Identification of 2-amino-5-aryl-pyrazines as inhibitors of human lactate dehydrogenase

Article abstract of DOI:10.1016/j.bmcl.2013.08.060

A 2-amino-5-aryl-pyrazine was identified as an inhibitor of human lactate dehydrogenase A (LDHA) via a biochemical screening campaign. Biochemical and biophysical experiments demonstrated that the compound specifically interacted with human LDHA. Structural variation of the screening hit resulted in improvements in LDHA biochemical inhibition and pharmacokinetic properties. A crystal structure of an improved compound bound to human LDHA was also obtained and it explained many of the observed structure-activity relationships.

Full text of DOI:10.1016/j.bmcl.2013.08.060

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5533–5539
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of 2-amino-5-aryl-pyrazines 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, Anthony M. Giannetti a, Thomas Hunsaker a, Sharada Labadie a, Yichin Liu a,
Yingchun Liu b, Shiva Malek a, David Peterson a, Keith Pitts a, Steve Sideris a, Mark Ultsch a,
Erica VanderPorten a, Jing Wang b, BinQing Wei a, Ivana Yen a, Qin Yue 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:
Received 9 July 2013
Revised 9 August 2013
Accepted 13 August 2013
Available online 22 August 2013
A 2-amino-5-aryl-pyrazine was identified as an inhibitor of human lactate dehydrogenase A (LDHA) via a
biochemical screening campaign. Biochemical and biophysical experiments demonstrated that the com-
pound specifically interacted with human LDHA. Structural variation of the screening hit resulted in
improvements in LDHA biochemical inhibition and pharmacokinetic properties. A crystal structure of
an improved compound bound to human LDHA was also obtained and it explained many of the observed
structure–activity relationships.
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
Lactate dehydrogenase
X-ray crystal structure
Pyrazine
Tumor metabolism
Since the early 20th century, it has been known that many
tumors exhibit altered metabolic characteristics relative to normal
tissues.1 One of the major differences between cancerous and
normal tissues is how they metabolize glucose. Some cancer cells
primarily metabolize glucose by glycolysis, whereas most normal
cells catabolize glucose by oxidative phosphorylation.2 Such differ-
ences in normal and tumor cell metabolism represent attractive
therapeutic opportunities to selectively target tumor cells.3
The shift toward anaerobic glycolysis with a concomitant in-
crease in lactate production is a hallmark of rapidly-proliferating
cells (known as the Warburg Effect).4 The conversion of both glu-
cose and glutamine to lactate under anaerobic conditions involves
the enzyme lactate dehydrogenase A (LDHA). It has been known for
several decades that LDHA is over-expressed in human tumor tis-
sue.5 Additionally, shRNA knockdown of LDHA expression in tumor
cell lines elicited a decrease in cell proliferation under hypoxic con-
ditions.6 These observations, coupled with viability of human ge-
netic knock-outs, make LDHA an attractive target for inhibiting
tumor cell proliferation.7,8
Biochemical screening efforts at Genentech identified a series of
2-thio-6-oxo-1,6-dihydropyrimidines, which we disclosed in an
earlier publication (Fig. 1, compound 1).14 We now describe the
identification, validation, and structure–activity relationships of
an amino-pyrazine biochemical screening hit from the Genen-
tech/Roche corporate compound collection (Fig. 1, compound 2).
Compound 2 was identified using a human LDHA biochemical
assay that monitored the disappearance of the NADH co-factor dur-
ing enzymatic conversion of pyruvate to lactate (IC50 = 4 l
M).15,16
Compound 2 also demonstrated a favorable ligand-efficiency (LE)
value in our LDHA biochemical assay (LE = 0.30).17 Importantly, a
similar IC50 value was achieved when LDHA biochemical inhibition
was quantified using mass spectrometry, suggesting that the
observed biochemical inhibition did not result from spectrophoto-
metric assay artifacts. Biochemical assay using structurally-related
human lactate dehydrogenase B (LDHB) also demonstrated a simi-
lar biochemical potency (IC50 = 12 l
M).18
In addition to biochemical assays, compound 2 was profiled
using a surface plasmon resonance (SPR) assay.16 Analysis of hu-
man LDHA and compound 2 in the absence of NADH provided a
Some inhibitors of human LDHA have been reported in the
literature.9–13 The most potent and well-characterized of these
inhibitors resulted from fragment screening campaigns.10,11
very weak SPR KD value (KD = 857
2 associated with LDHA in the presence of NADH and with a KD that
closely matched the biochemical IC50 value (KD = 7 M). These dif-
lM). In comparison, compound
l
fering SPR binding results illustrated the cooperative binding of the
NADH co-factor and compound 2 in a manner that might parallel
⇑
Corresponding author.
E-mail address: benjampf@gene.com (B.P. Fauber).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.08.060

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B. P. Fauber et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5533–5539
O
CN
HN
H
N
HO
N
N
NH
S
N
H2N
O
O
S
Cl
NH2
O
O
1
2
Figure 1. LDHA inhibitors identified via biochemical screening campaigns at Genentech.
events occurring during the catalytic conversion of pyruvate to
lactate.19 Taken collectively, these biochemical and biophysical re-
sults suggested that compound 2 was a validated human lactate
dehydrogenase inhibitor worthy of additional investigation.
It was recognized that the carboxylic acid motif present in com-
pound 2 could result in poor in vivo pharmacokinetics due to phase
II metabolism of the acid moiety.20 Thus, structural exploration
commenced with modification of the 5-position of the 2-amino-
pyrazine core in an effort to explore carboxylic acid replacements
while maintaining favorable potency (Table 1). Replacement of
the aryl carboxylic acid with an aryl ester eroded activity, as did
replacement of the carboxylic acid with an amide or acyl-sulfon-
amide group (Table 1, compounds 3–5, respectively). Use of a
3-carboxy-4-pyridyl ring resulted in some loss of potency (Table 1,
compound 6). Optimal placement of the carboxylic acid group was
probed by moving it to the 2-position of the arene, which was not
tolerated (Table 1, compound 7), whereas movement of the acid
group to the 4-position of the arene resulted in only a modest
reduction in potency (Table 1, compound 8). Introduction of a 3-
phenol adjacent to the 4-carboxylic acid to facilitate an intramolec-
ular hydrogen bond between the groups, and thus limit rotation
about the carbonyl–aryl CÀC bond and make it co-planar with
the aromatic ring, did not provide a significant improvement in
potency versus the parent 4-carboxylic acid analog (Table 1, com-
pounds 9 and 8, respectively). Conversion of the arene to a 4-car-
upset the aforementioned carboxylic acid group’s interaction with
the histidine residue in the LDHA binding pocket.
We then turned our attention to exploration of the (R)-methyl-
benzyl amine region of the molecule. Removal of the chiral
a-methyl group resulted in a slight loss of potency, while the (S)-
methyl-benzyl amine produced an inactive molecule (Table 3,
compounds 19 and 20, respectively). A rationalization for the loss
in potency was subsequently explained by the crystallographic
observation that the benzylic group fills a hydrophobic region of
the pocket where there is limited space to accommodate the oppo-
site torsional orientation of the benzylic group that would be in-
duced by the (S)-enantiomer (see below).
We explored chlorine substitution on the phenyl ring of the (R)-
methyl-benzyl amine and found the 4-chloro-group provided an
approximate twofold improvement in potency (Table 3, compound
21). Methylation of the NÀH moiety resulted in an inactive mole-
cule, as did replacement of the secondary amine with an ether link-
age to the pyrazine core (Table 3, compounds 22 and 23,
respectively). Taken collectively, these data points suggested a
strict steric requirement for the positioning of the benzylic group.
Saturation of the benzylic ring, along with inclusion of another sat-
urated ring analog, was tolerated (Table 3, compounds 24 and 25).
However, an amino-ether analog was not permitted, indicating a
preference for lipophilic groups in this region of the binding site
(Table 3, compound 26).
boxy-N-pyrazole retained potency, while conversion to
carboxy-thiophene displayed less biochemical activity (Table 1,
compounds 10 and 11, respectively).
After analyzing the analogs in Table 1, it was clear that the
acidic functionality present in compound 2 was preferred. A simi-
lar preference has also been noted in other LDHA inhibitors.9–13
The aforementioned SAR preferences were subsequently explained
by the crystallographic observation that the carboxylic acid group
makes a hydrogen bond interaction with a histidine residue in the
LDHA binding pocket (see below).
The next phase of exploration focused on substitution of the 3-
benzoic acid ring while maintaining the important acidic function-
ality. Systematic exploration of the 2-, 4-, 5-, and 6-positions of the
ring are detailed in Table 2. Methylation of the various positions
about the ring indicated a preference for the 6-position (Table 2,
compounds 12–15). Additional 6-position groups were explored,
including the larger iso-propyl group, as well as electron-donating
and electron-withdrawing groups (Table 2, compounds 16–18). All
6-position changes had minimal impact on the potencies of the
molecules, indicating a steric preference for substitution at the 6-
position of the aryl ring with minimal substituent-induced elec-
tronic effects. The 6-position group was subsequently shown by
crystallography to make a lipophilic interaction with the protein
pocket, explaining the preference for the 6-position over the other
locations on the aromatic ring (see below). Additionally, the disfa-
vored substitution at the 2- and 4-positions of the ring could be
rationalized as disrupting the bond angle between the carboxylic
acid and the arene. These bond angle disruptions could in turn
a
Having explored the peripheral regions of the 2-amino-pyrazine
hit, we then focused our effort on the pyrazine core present in 2.
Single-point changes were conducted to understand the contribu-
tions of each core element to the overall potency of the molecule.
Systematic removal of the pyrazine ring nitrogens indicated a
requirement for the 1-position nitrogen (Table 4, compounds 27
and 28). Removal of the 2-amino group, or conversion to a 2-phe-
nol group, resulted in a loss of potency (Table 4, compounds 29 and
30).21 Likewise, further changes to the 2-amino position including
methylation, acylation, and sulfonylation of the amino-group were
not tolerated (Table 4, compounds 31–33). Inclusion of a 6-methyl-
group on the 2-amino-pyrazine core resulted in a loss of potency
(Table 4, compound 34). Taken collectively, these data points indi-
cated a strong binding site preference for the hydrogen bond do-
nor–acceptor motif present in the 2-amino-pyrazine scaffold.
To further explain the observed SAR in this series, we obtained a
2.05 Å resolution crystal structure of an improved inhibitor bound
to human LDHA in the presence of NADH.22 As shown in Figure 2a,
compound 18 binds near several conserved residues involved in
the catalytic processing of lactate dehydrogenase substrates (i.e.,
Arg168 and His192).23 The NADH co-factor was also observed in
the crystal structure, adjacent to compound 18 and in a configura-
tion similar to that previously described in the literature.24 The
hydrogen bonding interactions of compound 18 are summarized
in Figure 2b. The compound formed a hydrogen bond via its car-
boxylic acid group to His192, but did not interact with Arg168
(>5 Å distance). This interaction was consistent with the require-
ment for the carboxylic acid group on the 3-position ring (Table 1).

B. P. Fauber et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5533–5539
5535
Table 1
Table 2
Structure–activity relationships of the 5-position of the 2-amino-pyrazine
Structure–activity relationships of the benzoic-acid region of the 2-amino-pyrazine
5
R
4
6
HO
N
N
NH
R
N
N
NH
2
O
NH2
NH2
a
b
c
Compd R-group
LDHA
LDHA SPR LDHB
LDHA
LEd
Compd R-
group
LDHA IC50
M)
LDHA SPR KD
M)
LDHB IC50
M)
LDHA
LEd
a
b
c
IC50
M)
KD
(l
M)
IC50
M)
(
l
(l
(l
(l
(
l
12
13
14
15
16
17
2-Me
4-Me
5-Me
6-Me
6-iPr
6-
35
15
4
2
2
>100
29
7
3
5
44
40
7
6
7
0.24
0.26
0.29
0.31
0.29
0.29
HO
2
4
7
12
0.30
—
O
2
3
7
OMe
6-Cl
18
2
5
8
0.31
EtO
H2N
3
4
5
6
>100
>100
>100
24
>100
>100
>100
32
ND
92
See the Supplementary data for experimental details associated with each assess-
ment. All biochemical assay results are reported as the arithmetic mean of two
separate runs (n = 2). SPR data are n = 1. ND = not determined.
O
O
Human LDHA biochemical inhibition.15
a
b
—
Human LDHA dissociation constant as determined by surface plasmon reso-
nance (SPR).
c
Human LDHB biochemical inhibition.
Ligand efficiency (LE) was calculated using the human LDHA biochemical IC50
d
value.17
H
N
>100
48
—
S
O
O
O
N
presence of the co-factor. Additionally, this interaction also ex-
plained the loss of affinity described earlier in this work that re-
sulted from the conversion of the benzylic NÀH linker into a
benzylic ether linkage. The ether linkage would have created a
repulsing lone-pair lone-pair effect between the ether oxygen
HO
0.26
O
and the a-phosphate oxygen of NADH.
7
>100
>100
>100
—
A hydrogen bond acceptor–donor interaction motif between
Thr247 and the 2-amino-pyrazine core of compound 18 was also
observed. This observation was consistent with the strict SAR
requirements we noted in our core change analogs that systemat-
ically disrupted the acceptor–donor motif (Table 4). Compound 18
did not make any detectable interactions with water molecules in
the binding site.
The 6-chloro-group on the benzoic acid ring and the 3-(R)-
methyl-benzylamine group formed a hydrophobic interaction with
Ile241 in the binding site (Fig. 2a). Again, these structural observa-
tions were consistent with the previously discussed SAR studies
and stereochemical requirements at the 3-position of the pyrazine
core (Table 3). The (R)-methyl-benzylamine group made several
close contacts with the surrounding protein residues. This binding
mode was consistent with the observation that the (S)-methyl ana-
log was detrimental to potency, presumably due to a lack of space
to accommodate other rotational orientations of the benzylic
group induced by the (S)-enantiomer.
HO
OH
O
8
9
14
10
ND
ND
16
16
0.27
0.27
O
OH
O
HO
O
N
N
10
11
15
50
ND
33
15
0.28
0.25
HO
O
S
>100
HO
See the Supplementary data for experimental details associated with each assess-
ment. All biochemical assay results are reported as the arithmetic mean of two
separate runs (n = 2). SPR data are n = 1. ND = not determined.
In an effort to combine preferred fragments identified by the
above activities into one molecule, we synthesized a 6-methyl-3-
benzoic acid analog bearing a 4-chloro-(R)-methyl-benzylamine
(Fig. 3, compound 35). The combination of these features resulted
Human LDHA biochemical inhibition.15
a
b
Human LDHA dissociation constant as determined by surface plasmon reso-
nance (SPR).
c
Human LDHB biochemical inhibition.
Ligand efficiency (LE) was calculated using the human LDHA biochemical IC50
d
in a human LDHA inhibition IC50 of 0.50 lM. Additionally, com-
value.17
pound 35 was fourfold selective versus human LDHB, 10-fold
selective versus human lactate dehydrogenase C (LDHC), and
>500-fold selective versus the structurally-related human malate
dehydrogenase 1 (MDH1) and human malate dehydrogenase 2
(MDH2) enzymes.25 compound 35 was not profiled against a ki-
nase selectivity panel, but it is anticipated that it could possess
some kinase binding affinity due to its hydrogen bond donor–
acceptor motif (i.e., 2-amino-pyrazine) commonly found in kinase
inhibitors.26
In addition to interacting with His192, compound 18 also formed a
hydrogen bond between the NÀH of the 3-(R)-methyl-benzyl-
amine group and the
a-phosphate oxygen of the NADH co-factor.
This crystallographic ligand interaction with the NADH co-factor
was consistent with the SPR result discussed earlier in which a re-
lated compound exhibited a much higher affinity for LDHA in the

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B. P. Fauber et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5533–5539
Table 3
Table 4
Structure–activity relationships of the 3-position of the 2-amino-pyrazine
Structure–activity relationships of the 2-amino-pyrazine core
HO
N
N
R
HO
NH
O
NH2
Het.
O
Compd R-group
LDHA
IC50
LDHA SPR
LDHB
IC50
(lM)
LDHA
LEd
a
b
c
KD
(l
M)
Compd Heterocycle
LDHA
LDHASPR
LDHB
IC50
LDHA
LEd
(l
M)
a
b
c
IC50
M)
KD
(
lM)
(
l
(lM)
19
12
22
28
0.29
—
27e
18
29
56
0.27
NH
N
N
NH2
NH2
28
>100
25
>100
>100
>100
ND
—
20
>100
>100
ND
NH
N
Cl
29
23
0.27
—
N
N
21
2
5
5
0.31
NH
3021
>100
>100
N
N
OH
22
>100
>100
3
>100
>100
7
ND
—
31
32
73
>100
ND
>100
>100
0.22
—
N
N
N
N
H
23
>100
16
—
>100
O
N
N
N
H
O
O
24
0.31
33
34
71
91
ND
>100
>100
0.20
0.22
S
N
N
N
NH
O
H
>100
25
19
18
80
0.29
—
N
NH2
NH
See the Supplementary data for experimental details associated with each assess-
ment. All biochemical assay results are reported as the arithmetic mean of two
separate runs (n = 2). SPR data are n = 1. ND = not determined.
O
26
>100
>100
ND
NH
Human LDHA biochemical inhibition.15
a
See the Supplementary data for experimental details associated with each assess-
ment. All biochemical assay results are reported as the arithmetic mean of two
separate runs (n = 2). SPR data are n = 1. ND = not determined.
b
Human LDHA dissociation constant as determined by surface plasmon reso-
nance (SPR).
c
Human LDHB biochemical inhibition.
Ligand efficiency (LE) was calculated using the human LDHA biochemical IC50
Human LDHA biochemical inhibition.15
d
a
b
Human LDHA dissociation constant as determined by surface plasmon reso-
value.17
nance (SPR).
c
Human LDHB biochemical inhibition.
d
Ligand efficiency (LE) was calculated using the human LDHA biochemical IC50
value.17
kg), and 41% oral availability in male Sprague–Dawley rats
(Table 6).
We also assessed the ability of compound 35 to inhibit the pro-
Compounds 2 and 35 were profiled against a panel of in vitro
DMPK assays. In addition to compound 35 being a more potent
inhibitor of LDHA than compound 2, there were also improve-
ments in the liver microsomal stabilities in humans and rats (Ta-
ble 5). Both compounds also exhibited favorable aqueous kinetic
solubility and cell permeability in the Madin–Darby canine kidney
(MDCK) cell permeability model.27 Additionally, neither compound
inhibited the major cytochrome P450 (CYP) isoforms up to com-
duction of lactate in MCF7 cells.16 No dose–response was observed
in the assay but at the highest compound concentration of 50 lM,
we observed a 40% reduction in lactate production with no effect
on cell viability. The reasons for the minimal cellular activity are
currently unknown, but could be related to insufficient biochemi-
cal potency or high plasma–protein binding (>99% bound in human
plasma and 99% bound in rodent plasma). Additional experiments
are ongoing to clarify which, if any, of these potential liabilities is
responsible for the poor cell-based activity exhibited by compound
35.
pound concentrations of 10 l
M.28 Compound 35 was profiled in
an in vivo single-dose rat pharmacokinetic experiment (1.5 mg/
kg po and 0.5 mg/kg iv) and demonstrated high in vivo clearance
(Clp = 60 mL/min/kg), a modest volume of distribution (Vd = 1.4 L/
Syntheses of analogs that explored the 3- and 5-positions of the
2-amino-pyrazine core are described in Scheme 1. The route com-

B. P. Fauber et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5533–5539
5537
Table 6
Single-dose rat in vivo pharmacokinetic properties of compound 35
Compd Clp (mL/min/kg) Vd (L/kg) Cmax(iv)
35a
60 1.4 2.1
(l
M) AUCinf(po) (h
0.55
l
M) t1/2 (h) % Fb
2.8 41
See the Supplementary data for experimental details associated with each assess-
ment. Data reported are the arithmetic means from the dosing cohorts.
a
Male Sprague–Dawley rats (n = 3), 1.5 mg/kg po (26/74 suspension of DMSO/
MCT), 0.5 mg/kg iv (18/60/22 solution of DMSO/PEG400/saline).
b
Bioavailability was calculated according to the equation % F = (AUCpo  Doseiv
/
AUCiv  Dosepo)  100.
menced with the SNAr reaction of various amines on the 3,5-dibro-
mo-2-amino-pyrazine scaffold (Scheme 1, Intermediate 36).29 The
pyrazine–aryl bond was formed under Suzuki–Miyaura cross-cou-
pling conditions, or Buchwald–Hartwig amination conditions for
analogs that contained a pyrazine-N-heteroaryl bond.30,31 Final
compounds containing a carboxylic acid were carried through
the synthetic sequences as the corresponding ethyl ester, then
saponified in the final step to reveal the carboxylic acid (Scheme 1,
compounds 2–18).
Figure 2a. Co-crystal structure of compound 18 (yellow) in complex with LDHA
(grey). NADH is also present (gold). Nearby side-chain residues involved in the
catalytic processing of lactate dehydrogenase substrates are also shown in grey
(Arg168 and His192). Ile241 (grey) makes a hydrophobic contact with compound
18. Compound 18 does not make any direct interactions with water molecules, thus
they have been omitted for clarity. Hydrogen bonds are not depicted in this figure.
The resolution of the structure is 2.05 Å.
Changes to the pyrazine core are summarized in Scheme 2. Pyr-
idine compound 27 was made from the alkylation of 5-bromopyr-
idine-2,3-diamine with (1-bromoethane)benzene to yield the
corresponding racemic product (Scheme 2, compound 40).32 A Su-
zuki–Miyaura coupling of the remaining halide, followed by sapon-
ification provided the final compound (27). The pyridine isomer of
the aforementioned analog was synthesized via a SNAr reaction
with (R)-methyl-benzylamine to produce intermediate 42,33 fol-
lowed by a Suzuki–Miyaura coupling. Subsequent reduction of
the nitro-group and saponification of the ester revealed pyridine
compound 28. Compounds 29 and 30 were made using varied
starting materials and
a similar procedure as described in
Scheme 1. Compound 3 was further elaborated into the N-methyl
analog using reductive methylation, as well as N-acetyl and
N-sulfonyl analogs under standard conditions, followed by sapon-
ification to reveal the final compounds (Scheme 2, compounds
31-33). The 2-amino-6-methylpyrazine core was generated via
the dibromination of 2-amino-6-methylpyrazine,34 followed by a
SNAr reaction, arylation, and saponification to complete the
synthesis of compound 34.
Figure 2b. Alternate view of co-crystal structure of compound 18 (yellow) in
complex with LDHA (grey). NADH is also present (gold). Hydrogen bonds are
depicted as dashed lines (black), with distances ranging from 2.9 to 3.1 Å.
In summary, a new class of 2-amino-pyrazine LDHA inhibitors
was identified via biochemical screening and validated using bio-
chemical and biophysical assays. The crystal structure of an im-
proved inhibitor indicated that the molecules bind in the active
site in the presence of NADH and interact with one of the major
catalytic residues involved in substrate catalysis. Structural modi-
fications of the original hit improved the biochemical potency and
ligand-efficiency, while also improving the DMPK properties of the
compounds. Additional efforts to obtain potent and cell-active
LDHA inhibitors will be reported in due course.
Cl
HO
N
NH
O
N
NH2
35
Figure 3. Combination of the most potent structural features in one molecule.
Table 5
In vitro DMPK properties of compounds 2 and 35
Compd
HLMa Clhep
(mL/min/kg)
RLMb Clhep
(mL/min/kg)
Human/rat plasma-protein
binding (% bound)
MDCK permeabilityc
A–B (10À6 cm/s)
MDCK permeabilityc
B–A (10À6 cm/s)
Solubilityd
M)
(l
2
35
5
1
9
4
99/96
>99/99
9.6
12.1
4.0
10.3
137
81
See the Supplementary data for experimental details associated with each assessment.
a
Human in vivo clearance value extrapolated from in vitro human liver microsome (HLM) experiment.
Rat in vivo clearance value extrapolated from in vitro rat liver microsome (RLM) experiment.
Madin–Darby canine kidney (MDCK) cell permeability assay to assess membrane permeability properties.27
Aqueous kinetic solubility at pH 7.4 (estimated from a high-throughput assay).
b
c
d

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B. P. Fauber et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5533–5539
HO
N
N
N
N
NH
O
NH2
10
c,d
Br
N
Br
f,b,d
a
b,d
Br
N
N
NH
Ar
N
N
NH
HO
N
N
O
N
NH2
O
NH2
NH2
NH2
23
36
37
2
3
10
11
Ar = 3-CO2H-Ph
Ar = 3-CO2Et-Ph
Ar = 4-CO2H-N-pyrazole
Ar = 5-CO2H-3-thiophene
4 Ar = 3-CONH2-Ph
12 Ar = 2-Me-3-CO2H-Ph
e
5 Ar = 3-MeSO2NHCO-Ph 13 Ar = 3-CO2H-4-Me-Ph
6 Ar = 3-CO2H-(4-pyridyl)
14 Ar = 3-CO2H-5-Me-Ph
7
8
15
16
Ar = 2-CO2H-Ph
Ar = 4-CO2H-Ph
Ar = 3-CO2H-6-Me-Ph
R1
R2
N
R2
N
Ar = 3-CO2H-6-i-Pr-Ph
9 Ar = 4-CO2H-3-OH-Ph
17 Ar = 3-CO2H-6-OMe-Ph
18 Ar = 3-CO2H-6-Cl-Ph
b,d
Br
N
HO
N
N
R3
R3
O
N
NH2
NH2
19
R
1 = R2 = H, R3 = Bn
38a-f
20 R1 = R2 = H, R3 = (S)-Me-Bn
21 R1 = R2 = H, R3 = (R)-Me-(4-Cl-Bn)
22 R1 = H, R2 = Me, R3 = (R)-Me-Bn
24
25
R
R
1 = R2 = H, R3 = (R)-Me-(c-Hex)
1 = R2 = H, R3 = CH2-(c-Pentyl)
26 R1 = R2 = H, R3 = (CH2)2OMe
35 R1 = Me, R2 = H, R3 = (R)-Me-(4-Cl-Bn)
Scheme 1. Reagents and conditions: (a) (R)-methyl-benzylamine, N,N-diisopropylethylamine, n-BuOH, M.W. 150 °C, 6 h, 86% yield; (b) Pd(dppf)Cl2 (10 mol %), ArB(OH)2 or
ArB(pin), Cs2CO3, 1,4-dioxane, water, M.W. 120 °C, 30 min, 5–20% yield; (c) ethyl 1H-pyrazole-4-carboxylate, CuI (10 mol %), N1,N2-dimethylethane-1,2-diamine (20 mol %),
Cs2CO3, 1,4-dioxane, reflux, 1 h, 39% yield; (d) LiOH, MeOH, water, 23–60 °C, 12 h, 5–40% yield; (e) primary or secondary amine, N,N-diisopropylethylamine, n-BuOH, reflux,
1 h, 60% yield; (f) NaH, (R)-1-phenylethanol, THF, 0 °C ? reflux, 2 h, 16% yield.
Br
Br
NH2
NH2
a
d
f
b,c
b,e,c
b
Br
Br
Br
NH
HO
HO
EtO
NH
N
O
O
O
N
N
NH2
N
NH2
39
40
42
27
N
Br
NH
N
NH
NO2
NO2
NH2
41
28
Br
N
Br
R
c
N
NH
R
N
N
NH
R
HO
N
NH
R
O
N
N
N
43
45
47
48
3
29 R = H
R = H
R = H
R = H
30
31
44 R = OH
46 R = OH
R = OH
R = OH
R = NH2
g,c
R = NHMe
h,c
i,c
32
33
R = NHCOMe
Br
Br
NH2
j
f,b,c
HO
NH
R = NHSO2Me
N
NH2
N
O
N
NH2
49
50
34
Scheme 2. Reagents and conditions: (a) (1-bromoethyl)benzene, EtOH, reflux, 18 h, 7% yield; (b) Pd(dppf)Cl2 (10 mol %), 3-(ethoxycarbonyl)phenylboronic acid, Cs2CO3, 1,4-
dioxane, water, reflux, 1 h, 5-32% yield; (c) LiOH, MeOH, water, 23 °C, 12 h, 5-40% yield; (d) (R)-methyl-benzylamine, triethylamine, EtOH, 23 °C, 12 h, 41% yield; (e) Pd/C
(10 mol %), H2 (1 atm), EtOH, 23 °C, 18 h 50% yield; (f) (R)-methyl-benzylamine, N,N-diisopropylethylamine, n-BuOH, reflux, 1 h, 60% yield; (g) 37% formalin, AcOH,
NaBH(OAc)3, 23 °C, 12 h, 45% yield; (h) Ac2O, ZnCl2 (10 mol %), 23 °C, 2 h, 80% yield; (i) MeSO2Cl, pyridine, 23 °C, 0.5 h, 30% yield; (j) N-bromosuccinimide, benzoylperoxide
(1 mol %), CCl4, 23 °C, 2 h, 28% yield.

B. P. Fauber et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5533–5539
5539
D.; Pitts, K.; Purkey, H. E.; Sideris, S.; Ultsch, M.; VanderPorten, E.; Wei, B.; Xu,
Q.; Yen, I.; Yue, Q.; Zhang, H.; Zhang, X. Bioorg. Med. Chem. Lett. 2013, 23, 3186.
15. The standard error of the mean for reference compounds in the LDHA
biochemical assay was 50%. For more details, see: Vander Porten, E. Frick, L.
Turincio, R. Thana, P. La Marr, W. Liu, Y. Anal. Biochem., in press, dx.doi.org/
10.1016/j.ab.2013.07.003.
Acknowledgements
We thank Drs. Krista Bowman and Jian sheng Wu and their
respective Genentech research groups for performing protein
expression and purification activities. We also thank Dr. Peter Jack-
son for many helpful discussions regarding LDHA. In addition, we
thank Dr. Patrick Lupardus and Crystallographic Consulting, LLC
for 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.
16. See the Supplementary data section for details regarding the enzyme, SPR, and
cell assay methods.
17. Hopkins, A. L.; Groom, C. R.; Alex, A. Drug Discovery Today 2004, 9, 430.
18. LDHA preferentially catalyses the formation of lactate from pyruvate, whereas
LDHB preferentially catalyzes the reverse reaction. LDHA and LDHB share high
levels of binding site homology—all of the key catalytic residues are conserved.
Overall, the two enzymes share 83% homology when compared via sequence
alignment (see Ref. 11 for additional details).
19. NADH binding to LDHA prior to the binding of pyruvate is the accepted LDHA
catalytic mechanism. See Ref. 8 for additional details.
20. Kalgutkar, A. S.; Daniels, J. S. Carboxylic Acids and Their Bioisosteres. In
Metabolism, Pharmacokinetics and Toxicity of Functional Groups; Smith, D. A., Ed.,
1st ed.; The Royal Society of Chemistry: Cambridge, 2010; pp 126–143.
21. No attempts were made to identify which, if any, pyrazinone tautomer
dominated in organic and/or aqueous solutions. Compound 30 is arbitrarily
drawn and described as the phenolic tautomer.
22. 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 ‘W’/protein chain ‘D’ 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 ‘W’/protein
chain ‘D’ complex, although some subtle differences were apparent. See the
deposited PDB file for additional structural details: 4M49.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.08.
060.
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