Ryan Greenwood

Design and Synthesis of Novel Lactate Dehydrogenase A Inhibitors by Fragment-Based Lead Generation

Lactate dehydrogenase A (LDHA) catalyzes the conversion of pyruvate to lactate, utilizing NADH as a cofactor. It has been identified as a potential therapeutic target in the area of cancer metabolism. In this manuscript we report our progress using fragment-based lead generation (FBLG), assisted by X-ray crystallography to develop small molecule LDHA inhibitors. Fragment hits were identified through NMR and SPR screening and optimized into lead compounds with nanomolar binding affinities via fragment linking. Also reported is their modification into cellular active compounds suitable for target validation work.

Discovery of 4-Amino-N-[(1S)-1-(4-chlorophenyl)-3-hydroxypropyl]-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide (AZD5363), an Orally Bioavailable, Potent Inhibitor of Akt Kinases.

Wide-ranging exploration of analogues of an ATP-competitive pyrrolopyrimidine inhibitor of Akt led to the discovery of clinical candidate AZD5363, which showed increased potency, reduced hERG affinity, and higher selectivity against the closely related AGC kinase ROCK. This compound demonstrated good preclinical drug metabolism and pharmacokinetics (DMPK) properties and, after oral dosing, showed pharmacodynamic knockdown of phosphorylation of Akt and downstream biomarkers in vivo, and inhibition of tumor growth in a breast cancer xenograft model.

Structure-Guided Design of Highly Selective and Potent Covalent Inhibitors of Erk1/2.

The RAS/RAF/MEK/ERK signaling pathway has been targeted with a number of small molecule inhibitors in oncology clinical development across multiple disease indications. Importantly, cell lines with acquired resistance to B-RAF and MEK inhibitors have been shown to maintain sensitivity to ERK1/2 inhibition by small molecule inhibitors. There are a number of selective, noncovalent ERK1/2 inhibitors reported along with the promiscuous hypothemycin (and related analogues) that act via a covalent mechanism of action. This article reports the identification of multiple series of highly selective covalent ERK1/2 inhibitors informed by structure-based drug design (SBDD). As a starting point for these covalent inhibitors, reported ERK1/2 inhibitors and a chemical series identified via high-throughput screening were exploited. These approaches resulted in the identification of selective covalent tool compounds for potential in vitro and in vivo studies to assess the risks and or benefits of targeting this pathway through such a mechanism of action.

Discovery of AZD3147: A Potent, Selective Dual Inhibitor of mTORC1 and mTORC2

High throughput screening followed by a lead generation campaign uncovered a novel series of urea containing morpholinopyrimidine compounds which act as potent and selective dual inhibitors of mTORC1 and mTORC2. We describe the continued compound optimization campaign for this series, in particular focused on identifying compounds with improved cellular potency, improved aqueous solubility, and good stability in human hepatocyte incubations. Knowledge from empirical SAR investigations was combined with an understanding of the molecular interactions in the crystal lattice to improve both cellular potency and solubility, and the composite parameters of LLE and pIC50-pSolubility were used to assess compound quality and progress. Predictive models were employed to efficiently mine the attractive chemical space identified resulting in the discovery of 42 (AZD3147), an extremely potent and selective dual inhibitor of mTORC1 and mTORC2 with physicochemical and pharmacokinetic properties suitable for development as a potential clinical candidate.

Structure-Guided Discovery of Potent and Selective Inhibitors of ERK1/2 from a Modestly Active and Promiscuous Chemical Start Point.

There are a number of small-molecule inhibitors targeting the RAS/RAF/MEK/ERK signaling pathway that have either been approved or are in clinical development for oncology across a range of disease indications. The inhibition of ERK1/2 is of significant current interest, as cell lines with acquired resistance to BRAF and MEK inhibitors have been shown to maintain sensitivity to ERK1/2 inhibition in preclinical models. This article reports on our recent work to identify novel, potent, and selective reversible ERK1/2 inhibitors from a low-molecular-weight, modestly active, and highly promiscuous chemical start point, compound 4. To guide and inform the evolution of this series, inhibitor binding mode information from X-ray crystal structures was critical in the rapid exploration of this template to compound 35, which was active when tested in in vivo antitumor efficacy experiments.

Novel and versatile synthesis of disubstituted 1,2-dihydro-1,2,4-triazol-3-ones.

A novel method for the synthesis of a wide range of 1,5-disubstituted 1,2-dihydro-1,2,4-triazol-3-ones is described. The key step involves a reaction between a dilithiated BOC-hydrazine and a N-alkoxycarbonylcarboximidothioate. A broad range of aryl and alkyl functional groups are tolerated, providing a versatile route for the synthesis of triazolones.

Identification and optimisation of a series of N-(4-anilino-2-pyridyl)acetamide activin receptor-like kinase 1 (ALK1) inhibitors

A novel class of N-(4-anilino-2-pyridyl)amide based activin receptor-like kinase (ALK1) inhibitors are disclosed, which were rapidly optimised to a ligand efficient probe compound 21 with good potency in enzyme (4 nM) and cell (45 nM) assays and favourable physical and pharmacokinetic properties (24 h free cover over cell IC50 after a single 50 mg kg−1 dose in nude mice). This was achieved by identifying a small, ligand efficient group in the solvent channel (C2) whilst optimising the selectivity pocket (C4) group for enzyme and cell potency, using related SAR that has been observed previously for Src inhibitors.

Abstract 4478: Discovery of AZD5363, an orally bioavailable, potent ATP-competitive inhibitor of AKT kinases

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL AKT is a key node in the most frequently de-regulated signaling pathway in human cancer and has been shown to mediate resistance to a range of cytotoxic, anti-hormonal and targeted therapies. We decided to explore inhibitors of AKT as potential new anti-cancer therapeutics. Here we disclose for the first time the discovery and structure of AZD5363, an orally bioavailable, potent ATP-competitive inhibitor of AKT. We evaluated a range of chemical starting points arising from our previous collaboration with the Institute of Cancer Research and Astex Therapeutics Ltd. Ultimately AZD5363 was discovered following a long journey that started from a pyrrolopyrimidine series of compounds. Our first challenge was to improve potency and a second challenge was to improve ROCK selectivity. ROCK is an AGC kinase like AKT but is involved in regulation of vascular tone and thus blood pressure. Extensive SAR studies exploring the series revealed that achieving selectivity over ROCK while retaining AKT potency was quite challenging. Eventually we discovered ways which could improve both selectivity and potency. However, these compounds had significant activity against the hERG ion channel which is implicated in the development of Torsades de Pointes and cardiac death. The next phase of work therefore had to focus on reducing hERG activity, while at the same time not adversely impacting either AKT potency or ROCK selectivity. Finally we discovered that introduction of a key substituent group provided a compound that achieved reduced hERG potency and, surprisingly, also achieved a further small improvement in both AKT potency and ROCK selectivity. This compound was AZD5363. A crystal structure of AZD5363 bound to AKT has revealed some of the key interactions that may contribute to its potency. For example, the pyrrolopyrimidine appears to form hydrogen bonds to the hinge region of the kinase. AZD5363 inhibits all known AKT isoforms with a potency of <10 nM and inhibits phosphorylation of the AKT substrate, PRAS40 in BT474c cells with a potency of 0.31 μM. Activity in in vivo pharmacodynamic and xenograft models has also been demonstrated. A synthetic route suitable for scale-up has been developed. In conclusion, AZD5363 is a potent inhibitor of AKT in vitro and in cells. It has good hERG and ROCK selectivity. It has pharmacodynamic and xenograft activity in vivo. AZD5363 has potential in cancer therapy and is currently in phase 1 clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4478. doi:10.1158/1538-7445.AM2011-4478