Cell Active Hydroxylactam Inhibitors of Human Lactate Dehydrogenase with Oral Bioavailability in Mice

Article abstract of DOI:10.1021/acsmedchemlett.6b00190

A series of trisubstituted hydroxylactams was identified as potent enzymatic and cellular inhibitors of human lactate dehydrogenase A. Utilizing structure-based design and physical property optimization, multiple inhibitors were discovered with <10 μM lactate IC₅₀ in a MiaPaca2 cell line. Optimization of the series led to 29, a potent cell active molecule (MiaPaca2 IC₅₀ = 0.67 μM) that also possessed good exposure when dosed orally to mice.

Full text of DOI:10.1021/acsmedchemlett.6b00190

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Letter
Cell active hydroxylactam inhibitors of human lactate
dehydrogen-ase with oral bioavailability in mice
Hans E. Purkey, Kirk Robarge, Jinhua Chen, Zhongguo Chen, Laura B. Corson, Charles Z. Ding,
Antonio G DiPasquale, Peter S. Dragovich, Charles Eigenbrot, Marie Evangelista, Benjamin P. Fauber,
Zhenting Gao, Hongxiu Ge, Anna Hitz, Qunh Ho, Sharada S. Labadie, Kwong Wah Lai, Wenfeng Liu,
Yajing Liu, Chiho Li, Shuguang Ma, Shiva Malek, Thomas O'Brien, Jodie Pang, David Peterson, Laurent
Salphati, Steve Sideris, Mark Ultsch, Binqing Wei, Ivana Yen, Qin Yue, Huihui Zhang, and Aihe Zhou
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00190 • Publication Date (Web): 26 Aug 2016
Downloaded from http://pubs.acs.org on September 3, 2016
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Cell active hydroxylactam inhibitors of human lactate dehydrogen-
ase with oral bioavailability in mice
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Hans E. Purkeya*, Kirk Robargea, Jinhua Chenb, Zhongguo Chenb, Laura B. Corsona, Charles Z.
Dingb, Antonio G. DiPasqualec, Peter S. Dragovicha, Charles Eigenbrota, Marie Evangelistaa, Ben-
jamin P. Faubera, Zhenting Gaob, Hongxiu Geb, Anna Hitza, Qunh Hob, Sharada Labadiea, Kwong
Wah Laib, Wenfeng Liub, Yajing Liub, Chiho Lib, Shuguang Maa, Shiva Maleka, Thomas O’Briena,
Jodie Panga, David Petersona, Laurent Salphatia, Steve Siderisa, Mark Ultscha, BinQing Weia,
Ivana Yena, Qin Yuea, Huihui Zhangb, Aihe Zhoua
aGenentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA
bWuXi AppTec Co., Ltd. 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, P. R. China
cUniversity of California, Berkeley, College of Chemistry, Berkeley, California, 94720, USA
KEYWORDS: Lactate dehydrogenase, tumor metabolism, glycolysis, X-ray crystal structure, structure-based design.
ABSTRACT: A series of trisubstituted hydroxylactams was identified as potent enzymatic and cellular inhibitors of hu-
man lactate dehydrogenase A (LDHA). Utilizing structure based design and physical property optimization, multiple in-
hibitors were discovered with <10 ꢀM lactate IC50 in a MiaPaca2 cell line. Optimization of the series led to 29, a potent
cell active molecule (MiaPaca2 IC50 = 0.67 ꢀM) that also possessed good exposure when dosed orally to mice.
Tumor cells often show signs of metabolic alteration
when compared to normal cells.1 Instead of the mito-
chondrial tricarboxylic acid (TCA) cycle utilized by
healthy cells, they often rely on glycolysis for energy gen-
eration,2 even in the presence of normal oxygen levels.3
Lactate dehydrogenase A (LDHA) is critical to this pro-
cess by catalyzing the conversion of pyruvate to lactate in
the final step of glycolysis.4 Overexpression of LDHA is
found in many types of cancer cells,5,8 and shRNA medi-
ated LDHA depletion results in the inhibition of tumor
growth in glycolytically dependent cancer cell lines, xen-
ografts, and genetically engineered murine models.7,8 To-
gether, these facts have focused attention on the thera-
peutic potential of LDHA inhibition for the treatment of
cancer. A recent report from our laboratories identified
the lactate dehydrogenase B isoform (LDHB) as an essen-
tial gene for triple negative breast cancer tumors.9 Given
this linkage, we sought a pan-LDH inhibitor, if possible,
in order to maximize potential efficacy across the broad-
est set of tumors.
reported LDHA inhibitors containing a variety of scaf-
folds.17-21 To date, they have suffered from a lack of cellu-
lar activity, despite relatively potent biochemical inhibi-
tion. For example, 3,6-disubstituted dihydropyrones and
diketones, although possessing low double digit nanomo-
lar IC50s in vitro, failed to show activity against glycolytic
cell lines.18,21 This has been attributed to poor permeabil-
ity and high plasma protein binding, presumably due to
the acidic core of the compounds imparting poor physical
properties. Herein we report the optimization of a series
of hydroxylactam inhibitors and the resulting impact on
LDHA biochemical and cellular potency.
In order to further optimize this general class of dicar-
bonyl LDHA inhibitors, we sought to modify our medici-
nal chemistry approach in two ways. The first was to re-
place the diketone and dihydropyrone cores with a hy-
droxylactam. Previous matched pair comparison of these
three scaffolds demonstrated that the hydroxylactam,
exemplified by 1, was the least acidic of the set (pKa = 4.1,
versus 2.4 and 3.0, respectively, for the lactam and ke-
tone), while maintaining biochemical potency.18 We hy-
pothesized that this pKa modulation could lead to im-
proved physical properties of the compounds, leading to
improved permeability and reduced plasma protein bind-
ing, yielding cell active compounds if biochemical poten-
cy could be improved as well.
A number of LDHA inhibitors have recently been dis-
closed by several academic and industrial groups,10-16
however they have been hampered primarily by a lack of
cellular activity. An LDHA inhibitor with excellent cellu-
lar potency was disclosed, but poor pharmacokinetics
prevented its evaluation in vivo.15 Our group has recently
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Ph
Ph
H
4
18
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2
Ph
0.49
2.2
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4
5
Ph
4-Thiazolyl
3-Thiophenyl
3-Thiophenyl
3-Thiophenyl
0.40
0.12
>10
2.3
0.74
>10
Ph
2-Pyridyl
(2-F)Ph
(3-Br)Ph
(3-NH2)Ph
(3-OH)Ph
(4-CN)Ph
6
7
0.36
1.8
3-Thiophenyl 0.082
3-Thiophenyl 0.058
3-Thiophenyl 0.042
0.8
8
9
10
0.20
0.27
1.1
3-Thiophenyl
0.70
0.10
11 (4-Cyclopropyl)Ph 3-Thiophenyl
0.48
0.21
0.055
0.057
12
13
14
(4-OMe)Ph
(4-NMe2)Ph
(4-OH)Ph
3-Thiophenyl 0.065
3-Thiophenyl 0.015
3-Thiophenyl 0.014
16
17
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Figure 1. Overlay of previously disclosed X-ray structures of
LDHA/diketone-containing inhibitor complexes 4QO7 (cy-
an) and 4QO8 (white)19. Hydrogen bonds from 4QO7 are
shown as yellow dashed lines.
LDHA and LDHB enzymatic assay values are the geomet-
ric mean of at least 2 separate runs as previously described.19
Secondly, overlay of the crystal structures of two previ-
ously disclosed diketone compounds19 (Figure 1) suggest-
ed that simultaneous substitution of the axial and equato-
rial positions of the core might be possible. The resulting
diphenyl substituted compound (2) indicated that this
substitution pattern was tolerated and led to an eight-fold
improvement in biochemical potency. We therefore ex-
plored phenyl isosteres (3-4) and found that a 3-
thiophene (4) further improved potency by four fold.
Encouraged by these results, we held the thiophene con-
stant at R2 while exploring a variety of substituents on the
phenyl ring at R1 (5-14). Compounds were synthesized and
tested as racemic mixtures for synthetic convenience. We
held the 3 position on the hydroxylactam core constant,
as this group had proven ideal in previous optimization of
the series.18,19 2-Pyridyl substitution (5) resulted in the
complete loss of activity and 2-fluorophenyl (6) reduced
potency slightly. 3-Bromophenyl (7) improved LDHA po-
tency to 82 nM, however the selectivity for LDHA over
LDHB increased from 4-5 fold to 10 fold. This was not
desired as we sought a pan-LDH inhibitor. Meta amino
and hydroxy substitution (8 and 9, respectively) yielded
roughly equipotent analogs, each improving inhibition
slightly. Para substitution resulted in more variable SAR.
Electron withdrawing groups such as cyano (10) dimin-
ished affinity to 700 nM, while electron donating substit-
uents (11-14) maintained or improved potency. Except for
7, this set of compounds maintained a ~4-5 fold selectivity
for LDHA over LDHB relative to that of 1.
An X-ray co-crystal structure of 9 bound to LDHA (Fig-
ure 2) confirmed our hypothesis that simultaneous axial
and equatorial substitution could be accommodated by
the protein. The hydroxylactam core maintains the core
interactions previously observed for this series.18,19 The
lactam carbonyl hydrogen bonds the catalytic Arg168 and
the hydroxyl bridges His192 and Asn137. There is no hy-
drogen bond between the lactam NH and the protein,
although it points towards Thr247. The inhibitor stacks
on top of the NADH cofactor, occupying only the py-
ruvate binding site. We have previously demonstrated
that this class of compounds is non-competitive with
NADH binding in the LDHA active site.17 The thiophene
substituent occupies the axial position, forming an intra-
molecular edge-to-face π−π interaction with the 2-
chlorophenyl ring and a second edge-to-face π−π interac-
tion with Tyr238. The 3-hydroxyphenyl ring occupies an
equatorial position relative to the hydroxylactam core,
stacking against Ile241. The phenol OH is hydrogen bond-
ed to a water, which is in turn involved in an extensive
hydrogen bonding network with the backbone NH and
carbonyl of Thr247 and a phosphate oxygen of NADH.
Intriguingly, despite this highly coordinated water inter-
action, 9 is only 3 fold more potent against LDHA than
the parent compound 4.
Arg98 is just beyond the para- position of the phenyl
ring, with space available in between it and the inhibitor.
Given the excellent potency of analogs with para- polar
substituents (13-14), it was hypothesized that polar groups
in this region might be interacting with this arginine and
be responsible for the increased potency. This arginine is
in a structurally variable region of LDHA, differing among
individual subunits of the tetrameric crystallographic
asymmetric unit.
TABLE 1. Structure activity relationships for trisub-
stituted hydroxylactams
In order to probe this area, we explored further substi-
tution at the meta- and para- positions of the phenyl ring
(Table 2). Direct linkage of a morpholine (15) or ether (19-
LDHA
LDHB
Cmpd
R1
R2
IC50 (ꢀM) IC50 (ꢀM)
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17 NH-Cyclohexyl
H
0.004
0.003
0.069
0.037
0.025
0.052
0.077
0.035
0.66
0.19
1
2
3
4
5
6
7
18
19
20
21
NH-(4-F)Ph
O-THP
O-(4-F)Ph
H
H
H
H
Cyclohexyl
1-Piperazinyl
0.18
22
H
0.78
8
9
1-(4-Oxetane)
piperazinyl
23
24
H
H
0.047
0.016
0.39
10
11
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1-(4-OMe) Piperi-
dinyl
0.075
25
26
H
H
1-Piperidinyl
1-Pyrrolidinyl
0.009
0.007
0.049
0.050
1-(4-Acetyl)
piperazinyl
27
H
0.005
0.035
Figure 2. Compound 9 (cyan) co-crystalized with LDHA
(white) [PDB: 5IXS]. The NADH cofactor is shown in green
sticks, the crystallographic a red sphere, and Hydrogen
bonds are yellow dashed lines. Crystallization was carried out
as previously disclosed.21 Additional details can be found in
the supporting information.
28
H
H
H
1-Morpholino
1-Morpholino
1-Morpholino
0.009
0.003
0.055
0.030
0.005
0.14
29-R
30-S
Compounds are racemic mixtures, except where indicated.
20) at the meta position maintained LDHA potency, while
amino linkages to cyclic alkyl or aryl substituents (16-18)
improved LDHA potency to a single digit nanomolar lev-
el. Unfortunately, selectivity against LDHB increased to
~10-20 fold, in opposition to our goal of an inhibitor with
pan-LDH potency. Direct substitution of piperidines and
piperazines in the para position led to LDHA potencies in
the low to mid double digit nanomolar range (21-28). A
hydrogen bond donor in the 4- position of these heteroal-
kyl rings (22) was slightly inferior to either carbon (21, 25-
26) or hydrogen bond acceptors at the same position in
the ring (24, 27-28). This is consistent with the potential
interaction of these groups with Arg98. Of particular note
is morpholine substituted phenyl 28. In addition to its 9
nM potency, it has only a 3-fold potency shift between
LDHA and LDHB, one of the smallest ratios observed in
this series of biochemically potent compounds. We sepa-
rated 28 into its enantiomers and found that the R enan-
tiomer 29 was 18 fold more potent than S enantiomer 30.
We were able to solve crystal structures of each enanti-
omer bound to LDHA. An overlay of the two structures
(Figure 3) reveals they have the same binding mode in
terms of substituent disposition. Interestingly, the hy-
droxylactam core is rotated 180 degrees in these struc-
tures (exchanging the positions of the lactam NH and
unsubstituted sp3 carbon). More potent isomer 29 posi-
tions the lactam pointing towards Thr247. Although the
distance and Thr rotomer do not support a direct hydro-
gen bond, this conformation has the least potential clash-
es with the protein. In contrast, 30 points the lactam NH
toward an NADH hydroxyl and Asn137, but again is not
close enough to make direct hydrogen bonds. The sp3
carbon on the other side of the core is pointed towards
the Thr247 OH, a less favorable interaction, consistent
with the reduced LDHA potency observed for this com-
pound. This absolute stereochemistry assignment of 29 as
R and 30 as S is also supported by the small molecule
crystal structure of the more potent enantiomer from a
related pair of compounds (See Supporting Information).
As expected, the terminal morpholine ring is pointing
towards the guanidinium group of Arg98, however, the
geometry is not appropriate for hydrogen bond for-
mation. Instead, it interacts via a polar interaction be-
tween the morpholine oxygen and the positive charge of
the arginine residue.
TABLE 2. Structure activity relationships for 3- and 4-
position phenyl modifications on trisubstituted hy-
droxylactams
Encouraged by the favorable enzymatic potency of the-
se inhibitors, we tested selected analogs in a MiaPaca2
cell line for the inhibition of lactate production (Table 3,
21, 25, 29). All showed measurable inhibition, with IC50s
ranging from 0.67 -25 ꢀM. Given the sub-micromolar cel-
lular potency of 29, additional close-in analogs around the
morpholine ring functionality were prepared (31-36).
None of these alternatives improved either the enzy-
matic or cellular potency relative to 28 or 29. Adding po-
LDHA
LDHB
Cmpd
15 1-Morpholino
16 NH-THP
R1
R2
IC50 (ꢀM) IC50 (ꢀM)
H
H
0.080
0.009
0.25
0.16
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for a subset of the compounds and found several trends
larity to the morpholine ring via a carbonyl (32) or sulfone
1
2
3
4
5
6
7
8
(34) resulted in a complete loss of cellular potency.
(Table 4). MDCK permeability was moderate/high for the
smaller, less functionalized compounds 2 and 4. Addition
of a saturated or substituted aromatic ring onto the phe-
nyl through an NH linkage (16 and 18) increased the
number of hydrogen bond donors to 3, resulting in lower
permeability and hints of greater efflux. The additional
lipophilicity of 18, as measured by LogD7.4, likely led to an
increase of protein binding to >99.9% when compared to
2. Direct attachment of a heteroalkyl ring (24, 29, 31) re-
duced the hydrogen bond donor count, and when com-
bined with the neutral or decrease of LogD7.4 attributable
to the morpholine (29, 31), restored moderate MDCK
permeability. High plasma protein binding was observed
on all compounds containing either a saturated or aro-
matic fifth ring, with the exception of 29, which had
mouse plasma protein binding of 99.1%. This lower pro-
tein binding may be one of the contributors to the superi-
or cellular potency observed for this compound, as the
cellular assay conditions contain 10% FBS. Compound 29
inhibited proliferation in 37 of 347 cancer cell lines tested
at a potency cut off of 5 ꢀM (Figure S1). Interestingly, 2
chondrosarcoma (bone) cancer cell lines that harbored
IDH1 mutations were found to be especially sensitive to
29 (IC50 = 0.8 ꢀM). IDH1 catalyzes the conversion of mito-
chondrial isocitrate to α-ketoglutarate, so IDH1 mutant
tumors may have decreased mitochondrial fitness and
increased dependency on glycolysis.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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40
41
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45
46
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48
49
50
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52
53
54
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56
57
58
59
60
Figure 3. Overlay of the crystal structures 29 (white) [PDB:
4ZVV] and 30 (cyan) [PDB: 5IXY] bound to LDHA. Hydrogen
bonds are shown as yellow dashed lines.
TABLE 3. Structure activity relationships for 4 phenyl
modifications on trisubstituted hydroxylactams
TABLE 4. Physicochemical property effects on per-
meability and protein binding.
LDHALDHBMiaPaca2
Cmpd
21
R
IC50 IC50 Lactate LogD7.4
MDCK Papp A:B
HBD LogD7.4 (10-6 cm/sec) /
TPSA
MousePPB
(%)
(ꢀM) (ꢀM) IC50 (ꢀM)
Cmpd
(Å2)
Cyclohexyl
0.025 0.18
25
3.5
1.2
Ratio (A:B/B:A)
24 1-(4-OMe)Piperidinyl 0.016 0.075
2.9
2
4
46
46
67
58
49
58
58
2
2
3
3
2
2
2
1.4
0.8
-0.3
2.4
2.1
9.4 / 0.5
7.4 / 1.0
0.5 / 1.8
1.0 /2.8
1.7 / 0.9
3.4 / 0.9
6.0 / 1.4
99.3
ND
29-R
31
1-Morpholino
0.003 0.005 0.67
0.027 0.14 3.9
0.021 0.080 >50
0.6
2,6-
16
ND
1.3
0.4
1.3
Dimethylmorpholino
18
>99.9
>99.9
99.1
32
3-Oxomorpholino
25
29-R
31
2,2-
33
0.015 0.070
0.013 0.046
0.017 0.14
5.1
0.6
1.3
Dimethylmorpholino
>99.9
1,1-
34
35
>50
ND
The MDCK assay23 was used as an indicator of cell perme-
ability
Dioxidothiomorpholino
1-(4-Spiro-
oxetanyl)piperidinyl
9.9
6.1
0.7
0.8
Compound 29 proved to possess the optimal combina-
tion of strong enzymatic and MiaPaca2 cellular potency,
moderate permeability, and reduced plasma protein bind-
ing compared to other potent compounds in this series. It
was stable in liver microsomes with predicted hepatic
clearances (Clp) of 3.3, 8.1 and 14 mL/min/kg, based on
human, rat and mouse microsomes, respectively. We car-
ried out in vivo pharmacokinetic experiments in mice
with 29, and were gratified to observe that this compound
presented a low Clp, consistent with the predicted hepatic
Cl and also had high bioavailability when dosed orally at 5
mg/kg (Table 5, concentration vs. time plots in Figure S2).
36 1(4-Cyano)piperidinyl 0.010 0.060
MiaPaca2 cell assay values are the geometric mean of at
least 2 separate runs as previously described21. LogD7.4 was
measured via a high throughput method.22
By switching the core from a diketone or dihydropy-
rone to a hydroxylactam, we had hoped the reduced acidi-
ty would help improve the physical properties of the
compounds, yielding molecules with improved permea-
bility and plasma protein binding. We measured the
MDCK permeability and mouse plasma protein binding
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At higher oral doses, ranging from 50-200 mg/kg, 29 dis- In summary, structure based design was utilized to cre-
1
2
3
4
5
6
7
8
played greater exposure (Table 5 & Figure S3), confirming
the possibility of 29 serving as a tool compound for in
vivo efficacy studies in mice.
ate a novel series of substituted hydroxylactam LDHA
inhibitors. These compounds combined the occupancy of
multiple sites that had not been filled simultaneously by
previous inhibitors, along with a less acidic hydroxylac-
tam core. This strategy afforded a dramatic improvement
in enzymatic potency as well as inhibitors displaying
LDHA inhibition in MiaPaca2 cells. Compound 29 has
sub-micromolar MiaPaca2 potency and inhibits prolifera-
tion in 11% of cancer cell lines in a broad panel. Moreover,
29 combines potent activity with modest permeability,
lower plasma protein binding and good in vivo exposure
when dosed orally to mice.
To our knowledge, 29 is the first example of an LDHA
inhibitor possessing both sub-micromolar cellular poten-
cy and adequate pharmacokinetic properties that make it
suitable for use as an in vivo tool compound. Reports on
potency in cells, mechanism of action, tumor growth in-
hibition, and efficacy characteristics of this compound are
forthcoming from our laboratories.
TABLE 5. Pharmacokinetic parameters following IV
and PO administrations of 29 to mice
Cmax
F
AUC
Dose
(mg/kg)
T1/2 Vss
(hr) (L/kg)(ml/min/kg)
Clp
Route
(ꢀM)
(ꢀM*hr)
(%)
9
iv
1
1.5
5.1
2.5
1.0 1.4
21.1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
po
po
po
po
5
1.7 70
60.7
50
100
200
125
250
403
224
440
Scheme 1. Synthesis of substituted hydroxylactam
compounds
S
O
S
O
O
O
O
NH2
HN
R1
HCl
O
ASSOCIATED CONTENT
Supporting Information
Ti(OEt)4
S
R1 R2
N
O
R2
NaH
n-BuLi
THF, 0 °C, 2 h
MeOH, 0 °C, 1 h
O
THF, D, 16 h
R1 R2
O
37a-i
O
38a-i
39a-i
Additional figures and tables are available in the Supporting
Information. This material is available free of charge on the
ACS Publications website.
Cl
S
O
O
S
OH
Cl
Cl
O
O
S
K2CO3
MeOH, heat, 2 h
H2N
R1
R2
R2
R1
R2
R1
40a-i
N
H
O
K2CO3
MeCN, heat, 2 h
N
H
O
41a-i
3-7, 11, 12, 19, 42
AUTHOR INFORMATION
OH
Cl
1. NH2COOtBu
Amine or Phenol
BrettPhos-Admix
NaOt-Bu, Dioxane
S
Pd2(dba)3, t-Bu-Xphos
NaOt-Bu, Dioxane
120 oC, 1h
Corresponding Author
S
N
H
O
8
110 oC, 20 min, microwave
* Email: purkey.hans@gene.com
9*, 15-18, 20
14*, 13, 22-36
2. HCl-dioxne
7
Br
ACKNOWLEDGMENT
Zn(CN)2
Pd2(dba)3, X-phos
Cs2CO3
Amine
OH
Cl
We thank Drs. Krista Bowman and Jiansheng Wu and their
respective research groups for performing protein expression
and purification activities. We also thank the Genentech
Small Molecule analytical group for their support. We thank
the Genentech gCell group for cell line banking and mainte-
nance, members of the Genentech Cell Line Screening Initia-
tive and Dr. Richard Bourgon for assistance on cell-based
drug screens. We thank Crystallographic Consulting, LLC for
diffraction data collection. We acknowledge the use of syn-
chrotron X-ray sources at the Advanced Light Source and the
Stanford Synchrotron Radiation Lightsource supported by
the Department of Energy’s Office of Science under contracts
DE-AC02-05CH11231 and DE-AC02- 76SF00515, respectively.
We also acknowledge NIH Shared Instrumentation Grant
S10-RR027172 for the purchase of the diffractometer used for
small molecule crystal data collection.
t-BuONa, Brettphos, Pd2(dba)3
10
S
Dioxane, 120 °C, 16 h
Dioxane, 120 °C, 16 h
S
N
H
O
OH
B
OH Pd(dppf)Cl2, K2CO3
Dioxane, 110 °C, 16 h
1.
42
Br
21
2. Pd-C, AcOH, H2, 60 PSI
*9 and 14 were formed as byproducts in the phenol and amine coupling reactions.
Inhibitors were synthesized as shown in Scheme 1.
Condensation of ketones 37a-i with racemic tert-
butylsulfinamide gave sulfinamides 38a-i. Subsequent
addition of methyl 3-oxobutanoate to 38a-i afforded ke-
toesters 39a-i, which were then subjected to acid depro-
tection, resulting in amine intermediates 40a-i. Base me-
diated cyclization yielded ketolactams 41a-i, which were
then condensed with bis(2-chlorophenyl)disulfide to yield
final products 3-7, 11-12, 19 and 42. In the case were R1
was 3-bromophenyl (7), Buchwald cross-coupling reac-
tions with substituted amines or phenols yielded inhibi-
tors 8-9, 15-18 and 20. Under similar conditions, 42,
where R1 was 4-bromophenyl, yielded inhibitors 10, 13, 14,
22-28 and 31-36. Suzuki coupling of 42 with cyclohex-1-en-
1-ylboronic acid and subsequent hydrogenation afforded
21. For compounds 29 and 30, racemic compound 28 was
resolved by chiral SFC to afford the single stereoisomers.
Absolute stereochemistry was assigned based on the small
molecule X-ray crystal structure of 29 (Figure S4).
ABBREVIATIONS
LDHA, lactate dehydrogenase A; LDHB, lactate dehydrogen-
ase B; PPB, plasma protein binding; SFC, supercritical fluid
chromatography; MDCK, Madin-Darby canine kidney; AUC,
area under the curve, Vss, steady-state volume of distribu-
tion; T1/2, half life; Clp, plasma clearance; Cmax, maximum
concentration; F, oral bioavailability.
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OH
Cl
OH
Cl
1
2
S
S
3
4
5
6
O
N
N
H
O
N
H
O
S
7
8
9
1
29
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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27
28
29
30
31
32
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34
35
36
37
38
39
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60
LDHA IC50: 4 ꢀM
Not cell active
LDHA IC50:
MiaPaca2 IC50: 670 nM
Orally Bioavailable in Mice
3 nM
7
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