Identification of substituted 3-hydroxy-2-mercaptocyclohex-2-enones as potent inhibitors of human lactate dehydrogenase

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3764–3771

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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl

Identification of substituted 3-hydroxy-2-mercaptocyclohex-2enones as potent inhibitors of human lactate dehydrogenase Peter S. Dragovich a,⇑, Benjamin P. Fauber a, Jason Boggs a, Jinhua Chen b, Laura B. Corson a, Charles Z. Ding b, Charles Eigenbrot a, HongXiu Ge b, Anthony M. Giannetti a, Thomas Hunsaker a, Sharada Labadie a, Chiho Li b, Yichin Liu a, Yingchun Liu b, Shuguang Ma a, Shiva Malek a, David Peterson a, Keith E. Pitts a, Hans E. Purkey a, Kirk Robarge a, Laurent Salphati a, Steve Sideris a, Mark Ultsch a, Erica VanderPorten a, Jing Wang b, BinQing Wei a, Qing Xu b, Ivana Yen a, Qin Yue a, Huihui Zhang b, Xuying Zhang b, Aihe Zhou a a b

Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA 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

Article history: Received 7 June 2014 Revised 23 June 2014 Accepted 25 June 2014 Available online 1 July 2014 Keywords: Lactate dehydrogenase X-ray crystal structure Glycolysis Tumor metabolism

a b s t r a c t A novel class of 3-hydroxy-2-mercaptocyclohex-2-enone-containing inhibitors of human lactate dehydrogenase (LDH) was identified through a high-throughput screening approach. Biochemical and surface plasmon resonance experiments performed with a screening hit (LDHA IC50 = 1.7 lM) indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of this screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC50 = 0.18 lM). Two crystal structures of optimized compounds bound to human LDHA were obtained and explained many of the observed structure–activity relationships. In addition, an optimized inhibitor exhibited good pharmacokinetic properties after oral administration to rats (F = 45%). Ó 2014 Elsevier Ltd. All rights reserved.

The altered metabolism exhibited by many tumors is currently viewed as an attractive differentiator from normal tissues which can be exploited for the development of novel anti-cancer therapeutics.1 One such example is the utilization of glucose in which cancer cells metabolize this nutrient primarily via glycolysis as opposed to the more energy-efficient but oxygen-dependent mitochondrial oxidative phosphorylation process.2 Somewhat surprisingly, glycolytic glucose metabolism is known to occur in many tumors even in the presence of normal oxygen levels.2 In contrast, normal tissues typically employ glycolysis for energy production only when oxygen supplies limit oxidative phosphorylation (e.g., in strenuously working muscle). This difference suggests that inhibition of ‘aerobic glycolysis’ in cancer cells may preferentially impede tumor growth and proliferation while having minimal impacts on normal host tissues.3,4 The final step in glycolysis is catalyzed by lactate dehydrogenase A (LDHA; also known as LDH-M and LDH-5), a homotetrameric enzyme which converts pyruvate to lactate in the cell cytosol ⇑ Corresponding author. Tel.: +1 650 467 6854. E-mail address: [email protected] (P.S. Dragovich). http://dx.doi.org/10.1016/j.bmcl.2014.06.076 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

(Fig. 1).5–7 The enzyme utilizes the reduced form of nicotinamide adenine dinucleotide (NADH) as a co-factor to stereospecifically transfer a hydride to the pyruvate ketone moiety. This transformation can also be accomplished by lactate dehydrogenase B (LDHB; also known as LDH-H and LDH-1), an alternate LDH isoform, although this enzyme preferentially catalyzes the reverse reaction in which lactate is converted to pyruvate.5,7 As a HIF1a and Myc target gene, LDHA is induced by hypoxia or mutations in VHL, FH, SDH, or the RAS/PI3K/AKT signaling pathways.8,9 In addition, elevated LDHA levels are associated with many cancers and are often predictive of poor survival in many indications.8 Encouragingly, shRNA-mediated LDHA knock-down in glycolytic cancer cell lines results in significant inhibition of tumor growth, and more pronounced effects are observed under hypoxic conditions where cells rely primarily on glycolytic energy production.9a,c Importantly, humans lacking LDHA through hereditary deficiency display mild phenotypes suggesting that inhibition of the enzyme will not lead to significant intolerable side-effects.10 Collectively, these data implicate LDHA as an attractive target for the development of new anti-cancer agents for use against hypoxic tumors and/or those which display strong glycolytic phenotypes.

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O O

H

HO

O

Pyruvate

O

NH 2

Lactate

N

LDHA

NH2

N O HO

H O

H

O

NH2 RO

O

RO

O

O

N H 2N

NADH

NAD HO

OH

O O O P O P O O O

NADH

NH 2

N

OH

HO

OH

O -

H

N

HO

LDHB

O

H

H

N

N

Oxamate (inhibitor)

O O

OH

Figure 1. LDH biochemistry.

O HN

H N H2N O

S

Cl

S

CF 3

CN

O HN

N

O

Cl

O

OH

N

H2N

N

111

N OH

O

212

313 HO

F

F

S

S

H N

H N O

N

O

O

O

OH

O

OH

NH O O S N H N

N

OH

H3CO

N

OCH3

O

414

515 F Cl O

N HO

S

OH

OH

O N H

O OH

N

OH

OCH3

O

OH O

616 Figure 2. Structures of representative human LDHA inhibitors.

We11,12 and others13–17 have recently reported multiple examples of human LDHA inhibitors in the scientific and patent literature (compounds 1–6, Fig. 2). All of these molecules contain either a carboxylic acid moiety or some other acidic functional group which can ionize at physiological pH (e.g., the pKa of compound 1 was determined to be 3.9411). In the case of inhibitors 4 and 6, crystallographic studies determined that a carboxylic acid moiety present in each compound interacted with the basic sidechain of LDHA residue Arg-168 in a manner related to that observed for a substrate-derived inhibitor (oxamate; Fig. 1).14,16,18,19 Computational modeling suggested that similar interactions occurred between compound 3 and the LDHA active site.13 Although contacts between LDHA Arg-168 and the acidic moieties present in inhibitors 1 and 2 were not observed crystallographically, structure–activity relationships developed within each

series demonstrated the importance of the ionizable group for maintaining good LDHA inhibition activity.11,12 Thus, an acidic moiety that can ionize at physiological pH is an important pharmacophore component for the majority of known LDHA inhibitors. As part of our efforts to identify new LDHA inhibitors, we conducted high-throughput screening of the Roche and Genentech chemical archives using a fluorescence-based assay which monitored the disappearance of the NADH co-factor during enzymatic conversion of pyruvate to lactate.20 This screening effort identified the 3-hydroxy-2-mercaptocyclohex-2-enone 7 as a moderately potent LDHA inhibitor, and this activity was subsequently confirmed in lower-throughput biochemical assessments employing both spectrophotometric and mass spectrometry detection methods (IC50 = 1.7 lM and 0.88 lM, respectively; Table 1).20–22 Given the importance of acidic moieties in the pharmacophores of known

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Table 1 Structure and biological properties of compound 7

OH

NO2 S O

7 Assay description

a

Result (lM)

LDHA IC50 (UV endpoint) LDHA IC50 (MS endpoint) LDHA KD (SPR, +NADH) LDHA KD (SPR, NADH) LDHB IC50 (UV endpoint)

1.7 0.88 3.5 771 12.3

a See Supplementary data for experimental details associated with each assessment.

OH

NO 2

O

S

H

O

O

7 -H

7a +H

-

O

-H NO 2

S

+H O

-

O

7b

NO 2 S

NO 2 S O

7c

Scheme 1. Structure of compound 7 along with possible tautomeric and ionized forms.

LDHA inhibitors, we were intrigued by the possibility that compound 7 might ionize at physiological pH and thereby function as a carboxylic acid mimic. Specifically, we envisioned deprotonation of the hydroxy-cyclohexenone present in 7, or the cyclic 1,3-dicarbonyl moiety contained in the 7a tautomer, thereby affording the corresponding ionized forms (structures 7b and 7c; Scheme 1).23 The measured pKa of compound 7 (2.63) was consistent with this

hypothesis and indicated that such ionization under physiological conditions was indeed possible. Accordingly, additional biochemical and biophysical experiments were performed with 7 to both increase confidence that the molecule was a bona fide LDHA inhibitor and to further characterize its LDHA inhibition mechanism. Surface plasmon resonance (SPR) assessments performed in the presence and absence of NADH determined that the compound specifically associated with LDHA and that measured KD values were (1) close to the biochemical IC50 potency and (2) much stronger in the presence of the co-factor (compare Fig. 3A and B, Table 1).22 Similar SPR results were observed for several other hits that emerged from our LDHA screening efforts.11,12 As with these previously characterized inhibitors, the SPR findings suggested that binding of 7 to LDHA required prior association of the NADH co-factor.24 Compound 7 also inhibited the closely related LDHB isoform with an IC50 value similar to that observed for LDHA (Table 1), and the above characterization data collectively provided assurance that 7 was a specific and genuine anti-LDHA agent. To facilitate medicinal chemistry efforts aimed at improving the inhibition activity of 7, we obtained a crystal structure of the molecule in complex with the LDHA enzyme.25 As shown in Figure 4a, the compound bound in the LDHA active site in close proximity to the NADH co-factor that was also present in the structure. This orientation was consistent with the SPR data described above which indicated that the presence of NADH greatly improved the affinity of 7 for the LDHA protein. The enol moiety of 7 was expected to be deprotonated at the neutral pH employed to obtain the structure and this moiety interacted closely with the (presumably) protonated LDHA Arg-168 side chain (Figs. 4a and 4b).23,26 This residue is believed to interact in a related manner with the pyruvate carboxylate moiety during substrate catalysis by the enzyme.5 One of the nitro-group oxygen atoms was also observed nearby (3.0 Å) the Arg-168 side chain. However, the orientation of the nitro functionality was not consistent with the formation of a robust hydrogen bond. The ketone carbonyl moiety of 7 was noted crystallographically to form hydrogen bonds with the side chains of LDHA residues Asn-137 and His-192. The protonated form of His-192 is believed to transfer a proton to the pyruvate carbonyl moiety during substrate turnover.5 Interestingly, the sulfur atom present in 7 was noted in close spatial proximity (3.3 Å) to both the Arg-168 and the His-192 LDHA side chains. However, the importance of this proximity and whether the sulfur forms beneficial hydrogen bonds and/or polar interactions with these residues is not known with certainty. Collectively, these observations indicated that the ionized form of 7 can function as a carboxylic acid mimic and thereby effectively interact with several key catalytic residues in the LDHA active site.

Figure 3. SPR data depicting the binding of compound 7 to LDHA in the presence (A) and absence (B) of NADH. The KD determined from fitting the kinetics is reported in panel A. In panel B, the KD was estimated relative to a control using methods described in Supplementary data. In both panels, the top concentration is 25 lM with injections related by a twofold dilution series. See Supplementary data for additional experimental details.

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Table 2 Structure–activity relationships of 3-hydroxy-2-mercaptocyclohex-2-enone-containing compounds

R1

OH

R2 R3

S 5 O R

Figure 4a. Co-crystal structure of inhibitor 7 (cyan) in complex with LDHA (grey). NADH is also present and is depicted in dark blue. Hydrogen bonds are depicted as dashed yellow lines. The resolution of the structure is 2.15 Å.

ARG168 HN H2N

-

NH2

ALA237 O

N

O

VAL234

-

O

ALA237

ASP165 S

GLY193

O

LEU164 HN

TYR238 ILE241

O

ASN137

NH2

NH

-

O O

ASP165

HIS192

Figure 4b. Depiction of key protein–ligand interactions observed in 7-LDHA cocrystal structure. Hydrogen bonds are indicated as dashed lines (red = questionable/ unknown significance). The depicted protonation states of 7 and various LDHA residues are inferred. Alternate tautomers and/or resonance forms of 7 and the LDHA residues are not considered.

Further analysis of the 7-LDHA co-crystal structure revealed that the 2-nitro-phenyl moiety of 7 occupied a narrow cleft located between the side chains of the Arg-168 and His-192 residues (Figs. 4a and 4b). Binding of the 2-nitro-phenyl group in this cleft was facilitated by subtle movements in the side chains and/or backbone atoms of several of the surrounding LDHA residues relative to their positions noted in other published crystal structures (e.g., Asp-165, Arg-168, and Ala-237).11,12 The 2-nitro-phenyl moiety made favorable van der Waals contacts with many of these surrounding residues including Leu-164, Asp-165, Arg-168, Gly-193, Val-234, and Ala-237. Somewhat surprisingly, the other phenyl substituent present in 7 was observed crystallographically in an axial location relative to the 3-hydroxy-cyclohexenone ring. This orientation resulted in a close edge-to-face interaction between the axial phenyl group and the 2-nitro-phenyl moiety of the ligand. The axial location also allowed the phenyl group to contact the hydrophobic side chains of LDHA residues Ala-237, Tyr-238, and Ile-241. These favorable protein–ligand and intra-ligand interactions likely compensate for the high-energy geometry adopted by 7 when bound to LDHA relative to the corresponding unbound conformation.27 With the above structural information available, we initiated medicinal chemistry activities aimed at improving the LDHA inhibition activity of compound 7. As shown in Table 2, removal of the 2-nitro group from the thio-phenyl ring present in 7 resulted in significant loss of anti-LDHA potency (compare 8 with 7). Similarly,

R4

Compd

R1

R2

R3

R4

R5

LDHA IC50a (lM)

7 8 9 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

NO2 H F Cl Br CH3 CF3 CH2CH3 CH2OH OH OCH3 CN CO2H CO2CH3 CO2NH2 SO2CH3 H H H H H H H H H H H H H H H H H H H H H H H H H H H F F Cl Cl Cl Cl Cl CH3 CH3 CH3 OCH3

H H H H H H H H H H H H H H H H F Cl Br CH3 CF3 OCH3 OCF3 NH2 CO2NH2 SO2NH2 H H H H H H H H H H H H Cl OCH3 CH3 Cl CH3 H Cl Cl H H H H H H H H

H H H H H H H H H H H H H H H H H H H H H H H H H H F Cl Br CH3 CH2CH3 CF3 OH OCH3 CO2H SCH3 NH2 NHAc Cl OCH3 CH3 H H F H H H H H F CH3 H H H

H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H Cl CH3 H H H Cl H H H H CH3 H OCH3

H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H Cl CH3 H H H CH3 H

1.7 >100 53 4.6 53 >100 >66 >100 36 >100 >100 1.6 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 63 12 >100 >100 24 >100 >64 >100 >100

See Supplementary data for experimental details associated with each assessment. Biochemical results are reported as the arithmetic mean of at least 2 separate runs. a LDHA biochemical inhibition.

replacement of the 2-nitro group with a halogen was only tolerated when Cl was employed as the 2-substituent (compare 7 with 9–11). Alkyl moieties were not suitable 2-nitro replacements (12–14), and these included the methyl group which had the potential to function as a Cl isostere. Analogs of 7 bearing monosubstituted thio-phenyl rings which incorporated a variety of more polar 2-substituents were also explored (15–22), but only the

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nitrile 18 retained meaningful LDHA inhibition activity. A large number of molecules containing other mono-substituted thiophenyl rings which lacked a 2-substituent were also examined (23–49). Unfortunately, all of these compounds exhibited significant worsening of anti-LDHA activity relative to the original lead 7. These structure-activity relationships illustrated the importance of a 2-substituent on the thio-phenyl ring for obtaining good LDHA inhibition activity. Accordingly, we also explored molecules which preserved a 2-substituent on the thio-phenyl ring but also included one additional moiety (50–60; Table 2). Only two of these compounds displayed encouraging levels of LDHA inhibition potency (53 and 56), and both incorporated the 2-Cl-substituent identified in our initial SAR efforts (cf., compound 10). A large number of molecules were also assessed which replaced the 2-nitro-phenyl moiety present in lead 7 with other aliphatic (61–67), benzylic (68–75), aromatic (76–77), or heteroaromatic groups (78–99; Supplementary data, Table S1). However, all of these compounds displayed poor LDHA inhibition properties. Collectively, the structure activity relationships depicted in Tables 2 and S1 indicate that the region of LDHA occupied by the 2-nitro-phenyl moiety of 7 is relatively intolerant to both subtle changes in the phenyl substitution pattern as well as larger modifications which seek to alter the orientation of the contacting protein residues. Importantly, this initial SAR exploration also identified the Cl and CN groups as suitable replacements for the 2-nitro substituent present in lead 7 that could subsequently be incorporated into new inhibitor designs. Having thoroughly explored modification of the 2-nitro-phenyl moiety contained in 7, we turned our attention to variation of the other (axially situated) phenyl group present in the inhibitor structure. As shown in Table 3, this exploration was conducted using Cl and CN groups in place of the 2-nitro-substituent since use of the two former moieties facilitated compound synthesis and was perceived to impart more desirable bio-pharmaceutical properties to the resulting compounds. Systematic introduction of ortho, meta, and para-Cl groups on the phenyl moiety relative to the 3-hydroxy-cyclohexenone ring afforded minimal changes in LDHA inhibition activity with the para modification being the most detrimental (compounds 100–102). Incorporating a larger para substituent (103) further worsened anti-LDHA properties, consistent with the close contact of the axial phenyl group with Tyr-238. Interestingly, incorporation of a 2,6-di-Cl phenyl moiety into the inhibitor design resulted in a significant improvement in LDHA inhibitor potency (104). Several other compounds which contained various 2,6-disubstituted phenyl groups also exhibited improved anti-LDHA properties (106, 109, 111, 117 and 120; Table 3) although not every 2,6-combination afforded such enhancements (108, 110, 112 and 119, Table 3). A structural explanation for the potency improvement imparted by the various 2,6-disubstituted phenyl moieties is provided below. As we previously noted for other classes of LDHA inhibitors,11,12 the SPR KD’s measured for selected 3-hydroxy-cyclohexenones described in this work closely paralleled the corresponding biochemical IC50 values (Table 3). Similarly, LDHB inhibition activities were determined for the more active compounds and were 5–10 fold less potent than the corresponding LDHA potencies (Table 3). As described above, 2,6-disubstitution of the 5-phenyl moiety present in the 3-hydroxy-cyclohexenone-containing inhibitors afforded significant improvements in LDHA inhibition properties. We therefore obtained a crystal structure of one such molecule (104) in complex with LDHA to better understand the nature of these improvements.25 As shown in Figure 5, the 3-hydroxycyclohexenone ring of 104 bound to and interacted with LDHA in a manner that was nearly identical to that observed for compound 7 (c.f., Figs. 4a and 4b). However, the 2,6-dichlorophenyl moiety of 104 was situated in an equatorial location relative to the

Table 3 Structure–activity relationships of 3-hydroxy-2-mercaptocyclohex-2-enone-containing compounds

OH R

X S

1

R2

O

3

R 1

2

Compd

X

R

R

10 100 101 102 103 104 105 106 107 108 109 110 111 112 18 113 114 115 116 117 118 119 120

Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl CN CN CN CN CN CN CN CN CN

H Cl H H H Cl Cl Cl Cl F Br CH3 Cl CF3 H Cl H H Cl Cl Cl CH3 Br

H H Cl H H H H H H H H H Br H H H Cl H H H H H H

R R

3

H H H Cl OCH3 H H H H H H H H H H H H Cl H H H H H

4

R4

LDHA IC50a (lM)

LDHA KDb (lM)

LDHB IC50c (lM)

H H H H H Cl F Br CF3 F Br CH3 Cl CF3 H H H H Cl Br CF3 CH3 Br

4.6 5.6 5.6 18 19 0.87 3.7 0.58 1.0 7.4 0.27 2.3 0.50 15 1.6 3.1 6.8 17 0.83 0.45 0.64 2.1 0.18

4.3 8.2 7.1 27 13 1.8 ND 0.49 0.50 ND ND ND 0.37 ND 1.5 ND ND ND 0.43 0.24 ND ND ND

19 11 13 27 37 6.9 24 5.3 3.4 50 1.3 15 1.9 >10 7.4 6.6 >10 >10 4.9 2.0 2.8 7.8 0.86

See Supplementary data for experimental details associated with each assessment. LDHA biochemical results are reported as the arithmetic mean of at least 2 separate runs. LDHB and SPR results are n = 1. ND = not determined. a LDHA biochemical inhibition. b LDHA dissociation constant as determined by surface plasmon resonance. c LDHB biochemical inhibition. Table 4 In vitro properties for representative 3-hydroxy-2-mercaptocyclohex-2-enone-containing compounds Compd

c Log P

RLM Cla (mL/ min/ kg)

HLM Clb (mL/ min/ kg)

Rat PPBc (fu)

Human PPBd (fu)

MDCK (AB)e x10-6 cm/s

Solubilityf (lM)

10 18 104 107 112 116 117

4.4 3.5 5.7 5.9 6.2 4.8 4.8

14 5.2 27 37 38 24 31

3.9 1.6 14 17 18 12 15

0.002 0.01 0.002 ND ND ND 0.005

0.001 0.001 0.005 ND ND ND 0.002

18 4.1 17 ND ND ND 14

147 118 128 117 143 117 75

See Supplementary data for experimental details associated with each assessment. All assay results are reported as the arithmetic mean of at least 2 separate runs (exception: solubility determinations are n = 1). ND = not determined. a Rat hepatic clearance predicted from rat liver microsomes (stable, moderate, labile = <17, 17–39, >39 mL/min/kg, respectively). b Human hepatic clearance predicted from human liver microsomes (stable, moderate, labile = <6, 6–15, >15 mL/min/kg, respectively). c Rat plasma protein binding; fraction unbound (fu). d Human plasma protein binding; fraction unbound (fu). e Apparent permeability coefficients (Papp,A–B) across MDCK cell monolayers. f Aqueous solubility.

3-hydroxy-cyclohexenone ring in contrast to the axial orientation previously observed for the unsubstituted phenyl group present in compound 7. In addition, the plane of the 2,6-disubstituted phenyl ring was rotated 90° relative to the cyclohexenone ring. This

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Figure 5. Co-crystal structure of inhibitor 104 (cyan) in complex with LDHA (grey). NADH is also present and is depicted in dark blue. Hydrogen bonds are depicted as dashed yellow lines. The resolution of the structure is 2.00 Å.

Figure 6. Pharmacokinetic profile of 104 in rats following oral and iv administration. Error bars depict standard deviations (n = 3).

orientation (1) avoided steric clashes between the 2,6-dichlorosubstituents and the cyclohexenone ring, (2) afforded good van der Waals contacts between the phenyl ring and the side chain of LDHA residue Ile-241, and (3) positioned one of the Cl atoms in proximity to the NADH co-factor which was also observed in the co-crystal structure. The other Cl substituent contacted the side chain of LDHA residue Tyr-238, which moved closer to inhibitor 104 relative to its location in the LDHA-7 crystal structure. Interestingly, although inhibitor 104 contained a 2-Cl-thiophenyl moiety in place of the 2-nitro-thiophenyl group present in 7, the LDHA residues surrounding this portion of both inhibitors were crystallographically observed in virtually identical locations. These results were consistent with the structure–activity relationships described above which suggested that the cleft occupied by the 2-nitro/Cl-phenyl moiety was not conformationally flexible or

tolerant of most compound alterations. The LDHA-104 crystal structure illustrates that chemically similar inhibitors may adopt significantly different binding modes when complexed with the protein and therefore suggested that it would be prudent to continue to use crystallography to guide future compound optimization. A subset of the newly-identified compounds was profiled in various in vitro and in vivo assessments to better characterize the DMPK properties of this novel inhibitor series. As shown in Table 4, the tested compounds exhibited stabilities toward rat and human liver microsomes which generally correlated with their lipophilicities (lower lipophilicity = more stable). All tested molecules also displayed moderate or high permeability properties in MDCK assessments with the most polar of the compounds (18) providing the lowest value. The aqueous solubilities measured for the compounds in Table 4 were all favorable, and these characteristics were likely imparted by the acidic moieties contained within the molecules. Similarly, all tested compounds exhibited very high protein binding in plasma from humans and rats. In addition, all of the compounds in Table 4 did not display meaningful CYP P450 inhibition activity (IC50 >5 lM 3A4, 2D6, 2C9, 1A2; data not shown). As depicted in Figure 6 and Table 5, compound 104 exhibited good exposures and bioavailability in rats following oral administration with plasma levels persisting for extended time periods. The latter result was likely due to the compound’s high plasma protein binding properties which also afforded much lower free concentrations of the molecule. The PK data provide an initial assessment of how the in vitro DMPK results depicted in Table 4 may translate to in vivo outcomes and also highlight areas for possible subsequent compound improvement (e.g., increase free exposures). The described inhibitors were synthesized as depicted in Scheme 2.28 Condensation of acetone with benzaldehyde or various substituted analogs afforded olefins of general structure 121.29 The two-step conversion of these intermediates to the corresponding 3hydroxy-cyclohexenones 122 was subsequently accomplished using literature methods.30 Reaction of these cyclohexenones with N-bromosuccinimide afforded the bromides 123, and these entities were exposed to various thiols under slightly basic conditions to provide the desired products (general structure 124). In summary, a novel class of 3-hydroxy-2-mercaptocyclohex-2enone-containing LDHA inhibitors was identified using a high throughput screening approach. The crystal structure of a representative compound in complex with the protein indicated that these inhibitors bound in the LDHA active site in a manner that mimicked the pyruvate substrate. Structural modification of the initial high throughput screening hit afforded >10-fold improvement in LDHA biochemical potency. An optimized molecule (104) was not active against the structurally-related31 malate dehydrogenase 1 and malate dehydrogenase 2 enzymes (IC50 >100 lM) suggesting that the 3-hydroxy-2-mercaptocyclohex-2-enone inhibitor class would not indiscriminately interact with other dehydrogenase enzymes. The ability of some of the more potent compounds (104, 107, and 117) to inhibit the production of lactate in the HCC1954 cell line was also assessed.22 Unfortunately, none

Table 5 Pharmacokinetic parameters for compound 104 following iv and po administration to rats (n = 3) Route

Dosea (mg/kg)

AUCb (lM h)

Clplasmac (mL/min/kg)

Vssd (L)

T1/2e (h)

Ff (%) (lm)

iv po

0.5 2.0

60.3 142

0.35 NA

0.39 NA

13.2 10.7

NA 45

See Supplementary data for experimental descriptions. a Formulations: IV = 10:50:40 EtOH/PEG400/50 mM citrate pH = 3 (solution); PO = MCT (suspension). b Area under pharmacokinetic curve (0–24 h). c Clearance. d Volume of distribution (steady-state). e Half-life. f Bioavailability. NA = not applicable.

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O

O H

OH CH3

i

ii, iii

H

R

O

R

R

121

122 iv

OH

OH S

R'

Br

v

O R

124

O R

123

Scheme 2. Synthesis of 3-hydroxy-2-mercaptocyclohex-2-enone-containing compounds. Reagents and conditions: (i) acetone, 1% aq NaOH, 65 °C, 2 h; (ii) CH3ONa, diethyl malonate, CH3OH, reflux, 4 h; (iii) 6 N aq NaOH, THF, 80 °C, 1 h, then 12 M HCl, reflux, 1 h; (iv) NBS, CH3CN, 25 °C, 2 h; (v) R0 SH, piperidine, CH2Cl2, 0–25 °C, 12 h.

of the molecules displayed activity in this assay when tested up to the 50 lM concentration level. The reasons for this lack of cellbased activity are currently not known with certainty but may include: (1) insufficient biochemical potency, and/or (2) high protein binding. Additional efforts to improve the biochemical potency of the described inhibitors and to optimize their ability to inhibit LDHA in cell-culture experiments will be reported in due course. Acknowledgments We thank Drs. Krista Bowman and Jiansheng Wu and their respective Genentech research groups for protein expression and purification activities. We also thank Dr. Peter Jackson for many helpful discussion regarding LDHA. In addition, we thank Crystallographic Consulting, LLC for diffraction data collection. We acknowledge 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 Science under contracts DE-AC02-05CH11231 and DE-AC02-76SF00515, respectively. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.06. 076. References and notes 1. (a) Galluzzi, L.; Kepp, O.; Vander Heiden, M. G.; Kroemer, G. Nat. Rev. Drug Disc. 2013, 12, 829; (b) Ward, P. S.; Thompson, C. B. Cancer Cell 2012, 21, 297; (c) Vander Heiden, M. G. Nat. Rev. Drug Disc. 2011, 10, 671; (d) Zhao, Y.; Liu, H.; Riker, A. I.; Fodstad, O.; Ledoux, S. P.; Wilson, G. L.; Tan, M. Front. Biosci. 2011, 16, 1844; (e) Kaelin, W. G., Jr.; Thompson, C. B. Nature 2010, 465, 562; (f) Tennant, D. A.; Durán, R. V.; Gottlieb, E. Nat. Rev. Cancer 2010, 10, 267. 2. (a) Vander Heiden, M. G.; Cantley, L. C.; Thompson, C. B. Science 2009, 324, 1029; (b) Hsu, P. P.; Sabatini, D. M. Cell 2008, 134, 703. 3. (a) Warburg, O. Science 1956, 123, 309; (b) Bensinger, S. J.; Christofk, H. R. Semin. Cell Dev. Biol. 2012, 23, 352; (c) Koppenol, W. H.; Bounds, P. L.; Dang, C. V. Nat. Rev. Cancer 2011, 11, 325. 4. (a) Hamanaka, R. B.; Chandel, N. S. J. Exp. Med. 2012, 209, 211; (b) Jones, N. P.; Schulze, A. Drug Discovery Today 2011, 17, 232; (c) Pelicano, H.; Martin, D. S.; Xu, R.-H.; Huang, P. Oncogene 2006, 25, 4633. 5. Granchi, C.; Bertini, S.; Macchia, M.; Minutolo, F. Curr. Med. Chem. 2010, 17, 672. 6. Salaway, J. G. Metabolism at a Glance, 3rd ed.; Blackwell: Malden, 2004. pp 10– 25.

7. LDHA and LDHB are each homotetramers comprised of M and H subunits, respectively. LDH heterotetramers containing both M and H subunits are also known. For more information, see Ref. 5. 8. (a) Kolev, Y.; Uetake, H.; Takagi, Y.; Sugihara, K. Ann. Surg. Oncol. 2008, 15, 2336; (b) Koukourakis, M. I.; Giatromanolaki, A.; Sivridis, E.; Gatter, K. C.; Harris, A. L. J. Clin. Oncol. 2006, 24, 4301; (c) Koukourakis, M. I.; Giatromanolaki, A.; Sivridis, E.; Bougioukas, G.; Didilis, V.; Gatter, K. C.; Harris, A. L. Br. J. Cancer 2003, 89, 877. 9. (a) Seth, P.; Grant, A.; Tang, J.; Vinogradov, E.; Wang, X.; Lenkinski, R.; Sukhatme, V. P. Neoplasia 2011, 13, 60; (b) Qing, G.; Skuli, N.; Mayes, P. A.; Pawel, B.; Martinez, D.; Maris, J. M.; Simon, M. C. Cancer Res. 2010, 70, 10351; (c) Fantin, V. R.; St.-Pierre, J.; Leder, P. Cancer Cell 2006, 9, 425. 10. (a) Kanno, T.; Sudo, K.; Maekawa, M.; Nishimura, Y.; Ukita, M.; Fukutake, K. Clin. Chim. Acta 1988, 173, 89; (b) Kanno, T.; Sudo, K.; Takeuchi, I.; Kanda, S.; Honda, N.; Nishimura, Y.; Oyama, K. Clin. Chim. Acta 1980, 108, 267. 11. Dragovich, P. S.; Fauber, B.; Corson, L. B.; Ding, C. Z.; Eigenbrot, C.; Ge, H.; Giannetti, A. M.; Hunsaker, T.; Labadie, S.; Liu, Y.; Malek, S.; Pan, B.; Peterson, 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. 12. Fauber, B. P.; Dragovich, P. S.; Chen, J.; Corson, L. B.; Ding, C. Z.; Eigenbrot, C.; Giannetti, A. M.; Hunsaker, T.; Labadie, S.; Liu, Y.; Liu, Y. c.; Malek, S.; Peterson, D.; Pitts, K.; Sideris, S.; Ultsch, M.; Vander Porten, E.; Wang, J.; Wei, B. Q.; Yen, I.; Yue, Q. Bioorg. Med. Chem. Lett. 2013, 23, 5533. 13. Granchi, C.; Roy, S.; Giacomelli, C.; Macchia, M.; Tuccinardi, T.; Martinelli, A.; Lanza, M.; Betti, L.; Giannaccini, G.; Lucacchini, A.; Funel, N.; León, L. G.; Giovannetti, E.; Peters, G. J.; Palchaudhuri, R.; Calvaresi, E. C.; Hergenrother, P. J.; Minutolo, F. J. Med. Chem. 2011, 54, 1599. 14. Ward, R. A.; Brassington, C.; Breeze, A. L.; Caputo, A.; Critchlow, S.; Davies, G.; Goodwin, L.; Hassall, G.; Greenwood, R.; Holdgate, G. A.; Mrosek, M.; Norman, R. A.; Pearson, S.; Tart, J.; Tucker, J. A.; Vogtherr, M.; Whittaker, D.; Wingfield, J.; Winter, J.; Hudson, K. J. Med. Chem. 2012, 55, 3285. 15. (a) 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-Miller, L. A.; Mills, G. B.; Quinn, C. J.; Schneck, J. L.; Scott, G. F.; Shaw, A. N.; Waitt, G. M.; Wooster, R. F.; Duffy, K. J. Cancer Metab. 2013, 1, 19. http:// dx.doi.org/10.1186/2049-3002-1-19; (b) Xie, H.; Hanai, J.-i.; Ren, J.-G.; Kats, L.; Burgess, K.; Bhargava, P.; Signoretti, S.; Billard, J.; Duffy, K. J.; Grant, A.; Wang, X.; Lorkiewicz, P. K.; Schatzman, S.; Bousamra, M.; Lane, A. N.; Higashi, R. M.; Fan, T. W. M.; Pandolfi, P. P.; Sukhatme, V. P.; Seth, P. Cell Metab. 2014. http:// dx.doi.org/10.1016/j.cmet.2014.03.003; (c) Brown, K. K.; Chai, D.; Dodson, C. S.; Duffy, K. J.; Shaw, A. N. WO 2013/096151.; (d) Brown, K. K.; Chai, D.; Dodson, C. S.; Duffy, K. J.; Shaw, A. N. WO 2013/096153.; (e) Chai, D.; Colon, M.; Dodson, C.; Duffy, K. J.; Shaw, A. N. WO 2012/061557. 16. Kohlmann, A.; Zech, S. G.; Li, F.; Zhou, T.; Squillace, R. M.; Commodore, L.; Greenfield, M. T.; Lu, X.; Miller, D. P.; Huang, W.-S.; Qi, J.; Thomas, R. M.; Wang, Y.; Zhang, S.; Dodd, R.; Liu, S.; Xu, R.; Xu, Y.; Miret, J. J.; Rivera, V.; Clackson, T.; Shakespeare, W. C.; Zhu, X.; Dalgarno, D. C. J. Med. Chem. 2013, 56, 1023. 17. Inhibitors of Plasmodium falciparum LDH with potential for use as antimalarials have also been described: (a) Choi, S.-r.; Pradham, A.; Hammond, N. L.; Chittiboyina, A. G.; Tekwani, B. L.; Avery, M. A. J. Med. Chem. 2007, 50, 3841; (b) Choi, S.-r.; Beeler, A. B.; Pradham, A.; Watkins, E. B.; Rimoldi, J. M.; Tekwani, B.; Avery, M. A. J. Comb. Chem. 2007, 9, 292; (c) Cameron, A.; Read, J.; Tranter, R.; Winter, V. J.; Sessions, R. B.; Brady, R. L.; Vivas, L.; Easton, A.; Kendrick, H.; Croft, S. L.; Barros, D.; Lavandera, J. L.; Martin, J. J.; Risco, F.; García-Ochoa, S.; Gamo, F. J.; Sanz, L.; Leon, L.; Ruiz, J. R.; Gabarró, R.; Mallo, A.; Gómez de la Heras, F. J. Biol. Chem. 2004, 279, 31429.

P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3764–3771 18. The LDH residue numbering scheme used herein follows the convention of PDB entries 1I10 and 4AJP which does not account for the initiating methionine in the gene sequence reflected in UniProt entry P00338. This numbering scheme differs by one residue from that described elsewhere: Chung, F. Z.; Tsujibo, H.; Bhattacharyya, U.; Sharief, F. S.; Li, S. S. Biochem. J. 1985, 231, 537. 19. For the crystal structure of oxamate in complex with LDHA, see PDB entry 1I10 Read, J. A.; Winter, V. J.; Eszes, C. M.; Sessions, R. B.; Brady, R. L. Proteins 2001, 43, 175. 20. VanderPorten, E.; Frick, L.; Turincio, R.; Thana, P.; LaMarr, W.; Liu, Y. Anal. Biochem. 2013, 441, 115. 21. For the use of related dicarbonyl-containing compounds as inhibitors of HIV protease and HCV NS5B polymerase, see: (a) Li, H.; Tatlock, J.; Linton, A.; Gonzalez, J.; Borchardt, A.; Dragovich, P.; Jewell, T.; Zhou, R.; Blazel, J.; Parge, H.; Love, R.; Hickey, M.; Doan, C.; Shi, S.; Duggal, R.; Lewis, C.; Fuhrman, S. Bioorg. Med. Chem. Lett. 2006, 16, 4834; (b) Vara Prasad, J. V. N.; Boyer, F. E.; Domagala, J. M.; Ellsworth, E. L.; Gajda, C.; Hamilton, H. W.; Hagen, S. E.; Markoski, L. J.; Steinbaugh, B. A.; Tait, B. D.; Humblet, C.; Lunney, E. A.; Pavlovsky, A.; Rubin, J. R.; Ferguson, D.; Graham, N.; Holler, T.; Hupe, D.; Nouhan, C.; Tummino, P. J.; Urumov, A.; Zeikus, E.; Zeikus, G.; Gracheck, S. J.; Saunders, J. M.; VanderRoest, S.; Brodfuehrer, J.; Iyer, K.; Sinz, M.; Gulnik, S. V.; Erickson, J. W. Bioorg. Med. Chem. 1999, 7, 2775. 22. See Supplementary data for details regarding enzyme, SPR, and cell culture assay methods, associated errors, and controls. 23. No attempts were made to identify which, if any, compound tautomers predominated in organic and/or aqueous solutions. All new molecules described in this work are arbitrarily drawn and described as 3-hydroxy-cyclohexenones. 24. The LDHA inhibition mechanism of 7 may mimic substrate catalysis in which binding of NADH to the protein precedes binding of pyruvate. See Ref. 5 for additional details.

3771

25. See 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 deposited pdb files for additional structural details: compound 7 = 4QO7; compound 104 = 4QO8. 26. The described protein–ligand interactions can also be depicted using alternate tautomers and/or resonance forms of 7 and the corresponding LDHA side chains. 27. Quantum mechanical calculations were used to predict the preferred conformation of 5-phenyl of compound 7 in an unbound state [DFT-B3LYP with 6-31G⁄⁄ basis set either in gas phase or with a Poisson–Boltzmann continuum solvation model (PBF) as implemented in Jaguar (Schrödinger)]. In vacuum, the phenyl group of 7 was found to prefer an equatorial location over the axial alternative by 1.3 kcal/mol in conformational energy. This equatorial preference remained the same when solvation energy was taken into account, although the calculated energy difference was reduced to 0.6 kcal/mol. 28. Compound 7 can be purchased from eMolecules. 29. The condensation with benzaldehyde itself was carried out at 0 °C instead of at elevated temperature. 30. Tamura, Y.; Yoshimoto, Y.; Kunimoto, K.; Tada, S.; Tomita, T.; Wada, T.; Seto, E.; Murayama, M.; Shibata, Y.; Nomura, A.; Ohata, K. J. Med. Chem. 1977, 20, 709. 31. Human LDHA and human MDH-2 protein sequences share 20% similarity. Xray structures of their protomers can be superimposed using 173 pairs of Ca atoms with an rms deviation of 1.7 Å. Ca atoms within 5 Å of bound NADH can be superimposed with an rms deviation of 0.7 Å.