Optimization of 5-(2,6-dichlorophenyl)-3-hydroxy-2-mercaptocyclohex-2-enones as potent inhibitors of human lactate dehydrogenase

Article abstract of DOI:10.1016/j.bmcl.2014.11.008

Optimization of 5-(2,6-dichlorophenyl)-3-hydroxy-2-mercaptocyclohex-2-enone using structure-based design strategies resulted in inhibitors with considerable improvement in biochemical potency against human lactate dehydrogenase A (LDHA). These potent inhi

Full text of DOI:10.1016/j.bmcl.2014.11.008

Bioorganic & Medicinal Chemistry Letters 25 (2015) 75–82
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Optimization of 5-(2,6-dichlorophenyl)-3-hydroxy-2-
mercaptocyclohex-2-enones as potent inhibitors of human lactate
dehydrogenase
a
b
a
a
Sharada Labadie a, , Peter S. Dragovich , Jinhua Chen , Benjamin P. Fauber , Jason Boggs
⇑
Laura B. Corson a, Charles Z. Ding b, Charles Eigenbrot a, HongXiu Ge b, Qunh Ho b, Kwong Wah Lai b,
Shuguang Ma a, Shiva Malek a, David Peterson a, Hans E. Purkey a, Kirk Robarge a, Laurent Salphati a,
Steven 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, 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:
Optimization of 5-(2,6-dichlorophenyl)-3-hydroxy-2-mercaptocyclohex-2-enone using structure-based
design strategies resulted in inhibitors with considerable improvement in biochemical potency against
human lactate dehydrogenase A (LDHA). These potent inhibitors were typically selective for LDHA over
LDHB isoform (4–10 fold) and other structurally related malate dehydrogenases, MDH1 and MDH2 (>500
fold). An X-ray crystal structure of enzymatically most potent molecule bound to LDHA revealed two
additional interactions associated with enhanced biochemical potency.
Received 19 September 2014
Revised 31 October 2014
Accepted 3 November 2014
Available online 10 November 2014
Keywords:
Lactate dehydrogenase A
Glycolysis
Ó 2014 Elsevier Ltd. All rights reserved.
Tumor metabolism
Structure-based design
Tumor cells exhibit altered glucose metabolism compared to
normal cells.1 They often rely on glycolysis for energy generation
in contrast to more efficient mitochondrial tricarboxylic acid
(TCA) cycle utilized by healthy cells.2 This dependency on glycoly-
sis occurs even in the presence of normal oxygen concentrations.3
Lactate dehydrogenase A (LDHA) plays a critical role by catalyzing
the conversion of pyruvate to lactate in the last step of the pro-
cess.4 Over-expression of LDHA is detected in many types of cancer
cells5 and shRNA-mediated LDHA knock-down results in signifi-
cant inhibition of tumor growth in glycolytically dependent cancer
cell lines.6 Moreover, humans deficient in LDHA show only mild
phenotype suggesting that inhibition of the enzyme will not lead
to unwanted toxicities.4,7 Collectively, these facts suggest that
the selective inhibition of LDHA would provide new therapeutic
potential in cancer treatment.
have resulted in poor cellular potency.15–17 In the case of 3-
hydroxycyclohex-2-enone series 6, containing two lipophilic phe-
nyl rings and an acidic 1,3-dicarbonyl moiety, the lack of cell
potency15 was ascribed to a high cell-shift associated with the lipo-
philic acidic template.18 To overcome the cell shift associated with
these templates without altering the acidity of the molecule, fur-
ther improvement in biochemical potency was required relative
to previously disclosed15 inhibitors 6a and 6b. Herein, we describe
the optimization and evaluation of 3-hydroxycyclohex-2-enone
series 6 in biochemical and related cellular assays.
Distinct binding pockets observed in the crystal structure of 5-
(2,6-dichlorophenyl)-3-hydroxycyclohexen-2-one
analog,
6b,
bound to LDHA15 provided possible functionalization strategies
to further enhance the biochemical potency (Fig. 2). First, the
pocket near the nicotinamide adenine dinucleotide (NADH) bind-
ing site contained unoccupied space encompassed by the flexible
100’s loop. Favorable interactions with the polar side-chains of
the residues R98, Q99, Q100 and E103 of the 100’s loop might
therefore be realized by functionalization (Fig. 1) of the phenyl ring
A at C-3 and C-4. The substituents at C-3 might also point inward
toward the NADH and make interactions with the functionalities as
observed earlier.14 Secondly, the C-50 position of the phenyl ring B
A number of LDHA inhibitors have recently been disclosed
(Fig. 1 and 1–3).8–12 Optimization of high throughput screening
hits (Fig. 1 and 4–6) by our group have also been reported.13–15
To date, low enzyme potency and poor physiochemical properties
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2014.11.008
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

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S. Labadie et al. / Bioorg. Med. Chem. Lett. 25 (2015) 75–82
HO
CF3
NH
N
O
H
N
H
N
O
O
OH
OH
O
S
N
NH2
S
N
OH
O
O
OH
N
O
H3CO
N
OCH3
1
2
3
Y 2'
S
O
Cl
B
CN
5'
HN
H
N
HO
O
S
N
HN
N
N
OH
H2N
O
S
Cl
O
O
O
H2N
X
X
A
3
4
6a
6b
X = Br, Y = CN
X =Y =Cl
5
4
Figure 1. Previously disclosed LDHA inhibitors.
suggested that functionalization of C-3 and C-4 of the phenyl ring
A and C-50 of the phenyl ring B present in molecule, such as 6b,
were reasonable strategies to enhance biochemical potency.
Accordingly, several 6b analogs containing various H-bond
donor/acceptor functionalities were evaluated for their LDHA
enzyme potencies (Table 1). In comparison to 6b, the C-3 N-linked
morpholine analog (7) showed a modest four-fold gain in enzyme
potency. However, a reduction in potency was observed for the
compound with N-linked morpholine at C-4 (8). We then directed
our focus towards further exploration of C-3 modifications of the
phenyl ring A. N-linked piperazine analogs (9–12) displayed bio-
chemical potencies comparable to 7. Compounds 13 and 14, substi-
tuted with 4-aminotetrahydropyran and isopropyloxy moieties,
respectively, showed 15- and 10-fold improvement relative to
6b. We then explored a few C-linked analogs. Compounds contain-
ing phenyl (15), 2-chlorophenyl (16) and heteroaryls (17–19) in
this position of the molecule displayed similar potencies except
for the pyrimidine analog 19.
D194
Y238
E103
H192
Q100
Q99
I241
100s loop
R98
In order to understand the gain in potency from this explora-
tion, a co-crystal structure of a representative molecule, 13, bound
to LDHA (Fig. 3a) was obtained.19 As expected, the inhibitor was
bound in the substrate binding site and the neighboring side-
chains of residues R168, H192 and N137 were within the H-bond
distance from the 1,3-dicarbonyl portion of the ligand. The 2,6-
dichlorophenyl ring A was positioned in the equatorial orientation
as observed in the crystal structure of 6b.15 Nicotinamide adenine
dinucleotide (NADH) co-factor was also present in the complex.
The 4-aminotetrahydropyranyl group on the ligand appeared sol-
vent exposed and was not pointed inward toward NADH.14 Some
residues of the 100’s loop were disordered due to crystal packing
contacts. Overlay of the X-ray crystal structures of 6b and 13
(Fig. 3b) revealed that a portion of the 100’s loop was considerably
closer to the phenyl ring A in 13 compared to the phenyl ring A in
the crystal structure of 6b. However, the polar moieties on the
side-chains of the 100’s loop residues were not in close proximity
to make favorable interactions with the 4-aminotetrahydropyranyl
NADH
Figure 2. Depiction of the optimization strategy from the crystal structure of 6b
(cyan) bound to LDHA (grey). Accessible space in the binding pocket is shown with
blue ellipses. Red arrows indicate possible positions on ring
functionalization.
A and B for
was in close proximity to the acidic side-chain and backbone NH of
the D194 residue of LDHA. Previous SAR exploration from our
group showed that the substitution at this position was tolerated15
and additional favorable interactions with the protein could be
obtained.13 This indicated that incorporation of H-bond acceptor/
donor moieties at C-50 of the ring B might afford more favorable
interactions with LDHA. These structural features collectively

S. Labadie et al. / Bioorg. Med. Chem. Lett. 25 (2015) 75–82
77
Table 1
Structure–activity relationship of compounds 7–19
OH
Cl
S
Cl
O
R2
Cl
R1
Compd No.
R1
H
R2
H
LDHA IC50 (nM)
870
Compd No.
R1
R2
H
LDHA IC50 (nM)
60
6b
13
NH
O
N
O
7
8
H
240
14
15
H
H
90
O
N
H
2600
100
O
Cl
N
N
N
9
H
230
16
H
60
N
N
N
O
O
10
11
H
H
370
100
17
18
H
H
108
85
N
N
N
HN
N
12
H
170
19
H
1100
N
See Ref. 15 for experimental details associated with each assessment. LDHA biochemical inhibition results are reported as the average of 2 separate runs for compounds with
LDHA IC50 <100 nM. For others, the result is from a single run.
a
b
D194
D194
Y238
Y238
Q99
Q99
100s loop
R98 R98
Q99
H192
NADH
R98
NADH
Figure 3. (a) Co-crystal structure (3.2 Å, PDB code = 4R69) of the inhibitor 13 (cyan) in complex with LDHA (grey). Hydrogen bonds are depicted as dashed red lines. (b)
Overlay of co-crystal structures of inhibitor 13 (cyan) in complex with LDHA (grey) and inhibitor 6b (green) in complex with LDHA (green).
group of the ligand 13. The disorderliness and mobility of the 100’s
loop made it difficult to ascertain interactions that may be respon-
sible for potency enhancement for this molecule. We then directed
our efforts towards functionalization of C-50 of the aryl ring B.
To obtain interactions with the acid side-chain and backbone
NH of D194, as discussed earlier, a new set of compounds with
hydrogen-bond donor/acceptor moieties at C-50 of the phenyl ring
B were synthesized and evaluated in biochemical assay (Table 2
20–32). Compounds with the hydroxyl, methoxy, amino and met-
hyamino groups at C-5 (20–23) exhibited a modest potency gain
relative to 6b. Compound containing carboxylic acid20 (24) and
amides (25–27) were weaker than the corresponding carboxylic
acid esters20 (28–32). Among the esters, the (R)-2-phenethyl ester
31 was the most potent analog with a 145-fold improvement in
biochemical potency over 6b. This dramatic improvement in
potency for 31 was possibly due to the H-bond interaction between
the ester moiety and the backbone NH of D194 and additional
favorable interaction between the phenyl ring and the side-chain
of the residue encompassing the binding pocket (see below).
Encouraged by these results, we envisioned that incorporation of
functionalities on both rings A and B might produce synergistic
effects and provide an inhibitor with greatly improved biochemical
potency. Therefore, compounds with substituents at both the C-3 of
ring A and the C-50 of ring B were investigated (Table 3 33–39). The

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S. Labadie et al. / Bioorg. Med. Chem. Lett. 25 (2015) 75–82
Table 2
Structure–activity relationship of compounds 20–32
OH
Cl
R
S
Cl
O
Cl
Compd No.
R
LDHA IC50 (nM)
Compd No.
R
LDHA IC50 (nM)
6b
20
21
22
H
870
120
370
640
26
27
28
29
CH2CONMe2
CH2CONHCH2CH2OH
CH2COOMe
10,000
300
40
OH
OMe
NH2
CH2COOEt
130
O
23
NHMe
170
30
120
6
O
O
O
Me
Me
24
CH2COOH
3500
31
O
O
25
CH2CONHMe
1800
32
90
See Ref. 15 for experimental details associated with each assessment. Biochemical results as the arithmetic mean of at least 2 separate runs for compounds with LDHA IC50
<100 nM. Data for others is the result of a single run.
Table 3
Structure–activity relationship of compounds 33–39
OH
R3
R2
S
Cl
O
Cl
R1
Compd No.
R1
R2
R3
Cl
LDHA IC50 (nM)a
30
LDHB IC50 (nM)b
180
33
OH
O
O
O
O
34
35
36
OMe
OH
Cl
25
15
410
140
CN
CN
OMe
OMe
460
2400
N
37
38
Cl
Cl
35
30
230
120
Ph
CH2COOEt
OMe
39
Cl
30
490
NH
O
a
LDHA biochemical inhibition results are reported as the arithmetic mean of 2 separate runs.
LDHB inhibition data are from a single run.
b
Table 4
In vitro properties for representative examples
Compd No.
HLM_Clhep (ml/min/kg)
Human PPB (%)
MDCK Papp.A:B (ꢀ10ꢁ6 cm/s)
10.6
1.1
14
Kinetic solubilitya
(lM)
cLogP
LLEb
31
35
39
18
19
20
NDc
>99.9
>99.9
61
50
49
7.5
5.0
6.1
0.7
2.8
1.3
a
b
c
Kinetic solubility was measured at pH 7.4 in PBS buffer.
See Ref. 28.
PPB could not be determined as mass peak (M+H) was not detected.

S. Labadie et al. / Bioorg. Med. Chem. Lett. 25 (2015) 75–82
79
improvement with this was comparable to the other groups tested
(Table 1). This moiety was combined with hydroxyl and methoxy on
C-50 of ring B. These combinations improved enzyme potency only
3-fold over 14 (Table 1). A slight improvement in enzyme potency
was observed when the C-20 Cl was replaced by CN providing inhib-
itor 35. Other combinations, 4-pyridyl at C-3 and methoxy at C-50
(37), phenyl at C-3 and ethyl acetic acid ester at C-50 (38) and 4-
aminotetrahydropyranyl group at C-3 and methoxy at C-50 (39)
showed only modest potency improvement relative to the mono-
substituted analogs. Generally, compounds with substituents both
at C-3 of the phenyl ring A and the C-50 of the phenyl ring B were
more potent than the compounds with substituent on only one of
the rings. However, the dramatic synergistic effect was not realized.
A subset of the potent analogs was profiled in a panel of in vitro
DMPK assays and against structurally related dehydrogenases. As
shown in Table 4, the tested compounds displayed good solubility
in pH 7.4 buffered aqueous solutions and low to moderate MDCK
permeability. The inhibitors exhibited very high protein binding
in plasma from humans. All tested compounds displayed low sta-
bility in human liver microsomes because of high cLogPs.21 The
most potent analog 31 was stable in human plasma and unstable
in mouse plasma (72% of parent remained after 1 h in mouse
plasma at 37 °C). The inhibitors were, typically, 4–10 fold selective
for LDHA over LDHB isoform except for the analog 31 which dis-
played similar potency for both LDHA and LDHB, The inhibitors
D194
Y238
R105
R168
100s loop
NADH
Figure 4. Co-crystal structure (2.11 Å, PDB code = 4R68) of inhibitor 31 (cyan) in
complex with LDHA (grey). Hydrogen bonds are depicted as dashed red lines.
isopropyloxy substituents at C-3 of ring A was selected to test this
hypothesis because of the ease of synthesis and the potency
Cl
S
Cl
S
(a)
O
Cl
Cl
O
O
HO
Cl
O
HO
Cl
O
(iv), (v)
(i), (ii)
(iii)
ref. 15
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
R1
Br
40
41
42
7,9-13
Cl
S
(b)
O
O
O
Cl
Cl
Cl
HO
Cl
O
(vi)
(i), (ii)
(iii)
ref.15
Cl
Cl
O
Cl
Cl
Cl
Cl
O
O
O
OH
43
44
45
14
(c)
Cl
S
Cl
S
HO
O
HO
O
(vii)
Cl
Cl
Cl
Cl
R1
Br
42
15-19
Scheme 1. Synthesis of compounds 7–19. Reagents and reaction conditions: See Table 1 for R1 substituents. (a) (i) LDA 2 M in THF/heptane/diethylbenzene, ꢁ78 °C, 1 h; (ii)
DMF, ꢁ78 °C, 2 h; (iii) (2-ClPhS)2, K2CO3, AcCN, 80 °C, 2 h; (iv) amine, Cs2CO3, Pd2(dba)3, Xanthphos, toluene, 110 °C, 16 h; (v) NaOH, water, 60 °C. (b) (i–iii) are the same as in
(a); (vi) 2-bromopropane, NaOH, MeCN, 80 °C, 12 h. (c) (vii) R1B(OH)2, Pd(PPh3)4, K2CO3, 1,4-dioxane, 6 h.

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S. Labadie et al. / Bioorg. Med. Chem. Lett. 25 (2015) 75–82
Cl
O
O
S
R
Cl
R
HO
Cl
O
SH
(i)
+
Cl
Cl
Cl
47a
R = OH
46
20-24
47b R = OMe
47c R = NH2
47d R = NHMe
47f R = CH2COOH
Cl
R = CH2COX
25 X = NHMe
26 X = NMe2
27 X = NHCH2CH2OH
28 X = OMe
29 X = OEt
Cl
O
R
S
OH
S
HO
Cl
O
HO
Cl
O
(ii) or (iii)
Cl
Cl
30 X =
O
25-32
O
31
32
X =
X =
24
O
Scheme 2. Synthesis of compounds 20–32. Reagents and reaction conditions: (i) K2CO3, acetonitrile, 80 °C, 14 h; (ii) amine, HATU, DIPEA, DMF, 16 h; (iii) alcohol, PPh3, DEAD,
PhI(AcO)2, THF, rt, 16 h.
R3
(a)
R2
S
O
O
HO
Cl
O
R3
R2
(i)
SH
+
Cl
Cl
O
Cl
O
2
3
47a
47b
R = OH, R = Cl
45
33 - 36
2
3
R = OMe, R = Cl
47g R2 = OH, R3 = CN
(b)
Cl
2
3
47h
R = OMe, R = CN
S
R2
O
O
O
O
HO
Cl
O
47b 47e
or
(ii)
(i)
Cl
Cl
Cl
Cl
Br
Cl
R1
R1
48
41
37, 38
(i)
47b
O
Cl
O
OH
Cl
S
S
Cl
Cl
Br
(iii)
O
O
O
Cl
Cl
NH
49
39
O
Scheme 3. Synthesis of compounds 33–39. Reagents and reaction conditions: See Table 3 for R1 and R2 and R3 substituents: (i) K2CO3, acetonitrile, 80 °C, 14 h (ii) R1B(OH)2,
Pd(dppf)Cl2, Cs2CO3, DMF, 1 h, 110 °C microwave; (iii) 4-aminotetrahydropyran, NaOt-Bu, Pd2(dba)3, Brettphos, toluene, 110 °C, 12 h.

S. Labadie et al. / Bioorg. Med. Chem. Lett. 25 (2015) 75–82
81
were inactive (IC50 = >10
l
M) against related malate dehydroge-
Therefore, in some cases, interactions that may be responsible for
affinity enhancement could not be observed in the crystal struc-
ture. Additional efforts to identify inhibitors, in the related scaffold,
with improved ability to inhibit LDHA in cell-culture experiments
were undertaken and will be reported in due course.
nase 1 (MDH1) and malate dehydrogenase 2 (MDH2) enzymes.
A few potent analogs were tested in MIA PaCa2 cells to assess
their ability to inhibit lactate production.22 All tested compounds
were inactive under normal serum conditions (10% fetal bovine
serum) at inhibitor concentrations of up to 10 lM. The reasons
Acknowledgments
for lack of cell potency for these inhibitors are unknown but it
could be potentially due to high protein binding and the high
cell-shift associated with the lipophilic acidic templates18 and high
intracellular LDHA concentration.17
We thank Drs. Krista Bowman and Jiansheng Wu and their
respective Genentech research groups for performing protein
expression and purification activities. We also thank the Discovery
Chemistry Small Molecule analytical group for their support and
Dr. Peter Jackson for many helpful discussions regarding LDHA.
We thank Crystallographic Consulting, LLC for diffraction data col-
lection. We acknowledge the use of synchrotron X-ray sources at
the Advanced Light Source and the Stanford Synchrotron Radiation
Lightsource supported by the Department of Energy’s Office of Sci-
ence under contracts DE-AC02-05CH11231 and DE-AC02-
76SF00515, respectively.
A co-crystal structure of the most potent analog 31 bound to
LDHA was obtained to understand the considerable improvement
in enzyme potency over 6b (Fig. 4). The inhibitor was bound in
the substrate binding pocket similar to that observed for com-
pounds 6b and 13. The usually mobile and disordered R105 was
well ordered in this complex and the guanidinium moiety of the
side-chain was within the H-bond distance (2.6 Å) from the acid
side-chain of D194.19,23 This interaction positioned the guanidi-
nium moiety in close proximity to the phenyl ring of the ester at
C-50 of 31 enabling favorable partial
p
–cation interaction.24 In
Supplementary data
addition, the carbonyl of the ester moiety was within the H-bond
distance (2.8 Å) from the backbone NH of D194. We believe, these
two additional favorable interactions with the protein were
responsible for the observed gain in potency for the inhibitor 31.
Syntheses25 of the described inhibitors with substitutions on
ring A are shown in Scheme 1a–c. Deprotonation of 1-bromo-2,4-
dichlorobenzene with LDA followed by DMF quench provided the
intermediate aldehyde 40. The aldehyde was converted to 41 as
previously described.15 Treatment of 41 with the 1,2-bis(2-chloro-
phenyl)disulfane in the presence of potassium carbonate formed
42. Coupling of 42 with various secondary amines in the presence
of Pd2(dba)3, Xantphos and cesium carbonate in toluene afforded
the amine substituted analogs 7, 9–11 and 13. Deacetylation of
11 under basic conditions yielded 12. Compound 8 was prepared,
in a similar fashion to 7, starting from 3,5-dichlorobromobenzene.
Alkylation of 2,4-dichlorophenol with 2-iodopropane produced 43.
Deprotonation of 43 with n-BuLi in hexanes at ꢁ78 °C and subse-
quent DMF quench produced the aldehyde 44 which was con-
verted to 14 via 45 as previously described.15 Compounds 15–19
were obtained by Suzuki couplings between 42 and boronic acids
in the presence of a palladium catalyst.
The compounds with substitutions on the ring B were synthe-
sized as described in Scheme 2. 5-(2,6-Dichlorophenyl)cyclohex-
ane-1,3-dione 4615 was treated with various thiols, 47a–d and
47f26,27 in the presence of potassium carbonate in acetonitrile to
generate compounds 20–24. The carboxylic acid 24 was converted
to esters and amides (25–32) using either Mitsunobu or HATU cou-
pling conditions.
Scheme 3 describes the synthesis of compounds with substitu-
tions on both rings A and B. Treatment of the intermediate 45 with
the thiols 47a,b,g and h provided 33–36. Suzuki coupling on the
intermediate 41 and subsequent treatment of the product 48 with
thiols 47a and 47e provided compounds 37 and 38, respectively.
Intermediate 41 was converted to 39 via the intermediate 49.
In summary, structure based design strategies were applied to
obtain analogs with considerable improvement in biochemical
potency relative to 6b. These inhibitors were substrate competitive
and were typically selective for LDHA over LDHB and other struc-
turally related malate dehydrogenases, MDH1 and MDH2. A co-
crystal structure of the most potent inhibitor 31 revealed that
the carbonyl and the phenyl group of the ester moiety in 31 were
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.1
1.008.
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in close proximity to allow H-bond and partial
p–cation interac-
tions, respectively, with the protein. Because of the flexibility of
the 100’s loop, it was difficult to efficiently fill the binding pocket.
In addition, the 100’s loop was very disordered near the inhibitor
binding site, perhaps arising from the crystal packing interactions.
15. Dragovich, P. S.; Fauber, B. P.; Boggs, J.; Corson, L. B.; Ding, C. Z.; Eigenbrot, C.;
Ge, H.; Giannetti, A. M.; Ho, Q.; Hunsaker, T.; Labadie, S.; Liu, Y.; Ma, S.; Malek,

82
S. Labadie et al. / Bioorg. Med. Chem. Lett. 25 (2015) 75–82
S.; Pan, B.; Peterson, D.; Pitts, K.; Purkey, H. E.; Robarge, K.; Salphati, L.; Sideris,
reservoir (20% (w/v) PEG 3350, 0.2 M sodium malonate pH 7.0, 2 mM NADH).
Inhibitors were incorporated by placing crystals in a solution lacking malonate
ion (30% (w/v)PEG 3350, 0.1 M HEPES pH 7.2, 1 mM NADH) made 5 mM in
inhibitor using a 50 mM DMSO stock solution and waiting 5–7 days. See
Supplementary material and deposited pdb file for additional structural
details.
S.; Ultsch, M.; VanderPorten, E.; Wei, B.; Xu, Q.; Yen, I.; Yue, Q.; Zhang, H.;
Zhang, X.; Zhou, A. Bioorg. Med. Chem. Lett. 2014, 24, 3764.
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Bousamra, M., II; Lane, A. N.; Higashi, R. M.; Teresa, W. M.; Fan, T. W. M.;
Pandolfi, P. P.; Sukhatme, V. P.; Seth, P. Cell Metab. 2014, 19, 1.
20. The benzoic acid (R = COOH) and the corresponding methyl ester (R = COOMe)
17. Billiard, J.; Dennison, J. B.; Briand, J.; Annan, R. S.; Chai, D.; Colón, M.; Dodson, C.
S.; Gilbert, S. A.; Greshock, J.; Jing, J.; Lu, H.; McSurdy-Freed, J. E.; Orband-
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analogs were inactive against LDHA (IC50 = >4 lM, data not shown). The
required thiol was prepared according to Godt, H. C.; Wann, R. E. J. Org. Chem.
1961, 26, 4047.
21. The partition coefficient (cLogP) was calculated with internal software using
the VolSurf approach. For additional details, see: Cruciani, G.; Crivori, P.;
Carrupt, P.-A.; Testa, B. J. Mol. Struct.-THEOCHEM 2000, 503, 17.
22. See Supplementary material for experimental details.
23. Two of the four protomers in the co-crystal complex with LDHA of compound
31 showed well-ordered R105.
24. Paulini, R.; Müller, K.; Diederich, F. Angew. Chem., Int. Ed. 2005, 44, 1788.
25. Compounds were purified by rHPLC and analyzed by LC/MS. All compounds
were of >95% purity.
26. See Supplementary material for the scheme describing preparation of various
thiols.
27. (a) Itoh, T.; Mase, T. Org. Lett. 2004, 6, 4587; (b) Godt, H. C.; Wann, R. E. J. Org.
Chem. 1966, 26, 4047.
18. For related dicarbonyl-containing compounds as inhibitors of HIV protease and
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19. Most procedures to produce and crystallize LDHA/inhibitor complex crystals
were as described in Ref. 15. LDHA crystals in space group P2(1) were grown by
vapor diffusion at 18 °C using a 50:50 mixture of protein stock (10 mg/mL) and
28. (a) Leeson, P. D.; Springthorpe, B. Nat. Rev. Drug Disc. 2007, 6, 881; (b) Leach, A.
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