Identification of 3,6-disubstituted dihydropyrones as inhibitors of human lactate dehydrogenase

Article abstract of DOI:10.1016/j.bmcl.2014.10.067

A series of 3,6-disubstituted dihydropyrones were identified as inhibitors of human lactate dehydrogenase (LDH)-A. Structure activity relationships were explored and a series of 6,6-spiro analogs led to improvements in LDHA potency (IC₅₀ <350 nM). An X-ray crystal structure of an improved compound bound to human LDHA was obtained and it illustrated additional opportunities to enhance the potency of these compounds, resulting in the identification of 51 (IC₅₀ = 30 nM).

Full text of DOI:10.1016/j.bmcl.2014.10.067

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5683–5687
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of 3,6-disubstituted dihydropyrones as inhibitors of
human lactate dehydrogenase
a
b
a
b
Benjamin P. Fauber a, , Peter S. Dragovich , Jinhua Chen , Laura B. Corson , Charles Z. Ding ,
Charles Eigenbrot a, Sharada Labadie a, Shiva Malek a, David Peterson a, Hans E. Purkey a, Kirk Robarge a,
Steve Sideris a, Mark Ultsch a, BinQing Wei a, Ivana Yen a, Qin Yue a, Aihe Zhou a
⇑
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 series of 3,6-disubstituted dihydropyrones were identified as inhibitors of human lactate dehydroge-
nase (LDH)-A. Structure activity relationships were explored and a series of 6,6-spiro analogs led to
improvements in LDHA potency (IC50 <350 nM). An X-ray crystal structure of an improved compound
bound to human LDHA was obtained and it illustrated additional opportunities to enhance the potency
of these compounds, resulting in the identification of 51 (IC50 = 30 nM).
Received 15 September 2014
Revised 18 October 2014
Accepted 20 October 2014
Available online 27 October 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
LDHA
X-ray crystal structure
Tumor metabolism
Abnormal cellular metabolism in tumor cells is one of the
emerging hallmarks of cancer.1 Tumor cells can produce ATP using
an inefficient mitochondrial-independent glycolytic pathway
under normoxic and hypoxic conditions,2 unlike normal tissues
which typically produce ATP using the more effective mitochon-
drial tricarboxylic acid (TCA) cycle3 (also known as the Krebs
cycle). Tumor cells’ catabolism of glucose generates several reac-
tion products including pyruvate and NADH.4 Pyruvate is further
converted into lactate by the NADH-dependent enzyme lactate
dehydrogenase (LDH)-A, thus completing the glycolytic cycle.5 Ele-
vated lactate levels are noted across several classes of human
tumors,6 indicating the potential increased activity of LDHA in can-
cerous tissues. Additional studies have shown that LDHA is over-
expressed in human tumors.7 Cancer cell lines with increased
LDHA expression exhibit a greater dependence on glycoloysis.8
Furthermore, a recent study demonstrated that the ablation of
LDHA with an inducible genetic knock-out mouse was efficacious
in a mouse non-small cell lung cancer (NSCLC) model.9 Collec-
tively, these results suggest that LDHA is an attractive target for
the treatment of human cancer.10
screen (HTS) of the Genentech/Roche corporate compound collec-
tion (Fig. 1).14 Compounds 113c and 213b were moderately potent
in the LDHA biochemical assay, yet they lacked suitable in vitro
and in vivo ADME properties for progression as lead series. Our
recent disclosure of a 1,3-dicarbonyl compound (3) and related
analogs13a illustrated this series’ NADH non-competitive binding
mode in the LDHA substrate binding site, its high ligand-efficiency
(LE),15 and favorable in vivo PK properties. The 1,3-dicarbonyl
motif of 3 resided in a tautomer consistent with the structure
shown in Figure 1 and was ionized at physiological pH (measured
pKa = 2.63). Furthermore, the binding mode of 3 in the LDHA X-ray
co-crystal structure was consistent with the enolate tautomer of 3
[PDB: 4QO7].13a To further explore the potential of the 1,3-dicar-
bonyl series, we investigated other core motifs and their impact
on the LDHA biochemical potency.16
A variety of core changes were introduced while holding the 3-
and 6-position substituents constant (Table 1). It was noted that
changing the core atom adjacent to one of the carbonyls (4–6)
did not affect the LDHA biochemical potency (IC50 = 4.0 lM). Yet,
these same core changes did impact the measured pKa values,
apparent cellular permeability (Papp) as assessed by the Madin-
Darby canine kidney (MDCK) assay,17 and human plasma–protein
unbound fraction. Further exploration of the 3-position linker in
tandem with the aforementioned core changes led to a matrix of
analogs (7–12) with the 3-position thioether being preferred for
LDHA potency (4–6). We presumed that the 3-position thioether
was preferred due to its ability to interact with the Arg168 residue
Several groups have identified LDHA inhibitors11 with some of
the most potent and well characterized inhibitors originating from
fragment screening campaigns.12 Our group discovered several dis-
tinct chemical classes of LDHA inhibitors13 from a high-throughput
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2014.10.067
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

5684
B. P. Fauber et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5683–5687
O
N
OH
O
O
CN
a
HN
H
N
R
S
O
R
H2N
O
S
Cl
13
14
O
O
1
2
OH
O
Cl
S
b
HO
N
N
NH
O
R
O
NH2
15
Scheme 1. First generation synthesis of 3,6-disubstituted dihydropyrones.
Reagents and conditions: (a) methyl acetoacetate, NaH, n-BuLi, THF 0 °C, 45–80%
yield; (b) 2-chlorothiophenol, CBr4, NaOH, THF, 0 ? 23 °C, 5–35% yield.
OH
NO2
S
O
OH
Cl
Cl
3
a
S
S
Me
MeO
Figure 1. LDHA inhibitors identified by HTS campaigns at Genentech.
O
2
16
17
Table 1
OH
O
Cl
Structure–activity relationships of the 1- and 3-positions of the 3,6-disubstituted
cores
S
b,c
R1
R2
OH
3
Cl
O
Y
5
6
18
X
1
O
Scheme 2. Second generation synthesis of 3,6-disubstituted dihydropyrones,
allowing for late-stage introduction of the 6-position substituent(s). Reagents and
conditions: (a) methyl acetoacetate, K2CO3, DMF, 90 °C, 78% yield; (b) lithium
diisopropyl amide (2.2 equiv), THF, 0 °C, followed by addition of R1COR2; (c) K2CO3,
MeOH, 23 °C, 28–40% yield over two steps.
b
Compd
X
Y
LDHA
IC50
pKa
MDCK
Human/rat
plasma–protein
binding
a
permeabilityc
A ? B/B ? A
(10ꢀ6 cm/s)
(lM)
(%unbound)
4
5
6
7
8
O
S
S
S
O
O
O
4.0
4.0
4.0
11
26
11
2.4
3.0
4.1
3.4
3.7
5.3
4.2
4.6
6.0
3/5
12/18
7/9
0.8/0.8
0.1/0.2
0.3/0.6
0.6/1.3
1.1/1.2
2.2/2.3
ND
CH2
NH
O
CH2
NH
O
(Scheme 2). Methyl acetoacetate was subjected to 2-chloro-ben-
zene disulfide (16) under basic conditions to produce the methyl
4/6
acetoacetate
a-thioether product (17). Treatment of 17 with two
11/16
11/12
8/10
4/10
19/30
equivalents of lithium diisopropyl amide, followed by the addition
of an aldehyde or ketone, then subjection to alkaline cyclization
conditions formed the dihydropyrone product (18).
9
10
11
12
NH 13
CH2 NH 25
NH NH 66
ND
ND
We took advantage of the modified synthetic route to explore
several 6-position analogs (Table 2) as dihydropyrone matched
molecular pairs22 to the previously reported13a all-carbon-core
analogs. The LDHA biochemical potency of the ortho-, meta-, and
para-chlorophenyl 6-position analogs (19–21, respectively) were
very similar to their all-carbon-core analogs, indicating compara-
ble SAR between the two cores. There was a six-fold drop in LDHA
potency when comparing the 20,60-disubstituted analogs in the
dihydropyrone core (22–23) against their matched molecular pairs
in the all carbon core. This change in potency was not initially
anticipated but it could be retrospectively attributed to the subtle
difference in the 6-position vector between the two core types.23
The favorable potency of the ortho- and meta-substituted analogs
(19 and 20) led us to explore other substituents on the 6-position
arene. The cyano-substituted analogs 24 and 25 were less potent
than the chloro-substituted matched molecular pairs, and the eth-
oxy substitution (26–27) provided only a slight improvement in
potency at the meta-position (LDHA IC50 = 2.2 lM) over the parent
molecule (4). Replacement of the 6-position phenyl ring with a
variety of alkyl and aliphatic heterocycles (exemplified by 28–29)
was poorly tolerated (LDHA IC50 >100 lM). Exploration of 6-posi-
tion heterocyclic aromatic groups revealed detectable LHDA
potency for the methylated pyrazole (30) and pyrimidine analogs
(31), whereas the 2-pyridyl (32) was the most potent (LDHA
All assay results are reported as the geometric mean of at least two separate runs.
ND = not determined.
a
Human LDHA biochemical inhibition.
Measured pKa value.
b
c
Madin–Darby canine kidney (MDCK) cell permeability assay to assess mem-
brane permeability properties.17
in the LDHA substrate binding site.13a,18 Changes to the 3-position
linker atom negatively affected this Arg168 interaction and led to
diminished LDHA affinity. The loss of LDHA affinity was most pro-
nounced in a 3-position benzylic analog (Table 1, X = Y = CH2, IC50
>100 lM) even though the molecule could adopt an similar ligand
confirmation to that of the more potent thioether analog (5).19
The identical LDHA potency values of 4 and 5 presented an
opportunity to further explore the 6-position SAR of the dihydro-
pyrone core. The initial dihydropyrone analogs were synthesized
using a previously described route20 in which a benzaldehyde
(13) was reacted with Weiler’s dianion21 to form the dihydropy-
rone core (14, Scheme 1). The 3-position thioether was then
installed under basic conditions to yield the final compounds
(15). This initial synthetic route was robust and allowed for the
generation of several dihydropyrone analogs, yet it required the
introduction of the 6-position substituent early in the synthesis.
In an effort to introduce the 6-position substituent late in the syn-
IC50 = 3.5
lM) of all the pyridine isomers (32–34). Compound 32
thesis, we devised
a modified route to the dihydropyrones
was equipotent to 4 in the LDHA biochemical assay, yet 32 was less

B. P. Fauber et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5683–5687
5685
Table 2
analogs with modest LDHA biochemical potency but no further
improvements beyond 35. The secondary amide analog (40) was
equipotent to the primary amide (39), whereas the tertiary amide
(41) was less potent. Based on these results, we concluded that
there were some steric requirements to maintain potency in this
region yet there were no notable improvements in the ligand affin-
ities. Homologation of 40 to a methylene-spaced secondary amide
analog (42) diminished the LDHA affinity. Additional directly-
linked and methylene-spaced 6-position amide analogs were
explored (data not shown), and none provided improvements in
potency over the parent methyl compound (35).
Structure–activity relationships of the 6-position group on 3,6-disubstituted dihyd-
ropyrones
OH
Cl
S
R
O
O
a
Compd
R-group
LDHA IC50
(lM)
LDHA LLEb
cLogP
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
(2-Cl)Ph
(3-Cl)Ph
(4-Cl)Ph
(2,6-Dichloro)Ph
(2,6-Dimethyl)Ph
(2-CN)Ph
(3-CN)Ph
(2-OEt)Ph
(3-OEt)Ph
i-Pr
5.5
3.9
16
5.0
17
>100
9.5
3.8
2.2
>100
>100
35
1.7
1.6
1.0
1.0
0.9
—
2.2
2.0
2.0
—
3.6
3.8
3.8
4.3
3.9
2.8
2.8
3.4
3.6
2.9
2.3
1.5
1.2
1.8
1.9
1.9
The limited success in identifying potent substituted 6-aryl and
6,6-disubstituted dihydropyrone analogs led us to revisit the axi-
ally-oriented 6-aryl analogs that were identified in our earlier
work.13a We envisioned that we could force the 6-position phenyl
into an axial orientation on the dihydropyrone scaffold with a spi-
rocyclic ring system. These spirocyclic analogs were synthesized
using the route described in Scheme 2 and the corresponding inda-
none and tetralone reagents. The resulting spirocyclic products
were separated into single unknown stereoisomers and tested as
single enantiomers, with one enantiomer typically 6–20-fold more
potent that the other enantiomer (Table 4). The favored enantio-
mers of the 40,40-linked dihydrochromene (43) and the 10,10-linked
tetralin (45) analogs were essentially equipotent to the parent
molecule (4), with 45 displaying the best LDHA potency
4-THP
—
3-(1-Me-pyrazole)
5-Pyrimidyl
2-Pyridyl
3.0
2.9
3.7
2.9
2.5
73
3.5
15
3-Pyridyl
4-Pyridyl
43
All assay results are reported as the geometric mean of at least two separate runs.
a
Human LDHA biochemical inhibition.
b
Ligand-lipophilicity efficiency (LLE)25 was calculated using the LDHA biochem-
ical IC50 and calculated logP (cLogP)24 values.
(IC50 = 2.1 l
M). Exploration of the 10,10-linked indane (47) led to
an approximate 10-fold improvement in LDHA potency
(IC50 = 360 nM) over 45.
To further explain the improved potency of 47, we obtained a
1.91 Å resolution co-crystal structure of 47 bound to human LDHA
in the presence of NADH [PDB: 4RLS].27 As shown in Figure 2, the
dihydropyrone core of 47 bound to LDHA in a similar confirmation
to the related all-carbon-core analogs previously reported by our
group.13a Based on the proximity of hydrogen bonding residues
near the ligand, we determined that the ester oxygen of the dihyd-
ropyrone core formed a hydrogen bond28 with Thr247 (2.9 Å). This
conformation of the core was most sensible given that the other
enantiomer would create a clash between the methylene on the
ligand’s core and the hydroxyl group of Thr247. This observation
also allowed us to establish the absolute stereochemistry of 47 as
the (R)-configuration at the 6-position of the dihydropyrone core.
Table 3
Structure–activity relationships at the 6-position of 3,6-disubstituted dihydropyrones
OH
Cl
S
R
O
O
Compd
R-group
LDHA IC50
(l
M)
LDHA LLEb
cLogP
a
35
36
37
38
39
40
41
42
Me
c-Pr
4.1
13
5.6
46
6.6
7.4
22
2.2
1.4
3.3
1.9
3.6
3.1
2.4
2.4
3.2
3.5
2.0
2.4
1.6
2.0
2.3
1.8
CH2OH
CO2H
CONH2
CONHMe
CONMe2
CH2CONHMe
Table 4
Structure–activity relationships of 6-spiro analogs of the 3,6-disubstituted dihydro-
pyrones
59
All assay results are reported as the geometric mean of at least two separate runs.
OH
Cl
a
Human LDHA biochemical inhibition.
S
b
Ligand-lipophilicity efficiency (LLE)25 was calculated using the LDHA biochem-
R
ical IC50 and calculated logP (cLogP)24 values.
O
n
O
X
lipophilic (as assessed by the cLogP)24 and possessed an improved
ligand-lipophilic efficiency (LLE)25 value (LLE = 3.7) compared to 4
(LLE = 2.4).
To further explore the 6-position SAR, we investigated 6,6-
disubstituted dihydropyrone analogs (Table 3) using the LDHA
co-crystal structure of 4 [PDB: 4QO7]13a to guide our designs.
Based on this co-structure, we assumed that the 6-position phenyl
ring would sit in an axial orientation on the dihydropyrone core
and the additional 6-position substituent would access the equato-
rial vector.26 The 6-position methyl group (35) was tolerated and
equipotent to 4, whereas the cyclopropyl group was less potent
b
Compd
R
X
n
Enantiomera LDHA IC50
LDHA
LLEc
cLogP
(lM)
43
44
45
46
47
48
49
50
51
52
H
H
H
H
H
H
O
O
CH2
CH2
CH2
CH2
1
1
1
1
0
0
0
0
0
0
A
B
A
B
A
B
A
B
A
B
3.3
28
2.1
22
0.36
2.6
0.35
2.1
2.8
1.9
2.2
1.2
3.1
1.5
2.6
1.8
2.5
1.2
2.7
2.7
3.5
3.5
3.1
3.1
3.9
3.9
5.0
5.0
Br CH2
Br CH2
Ph CH2
Ph CH2
0.030
0.61
(36) (LDHA IC50 = 13 lM). Using structure-based drug design prin-
All assay results are reported as the geometric mean of at least two separate runs.
ciples and docking studies, we envisioned adding substituents
along the same vector as the methyl group on 35 to engage the
nearby Thr247 or 30-hydroxyl group of the NADH ribose in a hydro-
philic interaction. The introduction of a 6-position hydroxymethyl
moiety (37), carboxylic acid (38), and primary amide (39) provided
a
Compounds were separated into single unknown stereoisomers and identified
as enantiomer A and B.
b
Human LDHA biochemical inhibition.
c
Ligand-lipophilicity efficiency (LLE)25 was calculated using the LDHA biochem-
ical IC50 and calculated logP (cLogP)24 values.

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B. P. Fauber et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5683–5687
We also assessed the ability of 51 to inhibit the production of
lactate in MCF7 cells.16 No dose–response was observed in the
assay at the highest compound concentration of 50 M under stan-
dard serum conditions (EC50 >50 M). When the MCF7 cellular
l
l
assay was conducted in the absence of serum we observed a reduc-
tion in lactate production with no effect on cell viability
(EC50 = 32 lM). This result illustrated that the weak cellular
potency of 51 was potentially related to insufficient biochemical
potency and/or high plasma–protein binding. We committed sub-
stantial efforts toward improving these two parameters and they
will be the focus of future disclosures.
In summary, we identified a new class of 3,6-disubstituted
dihydropyrone LDHA inhibitors. The synthesis of 6-substituted
and 6,6-disubstituted dihydropyrone analogs led to limited
potency improvements, whereas the 6,6-spirocyclic analogs led
to a potency breakthrough as exemplified by compound 47. The
co-crystal structure of LDHA with 47 illustrated additional oppor-
tunities for improving the ligand affinity and these were realized
with the synthesis of 51 as a potent inhibitor of human LDHA.
Figure 2. A 1.91 Å resolution co-structure of 47 (yellow) in complex with LDHA
(grey) in the presence of NADH (orange) [PDB: 4RLS]. Hydrogen bonds are dashed
lines (black). The side-chain residues and water molecules have been omitted for
clarity. Compound 47 formed hydrogen bonds with His192 (2.6 Å), Thr247 (2.9 Å),
and a bidentate hydrogen bond with Arg168 (2.6–3.1 Å).
Acknowledgments
We thank Dr. Peter Jackson for many helpful discussions
regarding LDHA. In addition, we thank J.L. Stillwell and Crystallo-
graphic Consulting, Ltd. for diffraction data collection. Use of the
Stanford Synchrotron Radiation Lightsource was supported by
the U.S. Department of Energy, Office of Science (BES), under Con-
tract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biol-
ogy Program was supported by the DOE Office of Biological and
Environmental Research, and by the National Institutes of Health,
National Institute of General Medical Sciences (including
P41GM103393).
One of the carbonyl groups on the dihydropyrone core engaged the
neighboring His192 residue in a hydrogen bond (2.6 Å), and His192
was further stabilized by a hydrogen bond network with the
nearby Asp165 (2.7–3.1 Å). As noted with the prior all-carbon
ligand LDHA co-crystal structures from our group,13a the thioether
and remaining carbonyl moiety on the dihydropyrone core
engaged Arg168 in a bidentate hydrogen bonding interaction
(2.6–3.1 Å).
The solvent accessible surface for the LDHA co-structure of 47
revealed some opportunities for additional substitution at the 70-
position of the indane group. The 70-position pointed toward the
so-called ‘100’s loop’ region of the protein where we and others
have observed side-chain and backbone mobility.11–13 The 70-posi-
tion vector would also allow us to target an interaction with Ile241
(Fig. 2). A previous publication from our group13b demonstrated
that a hydrophobic interaction with Ile241 in the LDHA substrate
binding pocket can lead to improvements in potency. Thus, we
synthesized a (70-Br)indane analog, and the favored enantiomer
(49) was equipotent with 47. This result confirmed our hypothesis
that there was an opportunity to add substituents using this vec-
tor. Compound 49 was subjected to Suzuki–Miyaura cross-cou-
pling conditions29 to provide the (70-phenyl)indane analogs. The
favored enantiomer of the (70-phenyl)indane (51) was a very
potent biochemical inhibitor of LDHA (IC50 = 30 nM). Although 51
was fairly lipophilic (cLogP = 5.0), the LLE and LE values of 51
(LLE = 2.5, LE = 0.35) were comparable to 4. Compound 51 also rep-
resented the first example in which we successfully merged the
SAR of the dihydropyrone series with the favored ligand–protein
interactions observed with our previously described 2-aminopyr-
azine series (exemplified by 2).13b
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
10.067.
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assays. The molecule was very soluble in pH 7.4 buffered aqueous
solution (kinetic solubility = 169 lM). The compound had low
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MDCK cell permeability (Papp(A?B) = 1.6 10ꢀ6 cm/s) and it was also
highly bound to human and rodent plasma proteins (<1% free in
both species). When 51 was incubated with human and rat liver
microsomes, we observed high and moderate hepatic clearance
(Clhep) values, respectively (Clhep = 20 and 41 mL/min/kg, respec-
tively). The high plasma–protein binding, low Papp, and high-to-
moderate in vitro Clhep values were presumably due to the lipo-
philicity of 51.30

B. P. Fauber et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5683–5687
5687
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