed and
the labeling mixture was heated at 120 ꢂC for 15 min for the 18F-
fluoroethylation reaction. The crude mixture was diluted with
water (1.5 mL) and injected onto an XBridge® C18 column (5
mm,
4.6 ꢃ 150 mm) for purification. The product was eluted with 45%
EtOH in 0.05 M sodium acetate buffer (pH 5.5) at a flowrate of 1 mL/
min (tR ¼ 18.0 min). The identity of the purified [18F]4 was
confirmed by co-elution with nonradioactive analog 4 on a RP-
For inhibition assays, cells were seeded in duplicate in standard
12-well plates at a density of 30,000 per well. Upon reaching full
confluency, the incubation media was replaced with fresh media
containing the unlabeled compounds 1 or 4 (50
m
M, n ¼ 2/

S.K. Chitneni et al. / European Journal of Medicinal Chemistry 119 (2016) 218e230
229
experiment). The drug-containing media was prepared from
Appendix A. Supplementary data
20 mM stock solutions of compounds (DMSO). Cells were then
incubated under standard cell culture conditions and aliquots
(0.1 mL) of supernatant were removed at 4 and 8 h after incubation.
The samples were stored at ꢁ80 ꢂC until analysis by LC-MS/MS. The
concentration of D-2-HG in the samples was measured by LC-MS/
MS as described before [14]. The WT-IDH1 cell line and IDH1-
132H cells that were treated with vehicle alone served as controls.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.04.066.
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