Structure-based optimization of hydroxylactam as potent, cell-active inhibitors of lactate dehydrogenase.

Structure-based design was utilized to optimize 6,6-diaryl substituted dihydropyrone and hydroxylactam to obtain inhibitors of lactate dehydrogenase (LDH) with low nanomolar biochemical and single-digit micromolar cellular potencies. Surprisingly the replacement of a phenyl with a pyridyl moiety in the chemical structure revealed a new binding mode for the inhibitors with subtle conformational change of the LDHA active site. This led to the identification of a potent, cell-active hydroxylactam inhibitor exhibiting an in vivo pharmacokinetic profile suitable for mouse tumor xenograft study.

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

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.

Metabolic plasticity underpins innate and acquired resistance to LDHA inhibition.

Metabolic reprogramming in tumors represents a potential therapeutic target. Herein we used shRNA depletion and a novel lactate dehydrogenase (LDHA) inhibitor, GNE-140, to probe the role of LDHA in tumor growth in vitro and in vivo. In MIA PaCa-2 human pancreatic cells, LDHA inhibition rapidly affected global metabolism, although cell death only occurred after 2 d of continuous LDHA inhibition. Pancreatic cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis were inherently resistant to GNE-140, but could be resensitized to GNE-140 with the OXPHOS inhibitor phenformin. Acquired resistance to GNE-140 was driven by activation of the AMPK-mTOR-S6K signaling pathway, which led to increased OXPHOS, and inhibitors targeting this pathway could prevent resistance. Thus, combining an LDHA inhibitor with compounds targeting the mitochondrial or AMPK-S6K signaling axis may not only broaden the clinical utility of LDHA inhibitors beyond glycolytically dependent tumors but also reduce the emergence of resistance to LDHA inhibition.

Discovery of (S)-1-(1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)pyridin-2(1H)-one (GDC-0994), an Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibitor in Early Clinical Development.

The extracellular signal-regulated kinases ERK1/2 represent an essential node within the RAS/RAF/MEK/ERK signaling cascade that is commonly activated by oncogenic mutations in BRAF or RAS or by upstream oncogenic signaling. While targeting upstream nodes with RAF and MEK inhibitors has proven effective clinically, resistance frequently develops through reactivation of the pathway. Simultaneous targeting of multiple nodes in the pathway, such as MEK and ERK, offers the prospect of enhanced efficacy as well as reduced potential for acquired resistance. Described herein is the discovery and characterization of GDC-0994 (22), an orally bioavailable small molecule inhibitor selective for ERK kinase activity.

Discovery of imidazo[1,5-a]pyridines and -pyrimidines as potent and selective RORc inverse agonists.

The nuclear receptor (NR) retinoic acid receptor-related orphan receptor gamma (RORγ, RORc, or NR1F3) is a promising target for the treatment of autoimmune diseases. RORc is a critical regulator in the production of the pro-inflammatory cytokine interleukin-17. We discovered a series of potent and selective imidazo[1,5-a]pyridine and -pyrimidine RORc inverse agonists. The most potent compounds displayed >300-fold selectivity for RORc over the other ROR family members, PPARγ, and NRs in our cellular selectivity panel. The favorable potency, selectivity, and physiochemical properties of GNE-0946 (9) and GNE-6468 (28), in addition to their potent suppression of IL-17 production in human primary cells, support their use as chemical biology tools to further explore the role of RORc in human biology.

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

Optimization of 5-(2,6-dichlorophenyl)-3-hydroxy-2-mercaptocyclohex-2-enone using structure-based design strategies resulted in inhibitors with considerable improvement in biochemical potency against human lactate dehydrogenase A (LDHA). These potent inhibitors were typically selective for LDHA over LDHB isoform (4­10 fold) and other structurally related malate dehydrogenases, MDH1 and MDH2 (>500 fold). An X-ray crystal structure of enzymatically most potent molecule bound to LDHA revealed two additional interactions associated with enhanced biochemical potency.

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

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

Structure based design of novel 6,5 heterobicyclic mitogen-activated protein kinase kinase (MEK) inhibitors leading to the discovery of imidazo[1,5-a] pyrazine G-479.

Use of the tools of SBDD including crystallography led to the discovery of novel and potent 6,5 heterobicyclic MEKi's [J. Med. Chem.2012, 55, 4594]. The core change from a 5,6 heterobicycle to a 6,5 heterobicycle was driven by the desire for increased structural diversity and aided by the co-crystal structure of G-925 [J. Med. Chem.2012, 55, 4594]. The key design feature was the shift of the attachment of the five-membered heterocyclic ring towards the B ring while maintaining the key hydroxamate and anilino pharamcophoric elements in a remarkably similar position as in G-925. From modelling, changing the connection point of the five membered ring heterocycle placed the H-bond accepting nitrogen within a good distance and angle to the Ser212 [J. Med. Chem.2012, 55, 4594]. The resulting novel 6,5 benzoisothiazole MEKi G-155 exhibited improved potency versus aza-benzofurans G-925 and G-963 but was a potent inhibitor of cytochrome P450's 2C9 and 2C19. Lowering the logD by switching to the more polar imidazo[1,5-a] pyridine core significantly diminished 2C9/2C19 inhibition while retaining potency. The imidazo[1,5-a] pyridine G-868 exhibited increased potency versus the starting point for this work (aza-benzofuran G-925) leading to deprioritization of the azabenzofurans. The 6,5-imidazo[1,5-a] pyridine scaffold was further diversified by incorporating a nitrogen at the 7 position to give the imidazo[1,5-a] pyrazine scaffold. The introduction of the C7 nitrogen was driven by the desire to improve metabolic stability by blocking metabolism at the C7 and C8 positions (particularly the HLM stability). It was found that improving on G-868 (later renamed GDC-0623) required combining C7 nitrogen with a diol hydroxamate to give G-479. G-479 with polarity distributed throughout the molecule was improved over G-868 in many aspects.

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

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 µM) 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 µM). 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%).

Discovery and preclinical development of vismodegib.

INTRODUCTION: Vismodegib is the first Hedgehog (Hh) pathway inhibitor approved in the US for the treatment of adults with metastatic or locally advanced basal cell carcinoma (BCC). It was approved by the US FDA on 30 January 2012, and by the European Commission on 12 July 2013, for the treatment of adult patients with symptomatic metastatic BCC, or locally advanced BCC inappropriate for surgery or radiotherapy. Vismodegib selectively inhibits the Hh signaling pathway, binding to and inhibiting a critical signal-transducing component of the pathway, Smoothened (SMO). Vismodegib was discovered by Genentech, Inc., under a collaboration agreement with Curis, Inc. AREAS COVERED: This article reviews the development of vismodegib from its discovery, preclinical pharmacology and validation to the clinical pharmacokinetics and validation in Phase I and II clinical investigations. We also provide a survey of other Hh pathway inhibitors in clinical development. EXPERT OPINION: The authors' experience in target-based drug discovery suggests that vismodegib's path to the clinic deserves some reflection to identify key steps that have contributed to its success. Targeting the Hh pathway with vismodegib blocks the abberant signaling caused by mutational inactivation of the negative regulator PTCH1 or mutational activation of SMO. Vismodegib gives physicians a treatment option for patients with locally advanced or metastatic BCC for whom surgery or radiation is not recommended.

A hit to lead discovery of novel N-methylated imidazolo-, pyrrolo-, and pyrazolo-pyrimidines as potent and selective mTOR inhibitors.

A series of N-7-methyl-imidazolopyrimidine inhibitors of the mTOR kinase have been designed and prepared, based on the hypothesis that the N-7-methyl substituent on imidazolopyrimidine would impart selectivity for mTOR over the related PI3Kα and δ kinases. The corresponding N-Me substituted pyrrolo[3,2-d]pyrimidines and pyrazolo[4,3-d]pyrimidines also show potent mTOR inhibition with selectivity toward both PI3α and δ kinases. The most potent compound synthesized is pyrazolo[4,3-d]pyrimidine 21c. Compound 21c shows a Ki of 2 nM against mTOR inhibition, remarkable selectivity (>2900×) over PI3 kinases, and excellent potency in cell-based assays.

Preclinical disposition of GDC-0973 and prospective and retrospective analysis of human dose and efficacy predictions.

[3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-((S)-3-hydroxy-3-piperidin-2-yl-azetidin-1-yl)-methanone (GDC-0973) is a potent and highly selective inhibitor of mitogen-activated protein kinase(MAPK)/extracellular signal-regulated kinase (ERK) 1/2 (MEK1/2), a MAPK kinase that activates ERK1/2. The objectives of these studies were to characterize the disposition of GDC-0973 in preclinical species and to determine the relationship of GDC-0973 plasma concentrations to efficacy in Colo205 mouse xenograft models. The clearance (CL) of GDC-0973 was moderate in mouse (33.5 ml · min(-1) · kg(-1)), rat (37.9 ± 7.2 ml · min(-1) · kg(-1)), and monkey (29.6 ± 8.5 ml · min(-1) · kg(-1)). CL in dog was low (5.5 ± 0.3 ml · min(-1) · kg(-1)). The volume of distribution across species was large, 6-fold to 15-fold body water; half-lives ranged from 4 to 13 h. Protein binding in mouse, rat, dog, monkey, and human was high, with percentage unbound, 1 to 6%. GDC-0973-related radioactivity was rapidly and extensively distributed to tissues; however, low concentrations were observed in the brain. In rats and dogs, [(14)C]GDC-0973 was well absorbed (fraction absorbed, 70-80%). The majority of [(14)C]GDC-0973-related radioactivity was recovered in the bile of rat (74-81%) and dog (65%). The CL and volume of distribution of GDC-0973 in human, predicted by allometry, was 2.9 ml · min(-1) · kg(-1) and 9.9 l/kg, respectively. The predicted half-life was 39 h. To characterize the relationship between plasma concentration of GDC-0973 and tumor growth inhibition, pharmacokinetic-pharmacodynamic modeling was applied using an indirect response model. The KC(50) value for tumor growth inhibition in Colo205 xenografts was estimated to be 0.389 µM, and the predicted clinical efficacious dose was ∼10 mg. Taken together, these data are useful in assessing the disposition of GDC-0973, and where available, comparisons with human data were made.

Potent, selective, and orally bioavailable inhibitors of mammalian target of rapamycin (mTOR) kinase based on a quaternary substituted dihydrofuropyrimidine.

A series of inhibitors of mTOR kinase based on a quaternary-substituted dihydrofuropyrimidine was designed and synthesized. The most potent compounds in this series inhibited mTOR kinase with K(i) < 1.0 nM and were highly (>100×) selective for mTOR over the closely related PI3 kinases. Compounds in this series showed inhibition of the pathway and antiproliferative activity in cell-based assays. Furthermore, these compounds had excellent mouse PK, and showed a robust PK-PD relationship in a mouse model of cancer.

Small molecule inhibition of GDC-0449 refractory smoothened mutants and downstream mechanisms of drug resistance.

Inappropriate Hedgehog (Hh) signaling has been directly linked to medulloblastoma (MB), a common malignant brain tumor in children. GDC-0449 is an Hh pathway inhibitor (HPI) currently under clinical investigation as an anticancer agent. Treatment of a MB patient with GDC-0449 initially regressed tumors, but this individual ultimately relapsed with a D473H resistance mutation in Smoothened (SMO), the molecular target of GDC-0449. To explore the role of the mutated aspartic acid residue in SMO function, we substituted D473 with every amino acid and found that all functional mutants were resistant to GDC-0449, with positively charged residues conferring potential oncogenic properties. Alanine scan mutagenesis of SMO further identified E518 as a novel prospective mutation site for GDC-0449 resistance. To overcome this form of acquired resistance, we screened a panel of chemically diverse HPIs and identified several antagonists with potent in vitro activity against these GDC-0449-resistant SMO mutants. The bis-amide compound 5 was of particular interest, as it was able to inhibit tumor growth mediated by drug resistant SMO in a murine allograft model of MB. However, focal amplifications of the Hh pathway transcription factor Gli2 and the Hh target gene cyclin D1 (Ccnd1) were observed in two additional resistant models, indicating that resistance may also occur downstream of SMO. Importantly, these HPI resistant MB allografts retained their sensitivity to PI3K inhibition, presenting additional opportunities for the treatment of such tumors.

Second generation 2-pyridyl biphenyl amide inhibitors of the hedgehog pathway.

Potent and efficacious inhibitors of the hedgehog pathway for the treatment of cancer have been prepared using the 2-pyridyl biphenyl amide scaffold common to the clinical lead GDC-0449. Analogs with polar groups in the para-position of the aryl amide ring optimized potency, had minimal CYP inhibition, and possessed good exposure in rats. Compounds 9d and 14f potently inhibited hedgehog signaling as measured by Gli1 mRNA and were found to be equivalent or more potent than GDC-0449, respectively, when studied in a Ptch(+/-) medulloblastoma allograft model, that is, highly dependent on hedgehog signaling.

Preclinical disposition and pharmacokinetics-pharmacodynamic modeling of biomarker response and tumour growth inhibition in xenograft mouse models of G-573, a MEK inhibitor.

The mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) pathway is a key signalling pathway that regulates cell proliferation. G-573 is an allosteric inhibitor of MEK that is both potent and selective. The objectives of these studies were to characterize the disposition of G-573 in preclinical species and to determine the relationship of G-573 plasma concentrations to pERK (phosphorylated ERK) and to tumour growth inhibition in HCT116 and H2122 mouse xenograft models. The clearance of G-573 was low in mouse (7.7 ml/min/kg), rat (2.24 ml/min/kg), dog (10 ml/min/kg), and cynomolgus monkey (0.754 ml/min/kg) while volumes of distribution (0.114-1.77 l/kg) was low to moderate, resulting in moderate half-lives across species (~2-9 h). Indirect response models were used to characterize the relationship between plasma concentration of G-573 to both pERK inhibition and tumour growth inhibition. The IC(50) value for pERK inhibition in HCT116 tumours by G-573 was estimated to be 0.406 µM. The IC(50) values for tumour growth inhibition in HCT116 and H2122 were estimated to be 3.43 and 2.56 µM, respectively. ED(50) estimates in HCT116 and H2122 mouse xenograft models were estimated to be ~4.6 and 1.9 mg/kg/day, respectively. The information from these studies provides useful information when characterizing candidates for potential further clinical testing.

GDC-0449-a potent inhibitor of the hedgehog pathway.

SAR for a wide variety of heterocyclic replacements for a benzimidazole led to the discovery of functionalized 2-pyridyl amides as novel inhibitors of the hedgehog pathway. The 2-pyridyl amides were optimized for potency, PK, and drug-like properties by modifications to the amide portion of the molecule resulting in 31 (GDC-0449). Amide 31 produced complete tumor regression at doses as low as 12.5mg/kg BID in a medulloblastoma allograft mouse model that is wholly dependent on the Hh pathway for growth and is currently in human clinical trials, where it is initially being evaluated for the treatment of BCC.

A selective, slow binding inhibitor of factor VIIa binds to a nonstandard active site conformation and attenuates thrombus formation in vivo.

The serine protease factor VIIa (FVIIa) in complex with its cellular cofactor tissue factor (TF) initiates the blood coagulation reactions. TF.FVIIa is also implicated in thrombosis-related disorders and constitutes an appealing therapeutic target for treatment of cardiovascular diseases. To this end, we generated the FVIIa active site inhibitor G17905, which displayed great potency toward TF.FVIIa (Ki = 0.35 +/- 0.11 nM). G17905 did not appreciably inhibit 12 of the 14 examined trypsin-like serine proteases, consistent with its TF.FVIIa-specific activity in clotting assays. The crystal structure of the FVIIa.G17905 complex provides insight into the molecular basis of the high selectivity. It shows that, compared with other serine proteases, FVIIa is uniquely equipped to accommodate conformational disturbances in the Gln217-Gly219 region caused by the ortho-hydroxy group of the inhibitor's aminobenzamidine moiety located in the S1 recognition pocket. Moreover, the structure revealed a novel, nonstandard conformation of FVIIa active site in the region of the oxyanion hole, a "flipped" Lys192-Gly193 peptide bond. Macromolecular substrate activation assays demonstrated that G17905 is a noncompetitive, slow-binding inhibitor. Nevertheless, G17905 effectively inhibited thrombus formation in a baboon arterio-venous shunt model, reducing platelet and fibrin deposition by approximately 70% at 0.4 mg/kg + 0.1 mg/kg/min infusion. Therefore, the in vitro potency of G17905, characterized by slow binding kinetics, correlated with efficacious antithrombotic activity in vivo.