Identification of substituted 2-thio-6-oxo-1,6-dihydropyrimidines as inhibitors of human lactate dehydrogenase

Article abstract of DOI:10.1016/j.bmcl.2013.04.001

A novel 2-thio-6-oxo-1,6-dihydropyrimidine-containing inhibitor of human lactate dehydrogenase (LDH) was identified by high-throughput screening (IC ₅₀ = 8.1 μM). Biochemical, surface plasmon resonance, and saturation transfer difference NMR experiments indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of the screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC₅₀ = 0.48 μM). A crystal structure of an optimized compound bound to human LDHA was obtained and explained many of the observed structure-activity relationships.

Full text of DOI:10.1016/j.bmcl.2013.04.001

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3186–3194
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of substituted 2-thio-6-oxo-1,6-dihydropyrimidines
as inhibitors of human lactate dehydrogenase
a
a
b
a
Peter S. Dragovich a, , Benjamin P. Fauber , Laura B. Corson , Charles Z. Ding , Charles Eigenbrot ,
HongXiu Ge b, Anthony M. Giannetti a, Thomas Hunsaker a, Sharada Labadie a, Yichin Liu a, Shiva Malek a,
Borlan Pan a, David Peterson a, Keith Pitts a, Hans E. Purkey a, Steve Sideris a, Mark Ultsch a,
Erica VanderPorten a, BinQing Wei a, Qing Xu b, Ivana Yen a, Qin Yue a, Huihui Zhang b, Xuying Zhang b
⇑
a Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA
b WuXi AppTec Co., Ltd, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel 2-thio-6-oxo-1,6-dihydropyrimidine-containing inhibitor of human lactate dehydrogenase (LDH)
Received 16 March 2013
Revised 28 March 2013
Accepted 1 April 2013
Available online 10 April 2013
was identified by high-throughput screening (IC50 = 8.1 lM). Biochemical, surface plasmon resonance,
and saturation transfer difference NMR experiments indicated that the compound specifically associated
with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural var-
iation of the screening hit resulted in significant improvements in LDHA biochemical inhibition activity
(best IC50 = 0.48
lM). A crystal structure of an optimized compound bound to human LDHA was obtained
Keywords:
and explained many of the observed structure–activity relationships.
Lactate dehydrogenase
X-ray crystal structure
Glycolysis
Ó 2013 Elsevier Ltd. All rights reserved.
Tumor metabolism
It has been known since the early 1900s that many tumors exhi-
bit altered metabolic characteristics relative to normal, non-trans-
formed tissues.1 One example of such altered metabolism is
related to the utilization of glucose. Many tumors increase the rate
of glucose uptake relative to normal cells and metabolize this nutri-
ent primarily via glycolysis as opposed to the more energy-efficient
but oxygen-dependent mitochondrial oxidative phosphorylation
process.2 In contrast to normal tissues which typically employ gly-
colysis only when oxygen supplies limit oxidative phosphorylation
(e.g., strenuously working muscle), such glycolytic glucose con-
sumption occurs in cancer cells even in the presence of abundant
oxygen levels.2 Originally described by Warburg,3 ‘aerobic glycoly-
sis’ is currently viewed as an attractive differentiator between tu-
mors and healthy tissues that can potentially be exploited for the
development of new anti-cancer agents.4
Lactate dehydrogenase A (LDHA; also known as LDH-M and
LDH-5) is a homotetrameric enzyme that catalyzes the cytosolic
conversion of pyruvate to lactate in the final step of glycolysis
(Fig. 1).5–7 This process involves a stereospecific hydride transfer
from the reduced form of the associated nicotinamide adenine
dinucleotide co-factor (NADH) to the pyruvate ketone moiety. An
alternate lactate dehydrogenase isoform (LDHB; also known as
LDH-H and LDH-1) can also effect this transformation although it
preferentially catalyzes the reverse reaction in which lactate is
converted to pyruvate.5,7 LDHA is a HIF1
a and Myc target gene in-
duced by hypoxia or mutations in VHL, FH, SDH, or the RAS/PI3K/
AKT signaling pathways, and elevated LDHA levels are prevalent
and associated with poor survival in many cancer indications.8
These observations suggest that LDHA may be an important con-
tributor to the metabolic alterations required for the growth and
proliferation of certain tumors. Indeed, shRNA-mediated LDHA
knockdown in glycolytic cancer cell lines results in significant inhi-
bition of tumor growth.9 Consistent with the function of LDHA in
glycolysis, this growth reduction is more pronounced under hyp-
oxic conditions where cells rely primarily on glycolytic energy pro-
duction for survival.9a,c Similarly, an LDHA inhibitor (1; FX-11,
Fig. 2) exhibited in vivo activity against glycolytically dependent
tumor xenograft models,10 although specific inhibition of the LDHA
enzyme by this compound was not confirmed in recent experi-
ments by others.11 Importantly, humans who lack LDHA through
hereditary deficiency display mild phenotypes suggesting that
inhibition of the enzyme will not lead to significant intolerable
side-effects.12 Collectively, these data make LDHA an attractive tar-
get for the development of new anti-cancer agents for use against
hypoxic and/or highly glycolytic tumors.
Several examples of human LDHA inhibitors previously re-
ported in the literature are depicted in Figure 2.10,11,13,14 Some of
⇑
Corresponding author. Tel.: +1 650 467 6854.
E-mail address: dragovich.peter@gene.com (P.S. Dragovich).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.001

P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3186–3194
3187
O
O
-
-
O
O
H
HO
O
Pyruvate
Lactate
NH2
O
H
H
O
LDHA
LDHB
N
N
N
NH2
N
N
O
-
OH
OH
HO
O
H
H
O
H
HO
O
O
NH2
NH2
O
P O P
O
O
-
O
N
N
RO
RO
O
O
NADH
NADH
NAD
HO
OH
HO
OH
Figure 1. LDH biochemistry.
O
OH
CF3
HO
HO
O
O
NH2
HO
N
OH
OH
O
10
213
3
1
(oxamate)
(FX-11)
H
N
H
N
O
OH
OH
S
N
S
O
O
O
411
Figure 2. Structures of representative human LDHA inhibitors.
Table 1
these molecules (e.g., compounds 1 and 2) were recently described
to exhibit ambiguous and/or weak LDHA associations11 suggesting
that the enzyme’s biochemical activity may be susceptible to non-
specific inhibition effects. Our own efforts to identify LDHA inhib-
itors began with high-throughput screening of the Roche and
Genentech compound collections using a biochemical assay which
spectrophotometrically monitored the disappearance of the NADH
co-factor during enzymatic conversion of pyruvate to lactate.15,16
This screening identified a 2-thio-6-oxo-1,6-dihydropyrimidine17
Structure and biological properties of compound 5
O
CN
5
HN
6
H
N
S
N
3
4
2
H2N
O
S
Cl
O
O
5
as
a
moderately potent LDHA inhibitor (compound 5,
M; Table 1). Importantly, a similar IC50 value was ob-
Assay descriptiona
Result (lM)
IC50 = 8.8
l
LDHA IC50 (UV endpoint)
LDHA IC50 (MS endpoint)
LDHA KD (SPR, +NADH)
LDHA KD (SPR, ꢀNADH)
LDHB IC50 (UV endpoint)
MDH-1 IC50 (UV endpoint)
MDH-2 IC50 (UV endpoint)
8.8
1.9
6.1
384
11.1
ꢁ10b
ꢁ10b
served for the molecule when LDHA biochemical inhibition was
quantitated via mass spectrometry, suggesting that the observed
effects did not result from spectrophotometric artifacts (Table 1).16
In addition, biophysical surface plasmon resonance (SPR; Biacore)
experiments conducted in the presence of NADH determined that
the compound specifically associated with LDHA with a KD that
closely matched the biochemical IC50 value (Fig. 3A, Table 1).16
These results collectively suggested that compound 5 was a bona
fide LDHA inhibitor worthy of further characterization.
a
See Supplementary data for experimental details associated with each assess-
ment. All biochemical and SPR assay results are reported as the arithmetic mean of
2 separate runs (n = 2).
b
No inhibition of either MDH-1 or MDH-2 was observed at the highest con-
Accordingly, additional biophysical experiments were con-
ducted with 5 to better elucidate its LDHA inhibition mechanism.
SPR assessments performed in the absence of NADH determined
that the compound associated much more weakly with LDHA as
compared with binding experiments conducted in the presence
of the co-factor (compare Fig. 3A and B, Table 1). These results
centration tested (10 lM). MDH = malate dehydrogenase.
suggested that optimal binding of 5 to LDHA required prior associ-
ation of the NADH co-factor in a manner that might parallel events
occurring during the catalytic conversion of pyruvate to lactate.18

3188
P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3186–3194
Figure 3. SPR data depicting the binding of compound 5 to LDHA in the presence (A) and absence (B) of NADH. The KD’s determined from fitting the kinetics or the
equilibrium dose response (inset) are reported in panel A. In panel B, the KD was estimated relative to a control using methods described in the Supplementary data. In both
panels, the top concentration is 25 lM with injections related by a twofold dilution series. See Supplementary data for additional experimental details.
Saturation transfer difference (STD) NMR experiments showed a
strong NADH-related signal which disappeared upon addition of
compound 5 to the NADH/LDHA mixture (Fig. 4).16 This behavior
is consistent with the simultaneous binding of 5 and NADH to
LDHA in a manner which inhibits the fast on-off binding exchange
of the co-factor.19 A similar result was obtained when oxamate was
added to the STD experiment (Fig. 4) suggesting that this entity did
not interfere with the binding of 5 to LDHA. Consistent with these
observations, the co-crystal structure of a related 2-thio-6-oxo-1,6-
dihydropyrimidine with LDHA suggested that the inhibitor, NADH,
and oxamate could simultaneously bind to the protein (see below).
In addition to the detailed characterization activities described
above, we also examined the ability of compound 5 to inhibit the
biochemical activity of other dehydrogenase enzymes. As shown
in Table 1, the molecule inhibited the closely related LDHB isoform
with an IC50 value nearly identical to that observed for LDHA. This
result was not entirely surprising given the close structural homol-
ogy between these two enzymes.20 Encouragingly, compound 5
displayed only weak inhibition of two other structurally-related21
dehydrogenases (malate dehydrogenase 1 and malate dehydroge-
nase 2, Table 1) suggesting that the molecule would not indiscrim-
inately inhibit this class of enzymes.16
Intrigued by the potency and specificity of 5, we initiated
medicinal chemistry activities aimed at improving the compound’s
LDHA inhibition properties. As shown in Table 2, methylation of
the sulfonamide functional group contained in 5 resulted in signif-
icant loss of anti-LDHA activity (compare 6 with 5). Similarly,
replacement of the para-H2NSO2-aniline moiety present in 5 with
other substituted anilines reduced LDHA inhibition potency to
varying degrees (compounds 7–18, Table 2). These potency
Figure 4. Saturation transfer difference NMR experiments performed with LDHA (3
l
M), NADH (500
lM; blue trace), compound 5 (500
lM; red trace), compound 5 and
NADH (500 and 500 lM, respectively; green trace) and compound 5 with NADH and oxamate (500, 500 lM and 2.5 mM, respectively; magenta trace). See Supplementary
data for additional experimental details.

P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3186–3194
3189
Table 2
Structure–activity relationships of 6-oxo-1,6-dihydropyrimidine-containing compounds
O
N
CN
R3
H
HN
R2
R1
N
S
O
Cl
M)
a
b
c
Compd
R1
R2
R3
LDHA IC50
(l
LDHA KD
(lM)
LDHB IC50 (lM)
5
6
7
8
H2NS(O2)–
MeNHS(O2)–
H
H
H
H
H
H
H
Cl
Cl
Me
Cl
Cl
Cl
Me
H
H
H
H
Me
OMe
Et
H
Me
Me
H
8.8
6.1
11.1
>100
>100
98
ND
ND
>100
55.7
22.6
>100
>100
>100
55.8
50.2
>100
46.8
27.6
26.5
38.6
532
570
552
1011
1105
1215
386
462
1055
314
466
640
552
Cl
iPr
H
9
10
11
12
13
14
15
16
17
18
H
H
H
H
H
F
Me
MeO
Me
ND
>100
>100
ND
>100
93
H
H
H
>100
>100
See supplementary data for experimental details associated with each assessment. All biochemical and SPR assay results are reported as the arithmetic mean of 2 separate
runs (n = 2). ND = not determined.
a
LDHA biochemical inhibition.
LDHA dissociation constant as determined by surface plasmon resonance.
LDHB biochemical inhibition.
b
c
reductions were subsequently explained by the crystallographic
observation of several critical protein–ligand interactions between
the para-H2NSO2 moiety of an inhibitor related to 5 and the LDHA
protein (see below). LDHB inhibition activities for compounds 6–
18 were generally weak and were typically two- to threefold less
potent than the corresponding LDHA values. Importantly, the
LDHA KD’s determined by SPR for the compounds in Table 1 in
the presence of NADH paralleled the IC50 measurements with less
potent biochemical inhibitors exhibiting weaker KD values. This
observation reinforced the belief that compound 5 and related
molecules derived their LDHA inhibition activities primarily
through specific interactions with the protein.
Having determined that the para-H2NSO2-aniline moiety pres-
ent in 5 was critical for anti-LDHA potency, we next explored mod-
ifying other portions of the inhibitor’s structure. As shown in
Table 3, substitution of the para-Cl-Ph moiety contained in 5 with
a simple phenyl group afforded slightly diminished LDHA inhibi-
tion activity (compare 19 with 5). However, similar replacement
with a meta-MeO-Ph group resulted in more dramatic loss of inhib-
itory potency (compound 20, Table 3). The described SAR is consis-
tent with subsequent crystallographic observations indicating the
lack of appropriate space to accommodate a relatively large
meta-Ph substituent in this portion of the 6-oxo-1,6-dihydropy-
rimidine moiety (see below). Substitution of the aliphatic methy-
lene present in 5 with a methyl group improved LDHA inhibition
activity by more than 10-fold (compare 21 with 5, Table 3). This
enhancement was largely retained when additional small substitu-
ents were added to the para-Cl-Ph moiety contained in 21 (com-
pounds 22 and 23). Interestingly, removal of the nitrile group
attached to the 2-thio-6-oxo-1,6-dihydropyrimidine ring system
resulted in drastic potency loss (compound 24, Fig. 5; also removes
para-Cl substituent relative to 21). The nitrile present in the related
inhibitor 22 was subsequently shown by crystallography to make a
hydrogen bond with a structural water molecule (see below) and
removal of this protein–ligand interaction may explain the ob-
served loss in activity. However, given the magnitude of the po-
tency reduction, it is possible that the nitrile also performs other
functions which favorably impact LDHA inhibition (e.g., influenc-
ing the 6-oxo-1,6-dihydropyrimidine tautomer distribution and/
or pKa).22 Replacement of the para-Cl-Ph moiety present in 21 with
a hydrogen atom afforded a less potent inhibitor (compound 25,
Fig. 5), although the observed activity reductions were not as pro-
nounced as those resulting from nitrile removal. Substituting the
methyl group present in 21 with an ethyl moiety afforded a mole-
cule with similar LDHA inhibition properties (compound 26,
Table 3). Incorporation of the same ethyl fragment into a com-
pound containing a meta-MeO-Ph 6-oxo-1,6-dihydropyrimidine
substituent improved LDHA inhibition activity approximately five-
fold (compare compounds 27 and 20, Table 3). A rationalization for
the potency improvements obtained by substituting the aliphatic
methylene present in 5 and/or 20 with methyl or ethyl groups is
provided in the crystallography discussion below.
As was observed for the compounds depicted in Table 2, the
LDHB activities of the inhibitors described in Table 3 were typically
several fold weaker than the corresponding LDHA values. Similarly,
the LDHA KD’s determined by SPR for the molecules in Table 3 par-
alleled their LDHA biochemical IC50 potencies with the most active
inhibitors exhibiting the strongest LDHA biophysical associations
(e.g., compounds 21–23, and 26). The majority of these compounds
were also stable in the presence of human liver microsomes and
afforded extrapolated human clearance values in the low to mod-
erate range. In addition, the aqueous solubilities of the most potent
inhibitors were measured and were determined to be acceptable
(Table 3, compounds 21–23, and 26).
We also assessed the ability of compounds 21–23 to inhibit the
production of lactate in HCC1954 cells.16 Unfortunately, none of
the molecules displayed activity in this assay when tested up to
the 50 lM concentration level. The reasons for this lack of cell-
based activity are currently not known with certainty but may
include: (1) insufficient biochemical potency, (2) poor cell perme-
ability, and/or (3) high protein binding.23 Additional experiments
are on-going to clarify which, if any, of these potential liabilities
is responsible for the poor cell-based activity exhibited by the de-
scribed 6-oxo-1,6-dihydropyrimidine-containing compounds.
To aid in the future optimization of this inhibitor series, a crys-
tal structure of compound 22 in complex with LDHA was obtained

3190
P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3186–3194
Table 3
Structure–activity relationships of 6-oxo-1,6-dihydropyrimidine-containing compounds
O
N
CN
R1 HN
H
N
R3
R2
S
H2N
O
S
O
O
R1
R2
R3
LDHA IC50
(
lM)
LDHA KD
(lM)
LDHB IC50
(l
M)
HLM Cld (mL/min/kg)
Solubilitye
(lM)
a
b
c
Compd
5
H
H
H
Me
Me
Me
Et
Cl
H
H
Cl
Cl
Cl
Cl
H
H
H
OMe
H
Cl
F
H
OMe
8.8
12.1
42.4
0.48
0.75
0.71
0.65
7.4
6.1
15.4
38.4
2.2
5.1
2.9
11.1
42.8
56.4
3.0
3.7
2.3
5.3
ND
ND
2.6
2.6
3.4
2.8
6.9
ND
ND
ND
58
30
64
19
20
21f
22f
23f
26f
27f
3.0
8.9
2.4
7.7
136
ND
Et
See Supplementary data for experimental details associated with each assessment. All biochemical and SPR assay results are reported as the arithmetic mean of 2 separate
runs (n = 2). HLM and solubility data are n = 1. ND = not determined.
a
LDHA biochemical inhibition.
LDHA dissociation constant as determined by surface plasmon resonance.
LDHB biochemical inhibition.
In vivo clearance value extrapolated from in vitro human liver microsome experiment (key: stable = <6.2; labile = >15).
Aqueous solubility (estimated from high-throughput assay).
Racemic.
b
c
d
e
f
O
N
O
N
CN
HN
HN
H
N
H
N
S
S
H2N
O
H2N
O
S
S
O
O
O
O
25
24
(racemic)
LDHA IC50 = >100 uM
(racemic)
LDHA IC50 = 12.5 uM
Figure 5.
entity formed hydrogen bonds with the NADH 30-OH group and the
backbone carbonyl of the LDHA Ala-97 residue. These interactions
between 22 and NADH are consistent with the SPR data described
earlier in this work which indicated that the compound exhibits
much greater affinity for the LDHA protein in the presence of the
co-factor.
Although inhibitor 22 was prepared in racemic form, only one
enantiomer (the R-Me isomer) was observed in the co-crystal
structure with LDHA. The methyl group present in 22 formed
favorable hydrophobic contacts with the LDHA Tyr-238 side chain,
suggesting why addition of aliphatic substituents to the methylene
moiety present in the original screening hit (5) improved LDHA
inhibition activity (Fig. 6b). Such substituents may also help en-
force the twisted conformation of the bound inhibitor observed
in the 22-LDHA co-crystal structure.28 In addition, hydrogen bonds
were observed between the aniline-amide NH of the inhibitor and
the side chain of LDHA residue Asp-194 as well as between the ani-
line-amide carbonyl moiety and a crystallographic water molecule
(Figs. 6b and 6d). These interactions presumably contribute to the
compound’s anti-LDHA activity.
(1.90 Å resolution).24 As shown in Figure 6a, the molecule bound to
the protein near several conserved residues involved in the cata-
lytic processing of LDH substrates (Arg-168, His-192 and Asp-
165).25 Interestingly, the inhibitor did not form any direct interac-
tions with these residues but was instead found contacting an
adjacent region of the protein known to undergo significant con-
formational changes during the enzyme’s catalytic cycle (key mo-
bile residue = Arg-105).5 The presence of compound 22 resulted
in a relatively ‘open’ conformation of this region in which a fourth
conserved residue required for enzyme catalysis (Arg-105) was
positioned well-removed from the substrate binding site. The
ꢂ7 Å distance between 22 and the Arg-168 and His-192 catalytic
residues suggested that small LDH substrate mimetics could simul-
taneously occupy the enzyme’s active site. This observation is con-
sistent with the STD NMR data described above indicating that a
related inhibitor (compound 5) and oxamate (a small substrate mi-
metic) do not interfere with each other’s binding to LDHA.
The NADH co-factor was observed in the crystal structure and
bound to LDHA adjacent to compound 22 in a location and orien-
tation similar to those previously described in the literature.26 As
shown in Figures 6b and 6d, the carbonyl of the inhibitor 6-oxo-
1,6-dihydropyrimidine moiety27 formed a hydrogen bond with
the NADH 20-OH group. In addition, the nitrile present in 22 was
located within H-bond distance of both the LDHA Gln-99 backbone
amide NH group and a crystallographic water molecule. The latter
As expected based on the SAR described in the preceding sec-
tion, the para-H2NSO2-aniline substituent contained in 22 formed
numerous favorable interactions with the LDHA protein. These
associations include: (1) a hydrogen bond between the sulfon-
amide NH2 moiety and the side chain of LDHA residue Asp-140,

P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3186–3194
3191
Figure 6a. Co-crystal structure of inhibitor 22 (cyan) in complex with LDHA (grey). NADH is also present and is depicted in pink. Key structural waters are also shown (red
spheres). Hydrogen bonds are not depicted in this figure. The resolution of the structure is 1.90 Å.
Figure 6b. Alternate view of co-crystal structure of inhibitor 22 (cyan) in complex with LDHA (grey). NADH is also present and is depicted in pink. Key structural waters are
also shown (red spheres). Hydrogen bonds are depicted as dashed yellow lines.

3192
P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3186–3194
Figure 6c. Alternate view of co-crystal structure of inhibitor 22 (cyan) in complex with LDHA (grey). Hydrogen bonds are depicted as dashed yellow lines.
VAL109
LEU108
PRO138
ARG105
Cl
GLN99
ASP194
Cl
O
O
-
GLU191
O
HN
-
O
O
O
CN
O
N
O
H
H
H
N
ALA97
N
H
S
LEU322
ILE141
OH
H2N
O
S
CH3
HO
O
O
OH
OR
NH2
NH
O
O
-
O
H2N
N
H
N
H2N
NADH
O
O
H
O
H
TYR238
ARG105
ASP140
Figure 6d. Depiction of key protein–ligand interactions observed in 22-LDHA co-crystal structure. Hydrogen bonds are depicted as dashed lines. An H-bond interaction
between a water molecule and the amide carbonyl of 22 is omitted for clarity.
(2) another hydrogen bond between the sulfonamide NH2 and the
Glu-191 side chain, (3) a hydrogen bond between a sulfonamide O-
atom and the backbone NH of Asp-140, and (4) a hydrogen bond
between the other sulfonamide O-atom and the side chain of
Arg-105 (Figs. 6c and 6d). These interactions explain the loss of
inhibition potency described earlier in this work that resulted
either from methylation of the para-H2NSO2-moiety or its removal
from the inhibitor structure. In addition, the sulfonamide group
was observed in close proximity to the side chains of LDHA resi-
dues Ile-141 and Leu-322 suggesting that accommodation of elon-
gated (i.e., alkylated) analogs would be difficult.
The 3,4-di-Cl-Ph moiety present in 22 was observed crystallo-
graphically to reside in a primarily hydrophobic cleft formed by
the side chains of LDHA residues Arg-105, Leu-108, Val-109, and
Pro-138 (Figs. 6c and 6d). The 3-Cl substituent on the phenyl ring
was oriented away from the ligand’s para-H2NSO2-aniline group,
presumably to avoid an intramolecular steric clash that would dis-
rupt the observed binding conformation of the inhibitor. The 3-Cl
substituent made close contacts with the surrounding LDHA resi-
dues, and this binding mode was consistent with the observation
that incorporation of a larger OMe group at the meta-position of
the inhibitor Ph ring was detrimental to anti-LHDA activity (see

P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3186–3194
3193
O
N
O
CN
S
EtO
HN
HS
R3
R2
i
CN
H
R3
R2
+
+
H2N
NH2
O
28a-c
29a-c
+
R1
NH2
H
N
O
ii
Br
Br
+
H2N
O
Cl
S
H2N
O
R1
30d-e
S
O
O
O
31d-e
iii
R2 = Cl; R3 = H
R2 = Cl; R3 = Cl
R2 = Cl; R3 = F
R1 = Me
R1 = Et
a
b
c
d
e
21-24, 26
Scheme 1. Synthesis of 2-thio-6-oxo-1,6-dihydropyrimidine-containing compounds. Reagents and conditions: (i) K2CO3, ethanol, reflux, 1 h or piperidine, ethanol, 5 h; (ii)
K2CO3, THF, reflux, 1 h; and (iii) K2CO3, ethanol, reflux, 1–24 h.
above). Collectively, the 22-LDHA co-crystal structure illustrates
that the inhibitor makes numerous hydrogen bonds with the LDHA
protein, the NADH co-factor, and crystallographic water molecules
along with several favorable hydrophobic protein–ligand interac-
tions. This structural information can be utilized to design new 2-
thio-6-oxo-1,6-dihydropyrimidine-containing LDHA inhibitors that
seek to improve the potency of the molecules detailed in this work.
The described inhibitors were either purchased from eMolecules
(5, 7–20, 25, 27) or were synthesized as depicted in Scheme 1 (6, 21–
24, 26). Synthesis commenced with the condensation of various
benzaldehydes (28a–c) with ethyl 2-cyanoacetate and thiourea to
give the corresponding 2-thio-6-oxo-1,6-dihydropyrimidines
(29a–c).29 Separately, bromides 31d–e were prepared via coupling
of 4-aminobenzenesulfonamide with acid chlorides 30d (commer-
cially available) and 30e (synthesized as described in the litera-
ture30). Appropriate condensation of the 2-thio-6-oxo-1,6-
dihydropyrimidines 29 with bromides 31 subsequently afforded
the desired LDHA inhibitors 21–24 and 26 in good yields. Compound
6 was prepared in an analogous manner with the required 4-amino-
N-methylbenzenesulfonamide being synthesized as described in
the literature.31
In summary, a novel class of 2-thio-6-oxo-1,6-dihydropyrimi-
dine-containing LDHA inhibitors was identified using a high
throughput screening approach. The crystal structure of a repre-
sentative compound in complex with the protein indicated that
these inhibitors bound in the LDHA active site but did not make di-
rect interactions with the majority of enzyme residues involved in
substrate catalysis. Structural modification of the initial high
throughput screening hit afforded >10-fold improvements in bio-
chemical potency, but none of the molecules studied were active
in cell culture experiments. Additional efforts to obtain potent,
cell-active LDHA inhibitors will be reported in due course.
diffraction data collection. The Advanced Light Source is supported
by the Director, Office of Science, Office of Basic Energy Sciences, of
the U.S. Department of Energy under Contract No. DE-AC02-
05CH11231.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.04.001.
References and notes
1. (a) Ward, P. S.; Thompson, C. B. Cancer Cell 2012, 21, 297; (b) Vander Heiden, M.
G. Nat. Rev. Drug Disc. 2011, 10, 671; (c) Zhao, Y.; Liu, H.; Riker, A. I.; Fodstad, O.;
Ledoux, S. P.; Wilson, G. L.; Tan, M. Front. Biosci. 2011, 16, 1844; (d) Kaelin, W.
G., Jr.; Thompson, C. B. Nature 2010, 465, 562; (e) Tennant, D. A.; Durán, R. V.;
Gottlieb, E. Nat. Rev. Cancer 2010, 10, 267.
2. (a) Vander Heiden, M. G.; Cantley, L. C.; Thompson, C. B. Science 2009, 324,
1029; (b) Hsu, P. P.; Sabatini, D. M. Cell 2008, 134, 703.
3. (a) Warburg, O. Science 1956, 123, 309; (b) Bensinger, S. J.; Christofk, H. R.
Semin. Cell Dev. Biol. 2012, 23, 352; (c) Koppenol, W. H.; Bounds, P. L.; Dang, C.
V. Nat. Rev. Cancer 2011, 11, 325.
4. (a) Hamanaka, R. B.; Chandel, N. S. J. Exp. Med. 2012, 209, 211; (b) Jones, N. P.;
Schulze, A. Drug Discovery Today 2011, 17, 232; (c) Pelicano, H.; Martin, D. S.;
Xu, R.-H.; Huang, P. Oncogene 2006, 25, 4633.
5. Granchi, C.; Bertini, S.; Macchia, M.; Minutolo, F. Curr. Med. Chem. 2010, 17, 672.
6. For a detailed description of glycolysis, see: Salaway, J. G. Metabolism at a
Glance, 3rd ed.; Blackwell Publishing: Malden, 2004. pp 10–25.
7. LDHA and LDHB are each homotetramers comprised of M and H subunits,
respectively. LDH heterotetramers containing both M and H subunits are also
known. For more information, see Ref. 5.
8. (a) Kolev, Y.; Uetake, H.; Takagi, Y.; Sugihara, K. Ann. Surg. Oncol. 2008, 15,
2336; (b) Koukourakis, M. I.; Giatromanolaki, A.; Sivridis, E.; Gatter, K. C.;
Harris, A. L. J. Clin. Oncol. 2006, 24, 4301; (c) Koukourakis, M. I.; Giatromanolaki,
A.; Sivridis, E.; Bougioukas, G.; Didilis, V.; Gatter, K. C.; Harris, A. L. Br. J. Cancer
2003, 89, 877.
9. (a) Seth, P.; Grant, A.; Tang, J.; Vinogradov, E.; Wang, X.; Lenkinski, R.;
Sukhatme, V. P. Neoplasia 2011, 13, 60; (b) Qing, G.; Skuli, N.; Mayes, P. A.;
Pawel, B.; Martinez, D.; Maris, J. M.; Simon, M. C. Cancer Res. 2010, 70, 10351;
(c) Fantin, V. R.; St.-Pierre, J.; Leder, P. Cancer Cell 2006, 9, 425.
10. Le, A.; Cooper, C. R.; Gouw, A. M.; Dinavahi, R.; Maitra, A.; Deck, L. M.; Royer, R.
E.; Vander Jagt, D. L.; Semenza, G. L.; Dang, C. V. Proc. Natl. Acad. Sci. 2010, 107,
2037.
Acknowledgments
We thank Drs. Krista Bowman and Jiansheng Wu and their
respective Genentech research groups for performing many associ-
ated protein expression and purification activities. We also thank
Dr. Peter Jackson for many helpful discussions regarding LDHA.
In addition, we thank Crystallographic Consulting, LLC for
11. 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. J. Med. Chem. 2012, 55, 3285.

3194
P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3186–3194
12. (a) Kanno, T.; Sudo, K.; Maekawa, M.; Nishimura, Y.; Ukita, M.; Fukutake, K.
Clin. Chim. Acta 1988, 173, 89; (b) Kanno, T.; Sudo, K.; Takeuchi, I.; Kanda, S.;
Honda, N.; Nishimura, Y.; Oyama, K. Clin. Chim. Acta 1980, 108, 267.
13. Granchi, C.; Roy, S.; Giacomelli, C.; Macchia, M.; Tuccinardi, T.; Martinelli, A.;
Lanza, M.; Betti, L.; Giannaccini, G.; Lucacchini, A.; Funel, N.; León, L. G.;
Giovannetti, E.; Peters, G. J.; Palchaudhuri, R.; Calvaresi, E. C.; Hergenrother, P.
J.; Minutolo, F. J. Med. Chem. 2011, 54, 1599.
14. (a) For additional examples of LDH inhibitors, see: 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, J.; 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. J. Med. Chem. http://dx.doi.org/10.1021/jm3014844.; (b) Chai,
D.; Colon, M.; Dodson, C.; Duffy, K. J.; Shaw, A. N. WO 2012/061557.; In
addition, inhibitors of Plasmodium falciparum LDH with potential for use as
anti-malarials have been described: (c) Choi, S.-R.; Pradham, A.; Hammond, N.
L.; Chittiboyina, A. G.; Tekwani, B. L.; Avery, M. A. J. Med. Chem. 2007, 50, 3841;
(d) Choi, S.-R.; Beeler, A. B.; Pradham, A.; Watkins, E. B.; Rimoldi, J. M.; Tekwani,
B.; Avery, M. A. J. Comb. Chem. 2007, 9, 292; (e) Cameron, A.; Read, J.; Tranter,
R.; Winter, V. J.; Sessions, R. B.; Brady, R. L.; Vivas, L.; Easton, A.; Kendrick, H.;
Croft, S. L.; Barros, D.; Lavandera, J. L.; Martin, J. J.; Risco, F.; García-Ochoa, S.;
Gamo, F. J.; Sanz, L.; Leon, L.; Ruiz, J. R.; Gabarró, R.; Mallo, A.; Gómez de la
Heras, F. J. Biol. Chem. 2004, 279, 31429.
15. VanderPorten, E.; Frick, L.; Turincio, R.; Thana, P.; LaMarr, W.; Liu, Y. Anal.
Biochem., submitted for publication.
16. See Supplementary data for details regarding enzyme, SPR, NMR, and cell
culture assay methods.
17. No attempts were made to identify which, if any, dihydropyrimidine tautomers
predominated in organic and/or aqueous solutions. All new compounds
described in this work are arbitrarily drawn and described as 6-oxo-1,6-
dihydropyrimidines.
18. Binding of NADH to LDHA precedes binding of the pyruvate in the accepted
LDHA catalytic mechanism. See Ref. 5 for additional details.
23. Compounds 21–23 bind strongly to human and animal plasma proteins (free
unbound fractions <1% in all cases examined). The apparent apical to
basolateral permeability of compound 21 determined using Madin–Darby
canine kidney (MDCK) cell monolayers was relatively low (Papp,
AꢀB = 0.30 ꢃ 10ꢀ6 cm/s) with minimal efflux noted (AꢀB/BꢀA = 0.69).
24. See the Supplementary data for experimental details associated with the
described co-crystal structure. All crystallographic descriptions are based on
analysis of the ligand chain ‘X’/protein chain ‘C’ LDHA protomer. Two other
LDHA protomers containing bound ligand were also observed in the tetramer
asymmetric unit. The protein–ligand interactions noted in these other
protomers were similar to those described for the ligand chain ‘X’/protein
chain ‘C’ complex, although some subtle differences were apparent. See
deposited PDB file for additional structural details: code = 4JNK.
25. The LDH residue numbering scheme used herein follows the convention of PDB
entries 1I10 and 4AJP which does not account for the initiating methionine in
the gene sequence reflected in UniProt entry P00338. This numbering scheme
differs by one residue from that described elsewhere: Chung, F. Z.; Tsujibo, H.;
Bhattacharyya, U.; Sharief, F. S.; Li, S. S. Biochem. J. 1985, 231, 537.
26. For other examples of LDH crystal structures containing NADH, see Ref. 20 and
Chaikuad, A.; Fairweather, V.; Conners, R.; Joseph-Horne, T.; Turgut-Balik, D.;
Brady, R. L. Biochemistry 2005, 44, 16221.
27. The resolution of the LDHA-22 co-crystal does not allow for precise
determination of the inhibitor tautomer that is bound to the protein. The
compound is arbitrarily described as
discussion of the co-crystal structure.
a 6-oxo-1,6-dihydropyrimidine in
28. To assess the effect of the methyl group on inhibitor conformation, a dihedral
angle scan of the C–S bond was performed on the bound conformation of
compound 22 and its des-methyl counterpart using MacroModel (Schrodinger
Maestro v9.2) with OPLS-2005 force field and default parameters. The results
show that, although the methyl group does not alter the global minima, there
is a competing local minimum if the methyl is absent, thus creating an entropic
penalty toward binding for the des-methyl molecule. See the Supplementary
data for additional details.
19. The STD NMR results are also consistent with complete displacement of NADH
from the LDHA protein by the binding of compound 5.
20. Read, J. A.; Winter, V. J.; Eszes, C. M.; Sessions, R. B.; Brady, R. L. Proteins 2001,
43, 175.
29. (a) Chen, W.; Huang, Y.-J.; Gundala, S. R.; Yang, H.; Li, M.; Tai, P. C.; Wang, B.
Bioorg. Med. Chem. 2010, 18, 1617; (b) Kambe, S.; Saito, K.; Kishi, H. Synthesis
1979, 287.
21. Human LDHA and human MDH-2 protein sequences share 20% similarity. X-
ray structures of their protomers can be superimposed using 173 pairs of Ca
atoms with an rms deviation of 1.7 Å. Ca atoms within 5 Å of bound NADH can
be superimposed with an rms deviation of 0.7 Å.
22. The pKa of compound 26 was measured and was determined to be relatively
acidic (3.94).
30. Boschi, F.; Camps, P.; Comes-Franchini, M.; Muñoz-Torrero, D.; Ricci, A.;
Sánchez, L. Tetrahedron: Asymmetry 2005, 16, 3739.
31. Khanna, I. K.; Weier, R. M.; Yu, Y.; Collins, P. W.; Miyashiro, J. M.; Koboldt, C. M.;
Veenhuizen, A. W.; Currie, J. L.; Seibert, K.; Isakson, P. C. J. Med. Chem. 1997, 40,
1619.