Discovery and Optimization of Potent, Cell-Active Pyrazole-Based Inhibitors of Lactate Dehydrogenase (LDH)

Article abstract of DOI:10.1021/acs.jmedchem.7b00941

We report the discovery and medicinal chemistry optimization of a novel series of pyrazole-based inhibitors of human lactate dehydrogenase (LDH). Utilization of a quantitative high-throughput screening paradigm facilitated hit identification, while structure-based design and multiparameter optimization enabled the development of compounds with potent enzymatic and cell-based inhibition of LDH enzymatic activity. Lead compounds such as 63 exhibit low nM inhibition of both LDHA and LDHB, submicromolar inhibition of lactate production, and inhibition of glycolysis in MiaPaCa2 pancreatic cancer and A673 sarcoma cells. Moreover, robust target engagement of LDHA by lead compounds was demonstrated using the cellular thermal shift assay (CETSA), and drug-target residence time was determined via SPR. Analysis of these data suggests that drug-target residence time (off-rate) may be an important attribute to consider for obtaining potent cell-based inhibition of this cancer metabolism target.

Full text of DOI:10.1021/acs.jmedchem.7b00941

Article
pubs.acs.org/jmc
Cite This: J. Med. Chem. XXXX, XXX, XXX-XXX
Discovery and Optimization of Potent, Cell-Active Pyrazole-Based
Inhibitors of Lactate Dehydrogenase (LDH)
Ganesha Rai,*,†,∞ Kyle R. Brimacombe,†,∞ Bryan T. Mott,† Daniel J. Urban,† Xin Hu,† Shyh-Ming Yang,†
Tobie D. Lee,† Dorian M. Cheff,† Jennifer Kouznetsova,† Gloria A. Benavides,‡ Katie Pohida,†
Eric J. Kuenstner,†,◆ Diane K. Luci,† Christine M. Lukacs,§,¶ Douglas R. Davies,§ David M. Dranow,§
Hu Zhu,† Gary Sulikowski,⊥ William J. Moore,# Gordon M. Stott,# Andrew J. Flint,# Matthew D. Hall,†
Victor M. Darley-Usmar,‡ Leonard M. Neckers,∥ Chi V. Dang,∇ Alex G. Waterson,⊥ Anton Simeonov,†
Ajit Jadhav,† and David J. Maloney*,†,○
†National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville,
Maryland 20850, United States
‡Mitochondrial Medicine Laboratory, Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294,
United States
§Beryllium Discovery Corp., 7869 Day Road West, Bainbridge Island, Washington 98110, United States
∥Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, 9000 Rockville Pike, Bethesda, Maryland 20892,
United States
⊥Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee 37232, United States
#NExT Program Support, Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National
Laboratory for Cancer Research, Frederick, Maryland 21702, United States
∇Abramson Cancer Center, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania,
Philadephia, Pennsylvania 19104, United States
S
* Supporting Information
ABSTRACT: We report the discovery and medicinal chemistry
optimization of a novel series of pyrazole-based inhibitors of human
lactate dehydrogenase (LDH). Utilization of a quantitative high-
throughput screening paradigm facilitated hit identification, while
structure-based design and multiparameter optimization enabled
the development of compounds with potent enzymatic and cell-
based inhibition of LDH enzymatic activity. Lead compounds such
as 63 exhibit low nM inhibition of both LDHA and LDHB,
submicromolar inhibition of lactate production, and inhibition of
glycolysis in MiaPaCa2 pancreatic cancer and A673 sarcoma cells.
Moreover, robust target engagement of LDHA by lead compounds
was demonstrated using the cellular thermal shift assay (CETSA),
and drug−target residence time was determined via SPR. Analysis
of these data suggests that drug−target residence time (off-rate) may be an important attribute to consider for obtaining potent
cell-based inhibition of this cancer metabolism target.
oxidation of pyruvate in mitochondria for a comparatively
greater energy payoff. Though aerobic glycolysis is an inefficient
way to generate ATP, it has been proposed that rapidly
proliferating cancer cells have adapted this approach to facilitate
the production of essential building blocks like nutrients such as
amino acids, lipids, and nucleotides to support rapid cell growth
rather than efficient ATP production.2 LDH is a key glycolytic
enzyme that catalyzes the final step in the glycolytic pathway,
INTRODUCTION
■
Tumor cells are often dependent on glycolysis for adenosine 5′-
triphosphate (ATP) biosynthesis, even in the presence of
sufficient oxygen to support oxidative phosphorylation, a
process termed aerobic glycolysis and classically known as the
“Warburg effect”.1 In such cancers, tumor cells exhibit a high
rate of glycolysis, metabolizing glucose into pyruvate, which
instead of entering mitochondria is reduced by lactate
dehydrogenase (LDH) to lactate and excreted by the cells.
This is in stark contrast to classic aerobic metabolism, in which
cells demonstrate low rates of glycolysis and instead rely on the
Received: July 5, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.7b00941
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Figure 1. Representative previously described LDH inhibitors and qHTS “hit” 5.
a
Scheme 1. Syntheses of Analogs 5, 9, and 12−15
a
Reagents and conditions: (a) NH2CSNHNH2, EtOH, reflux, 12 h, 55−75%; (b) (i) BrCH2COCO2Et, EtOH, reflux, 1 h; (ii) EtOH, H2SO4, reflux,
12 h, 24−35%; (c) (i) reversed-phase chromatography separation of regioisomers; (ii) HCl, AcOH, 120 °C, 1 h.
reducing pyruvate to lactate and regenerating NAD+ equiv-
alents necessary for continued glycolysis. Expression of the
LDHA gene is upregulated in many cancers to support the high
glycolytic activity in these cells.3,4 The LDH enzyme is a
tetramer composed of M subunits encoded by the LDHA gene
or H subunits encoded by the LDHB gene. In cancer cells, the
enzyme composed of 4 M subunits known as LDH-5 is thought
to predominate. Throughout this manuscript we will refer to
the enzyme as LDHA. Reduction of LDH activity through
knockdown or silencing of the LDHA gene has been shown to
reduce tumor cell growth in vitro under hypoxic conditions and
to suppress growth in tumor xenograft models.5 In addition,
high levels of LDHA expression have been correlated with poor
clinical outcome for a number of cancer types.6 Among cancers
with unmet therapeutic need, glioblastoma,7 pancreatic,8 and
advanced stage and rare hereditary kidney cancers9 are all
highly glycolytic and thus represent potential opportunities for
LDH inhibitors to provide clinical benefit.
However, despite its promise, LDHA has proven to be a
relatively intractable drug target. The enzyme active-site has a
highly mobile loop that caps the binding site for the small polar
organic anion substrate (pyruvate or lactate) and an extended
solvent exposed channel that binds cofactor. These features,
combined with the high protein levels of LDH in cancer cells,
necessitate a small molecule inhibitor that binds with
remarkable efficiency while simultaneously maintaining druglike
properties. Initial disclosures of LDH inhibitors emerged out of
academic laboratories (e.g., FX-115b and NHI-210) with efforts
from biotech11 and pharmaceutical companies, such as
AstraZeneca (e.g., 1),12 emerging later. To date, no clinical-
stage inhibitors of LDH have been reported; molecules from
GlaxoSmithKline (GSK) (2)13 and Genentech (3)14 have
shown modest cellular potency in vitro (e.g., inhibition of
B
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a
Scheme 2. Syntheses of Precursors I, II, and III and Analogs 7 and 10
a
Reagents and conditions: (a) BF3·OEt2, CH2Cl2−THF, 12 h, 88%; (b) N2H4, EtOH, reflux, 2 h, 82%; (c) EtOH, reflux, 5 h; (d) EtOH, AcOH,
reflux, 12 h, 77%; (e) TFA, CH2Cl2; (f) Ac2O, pyridine, 100 °C.
a
Scheme 3. Synthesis of 11, 16−32, and 33−41
a
Reagents and conditions: (a) CH3CO2Et, LDA, THF, −78 °C, 2−6 h, 15−90%; (b) II, AcOH, EtOH, reflux, 12 h, 12−70%; (c) TFA, CH2Cl2, 1 h;
b
(d) NaH, dioxane, 1 h, 0 °C, 60−75%; (e) III, EtOH, TsOH, MW, 15 min, 46−86%; (f) LiOH, THF/MeOH/H2O, 1 h. Commercially available.
lactate production) but no appreciable in vivo activity and do
not appear to have progressed into clinical studies.
be a particularly strong predictor of cell-based inhibition of the
target. In this report, we describe the discovery and medicinal
chemistry optimization of a novel series of pyrazole-based LDH
inhibitors. Compound 63 has proven to be a promising lead
compound worthy of further optimization, given its sub-μM
inhibition of cellular lactate production, demonstrated cellular
target engagement, slow in vitro off-rate, good microsomal
stability and aqueous solubility.
We designed and performed a quantitative high-throughput
screening (qHTS) campaign, and utilized structure-based
design and hit-to-lead optimization to discover novel
compounds that are potent inhibitors of LDH enzyme activity,
cellular lactate output, and cancer cell line growth. Lead
compounds from our work exhibit low nM inhibition of
LDHA/LDHB and sub-μM inhibition of lactate production in
MiaPaCa2 and A673 cells. Further, robust target engagement of
LDHA with these lead compounds was demonstrated by
cellular thermal shift assay (CETSA), and drug−target
residence time was determined via SPR. Among these
parameters, drug−target residence time (off-rate) appears to
CHEMISTRY
■
The qHTS identified trifluoromethylpyrazole compound 5 as a
hit candidate that was evaluated via extensive SAR studies.
Initial medicinal chemistry efforts focused on the pyrazole
substitutents. The syntheses of 5 and related analogs 9, 12−15
C
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a
Scheme 4. Synthesis of 43−45
a
Reagents and conditions: (a) ethyl 2-bromothiazole-4-carboxylate, K2CO3, 120 °C, 3−4 h, 27−78%; (b) bis(pinacolato)diboron, PdCl2(dppf),
AcOK, 1,4-dioxane, 95 °C, overnight 49%; (c) 4-(bromomethyl)benzenesulfonamide, 2N Na2CO3 (aq), Pd(Ph3P)4, tolune/EtOH (3/1), 80 °C, 2 h,
64−95%; (d) 1.5N LiOH (aq), THF, 2 h; (e) 4-hydroxybenzenesulfonamide, K2CO3, acetone, 20 h, 92%; (f) 1,1-dimethoxy-N,N-
dimethylmethanamine, 90 °C, overnight, then N2H4, EtOH, 60 °C, 4 h, 27%.
were accomplished following slight modifications to a known
literature method.15 As outlined in Scheme 1, commercially
available trifluoromethyl-β-diketones were condensed with
thiosemicarbazide to obtain key intermediates 5b, 9b, 12b−
15b and 5c, 9c, 12c−15c as a mixture of regioisomers, as
reported previously.15 The unseparated mixture of the
regioisomers was condensed with ethyl 3-bromo-2-oxopropa-
noate in the presence of sulfuric acid to obtain a ∼50/50
mixture of regioisomers 5d, 9d, 12d−15d and 5e, 9e, 12e−15e,
which were separated using reversed-phase chromatography.
The regioisomers were distinguished by their differences in
carbon and fluorine NMR as reported for similar compounds in
the literature16 and via LC/MS co-injection with a reference
compound obtained from commercial source. The desired 3-
aryl-substituted regioisomer is slightly less polar in nature and
elutes as a second peak in reversed-phase HPLC. The resulting
product was subsequently hydrolyzed with concentrated
hydrochloric acid to obtain analogs 5, 9, and 12−15.
The synthesis of the key precursors I, II, and III along with
analogs 7 and 10 is outlined in Scheme 2. Key intermediate tert-
butyl 2-hydrazinylthiazole-4-carboxylate (II) was synthesized
by esterification of the requisite 2-bromothiazole-4-carboxylic
acid (Ia) to form the tert-butyl ester (I). Subsequent
displacement of the bromide with hydrazine at reflux provided
II in 82% yield. Condensation of the acetylthiosemicarbazide
(IIIa) with ethyl bromopyruvate (IIIb) in ethanol initially
forms an acetyl derivative of ethyl 2-hydrazinylthiazole-4-
carboxylate that is eventually cleaved in situ by the hydrogen
bromide generated during the reaction to form ethyl 2-
hydrazinylthiazole-4-carboxylate (III) as a HBr salt. Subsequent
acid catalyzed reaction of II with benzoylacetonitrile in ethanol
followed by deprotection of the tert-butyl ester group gave 7, or
acetylation of the formed aminopyrazole intermediate followed
by tert-butyl deprotection provided 10 as shown in Scheme 2.
The synthetic route for hydroxypyrazole analogs 11 and 16−
41 listed in Tables 1 and 2 is described in Scheme 3.
Accordingly, direct condensation of the β-keto esters (11b,
16b−32b) in the presence of acetic acid with II and subsequent
cleavage of the tert-butyl ester group with TFA afforded analogs
11 and 16−32. For the synthesis of analogs 33−41, the β-keto
esters (11b, 27b, 31b, 32b and 36a) were alkylated with
appropriately substituted benzyl bromides (33a−35a and 39a−
41a) in the presence of sodium hydride in dioxane. The
alkylated β-keto esters (33b−41b) are then heated with III
under microwave irradiation in the presence of catalytic amount
of tosic acid to obtain the cyclized esters. Interestingly,
cyclization catalyzed by acetic acid in this case mostly produced
a major unidentified byproduct. However, switching to tosic
acid exclusively formed the desired 5-hydroxypyrazole deriva-
tive. Finally, analogs 33−41 were obtained by the LiOH
hydrolysis of the corresponding ethyl esters in THF-methanol−
water solvent. Commercially unavailable β-keto esters (21b and
27b−32b) and 4-(bromomethyl)benzenesulfonamide (33a)
were prepared according to the literature method.17
Scheme 4 illustrates a general strategy for the synthesis of
analogs 43−45. Potassium carbonate-catalyzed reaction of ethyl
2-bromothiazole-4-carboxylate with the requisite commercially
available bromopyrazole derivatives (43a and 44a) in DMSO at
120 °C gave intermediates 43b and 44b. The formed thiazole-
containing intermediates 43b and 44b were then converted to
corresponding boronic acid pinacol esters 43c and 44c by
PdCl2(dppf) catalyzed cross coupling with bis (pinacolato)-
diboron using potassium acetate as base at 80 °C. Suzuki
coupling of 43c and 44c with 4-(bromomethyl)benzene-
sulfonamide (33a), following a standard protocol catalyzed by
tetrakis(triphenylphosphine)palladium(0) in the presence of
aqueous sodium carbonate base and subsequent ester
hydrolysis using LiOH, provided analogs 43 and 44.
Preparation of analog 45 commenced with potassium
carbonate-assisted alkylation of commercially available phenacyl
bromide (45a) with 4-hydroxybenzenesulfonamide to provide
45b in 92% yield. Intermediate 45b was dissolved in a solution
D
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a
Scheme 5. Synthesis of 46 and 47
a
Reagents and conditions: (a) i-Pr2NEt, CH2Cl2, 0 °C, 1 h, 96%; (b) Fe, NH4Cl, EtOH, reflux, 94%; (c) t-BuBrettPhos, t-BuBrettPhos-palladacycle,
LHMDS, THF, 80 °C, 14 h, 62−83%; (d) K2CO3, 125 °C, 14 h, 60%; (e) TFA, CH2Cl2, 100 °C, MW, 15 min.
a
Scheme 6. Synthesis of 48−50
a
Reagents and conditions: (a) K2CO3, DMSO, 120 °C, 24 h, 25%; (b) HATU, i-Pr2NEt, DMF, 60 °C, 4 h; (c) NaBH3CN, AcOH, MeOH; (d)
TFA, CH2Cl2, 1 h; (e) K2CO3, DMSO, 125 °C, 3 h, 52%.
of DMF−DMA and heated to form the formyl derivative in situ
which was then refluxed with hydrazine in ethanol to give
pyrazole derivative 45c in 27% yield. Finally, coupling of 45c
with ethyl 2-bromothiazole-4-carboxylate using potassium
carbonate in DMSO was followed by LiOH hydrolysis of the
ester to provide analog 45.
critical for the success of the amination reaction. Several
attempts to couple the amine with the unprotected sulfonamide
were unsuccessful. Finally, potassium carbonate assisted
coupling of 46a and 47a with I in DMSO at 125 °C followed
by deprotection of the tert-butyl ester afforded analogs 46 and
47.
Access to analogs 46 and 47 was achieved using the synthetic
route outlined in Scheme 5. The key precursor, 4-
aminobenzenesulfonamide derivative IVd, was prepared
starting from p-nitrobenzenesulfonyl chloride (IVb) by
protecting the sulfonyl group with bis(3,4-dimethoxybenzyl)-
amine (IVa), followed by nitro reduction using iron/
ammonium chloride in ethanol. Subsequent Buchwald
amination of IVd was carried out with 4-bromo-3-arylpyrazole
(43a and 44a) using t-BuBrettPhos and t-BuBrettPhos
palladacycle catalyst system with LHMDS in THF to furnish
the intermediates 46a and 47a in good yield. The choice of
LHMDS as the base and protection of the sulfonamide were
A common synthetic strategy was used to synthesize analogs
48−50. As depicted in Scheme 6, potassium carbonate-assisted
SNAr bromide displacement on I with 3-phenyl-1H-pyrazol-4-
amine (Va) or 3-phenyl-1H-pyrazole-4-carbaldehyde (49a) in
DMSO at elevated temperatures gave intermediates V and 49b,
respectively. Notably, the SNAr reaction of I with any 3,4-
disubstituted pyrazoles was accomplished using the combina-
tion of potassium carbonate base, DMSO as a solvent, and an
optimal temperature between 120 and 130 °C depending on
the type of the substitution at 4-position of the pyrazole. As
exemplified for intermediate V, the reaction proceeds at 120 °C
to give the desired product in low yield. However, the product
E
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a
Scheme 7. Synthesis of 51−57
a
Reagents and conditions: (a) MeCN, LDA, −78 °C, 4 h, 78−97%; (b) Hantzsch ester, L-Pro, EtOH, 60 °C, 0.5 h, 86−98%; (c) III, TsOH, EtOH,
150 °C, MW, 15 min, 60−77%; (d) TsOH, NaNO2, KI, MeCN, 12 h, 36−47%. (e) 52: CuCN, DMSO, 160 °C, 0.5 h, 78%. (f) 51, 53−55: requisite
boronic acid, SiliCat-DPP-Pd, Na2CO3, DME, MW, 130 °C, 0.5 h. (g) 57: (1,10-phenanthroline)(trifluoromethyl)copper(I), DMF, 55 °C, 1 h, 97%;
(i) LiOH, THF−MeOH, 1 h.
completely decomposes at higher temperatures, yet the reaction
is sluggish at lower temperatures or using alternative
conditions. Intermediates V and 49b were then subjected to
amine coupling with either 4-sulfamoylbenzoic acid (48a) using
HATU or reductive amination with 4-formylbenzene-
sulfonamide (50a) or 1-piperazinesulfonamide (VI) using
sodium cyanoborohydride followed by TFA deprotection of
the tert-butyl ester group to obtain analogs 48−50.
hydrolyzed with lithium hydroxide and purified by HPLC to
obtain compound 56. With intermediates 51e and 56e in hand,
the iodide was then subjected to Suzuki coupling with the
appropriate boronic acid using SiliaCat DPP-Pd catalyst in the
presence of aqueous Na2CO3 in DME under microwave
irradiation conditions at 130 °C to provide the aryl (51, R1 =
Me; 53, R1 = Ph; 54, R1 = 3-pyridine; 55, R1 = 4-pyridine) after
ester hydrolysis with LiOH. For the synthesis of 52, the iodide
was displaced with cyanide by heating with CuCN in DMSO at
160 °C. For 57, Hartwig’s trifluoromethylator18 (1,10-
phenanthroline)(trifluoromethyl)copper(I) was used to install
the requisite trifluoromethyl group. As with the above-
mentioned analogs, the corresponding ethyl esters were
hydrolyzed using LiOH to furnish analogs 52 and 57.
A convergent synthesis via cross coupling of the iodide in
intermediate 51e or 56e was desired for more expedient analog
synthesis; however, that approach failed for a number of desired
analogs and more specifically with methylene cyclopropyl
analogs 62 and 63. Therefore, a linear synthetic route was
utilized, as shown in Scheme 8. Analogs 58 and 61 were
synthesized using two different methods and rigorously
analyzed to determine the correct regiochemistry of the
respective products. The synthesis commenced from reaction
of the requisite commercially available carboxylic acid with
1,2,3-benzotriazole to form N-acylbenzotriazole derivatives 58a
and 62a. The next step involved coupling of the formed N-
acylbenzotriazoles 58a and 62a to substituted acetophenones
59a and 61a via a magnesium bromide ethyl etherate-catalyzed
soft enolization method in the presence of Hunig’s base to form
1,3-diketones 58b and 61b−63b in 60−69%. The 1,3-diketones
58b and 61b−63b were then efficiently alkylated with 4-
(bromomethyl)benzenesulfonamide using cesium carbonate in
DMSO at room temperature to generate intermediates 58c and
61c−63c. Intermediates 59c and 60c were readily obtained
The syntheses of analogs 51−57 required an alternative and
novel synthetic strategy, as our initial attempts to access these
analogs from hydroxypyrazoles or alkylation of trifluoromethyl-
β-diketones were unsuccessful. As described in Scheme 7, our
synthesis began from LDA-catalyzed aroylation of acetonitrile
using commercially available aroyl chlorides 42a and 56a at
−78 °C to afford the requisite aroyl acetonitriles 42b and 56b.
A three-component Hantzsch ester-catalyzed reductive alkyla-
tion of 42b and 51b (commercially available), and 56b with 4-
formylbenzenesulfonamide (50a) at 60 °C in ethanol quickly
provided intermediates 42c, 51c, and 56c in good yield.
Subsequent tosic acid-catalyzed cyclocondensation of the
alkylated aroyl acetonitriles 42c, 51c, and 56c with ethyl 2-
hydrazinylthiazole-4-carboxylate (III) under microwave heating
at 150 °C for 15 min in ethanol gave the critical intermediates
42d, 51d, and 56d in 60−77% yield. Our initial attempts to
convert the amine functionality in compound 42d to a bromide
or chloride were unsuccessful due to undesired halogenation of
the thiazole at 5-position. However, using a sequential
diazotization−iodination procedure of the amines 42d, 51d,
and 56d in the presence of excess tosic acid with a premixed
solution of KI/NaNO2 in acetonitrile furnished the key
intermediates 42e, 51e, and 56e in moderate yield. A fortuitous
byproduct of this reaction was the des-amino (e.g., H-pyrazole)
derivative (R3 = 3,4-F), which after hydrolysis of the ethyl ester
afforded compound 42. A portion of the intermediate 56e was
F
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a
Scheme 8. Synthesis of 58−63
a
Reagents and conditions: (a) 1,2,3-benzotriazole, SOCl2, CH2Cl2, 4 h, 91−100%; (b) MgBr2·OEt, i-Pr2NEt, CH2Cl2, 12 h, 60−69%; (c) 4-
(bromomethyl)benzenesulfonamide, Cs2CO3, DMSO, 1 h, 55−83%; (d) TMS-Cl, NCS, CH2Cl2, 0 °C to rt, 3 h, 73−94%; (e) 4-
hydroxybenzenesulfonamide, K2CO3, DMSO, 50 °C, 1 h, 31−49%; (f) TsOH, EtOH, reflux, 12 h, 77−83% (mixture); (g) LiOH, THF−MeOH, 1 h.
from 58b−61b via chlorination with NCS followed by
potassium carbonate assisted alkylation of the 4-
hydroxybenzenesulfonamide. Tosic acid-catalyzed cycloconden-
sation of 2-alkylated 1,3-diketones 58c−63c with ethyl 2-
hydrazinylthiazole-4-carboxylate hydrobromide III generated a
mixture of both desired and undesired regioisomers. The ratio
of the formation of the regioisomers varies depending on the
nature of the substitution at R1. For example, the cyclization
ratio of 58c−61c was 50:50, whereas it decreased to 10:90
desired to undesired for 62c and 63c. The desired isomer is
slightly less polar (as judged by LC/MS analysis) and thus
elutes as the second peak with reversed-phase (C18)
chromatography. Of note, we were unable to separate these
isomers efficiently with normal phase silica gel chromatography.
The unseparated mixtures were then hydrolyzed with lithium
hydroxide in THF−methanol and separated in HPLC to give
58d−63d as first peak and 58−63 as second peak.
Analogs 64−68 were synthesized via functionalization at the
corresponding ethyl esters at the thiazole ring as shown in
Scheme 9. Heating the intermediate 64a with ammonia in a
sealed tube gave the amide analog 64, which upon dehydration
with trifluoroacetic anhydride (TFAA) in the presence of
Hunig’s base in dichloromethane followed by reaction with
sodium azide and ammonium chloride gave the tetrazole analog
67. The intermediate 64a was obtained by the condensation of
37b and III as shown in the Scheme 3. LAH reduction of the
intermediate 66a furnished analog 66 in high yield.
Trifluoromethylation of the intermediate 51e with (1,10-
phenanthroline)(trifluoromethyl)copper(I) in DMF as shown
in Scheme 7 provided intermediate 66a. Manganese dioxide
G
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a
Scheme 9. Synthesis of 64−68
a
Reagents and conditions: (a) NH3, EtOH, 60 °C, 18 h, 90%; (b) TFAA, i-Pr2NEt, CH2Cl2; (c) NaN3, NH4Cl, DMF, 125 °C, 2 h; (d) LiAlH4,
THF, 1 h; (e) MnO2, CHCl3, 2 h; (f) TMS-CF3, TBAF, THF, 4 h.
oxidation of the intermediate 68a and subsequent trifluor-
omethyl addition to the aldehyde using TBAF and TMS−CF3
provided analog 68. The intermediate 68a was readily obtained
from intermediate 43c using a Suzuki coupling with 4-
(bromomethyl)benzenesulfonamideas shown in Scheme 4.
concentrations concentrations above 20 μM, higher than
those typically screened (e.g., 10 μM). However, the multiple
dose qHTS format utilized at NCGC provides an opportunity
to identify even modest inhibitors, making it an effective hit-
identification platform for difficult targets like LDH.
Initial optimization efforts focused on systematic modifica-
tions to the CF3 group to explore variations in substituent size,
lipophilicity, and polarity, as shown in Table 1. Given the weak
potency and efficacy of these early analogs, tractable SAR was
not easily discernible, but some changes were clearly tolerated.
Replacement of CF3 (denoted as R1) with methyl (6), NH2
(7), or NHAc group (10) all led to compounds with similar
potency and efficacy as 5. In contrast, replacement with an i-Pr
(8) group or CHF2 (9) group resulted in a significant loss in
potency (>57 μM). Encouragingly, a drastic improvement in
potency (>100-fold) was observed when the CF3 group was
replaced by OH, to provide the hydroxylpyrazole compound
(11), with an IC50 of 0.144 μM. Additional hydroxyl analogs
(16−32, Table 1) all exhibited submicromolar activity to
further validate this SAR trend. Prior to the hydroxylpyrazole
discovery, we explored modifications to the phenyl group with
representative examples (12−15) shown in Table 1. For these
early analogs, a clear preference for halogens (e.g., Cl and F) at
the 4-position emerged as exemplified by the potency difference
between 12 and 13 (R2 = 2-Cl-Ph and 3-Cl-Ph, respectively),
compared to 14 and 15 (R2 = 4-Cl-Ph and 4-F-Ph). On the
basis of the improved potency of compound 11 (R1 = OH), we
held this group constant and expanded our SAR efforts of the
phenyl group (R2) with representative examples (16−32)
highlighted in Table 1. Single halogen substitutions with either
F or Cl substitution (16−19) were all well-tolerated, with the
RESULTS AND DISCUSSION
■
At the outset of our medicinal chemistry campaign, the leading
molecule reported in the literature, denoted as 1 (Figure 1),
was the result of a fragment-based screen and structure-guided
optimization campaign by AstraZeneca researchers.12 While
this compound had no appreciable cell-based activity (vide
infra), it provided reproducible and robust biochemical
inhibition of LDHA (IC50 = 1.02 μM, Table 1). Despite
conducting our screen with concentrations ranging from 13 nM
to 57 μM, very little inhibition was observed across a library of
approximately 400 000 compounds, an observation in agree-
ment with other screening efforts against this target. However,
our qHTS screening paradigm19 enabled the identification of
several compounds with modest potency (double digit μM)
and partial efficacy (30−60% maximum response) but
encouraging dose-dependent activity. One such compound, 2-
(3-phenyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)thiazole-4-car-
boxylic acid (5), was thus identified as a putative LDH inhibitor
and an attractive starting point for optimization based on its
relatively small size (MW = 339 g/mol) presence of a
carboxylic acid (could be docked to the pyruvate binding
site) and chemical tractability. This compound has been
previously reported as an EP1 receptor antagonist; the
compound was found to have no appreciable CYP inhibition
and was stable in human plasma, suggesting good drug-like
properties as a starting point for optimization.20 However, 5
exhibited only modest potency (22.2 μM, Table 1) and efficacy
(40−60%). It is worth noting this compound would likely have
been discarded as inactive in a traditional single-dose HTS
screening paradigm, as it only displayed inhibition at
4-F derivative (16) possessing the most potent activity (IC50
=
150 nM). Surprisingly, the preference for the 4-F group that we
observed for the CF3-substituted pyrazoles did not translate to
the hydroxypyrazole derivative, as compounds 18 and 19 (R2 =
2-F-Ph and 3-F-Ph, respectively) had comparable potencies of
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several biphenyl derivatives, with representative examples being
31 (R2 = (3-Ph)-Ph) and 32 (R2 = 3-(2-F-Ph)-Ph), which had
IC50 values of 266 nM and 95 nM, respectively. Though
encouraged by the progress in improving the inhibitory potency
of this series in the enzymatic assay, we were concerned that
none of these compounds demonstrated inhibition of lactate
production in cellular assays (data not shown), despite
biochemical potencies approaching 100 nM.
A majority of hydroxypyrazole based compounds reprodu-
cibly demonstrated nonclassic dose−response curves. Namely,
at most concentrations tested, these compounds yielded
sigmoidal dose−response curves, often with 100% efficacy.
However, at higher concentrations, inhibitory potency was
being diminished.
Table 1. LDHA Inhibition of 1, 5−32 with and without
a
LDHA
IC50
LDHA
SD (μM)
LDHA
(with EDTA)
analog
R1
NA
R2
(without EDTA)
1
NA
Ph
Ph
Ph
Ph
Ph
Ph
Ph
1.02 0.07
1.34 0.15
28.8 1.9
>57
b
5
CF3
Me
22.2 2.4
b
6
15.4 12
b
7
NH2
i-Pr
26.7
>57
>57
3
>57
One explanation for these aberrantly shaped IC50 curves
could be that at higher concentrations, the compounds were
not soluble and were precipitating out. However, these
compounds have very good aqueous solubility, and no evidence
of insolubility was observed. Alternatively, the compounds
being tested might also contain a lower potency activator of
LDHA that would overcome the inhibition at higher
concentrations. Multiple studies have previously reported that
several isoforms of LDHA, including human, demonstrate
metal-binding activity, notably with divalent cations such as
Mn2+, Co2+, and Zn2+. Incubation or complexation with certain
metals has been reported to maintain or potentiate LDHA
activity, while incubation with chelating agents such as EDTA
has been found to reversibly inhibit activity of the yeast LDHA
isoform.21 To test this hypothesis, the biochemical assay was
adapted and rerun in the presence of 100 μM EDTA to chelate
any (trace) metal present in the assay. Notably, in the presence
of EDTA, most of the hydroxylpyrazole derivatives (11, 16−
32) did not just lose the aberrant portion of the IC50 curve but
instead lost almost all inhibitory activity, with many
demonstrating IC50 values of >57 μM (Table 1). In contrast
to the hypothesis being tested, this result suggested that
inhibition by the hydroxylpyrazole compounds either required
or was potentiated by the presence of a trace metal. The activity
of the original hit 5 and the CF3-pyrazole analogs 14 and 15
was unaffected by EDTA, which suggested that inhibition by
this chemotype requires binding to LDHA that can be
enhanced by trace metals. Given the potential for metal
coordination by the hydroxypyrazole, in combination with the
thiazole nitrogen or sulfur and the carboxylic acid moiety,
additional studies to characterize the source and influence of
trace metals in this assay were conducted (see below for
additional details). These experiments pointed to the assay
buffer as the source of metal contamination, with trace amounts
of Zn2+ being the most likely contaminant affecting the assay.
Dialysis of individual assay components suggested that the trace
metal was present in the commercial Tris buffer used for the
assay rather than in the stocks of LDHA enzyme or inhibitor
(data not shown). Also, testing with several divalent cations
demonstrated that activity of the hydroxylpyrazole inhibitors
showed a dose-dependent potentiation with increasing metal
concentration and that the compounds were potentiated most
strongly by Zn2+ (added as zinc chloride, data not shown).
Metal impurities causing direct effects on LDHA has been
reported previously, as Ward et al. found that the presence of
silver resulted in inhibition of LDHA activity.12 Given these
observations, we sought to obtain the crystal structure of this
class of inhibitor bound to LDHA and carried our subsequent
rounds of LDHA inhibition assays under two conditions to fully
8
>57
9
CHF2
NHAc
OH
CF3
CF3
CF3
CF3
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
>57
b
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
9.57 1.8
>57
0.144 0.009
>57
>57
2-Cl-Ph
>57
3-Cl-Ph
>57
>57
4-Cl-Ph
26.6 0.1
27.7 1.9
32.3 2.1
>57
4-F-Ph
23.4 5.7
4-F-Ph
0.095 0.001
0.168 0.001
0.134 0.001
0.162 0.011
0.189 0.001
0.299 0.001
0.229 0.015
1.02 0.07
0.669 0.001
1.10 0.08
0.213 0.024
0.150 0.001
0.106 0.001
0.084 0.001
0.110 0.007
0.266 0.001
0.095 0.001
3-Cl-Ph
>57
2-F-Ph
>57
3-F-Ph
>57
3-CF3-Ph
3-OCF3-Ph
4-OMe-Ph
2-pyridine
4-pyridine
cyclohexyl
1-naphthyl
3,4-F-Ph
3,4-Cl-Ph
2,4-F-Ph
3,5-F-Ph
(3-Ph)-Ph
3-(2-F-Ph)-Ph
>57
>57
>57
>57
>57
>57
>57
>57
>57
>57
>57
>57
>57
a
IC50 values represent the half maximal (50%) inhibitory concen-
tration as determined in the HTS assay (n = 3) using a 22-dose
b
response in 1536-well format. Maximum response was less than 50%,
and thus IC50 values should be considered as lower confidence.
0.134 μM and 0.162 μM. Similar potency was also observed for
larger substituents on 20 (R2 = 3-CF3-Ph) and 21 (R2 = 3-
OCF3), which had IC50 values of 0.189 μM and 0.299 μM,
respectively. Incorporation of an electron donating group (22,
R2 = 4-OMe-Ph) was tolerated, while replacement of the
phenyl group with a 2-pyridine (23) or 4-pyridine (24) resulted
in a modest loss of activity (IC50 values of 1.02 and 0.669 μM,
respectively). Changing the phenyl to a cyclohexyl group (25)
gave potencies in line with the above-mentioned pyridine
derivatives (IC50 = 1.1 μM), while the naphthyl derivative (26)
had an IC50 value of 0.213 μM. Given data obtained from the
preliminary SAR described above, we sought to further explore
SAR of the pendent phenyl group at R2. Generally, bis-halogen
substituted were well tolerated, as exemplified by analogs 27−
30, with most compounds having comparable or improved
potency (IC50 values ranging from 84 nM to 150 nM) relative
to their monosubstituted counterparts. In an effort to explore
the tolerance for larger hydrophobic groups, we synthesized
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Figure 2. (A) Crystal structure of inhibitor 27 bound to LDHA in complex with zinc. The inhibitor is shown in sticks with salmon-colored carbons.
The protein is shown in ribbon representation, and the metal zinc is shown as a magenta sphere. A water molecule (red sphere) and protein residues
R168, H192, and T247 (yellow-colored carbons) are coordinated with Zn or form H-bonding interactions with the inhibitor. PDB code is 5W8I. (B)
Inhibitor 33 docked in the binding pocket of LDHA and overlaid with 4 (purple-colored carbons). The benzyl sulfonamide moiety is shown with
magenta-colored carbons.
account for any metal coordination effects: in the presence of
100 μM EDTA and in the presence of 100 μM ZnCl2.
increased steric bulk of the trifluoromethyl group (as compared
to the hydroxyl), the pyrazole is now tilted ∼30° out of plane.
In the absence of zinc, H192 now makes two hydrogen bonds
directly to the carboxylate and thiazole nitrogen of 15.
The X-ray crystal structure of inhibitor 27 bound to human
LDHA provided insight into the mechanism by which zinc
enhanced inhibitor potency. The complex was obtained by
transferring crystals grown with sodium malonate into a drop
without malonate but which contained citrate. To this solution,
27 and ZnCl2 were added to a final concentration of 2 mM and
allowed to soak for 3 days before crystals were harvested and
flash frozen. The 1.95 Å crystal structure contained compound
27 bound to three of the four subunits of the LDHA tetramer,
with malonate occupying the catalytic site of the remaining
monomer. The inhibitor is anchored by a salt bridge interaction
between its carboxylate and the guanidinium group of R168
(Figure 2A). A hydrogen bond also is observed between the
carboxylate and the side chain of T247. The thiazole ring and
the plane of N137 stack, and the distal difluorophenyl picks up
hydrophobic interactions with the planes of P138 and Q99.
The zinc is coordinated by H192, one water molecule, and
three atoms in-plane from 27 (the OH of the carboxylate, the
NH of the thiazole, and the OH of the hydroxylpyrazole group)
providing a clear rationale for the additional potency conferred
by zinc (Figure 2A, PDB code 5W8I). Our initial LDHA-
inhibitor complexes were formed in the absence of NADH;
however, the X-ray structure showed room below the inhibitor
for occupancy by cofactor. Subsequent structures included
NADH, which did not alter the position of the inhibitor but did
lead to substantial increases in binding affinity as determined by
SPR (vide infra).
With this experimental soaking method, we were then able to
determine a 1.8 Å crystal structure of the weaker CF3-
containing inhibitor 15 (Supporting Information Figure 2, PDB
code 5W8H). It was clear from the electron density in this
structure that the binding mode of 15 is essentially the same as
that of 27, with the carboxylate making the anchoring salt
bridge interaction with R168 and the thiazole rings being
superimposable between the two structures. Interestingly, the
published LDHA inhibitor NHI-2 was predicted to adopt
similar binding interactions in the active site, with the
carboxylate interacting with R168 (R169 in ref 10) and the
trifluoromethyl pointing to H192 (H193 in ref 10). The
trifluoromethyl is well ordered in the crystal structure, but
without the planar coordination enforced by zinc and with the
Despite the apparent requirement of a coordinating zinc ion
for optimal inhibitory potency, the similar binding orientation
observed for 15 and the ability of 15 to adjust in order to fill the
binding site and directly interact with H192 suggested that
potent analogs could be designed that would bind without zinc.
A different LDHA inhibitor crystal structure published by
Genentech (4, Figure 1, PDB code 4M49) revealed a
comparable binding orientation as 27 in our series14b and
suggested an opportunity to design a hybrid molecule. The
aminophenylsulfonamide substituent of 4 was reported to make
numerous critical hydrogen bonds with the enzyme (i.e.,
Asp140, Glu191, and lle141). Indeed, molecular modeling
overlay of the two structures (Figure 2B; purple = 4; tan and
magenta = postulated benzylsulfonamide analog of our
chemotype) suggested that incorporation of a similar benzyl-
sulfonamide substituent onto the 4-position of the pyrazole ring
could extend into the same pocket and pick up these same
interactions without significantly disrupting the binding
interactions of 27 (Figure 2B). Toward this end, compound
33 was synthesized, tested, and found to have an IC50 of 672
nM in the presence of EDTA (100 μM), as shown in Table 2.
This compound represented the first sub-μM inhibitor of this
chemical series that did not absolutely require zinc for potent
inhibition. However, since this molecule retained the hydroxyl-
pyrazole moiety, its activity could still be potentiated by zinc,
leading to low nM inhibition (data not shown). Following this
breakthrough, subsequent rounds of testing were conducted
with EDTA in the assay buffer to ensure that metal-
independent inhibition was driving SAR and further medicinal
chemistry optimization.
Concurrently, GSK reported the discovery and biological
characterization of 2, the first single/double-digit nM LDHA
inhibitor with appreciable cell-based activity (lactate production
assay), though the compound’s PK properties ultimately
precluded its use in vivo.12 Given this, 2 was used as a
benchmark compound for comparison purposes in both
biochemical (Table 2) and cell-based assays (Table 5).
Evaluation of parameters such as CLogP, ligand lipophilic
efficency (LLE), and ligand efficiency (LE) for prior art
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compounds 2 and 3 revealed an apparent preference for higher
lipophilic character, with ClogP values of 7.75 and 4.79 for 2
and 3, respectively (see Supporting Information Table 2). In
comparison, compound 5 and further optimized analog 33 have
ClogP values of 4.18 and 3.10, respectively. As a result, there is
a marked improvement in LLE for 33 (3.07) over 5 (0.36) and
prior art compounds 2 (−0.33) and 3 (1.58), as shown in Table
S2. To further explore the SAR of our benzylsulfonamide-
containing scaffold, we obtained high-resolution (1.6 Å) crystal
structures of 33 with (PDB code 5W8K) and without (PDB
code 5W8J) the NADH cofactor (Figure 3A). As anticipated
from the significantly improved inhibitory potency, the
sulfonamide moiety formed critical hydrogen bonds with
Asp140, Glu191, and I141. Compound 2 was less potent
when inhibition assays were carried out with 10× higher
NADH concentrations, whereas inhibition with our series of
inhibitors was largely unaffected by this change. However, the
crystal structure of the ternary complex with NADH and 33
suggested that inhibitor binding may be enhanced by the
presence of NADH (Figure 3B). Results from surface plasmon
resonance (SPR) studies (vide infra) substantiated this
hypothesis.
Table 2. LDHA Inhibition of 33−63 with Comparators 2 and
a
3
The importance of the sulfonamide and tolerance for
modification of this region was evaluated via the synthesis of
several analogs, with illustrative examples shown in Table 2.
Moving the sulfonamide group to the 3-position of the phenyl
ring (34), removing it completely (35), or adding a methyl to
the amine (39) or an amide (40) resulted in loss of inhibitory
activity (IC50 > 57 μM). Moreover, replacing the sulfonamide
with a carboxylic acid (41) led to decrease in potency with an
IC50 value of ∼34 μM. Given the tolerance and apparent benefit
of biphenyl derivatives (Table 1), the corresponding 3-Ph (37)
and 3-(2-F-Ph) (38) analogs were prepared and found to have
IC50 values of 349 nM and 755 nM, respectively. As the
hydroxypyrazole scaffold retains the ability to chelate Zn (and
likely other metals), we sought to explore pyrazole substituents
to identify analogs that alleviated this concern. The first series
of analogs (42−50) removed the hydroxyl group altogether (R1
= H). Encouragingly, several of these compounds (e.g., 42)
exhibited double digit nM IC50 values, comparable to the
potency of (2). As observed previously, the biphenyl moiety
(44; R3 = 3-Ph) was beneficial for improved inhibition in this
series as well (IC50 = 38 nM). However, given the sufficient
activity and ease of synthesis, the simple phenyl derivative (e.g.,
43) was frequently used to explore further SAR. Accordingly,
modification of the benzylmethylene group to either an oxygen
(45) or nitrogen (46 and 47) was well tolerated, but the
analogous amide (48) was not, with IC50 values of 124 nM, 57
nM, 91 nM, and >57 μM, respectively. Moreover, replacement
of the phenyl group with a piperazine moiety (49) led to a
significant loss of potency (IC50 = 17.3 μM), as did
incorporation of the 4-aminophenylsulfonamide group (50;
IC50 ≥ 57 μM). Taken together, these data support the notion
that this region is critical for potent inhibition and that
tolerance for structural modification is limited.
Additional modifications to the pyrazole substitutent (R1 in
Table 2) were explored next, as exemplified by analogs 51−63.
Installation of a methyl group (51, IC50 = 0.042 μM and nitrile
(52 IC50 = 0.115 μM) resulted in increased potency compared
to the parent hydroxypyrazole derivative (33). This region did
demonstrate some tolerance (albeit, with weaker potency) for
larger hydrophobic groups, with compound 53 (R1 = Ph)
possessing similar inhibition (IC50 = 0.139 μM). Interestingly,
a
IC50 values represent the half maximal (50%) inhibitory concen-
tration as determined in the HTS assay (n = 3) using a 22-dose
response in 1536-well format. Maximum response was less than 50%,
and thus IC50 values should be considered as lower confidence.
b
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Figure 3. (A) Crystal structure of LDHA bound with 33. The inhibitor is shown in sticks, and the protein is shown in surface representation. Key
protein residues Asp140, Glu191, and lle141 form H-bonding interactions with the sulfonamide moiety as shown by dotted red lines. PDB code is
5W8J. (B) Overlay of inhibitor 33 (salmon) and 4 shown in purple (Figure 1) bound in the binding pocket of LDHA.
a
the 3-pyridine (54) and 4-pyridine (55) derivatives had
significantly different potencies of 349 nM and 6.23 μM,
respectively, suggesting a deleterious interaction of the pyridine
nitrogen when in the 4-position (or beneficial interation of the
nitrogen in the 3-position). Incorporation of the an amino
group (56), and the original CF3 group (57) were both well
tolerated with IC50s values of 65 nM and 53 nM, respectively.
Next, we incorporated a cyclopropyl group (58−61) and later a
methylene cyclopropyl group (62 and 63) that were generally
more potent than previous analogs, displaying potencies
ranging from 9 to 110 nM. Extension of the cyclopropyl
group with a methylene spacer enabled a potential pseudo π−π
interaction between the π-like character of the cyclopropyl
group and an active-site tyrosine (Tyr238), as shown in
Supporting Information Figure 3, PDB code 5W8L. We also
tested the respective regioisomers (61d and 63d), and neither
of them showed any appreciable activity in enzymatic or cell-
based assays (data not shown). Notably, while the biochemical
potencies of the cyclopropyl and methylenecyclopropyl
derivatives were not significantly different from other potent
analogs, cellular assays provided a greater disparity in activity
(vide infra).
Thiazolecarboxylic acid replacements were investigated.
Efforts to replace the thiazole with any other ring structure,
including oxazole, pyridine, and other heterocycles, were all
markedly detrimental to activity (data not shown). Finally, to
determine the quantitative importance of the anchoring
interaction between the carboxylic acid moiety and R168, we
synthesized the corresponding amide (64), ethyl ester (65),
primary alcohol (66), tetrazole (67), and secondary alcohol
(68) derivatives. All showed a marked loss of potency (Table
3). The amide (64) and primary alcohol (66) analogs showed
modest inhibition of 27.7 μM and 25.5 μM, respectively, while
65, 67, and 68 were not inhibitory (IC50 ≥ 57 μM), illustrating
the requirement of the carboxylic acid moiety to maintain
potent inhibition.
Table 3. LDHA Inhibition of 64−68
LDHA (with EDTA) IC50 SD
analog
R1
R2
R3
(μM)
64
65
66
67
68
CONH2
OH
CF3
CF3
OH
H
3-Ph
3-Ph
H
27.7 1.9
>57
COOEt
b
CH2OH
25.5 6.2
>57
tetrazole
3-Ph
H
CH(OH)CF3
>57
a
IC50 values represent the half maximal (50%) inhibitory concen-
tration as determined in the HTS assay (n ≥ 3) using a 22-dose
response in 1536-well format. Maximum response was less than 50%,
b
and thus IC50 values should be considered as lower confidence.
Selectivity of representative analogs was determined by
assessing their biochemical activity against another LDH
isozyme, LDHB, and two “off-target” dehydrogenases, malate
dehydrogenase (MDH) and isocitrate dehydrogenase 1
(IDH1). While there was negligible activity against both
MDH and IDH1, most of the analogs revealed similar potency
for LDHB when compared to LDHA (Table 4).
To determine the cellular activity of analogs, we tested them
in a high-throughput fluorescence/absorbance-based lactate
production assay (1536-well) using the A673 human sarcoma
and MiaPaCa-2 human pancreatic cancer (see Table 5) cell
lines in 22-point dose−response. Both cell lines demonstrate a
highly glycolytic phenotype with robust production of lactate,
making them an ideal choice for these assays. 2, which was
reported to exhibit sub-μM inhibition of lactate production,
demonstrated an IC50 of 14.5 μM, a result consistent across a
number of cell lines. Though the reason for this discrepancy is
unclear (difference in assay platforms may be one possibility),
this observation underscored the need for novel inhibitors with
greater efficacy in cells. Compound 3 exhibited better potency
in this cell-based lactate production assay than did 2, with an
IC50 of 1.44 μM. Compounds from our chemical series, e.g., 61
and 63, exhibited comparable potency in these cell lines.
However, for many of the representative compounds shown in
Table 5, the potent biochemical inhibition translated into little
to no cell-based activity. To determine if cell permeability was a
Concurrently, Genentech published several papers describing
the discovery and optimization of 3 (Figure 1) as a potent
LDHA inhibitor.14c Compound 3 inhibited LDHA in our assay
with an IC50 of 424 nM, substantially less potent than the
reported values (IC50 < 10 nM). The discrepancy is likely due
to differences in the assay conditions and demonstrates the
importance of conducting head-to-head comparisons of
reference compounds when drawing conclusion about bio-
logical activity.
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appeared to reach appreciable intracellular levels (high μM)
and thus well in excess of their respective biochemical IC50
values (data not shown). Differential lactate inhibition by
compounds with comparable structures and presumably
physicochemical properties (e.g., 44 R1 = H and 61 R1 =
cyclopropyl) further suggested permeability was not the main
barrier to cellular efficacy; these compounds demonstrated
biochemical IC50 values of 38 nM and 27 nM, respectively, yet
61 was approximately 15-fold more potent in the lactate
production assay (0.983 μM (61) vs 15.2 μM (44)). Moreover,
the most potent compound in the lactate assay was compound
63 (IC50 = 0.517 μM), which possessed a methylene
cyclopropyl group at R1 and 3-Ph group at R3, yet was less
potent in the biochemical assay than the corresponding
cyclopropyl derivative (61). The cytotoxicity of compounds
against these glycolytic cell lines tracked reasonably well with
inhibition of lactate production, consistent with an on-target
mechanism of cell killing. All compounds that demonstrated
appreciable inhibition of lactate production (IC50 < 4 μM; 3,
60−63) also exhibited modest cytotoxicity (IC50 = 1−30 μM)
in a 48 h CellTiter-Glo assay in A673 and MiaPaCa-2 cells (see
Table 5).
In an effort to probe this roughly 100-fold offset between
biochemical and cellular potencies, we considered the concept
of drug−target residence time which has been championed by
Copeland and others.22 While this concept is usually brought
up in the context of in vivo efficacy, it has been reported to
impact cellular efficacy as well.23 Given the high concentration
of intracellular LDH (estimated to be in the range of 2−17
μM),11,24 we reasoned that longer residence times might be
necessary to achieve significant, sustained decreases in cellular
LDH function. As noted above, this approach could also be
beneficial in vivo, since drug concentration in systemic
Table 4. Selectivity against LDHB and Other
Dehydrogenases
a
IC50 (μM)
analog
LDHA
LDHB
MDH
IDH1wt
1
1.34
9.20
>57
37.8
>57
>57
>57
>57
>57
>57
>57
2
0.038
0.424
0.672
2.37
1.15
3
0.441
0.724
1.51
33
36
37
38
42
43
44
46
47
54
55
56
57
58
60
61
62
63
b
37.8
>57
33.7
>57
>57
48.6
b
0.349
0.754
0.095
0.177
0.038
0.057
0.091
0.349
6.23
0.424
0.911
0.129
0.204
0.049
0.078
0.096
0.259
2.82
47.4
>57
>57
>57
47.4
>57
>57
>57
>57
>57
b
b
37.8
>57
>57
>57
0.065
0.053
0.019
0.110
0.027
0.009
0.032
0.075
0.062
0.020
0.119
0.020
0.008
0.027
26.8
b
48.6
37.8
33.5
>57
26.6
33.5
47.4
29.8
>57
42.4
>57
b
37.8
a
IC50 values represent the half maximal (50%) inhibitory concen-
b
tration as determined in the HTS assay (n = 3). Maximum response
was less than 50%, and thus IC50 values should be considered as lower
confidence.
limiting factor, we measured the intracellular concentration of
several inhibitors using LC−MS/MS analysis. The compounds
Table 5. Cell-Based Activity of Representative Analogs
A673 cells
MiaPaCa-2 cells
a
c
a
c
IC50 SD (μM)
lactate inh
>57
IC50 (μM)
IC50 SD (μM)
IC50 (μM)
analog
cytotoxicity
long-term growth inh
lactate inh
>57
cytotoxicity
>57
long-term growth inh
1
>57
>20
0.84
>20
1.37
b
2
14.5 5.7
1.44 0.09
>57
>57
12.6 3.6
0.877 0.059
>57
28.4 11.2
1.24 0.08
>57
3
2.63 0.3
>57
33
36
37
38
42
43
44
46
47
54
55
56
57
58
60
61
62
63
>57
>57
>57
>57
b
26.8 3.1
>57
27.7 1.9
>57
b
b
30.0 3.5
>57
29.9 0.1
>57
b
30.8 2.1
>57
>57
>57
b
b
25.2 19.4
15.2 3.2
19.9 4.5
26.8 3.1
16.5 6.2
>57
>57
33.5 0.1
>57
>57
>20
>20
9.88 1.26
7.24 0.47
22.4 1.8
>57
>20
>20
>57
>57
>57
>57
b
>57
26.6 0.1
>57
>57
>57
>57
9.45 0.01
4.35 1.21
14.7 3.6
1.92 0.43
>57
17.7 6.6
4.80 2.47
17.5 1.2
1.96 0.13
1.34 0.15
5.12 0.33
0.854 0.059
>57
>57
>57
>57
>57
25.8 3.0
13.9 0.9
24.8 1.6
2.90 0.3
28.3 7.1
18.9 1.3
24.9 1.6
3.96 0.68
0.983 0.335
3.37 0.39
21.09
>20
8.00
>20
1.21
0.517 0.088
2.23
a
b
IC50 values represent the half maximal (50%) inhibitory concentration as determined in the HTS assay (n = 3). Maximum response was less than
c
50%, and thus IC50 values should be considered as lower confidence. IC50 values represent average of two assay replicates.
M
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Figure 4. Decrease in glycolytic flux caused by LDH inhibitors in A673 cells. The glycolysis stress test was performed in A673 cells. The extracellular
acidification rate (ECAR) over time, cellular basal ECAR, was measured, and then (A) 3, (B) 61, or (C) 63 was injected in a dose−response manner.
After 40 min, subsequent injections of glucose (10 mM), oligomycin (O at 1 μg/mL, reaching maximal glycolytic capacity), and 2-deoxyglucose (2-
DG at 50 mM, inhibition of glycolysis) were made. (D) Quantification of the maximal ECAR (% from control of maximal ECAR minus ECAR with
2-DG) of increasing concentrations of the five LDH inhibitors.
circulation decreases with time and LDH is an abundant,
ubiquitously expressed protein. Thus, we obtained SPR data
with representative compounds to examine this hypothesis.
For the initial study, four compounds (2, 33, 61, and 63)
were chosen based on their biochemical potency and
differential activity in the cell-based lactate output assay.25
Compounds from the internal chemical series displayed more
potent binding affinities and longer off-rates (koff) in the
presence of saturating NADH (500 μM) than without,
supporting the hypothesis that arose from the initial
crystallography studies. However, 2 was found to have greater
binding affinity in the absence of NADH (KD = 1.7 nM versus
59 nM with NADH), in agreement with its reported NADH
competitive mechanism of inhibition. Because inhibition of
LDH in cells results in increased intracellular concentrations of
NADH, a mechanism of inhibition that produces increased
affinity for LDH and greater duration of target occupancy when
NADH concentrations are elevated should be a more
efficacious mechanism than a NADH-competitive one. The
KD values for three representative compounds and a
comparator (2, 33, 61, and 63) in the presence of NADH
were 59 nM, 370 nM, 0.33 nM, and 0.11 nM, respectively.
Interestingly, the off-rates and corresponding residence times
measures the net glycolysis-dependent proton production in
the cells and, as such, is a marker of the inhibition of glycolysis
consequent on LDH inhibition. It is anticipated that as LDH
inhibition increases with the concentration of compound, the
reserve biochemical capacity of the enzyme for glycolysis is
exceeded, resulting in a depletion of NAD+ and, ultimately,
inhibition of the entire pathway. The extracellular acidification
rate (ECAR) was monitored in A673 cells before and after
injection of five compounds (2, 3, 33, 61, and 63) over a range
of concentrations. These data show a clear concentration-
dependent decrease in the glycolytic flux (Figure 4A−C),
consistent with the anticipated inhibition of the proton efflux
into the media. In addition, the capacity of the cell to switch
from oxidative phosphorylation to glycolysis following injection
of oligomycin, a specific inhibitor of ATP-synthase, is
suppressed by all LDH inhibitors. This can be clearly seen
from the quantification of % maximal ECAR (maximal
glycolytic capacity) in A673 cells for a panel of five LDHA
inhibitors, which shows that the inhibitory potency order is 63
> 61 > 33 > 3 > 2, which correlates with the cell-based lactate
inhibition assay (see Table 5), with the exception of 33. As
shown in Figure 4D, the leading compounds 63 and 61
completely inhibit lactate production/extracellular acidification
at concentrations of 2 and 5 μM, respectively, corroborating
that the incorporation of biphenyl and cyclopropyl (61) or a
methylene cyclopropyl (63) improved LDH inhibition in cell-
based in vitro assays.
−1
(τ) [calculated as 1/koff (s )] tracked well with the cellular
potency [2 (τ = 8 s), 33 (τ = 0.5 s), 61 (τ = 470 s), and 63 (τ =
1200 s)]. While rational optimization of drug−target residence
time has been reported,26 it can be difficult to delineate such
structure−activity relationships, as minor structural changes
may lead to surprisingly large differences in binding proper-
ties.27 As such, SPR analysis of binding kinetics will be
incorporated into our future SAR efforts as an important
optimization parameter and compound attribute, alongside
biochemical potency, cell-based potency, and ADME proper-
ties.
To obtain additional evidence of cellular target engagement
of LDHA by our compounds, CETSA (cellular thermal
stabilization assay), a technique that translates the principles
of biochemical thermal shift assays to a cellular context was
adapted and optimized for LDHA in a number of cell lines.28
Accordingly, a panel of representative compounds were tested
in A673 cells, spanning a wide range of potencies (Supporting
Information Figure 1a−e). Cellular binding and stabilization of
LDHA were observed with a number of top LDHA inhibitors
(Table 6) at concentrations as low as 100 nM. Compounds 62
The glycolysis stress test (GST) was performed on the
Seahorse XF analyzer in order to assess changes in glycolytic
flux resulting from LDH inhibition (Figure 4). This assay
N
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Table 6. CETSA LDHA Stabilization in A673 Cells
LDHA inh
lactate inh
IC50 (μM)
>57
CETSA: cellular LDHA stabilization
a
b
c
analog
IC50 (μM)
EC50 (μM)
max stab (normalized)
AUC (normalized)
1
1.020
0.038
0.424
22.200
0.672
0.038
0.053
0.027
0.009
0.032
1.60
8.18
1.26
>100
3.53
2.56
>100
0.25
0.07
0.17
64.8
52.4
79.0
8.5
62.08
43.22
72.96
13.84
53.66
89.77
26.76
70.66
100.00
81.95
2
3
12.60
5
33
44
57
61
62
63
>57
9.88
4.80
1.34
5.12
0.85
47.9
100.0
41.6
63.0
70.8
69.9
a
EC50 values represent the half maximal (50%) concentration (the concentration needed to stabilize half of the detectable LDHA protein at a set
b
melting temperature) as determined by CETSA. Maximum stabilization values represent the highest amount of stabilized LDHA detected for each
sample, as measured by Western blot band intensity. These values are scaled against the band with the greatest amount of stabilized LDHA
(maximum response of 44) to provide a relative measure of target binding. Area-under-the-curve (AUC) values demonstrate the total amount of
c
LDHA stabilized by each compound across all concentrations, scaled against the AUC of the compound with the greatest total amount of LDHA
stabilization (62) to provide a relative measure of LDHA engagement.
Figure 5. Incucyte long-term cellular growth data. (A) Cellular confluency of MiaPaCa-2 cells treated with LDHA inhibitors at 2.22 μM over 120 h.
(B) Dose−response relative growth of MiaPaCa-2 cells treated with 63 for 120 h. Relative growth of (C) MiaPaCa-2 and (D) A673 cells treated with
LDHA inhibitors for 120 h.
and 63 were particularly potent at stabilizing LDHA in A673
cells. Given its relatively direct readout on target engagement,
we examined the relationship of CETSA-based activity to other
measures of LDHA inhibitor potency. Though the compounds
with the most potent activity against LDHA in the biochemical
assay were also among the most potent in the CETSA assays,
no clear correlation emerged between the cellular stabilization
of LDHA via CETSA and either biochemical or cellular
activities of these molecules. Interestingly, 1 demonstrated
cellular stabilization of LDHA, despite showing no activity in
any prior cell-based LDHA assays. Conversely, 57 demon-
strated potent inhibition of LDHA and moderate inhibition of
cellular lactate, yet displayed comparatively little stabilization of
LDHA via CETSA, and no toxicity or growth inhibition of
either cell line tested. As such, CETSA-measured LDHA-
binding was utilized as one marker of activity in a panel of
many.
Having developed compounds with potent inhibition of
LDH (<20 nM) and lactate production (1−2 μM) and with
robust target engagement, as assessed via SPR and CETSA, we
investigated the cytotoxicity of these agents against MiaPaCa-2
and A673 cells. Initial efforts utilized a 48 h cytotoxicity assay
using ATP content as a viability surrogate (CellTiter-Glo), and
longer-term effects on cell growth were studied by monitoring
confluence over 5 days using an Incucyte ZOOM (see Table 5).
Early analogs had minimal to no effect on cell proliferation,
whereas 3 and 63 demonstrated suppression of cell growth over
time (Table 5, Figure 5A). Dose−response treatment of
MiaPaCa-2 cells with 63 showed effects on cellular proliferation
at concentrations as low as 250 nM and with nearly complete
arrest of cell growth at 20 μM (Table 5, Figure 5B). Area-
O
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under-the-curve (AUC) analysis was conducted on MiaPaCa2
and A673 dose−response data to enable comparison of LDH
inhibitors (Figure 5C and Figure 5D, respectively); 3 and 63
both exhibited single digit μM activity in both A673 and
MiaPaCa-2 cells. While inhibition of cell proliferation itself was
not exceptionally potent, it was encouraging that cytotoxicity
was positively correlated with target potency (enzymatic and
cellular lactate output), suggesting that the effects were target-
mediated.
To help define the utility of these initial compounds for
proof-of-concept animal studies, we performed in vivo PK and
in vitro ADME studies on representative inhibitors 61 and 63
(Table 7). Tier 1 ADME profiling comprised rat liver
Table 8. Pharmacokinetic Profiles of 61 and 63 in CD1
Mice
a
61
63
parameter
iv
po
iv
po
Cl (L h−1 kg−1
)
227
0.85
1.45
294
2.98
0.74
T1/2 (h)
1.94
1.20
0.25
3.2
3.75
1.78
0.25
2.5
b
b
Cmax (μM)
Tmax (h)
AUClast (μM·h)
Vss (L/kg)
F (%)
0.26
5.6
0.19
27
49
50
a
Values calculated from drug concentration in plasma following iv (2
mg/kg) and po (50 mg/kg) dosing. n = 3, 8 time points taken over 24
h. Compounds 61 and 63 were formulated as solution in PBS buffered
saline with 1.1 equiv of NaOH (final pH 7−8). Cmax = C0 (t = 0) for
IV administration. All pharmacokinetic studies were conducted at
Pharmaron Inc.
a
Table 7. Representative ADME Data for 61 and 63
b
T1/2
T1/2 (min)
mouse
(min)
PAMPA
permeability
kinetic
solubility
plasma
analog MLM HLM hepatocytes (10−6 cm/s)
(μg/mL) stability
distribution. These compounds were demonstrably stable in
mouse plasma, with no degradation observed over 120 min
(Table 7). Notably, compound 63 did exhibit a high volume of
distribution, possibly accounting for some of the observed high
clearance values. However, further studies are required to
determine whether renal clearance or erythrocyte sequestration
is a contributing factor, and ongoing medicinal chemistry
optimization around this chemotype may lead to compounds
with improved PK properties for use in animal studies.
Encouragingly, despite their high clearance and poor PAMPA
permeability, appreciable systemic exposure was achieved in the
range of cellular IC50 values following po dosing at 50 mg/kg.
Passive absorption was undoubtedly impacted by the presence
of a carboxylic acid moiety, yet bioavailability (F) was
determined to be ∼50% for both compounds, suggesting that
analogs with reduced clearance may lead to satisfactory
exposure following oral dosing. Given the SAR for this series,
which demonstrated the critical importance of the carboxylic
acid moiety, future efforts to improve PK properties will focus
on other areas of the molecule.
61
63
321
444
414
206
268
239
1
8
>82
>82
>120
>120
a
Aqueous kinetic solubility (PBS buffer) and PAMPA permeability
were conducted at NCATS. Mouse plasma stability studies were
conducted at Pharmaron Inc. and involved five time points. The
microsomal stability data [mouse liver microsomes (MLM), human
liver microsomes (HLM), and mouse hepatocytes] were conducted at
QuintaraBio and represent the stability in the presence of NADPH
and UDPGA. The parent compound was monitored at five time points
over 90 min.
microsome (RLM) stability, PAMPA permeability, and aqueous
solubility (pH 7.4). Both compounds exhibited high micro-
somal stabilities of >30 min (data not shown), the highest
estimable T1/2 from a single measurement at 15 min.29 Owing
to the presence of a carboxylic acid moiety, PAMPA
permeability was low ((1−8) × 10−6 cm/s) while solubility
was high (>82 μg/mL, the maximum concentration tested in
the assay). Time-course studies revealed that compounds were
also stable in mouse and human liver microsomes (T1/2 > 200
min for both compounds and species) and hepatocytes (mouse,
T1/2 ≈ 240 min) (Table 7). Given the presence of both
carboxylic acid and sulfonamide moieties in the lead molecules,
the potential to form glucuronide conjugates exists. As such, we
tested microsomal stability in the presence of NADPH/
UDPGA substrates. Encouragingly, neither 61 nor 63 showed
any evidence of glucuronidation when incubated in either
mouse or human liver microsomes, suggesting phase II
metabolism may not be a significant issue for this chemotype
(data not shown). This observation was further supported by
the long half-life in mouse hepatocytes.
As a result of the encouraging ADME data of lead
compounds, the in vivo PK properties of lead compounds
were determined. Both compounds showed clearance values
that far exceed hepatic blood flow (HBF) in mouse species (90
mL min−1 kg−1), with in vivo clearances of 294 and 227 mL
min−1 kg−1 for 61 and 63, respectively (Table 8). These data,
coupled with the long half-life in mouse liver microsomes,
suggested a mechanism other than liver metabolism was
contributing significantly to compound elimination in vivo.
Potential explanations for clearance much greater than hepatic
blood flow include compound instability in plasma, renal
clearance, sequestration in erythrocytes, and/or high volume of
CONCLUSION
■
LDH has long been implicated in the pathogenesis of cancer
and many other diseases and thus has received considerable
attention from both pharmaceutical companies and the
academic scientific community. Despite multiple efforts to
discover potent and drug-like inhibitors of LDHA, few viable
inhibitors emerged until the recent pioneering work by GSK
and Genentech. These published inhibitors possess potent
biochemical activity, yet their cellular effects remain modest and
pronounced in vivo activity remains elusive. Herein, we
describe the discovery and medicinal chemistry optimization
of a potent and cell-active pyrazole-based inhibitor of LDH.
The original “hit” molecule 5 was identified via a qHTS
campaign, and medicinal chemistry optimization was aided by
both crystallography efforts and a battery of biological assays.
As noted by other groups, despite obtaining potent biochemical
inhibition of LDH, early representatives in this series had very
modest cellular activity (e.g., inhibition of lactate production).
However, through utilization of target engagement assays
(CETSA) and biophysical characterization (SPR), we gained
insight into the drivers of cellular potency, with the target
residency times of these analogs being a key determinant. To
that end, we identified compound 63 as a chemical probe for
P
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59a or 61a (51.0 mmol, 1 equiv), magnesium bromide diethyl etherate
(32.9 g, 127 mmol, 2.5 equiv), and acylbenzotriazole derivative 58a or
62a (76 mmol, 1.5 equiv in CH2Cl2 (100 mL)) was added Hunig’s
base (26.7 mL, 153 mmol, 3 equiv) slowly (cooling is necessary for
large scale), and then the mixture was stirred at rt for 12 h. The
reaction mixture was cooled in an ice bath and quenched with 1 M
HCl. The product was extracted with CH2Cl2, and the organic layer
was subsequently washed with brine. After drying the organic layer
with MgSO4, the crude product was purified on a ISCO flash system
using a 220 g gold column, eluting with 0−30% ethyl acetate over 20
column volumes in hexanes to afford a yellow oil after removing the
solvent in 60−69% yield.
General Procedure for the Alkylation of 1-Aryl-3-Substi-
tuted Propane-1,3-diones (Method C). 1-Aryl-3-substituted
propane-1,3-dione (35.9 mmol, 1 equiv) and cesium carbonate
(14.05 g, 43.1 mmol, 1.2 equiv) in DMSO (25 mL) were stirred at
room temperature for 10 min. 4-(Bromomethyl)benzenesulfonamide
(10.78 g, 43.1 mmol, 1.2 equiv) was added in one portion and further
stirred at room temperature for another 1−2 h. The resulting mixture
was diluted with large excess ethyl acetate and filtered through Celite
to remove any solid impurities. The filtrate was washed with saturated
ammonium chloride 3 times and then with brine. The organic layer
was dried with Na2SO4, filtered, and concentrated under reduced
pressure. The residue was purified directly on flash system using a 220
g silica column, eluting with 20−60% ethyl acetate in hexanes over 16
column volumes to afford pure products 58c, 61c−63c in 55−83%
yield.
General Procedure for the Cyclization of 1-Aryl-2,3-
disubstituted Propane-1,3-diones and Hydrolysis (Method
D). A mixture of appropriate 1-aryl-2, 3-disubstituted propane-1,3-
dione (2.24 mmol, 1 equiv), ethyl 2-hydrazinylthiazole-4-carboxylate
hydrogen bromide III (0.600 g, 2.24 mmol, 1 equiv), and tosic acid
(0.425 g, 2.24 mmol, 1 equiv) in ethanol (10 mL) was heated in a
microwave for 15 min at 150 °C. The excess solvent was removed
using forced air, and the residue was taken in dichloromethane. The
crude suspension was then directly loaded to a 100 g silica column
fitted to a flash system and eluted with 20−40% ethyl acetate in
hexanes to get a mixture of regioisomers in 77−83% yield. This
mixture of products was taken as such in a THF−methanol (2:1)
mixture and treated with 1.5 M solution (5 equiv) of aqueous lithium
hydroxide. The reaction mixture was stirred for 1 h, the solvent was
removed and acidified with 1 M hydrochloric acid and then extracted
with ethyl acetate. After evaporation of the organic layer, the crude
material was taken in DMSO and the regioisomers were separated on a
preparative HPLC. The desired isomer was eluted as a second peak.
(1H-Benzo[d][1,2,3]triazol-1-yl)(cyclopropyl)methanone
(58a). This compound was prepared using Method A starting from
cyclopropanecarboxylic acid in 100% yield. LC−MS retention time:
(method 2) = 3.256 min, m/z (M + H)+ = 188.
LDH and a promising lead compound worthy of further
optimization, given its sub-μM inhibition of lactate production,
robust cellular target engagement, inhibition of glycolytic flux,
and favorable off-rate in SPR studies. While 63 possesses some
favorable ADME attributes (e.g., microsomal stability,
solubility), additional optimization will be required to achieve
a PK profile suitable for use in in vivo efficacy studies. Current
efforts are focused on implementing the lessons learned so far,
and outlined herein, to guide development of compounds with
better cellular potency and PK properties. Results from these
additional efforts, along with further biological characterization
of the compounds, will be reported in due course.
EXPERIMENTAL SECTION
■
General Methods for Chemistry. All air or moisture sensitive
reactions were performed under positive pressure of nitrogen or argon
with oven-dried glassware. Anhydrous solvents and bases such as
dichloromethane, N,N-dimethylforamide (DMF), acetonitrile, ethanol,
DMSO, dioxane Hunig’s base, and triethylamine were purchased from
Sigma-Aldrich. Palladium catalysts were purchased from Strem
Chemicals and used as such. SiliaCat heterogeneous catalyst DPP-Pd
(catalog no. R390-100) was purchased from SiliCycle Inc. Preparative
purification was performed on a Waters semipreparative HPLC system
using a Phenomenex Luna C18 column (5 μm, 30 mm × 75 mm) at a
flow rate of 45 mL/min. The mobile phase consisted of acetonitrile
and water (each containing 0.1% trifluoroacetic acid). A gradient of
10−50% acetonitrile over 8 min was used during the purification.
Fraction collection was triggered by UV detection (220 nm).
Analytical analysis was performed on an Agilent LC/MS (Agilent
Technologies, Santa Clara, CA). Method 1: A 7 min gradient of 4−
100% acetonitrile (containing 0.025% trifluoroacetic acid) in water
(containing 0.05% trifluoroacetic acid) was used with an 8 min run
time at a flow rate of 1 mL/min. A Phenomenex Luna C18 column (3
μm, 3 mm × 75 mm) was used at a temperature of 50 °C. Method 2:
A 3 min gradient of 4−100% acetonitrile (containing 0.025%
trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid)
was used with a 4.5 min run time at a flow rate of 1 mL/min. A
Phenomenex Gemini Phenyl column (3 μm, 3 mm × 100 mm) was
used at a temperature of 50 °C. Purity determination was performed
using an Agilent diode array detector for both method 1 and method
2. Mass determination was performed using an Agilent 6130 mass
1
spectrometer with electrospray ionization in the positive mode. H
NMR spectra were recorded on Varian 400 MHz spectrometers.
Chemical shifts are reported in ppm with undeuterated solvent
(DMSO-d6 at 2.49 ppm) as internal standard for DMSO-d6 solutions.
All of the analogs tested in the biological assays have purity greater
than 95%, based on both analytical methods. High resolution mass
spectrometry was recorded on Agilent 6210 time-of-flight LC/MS
system. Confirmation of molecular formula was accomplished using
electrospray ionization in the positive mode with the Agilent
Masshunter software (version B.02).
1-Cyclopropyl-3-phenylpropane-1,3-dione (58b). This com-
pound was prepared using method B starting from 58a and 59a in 60%
yield. LC−MS retention time: (method 2) = 3.52 min, m/z (M + H)+
= 189.
4-(2-Benzoyl-3-cyclopropyl-3-oxopropyl)benzene-
sulfonamide (58c). This compound was prepared using method C
starting from 58b in 61% yield. LC−MS retention time: (method 2) =
3.32 min, m/z (M + H)+ = 358.
2-(5-Cyclopropyl-3-phenyl-4-(4-sulfamoylbenzyl)-1H-pyra-
zol-1-yl)thiazole-4-carboxylic Acid (58). This compound was
prepared using general method D starting from 58c and III. LC−MS
retention time: (method 1) 4.905 min and (method 2) 3.323 min. 1H
NMR (400 MHz, DMSO-d6) δ 13.17 (s, 1H), 8.29 (s, 1H), 7.72−7.67
(m, 2H), 7.52−7.46 (m, 2H), 7.42−7.32 (m, 3H), 7.30−7.23 (m, 4H),
4.12 (s, 2H), 2.23 (tt, J = 8.5, 5.6 Hz, 1H), 1.00−0.91 (m, 2H), 0.68−
0.60 (m, 2H); HRMS (ESI) m/z (M + H)+ calcd for C23H21N4O4S;
481.0999 found 481.0992.
General Procedure for the Synthesis of Acylbenzotriazole
Derivatives (Method A). To a solution of 1H-benzo[d][1,2,3]-
triazole (23.8 g, 200 mmol, 4 equiv) in CH2Cl2 (150 mL) was added
thionyl chloride (3.65 mL, 49.9 mmol, 1 equiv), and the mixture was
stirred at room temperature for 0.5 h. Appropriate alkylcarboxylic acid
(49.9 mmol, 1 equiv) was then added slowly, and the mixture was
stirred for another 2 h. The reaction mixture was filtered, and the filter
cake was washed with CH2Cl2 (50 mL). The filtrate was neutralized
with bicarbonate solution slowly and stirred for 30 min and then
transferred to a separatory funnel. The organic layer was washed with
bicarbonate solution and then with brine, dried over MgSO4, filtered,
and concentrated. The residue was purified directly on a flash system
using 120 g silica column eluting with 0−20% ethyl acetate in hexanes
over 15 column volumes. The first peak was collected and dried to get
an oil or solid.
2-(5-Cyclopropyl-3-phenyl-4-(4-sulfamoylphenoxy)-1H-pyr-
azol-1-yl)thiazole-4-carboxylic Acid (59). This compound was
prepared using general method D starting from 59c and III. LC−MS
retention time: (method 1) 5.119 min and (method 2) 3.257 min. 1H
General Procedure for the Synthesis of 1-Aryl-3-substituted
Propane-1,3-diones (Method B). To a mixture of acetophenone
Q
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NMR (400 MHz, DMSO-d6) δ 13.16 (s, 1H), 8.35 (s, 1H), 7.83−7.71
(m, 4H), 7.45−7.31 (m, 3H), 7.29−7.14 (m, 4H), 2.63 (tt, J = 8.5, 5.5
Hz, 1H), 0.99−0.88 (m, 2H), 0.92−0.80 (m, 2H); HRMS (ESI) m/z
(M + H)+ calcd for C22H19N4O5S, 483.0791; found, 483.0809.
2-(3-([1,1′-Biphenyl]-3-yl)-5-cyclopropyl-4-(4-sulfamoylphe-
noxy)-1H-pyrazol-1-yl)thiazole-4-carboxylic Acid (60). This
compound was prepared using general method D starting from 60c
and III. LC−MS retention time: (method 1) 5.823 min and (method
Cell Lines. The MiaPaCa-2 and A673 cell lines were obtained from
American Type Culture Collection (ATCC, Manassas, VA, USA). All
cell lines were cultured in DMEM (Invitrogen 11965118) culture
medium supplemented with 10% fetal bovine serum and 100 units/mL
penicillin, 100 μg/mL streptomycin and maintained in a 37 °C, 5%
CO2/95% humidified air incubator.
Biochemical Assays. LDHA Biochemical Assay. Briefly, 3 μL of
human lactate dehydrogenase 5 (no. A38558H, Meridian Life Science,
Inc., Memphis, TN) in LDH assay buffer (200 mM Tris HCl, pH 7.4,
100 μM EDTA, and 0.01% Tween-20) was added to a black solid
bottom 1536-well assay plate (Greiner Bio-One) using a BioRAPTR
FRD dispenser (Beckman Coulter, Brea, CA). A 1536-well pintool
dispenser outfitted with 20 nL pins (Wako Automation, San Diego,
CA) was used to transfer 23 nL of DMSO-solubilized compound
(both library and vehicle controls) to each 1536-well assay plate.
Following compound transfer, 1 μL of substrate solution containing
NADH and sodium pyruvate (Sigma-Aldrich, St. Louis, MO) in LDH
assay buffer was dispensed via BioRAPTR FRD to initiate the reaction.
Final concentrations in the 4 μL reaction volume were 2 nM LDHA
enzyme, 0.06 mM NADH, and 0.2 mM sodium pyruvate. Following a
5 min incubation period at room temperature, 1 μL of detection
reagent (C. kluyveri diaphorase (Sigma-Aldrich) and resazurin sodium
salt (Sigma-Aldrich) in LDH assay buffer) was added to a total volume
of 5 μL. Final concentrations of detection reagents were 0.133 mg/mL
diaphorase and 37 μM resazurin. Plates were immediately transferred
to a ViewLux microplate imager (PerkinElmer, Waltham, MA), and
any resulting resorufin fluorescence was measured (ex 540 nm, em 590
nm) at 0 and 20 min. Fluorescence was normalized using enzyme-free
and DMSO-treated control wells on each plate.
1
2) 3.496 min. H NMR (400 MHz, DMSO-d6) δ 13.17 (s, 1H), 8.36
(s, 1H), 7.93 (td, J = 1.8, 0.5 Hz, 1H), 7.85−7.71 (m, 3H), 7.66 (ddd, J
= 7.8, 1.9, 1.1 Hz, 1H), 7.56−7.32 (m, 6H), 7.30−7.21 (m, 4H), 2.73−
2.61 (m, 1H), 1.02−0.90 (m, 2H), 0.93−0.83 (m, 2H); HRMS (ESI)
m/z (M + H)+ calcd for C28H23N4O5S2, 559.1104; found, 559.1127.
1-([1,1′-Biphenyl]-3-yl)-3-cyclopropylpropane-1,3-dione
(61b). This compound was prepared using method B starting from
58a and 61a in 66% yield. LC−MS retention time: (method 2) = 3.85
min, m/z (M + H)+ = 265.
4-(2-Benzoyl-3-cyclopropyl-3-oxopropyl)benzene-
sulfonamide (61c). This compound was prepared using method C
starting from 61b in 63% yield. LC−MS retention time: (method 2)
3.46 min, m/z (M + H)+ = 434.
2-(3-([1,1′-Biphenyl]-3-yl)-5-cyclopropyl-4-(4-sulfamoylben-
zyl)-1H-pyrazol-1-yl)thiazole-4-carboxylic Acid (61). This com-
pound was prepared using general method D starting from 61c and
III. LC−MS retention time: (method 1) 5.636 min and (method 2)
1
3.556 min. H NMR (400 MHz, DMSO-d6) δ 13.09 (s, 1H), 8.31 (s,
1H), 7.78−7.72 (m, 2H), 7.67 (ddd, J = 7.5, 1.9, 1.3 Hz, 1H), 7.60 (td,
J = 1.8, 0.6 Hz, 1H), 7.57−7.45 (m, 2H), 7.40 (d, J = 4.3 Hz, 4H),
7.36−7.30 (m, 3H), 7.28 (s, 2H), 4.17 (s, 2H), 2.28 (tt, J = 8.6, 5.6 Hz,
1H), 1.09−0.85 (m, 2H), 0.77−0.54 (m, 2H); HRMS (ESI) m/z (M +
H)+ calcd for C29H25N4O4S, 557.1312; found, 557.1320.
1-(1H-Benzo[d][1,2,3]triazol-1-yl)-2-cyclopropylethan-1-one
(62a). This compound was prepared using method A from 2-
cyclopropylacetic acid in 91% yield. LC−MS retention time: (method
2) 3.53 min, m/z (M + H)+ = 202.
4-Cyclopropyl-1-phenylbutane-1,3-dione (62b). This com-
pound was prepared using method B starting from 62a and 59a in
63% yield. LC−MS retention time: (method 2) = 3.76 min, m/z (M +
H)+ = 203.
4-(2-Benzoyl-4-cyclopropyl-3-oxobutyl)benzene-
sulfonamide (62c). This compound was prepared using method C
starting from 62b in 55% yield. LC−MS retention time: (method 2) =
3.35 min, m/z (M + H)+ = 372.
2-(5-(Cyclopropylmethyl)-3-phenyl-4-(4-sulfamoylbenzyl)-
1H-pyrazol-1-yl)thiazole-4-carboxylic Acid (62). This compound
was prepared using general method D starting from 62c and III. LC−
MS retention time: (method 1) 5.199 min and (method 2) 3.495 min.
1H NMR (400 MHz, DMSO-d6) δ 13.15 (s, 1H), 8.29 (s, 1H), 7.74−
7.66 (m, 2H), 7.60−7.53 (m, 2H), 7.44−7.36 (m, 3H), 7.32−7.25 (m,
4H), 4.14 (s, 2H), 3.15 (d, J = 6.9 Hz, 2H), 1.22−1.05 (m, 1H), 0.38−
0.28 (m, 2H), 0.26−0.17 (m, 2H); HRMS (ESI) m/z (M + H)+ calcd
for C24H23N4O4S2, 495.1155; found, 495.1174.
1-([1,1′-Biphenyl]-3-yl)-4-cyclopropylbutane-1,3-dione
(63b). This compound was prepared using method B starting from
62a and 61a in 69% yield. LC−MS retention time: (method 2) 4.02
min, m/z (M + H)+ = 279.
4-(2-([1,1′-Biphenyl]-3-carbonyl)-4-cyclopropyl-3-oxobutyl)-
benzenesulfonamide (63c). This compound was prepared using
method C starting from 63b in 83% yield. LC−MS retention time:
(method 2) 3.59 min, m/z (M + H)+ = 448.
LDHB Biochemical Assay. Human lactate dehydrogenase 1 (no.
A38155H, Meridian Life Science, Inc., Memphis, TN) was assayed as
described above for LDHA. Final concentrations in the 4 μL reaction
volume were 2 nM LDHB enzyme, 130 μM NADH, and 160 μM
sodium pyruvate.
MDH Biochemical Assay. Briefly, 3 μL of MDH solution
(containing 13.33 IU/mL malate dehydrogenase from porcine heart,
0.2 mM NAD, 0.067 mg/mL diaphorase, and 0.067 mM resazurin in
MDH assay buffer (50 mM Tris, pH 8.0, 5 mM MgCl2, 0.01% Brij 3))
was added to a black solid bottom 1536-well assay plate (Greiner Bio-
One) using a BioRAPTR FRD dispenser (Beckman Coulter, Brea,
CA). A 1536-well pintool dispenser outfitted with 20 nL pins (Wako
Automation, San Diego, CA) was used to transfer 23 nL of DMSO-
solubilized compound (Cherrypick plates) to each 1536-well assay
plate. Following compound transfer, plates were incubated at room
temperature for 10 min. 1 μL of substrate solution containing malic
acid (160 μM) was dispensed via BioRAPTR FRD to initiate the
reaction. Plates were immediately transferred to a ViewLux microplate
imager (PerkinElmer, Waltham, MA), and any resulting resorufin
fluorescence was measured (ex 540 nm, em 590 nm) at 0 and 5 min.
Well fluorescence was normalized using enzyme-free and DMSO-
treated control wells on each plate, and changes in fluorescence
(ΔRFU) were calculated using the difference in fluorescent signal for
each well at 5 versus 0 min.
IDH1 Biochemical Assay. IDH1 protocol was performed as
previously described.30 WT IDH1 (3 μL) in assay buffer (20 mM
Tris, pH 7.5, 10 mM MgCl2, 150 mM NaCl, and 0.05% protease-free
BSA) was added to the black solid bottom 1536-well assay plate using
a BioRAPTR flying reagent dispenser (BioRAPTR; Beckman Coulter).
A pintool (Kalypsys) was used to transfer 23 nL of compound solution
(library and control) to the 1536-well assay plates, and plates were
spun down at 1000 rpm for 1 min. After 30 min of incubation at room
temperature, 1 μL of substrate buffer was added to initiate the reaction
at final concentrations of 0.045 μg/mL enzyme, 2 mM BME, 240 μM
isocitrate, 180 μM NADP+, 60 μg/mL diaphorase, and 37.5 μM
resazurin. The plate was rapidly transferred to a ViewLux
(PerkinElmer), and the fluorescence product resorufin was measured
(excitation = 525 nm, emission = 598 nm) in kinetic mode. The plates
were read continuously from t = 0 to t = 5 min.
2-(3-([1,1′-Biphenyl]-3-yl)-5-(cyclopropylmethyl)-4-(4-
sulfamoylbenzyl)-1H-pyrazol-1-yl)thiazole-4-carboxylic Acid
(63). This compound was prepared using general method D starting
from 63c and III. LC−MS retention time: (method 1) 6.08 min and
1
(method 2) 3.521 min. H NMR (400 MHz, DMSO-d6) δ 13.11 (s,
1H), 8.28 (s, 1H), 7.75−7.69 (m, 2H), 7.68−7.62 (m, 2H), 7.59 (dt, J
= 7.7, 1.4 Hz, 1H), 7.52−7.45 (m, 1H), 7.45−7.37 (m, 4H), 7.35−7.29
(m, 3H), 7.27 (s, 2H), 4.16 (s, 2H), 3.16 (d, J = 6.9 Hz, 2H), 1.20−
1.03 (m, 1H), 0.37−0.28 (m, 2H), 0.24−0.18 (m, 2H); HRMS (ESI)
m/z (M + H)+ calcd for C30H27N4O4S, 571.1468; found, 571.1471.
Lactate Production Assays. A673 and MiaPaCa-2 cells were
cultured as described above and plated in 1536-well black clear bottom
R
DOI: 10.1021/acs.jmedchem.7b00941
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
tissue culture plates using a Multidrop Combi peristaltic dispenser
(ThermoFisher, Waltham, MA) at a density of 500 cells/well in 4 μL
of DMEM (Invitrogen 31053036) culture medium. A 1536-well
pintool dispenser outfitted with 20 nL pins (Wako Automation, San
Diego, CA) was used to transfer 23 nL of compound in DMSO to the
1536-well assay plates. After a 2 h incubation at 37 °C, 2 μL of
reconstituted lactate reaction mix (BioVision K607-100) was
dispensed into each well using a BioRAPTR FRD dispenser (Beckman
Coulter, Brea, CA). Plates were incubated at room temperature for 30
min, transferred to a ViewLux microplate imager (PerkinElmer,
Waltham, MA), and the fluorescence (Ex/Em 525/598 nm) and
absorbance (573 nm) were measured accordingly.
temperature, the test samples in the donor compartment were stirred
using the Gutbox technology (Pion Inc.) to reduce the unstirred water
layer. The test article concentrations in the donor and acceptor
compartments were measured using an UV plate reader (Nano Quant,
Infinite 200 PRO, Tecan Inc., Mannedorf, Switzerland). Permeability
̈
calculations were performed using Pion Inc. software and were
expressed in units of 10−6 cm/s.
Kinetic Solubility Test Assay. Pion’s patented μSOL assay for
kinetic solubility determination was used. In this assay, the classical
saturation shake-flask solubility method was adapted to a 96-well
microtiter plate format and a cosolvent method with n-propanol as the
reference compound was utilized. Test compounds were prepared in
10 mM DMSO solutions (45 μL) and diluted with the cosolvent to a
final drug concentration of 150 μM in the aqueous solution (pH 7.4).
Samples are incubated at room temperature for 6 h to achieve
equilibrium. The samples were then filtered to remove any precipitate
formed. The concentration of the compound in the filtrate was
measured by UV absorbance. The reference drug concentration of 17
μM was used for quantitation of unknown drug concentration in
filtrate. Spectroscopically pure 1-propanol was used as a cosolvent to
suppress precipitation in the reference solutions. The kinetic solubility
(μg/mL) was calculated with using the μSOL Evolution software.
Rat Liver Microsome Stability Assay. See ref 30 for details.
Mouse Pharmacokinetic Studies. Studies were conducted by
Pharmaron. Male CD1 mice (sourced from Si Bei Fu Laboratory
Animal Technology Co. Ltd.), approximately 6−8 weeks of age and a
weight of approximately 20−30 g, were dosed with 61 and 63 at 2 mg/
kg (iv) and 50 mg/kg (po). The formulation (0.1 M NaoH in PBS
buffered saline, adjusted with 1 N HCl to pH 7−8.5) was prepared on
the day of dosing or directly prior to dosing. Each cohort had three
mice, and plasma was collected at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h,
8 h, 12 h, and 24h postdose for iv and 15 min 30 min, 1 h, 2 h, 4 h, 8 h,
12 h, and 24 h for po. Approximately 0.025 mL of blood was collected
via the dorsal metatarsal vein at each time point. Blood samples were
then transferred into plastic microcentrifuge tubes containing
heparin−Na as anticoagulant. Samples were then centrifuged at
4000g for 5 min at 4 °C to obtain plasma. Plasma samples were then
stored in polypropylene tubes, quickly frozen, and kept at −75 °C
until analyzed by LC/MS/MS. The following pharmacokinetic
parameters were measured: T1/2, C0, Cmax, Tmax, CL, Vd, AUClast, and
F. Animals were also monitored during the in-life phase by once daily
cageside observations; any adverse clinical signs are noted as part of
the PK report.
Cellular Thermal Shift Assay (CETSA). The cellular thermal shift
assay and the isothermal dose response were run as previously
described.27 Additional details are described in supplemental methods.
Cytotoxicity Assay. A673 and MiaPaCa-2 cells were cultured as
described above and plated in 1536-well white solid tissue culture
plates using a Multidrop Combi peristaltic dispenser (ThermoFisher,
Waltham, MA) at a density of 500 cells/well in 5 μL of DMEM
(Invitrogen 11965118) culture medium supplemented with 10% fetal
bovine serum and 100 units/mL penicillin, 100 μg/mL streptomycin.
A 1536-well pintool dispenser outfitted with 20 nL pins (Wako
Automation, San Diego, CA) was used to transfer 23 nL of compound
in DMSO to the 1536-well assay plates. After a 48 h incubation at 37
°C, 2.5 μL of CellTiter-Glo (Promega) was dispensed into each well
using a BioRAPTR FRD dispenser (Beckman Coulter, Brea, CA).
Plates were incubated at room temperature for 10 min, transferred to a
ViewLux microplate imager (PerkinElmer, Waltham, MA), and the
ATP-coupled luminescence was measured using a 1 s exposure.
Incucyte Long-Term Growth Inhibition Assay. A673 and
MiaPaCa-2 cells were cultured as described above and plated in 384-
well black clear bottom tissue culture plates using an multidrop Combi
peristaltic dispenser (ThermoFisher, Waltham, MA) at a density of
1000 cells/well in 40 μL of DMEM (11965118) culture medium
supplemented with 10% fetal bovine serum and 100 units/mL
penicillin, 100 μg/mL streptomycin.. DMSO-solubilized compounds
were added using a 384-well pintool dispenser (Wako Automation,
San Diego, CA), and plates were incubated and measured in an
IncuCyte ZOOM live cell analysis system (Essen Bioscience, Ann
Arbor, MI) in a cell culture environment at 37 °C containing 5% CO2.
Cell confluency was assessed using high definition phase contrast every
4 h for a total of 120 h. Area under the curve and dose−response curve
analyses was performed using Prism (GraphPad Software).
Use of Animal Subjects. All animal studies included as part of this
manuscript were performed in accordance with institutional guidelines
as defined by Institutional Animal Care and Use Committee
(IACUC).
Glycolytic Stress Test Assay. A673 cells were cultured in
Dulbecco’s modified Eagle’s medium (ATCC catalog no. 302002)
supplemented with fetal bovine serum (10%). The cells were plated
into a XF96 cell culture microplate in the above medium and
maintained in a 5% CO2 incubator at 37 °C for 24 h prior to the
experiments. The day of the assay, compounds are diluted to the
appropriate concentration in freshly prepared assay medium (Seahorse
basic DMEM with 2 mM glutamine, pH 7.4 at 37 °C). The medium in
the plate with cells was then changed to assay medium and maintained
in a non-CO2 incubator at 37 °C for 1 h prior to the assay. The
Seahorse XF glycolysis stress test (GST) was conducted by injecting
the LDH inhibitors and then, at 40 min, subsequent injections of
glucose (10 mM final concentration), oligomycin (1 μg/mL final
concentration), and 2-deoxyglucose (2-DG; 50 mM final concen-
tration) as described in ref 31.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.7b00941.
■
S
Additional figures, experimental procedures, and spectro-
scopic data (1H NMR, LC/MS, and HRMS) for
representative compounds (PDF)
Molecular formula strings and some data (CSV)
PAMPA Permeability Assay. The stirring double-sink PAMPA
method patented by pION Inc. (Billerica, MA) was employed to
determine the permeability of compounds via PAMPA passive
diffusion. The PAMPA lipid membrane consisted of an artificial
membrane of a proprietary lipid mixture and dodecane (Pion Inc.),
optimized to predict gastrointestinal tract (GIT) passive diffusion
permeability, and was immobilized on a plastic matrix of a 96-well
“donor” filter plate placed above a 96 well “acceptor” plate. A pH 7.4
solution was used in both donor and acceptor wells. The test articles,
stocked in 10 mM DMSO solutions, were diluted to 0.05 mM in
aqueous buffer (pH 7.4), and the concentration of DMSO was 0.5% in
the final solution. During the 30 min permeation period at room
Accession Codes
Protein Data Bank codes are the following: 5W8I (27), 5W8H
(15), 5W8J (33), 5W8K (33), 5W8L (63). Authors will release
the atomic coordinates and experimental data upon article
publication.
AUTHOR INFORMATION
Corresponding Authors
*G.R.: phone, 301-217-0263; fax, 301-217-5736; e-mail,
bantukallug@mail.nih.gov.
■
S
DOI: 10.1021/acs.jmedchem.7b00941
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Journal of Medicinal Chemistry
Article
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*D.J.M.: phone, 301-857-1899; e-mail, dmaloney@inspyrtx.
com.
ORCID
Matthew D. Hall: 0000-0002-5073-442X
David J. Maloney: 0000-0002-5607-4178
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Present Addresses
○D.J.M.: Inspyr Therapeutics, 31200 Via Colinas, Suite 200,
Westlake Village, CA 91362.
◆E.J.K.: Department of Chemistry, University of California,
Irvine, CA 92697.
¶C.M.L.: Moderna, 500 Technology Square, Cambridge, MA,
02139.
Author Contributions
∞G.R. and K.R.B. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This project has been funded in whole or in part with federal
funds from the National Cancer Institute, National Institutes of
Health, under Contract HHSN261200800001E. The content of
this publication does not necessarily reflect the views or policies
of the Department of Health and Human Services, nor does
mention of trade names, commercial products, or organizations
imply endorsement by the U.S. Government. G.R., K.R.B.,
B.T.M., D.J.M., X.H., S.-M.Y., T.D.L., D.M.C., J.K., K.P., E.J.K.,
D.K.L., H.Z., M.D.H., A.S., A.J., and D.J.M. also gratefully
acknowledge funding by the Intramural Research Program,
National Center for Advancing Translational Sciences
(NCATS), National Institutes of Health (NIH). The authors
thank Sam Michael and Richard Jones for automation support;
Paul Shinn, Misha Itkin, Zina Itkin, and Danielle van Leer for
the assistance with compound management; William Leister,
Heather Baker, Elizabeth Fernandez, and Christopher Leclair
for analytical chemistry and purification support; Kimloan
Nguyen for in vitro ADME data. We also thank Genentech for
the generous donation of GNE140 for our studies, David
Myszka at Biosenor Tools for conducting the SPR experiments,
and Pharmaron Inc. for conducting the pharmacokinetic
studies.
(8) Blum, R.; Kloog, Y. Metabolism addition in pancreatic cancer.
Cell Death Dis. 2014, 5, e1065.
(9) Shuch, B.; Linehan, M. W.; Srinivasan, R. Aerobic glycolysis: A
novel target in kidney cancer. Expert Rev. Anticancer Ther. 2013, 13,
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(10) Granchi, C.; Roy, S.; Giacomelli, C.; Macchia, M.; Tuccinardi,
T.; Martinelli, A.; Lanza, A.; Betti, L.; Giannaccini, G.; Lucacchini, A.;
́
Funel, N.; Leon, L. G.; Giovannetti, E.; Peters, G. J.; Palchaudhuri, R.;
ABBREVIATIONS USED
■
Calvaresi, E. C.; Hergenrother, P. J.; Minutolo, F. Discovery of N-
hydroxyindole-based inhibitors of human lactate dehydrogenase
isoform A (LDH-A) as starvation agents againsts cancer cells. J.
Med. Chem. 2011, 54, 1599−1612.
LDH, lactate dehydrogenase; qHTS, quantitative high-
throughput screening; CETSA, cellular thermal shift assay;
SPR, surface plasmon resonance; SAR, structure−activity
relationship; TFA, trifluoroacetic acid; LHMDS, lithium
bis(trimethylsiyl)amide; HATU, 1-[bis(dimethylamino)-
methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexa-
fluorophosphate; MW, microwave; TBAF, tetra-n-butylammo-
nium fluoride; NCGC, NIH Chemical Genomics Center;
EDTA, ethylenediaminetetraacetic acid
(11) Kohlmann, A.; Zech, S. G.; Li, F.; Zhou, T.; Squillace, R. M.;
Commodore, L.; Greenfield, M. T.; Lu, X.; Miller, D. P.; Huang, W.-S.;
Qi, G.; Thomas, R. M.; Wang, Y.; Zhang, S.; Dodd, R.; Liu, S.; Xu, R.;
Xu, Y.; Miret, J. J.; Rivera, V.; Clackson, T.; Shakespeare, W. C.; Zhu,
X.; Dalgarno, D. C. Fragment growing and linking lead to novel
nanomolar lactate dehydrogenase inhibitors. J. Med. Chem. 2013, 56,
1023−1040.
(12) Ward, R. A.; Brassington, C.; Breeze, A. L.; Caputo, A.;
Critchlow, S.; Davies, G.; Goodwin, L.; Hassall, G.; Greenwood, R.;
Holdgate, G. A.; Mrosek, M.; Norman, R. A.; Pearson, S.; Tart, J.;
Tucker, J. A.; Vogtherr, M.; Whittaker, D.; Wingfield, J.; Winter, J.;
Hudson, K. Design and synthesis of novel lactate dehydrogenase A
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DOI: 10.1021/acs.jmedchem.7b00941
J. Med. Chem. XXXX, XXX, XXX−XXX