Structure-Guided Chemical Optimization of Bicyclic Peptide (Bicycle) Inhibitors of Angiotensin-Converting Enzyme 2.

Angiotensin-converting enzyme 2 (ACE2) is a metalloprotease that cleaves angiotensin II, a peptide substrate involved in the regulation of hypertension. Here, we identified a series of constrained bicyclic peptides, Bicycle, inhibitors of human ACE2 by panning highly diverse bacteriophage display libraries. These were used to generate X-ray crystal structures which were used to inform the design of additional Bicycles with increased affinity and inhibition of ACE2 enzymatic activity. This novel structural class of ACE2 inhibitors is among the most potent ACE2 inhibitors yet described in vitro, representing a valuable tool to further probe ACE2 function and for potential therapeutic utility.

BT8009; A Nectin-4 Targeting Bicycle Toxin Conjugate for Treatment of Solid Tumors.

Multiple tumor types overexpress Nectin-4 and the antibody-drug conjugate (ADC), enfortumab vedotin (EV) shows striking efficacy in clinical trials for metastatic urothelial cancer, which expresses high levels of Nectin-4, validating Nectin-4 as a clinical target for toxin delivery in this indication. Despite excellent data in urothelial cancer, little efficacy data are reported for EV in other Nectin-4 expressing tumors and EV therapy can produce significant toxicities in many patients, frequently leading to discontinuation of treatment. Thus, additional approaches to this target with the potential to extend utility and reduce toxicity are warranted. We describe the preclinical development of BT8009, a "Bicycle Toxin Conjugate" (BTC) consisting of a Nectin-4-binding bicyclic peptide, a cleavable linker system and the cell penetrant toxin mono-methylauristatin E (MMAE). BT8009 shows significant antitumor activity in preclinical tumor models, across a variety of cancer indications and is well tolerated in preclinical safety studies. In several models, it shows superior or equivalent antitumor activity to an EV analog. As a small hydrophilic peptide-based drug BT8009 rapidly diffuses from the systemic circulation, through tissues to penetrate the tumor and target tumor cells. It is renally eliminated from the circulation, with a half-life of 1-2 hours in rat and non-human primate. These physical and PK characteristics differentiate BT8009 from ADCs and may provide benefit in terms of tumor penetration and reduced systemic exposure. BT8009 is currently in a Phase 1/2 multicenter clinical trial across the US, Canada, and Europe, enrolling patients with advanced solid tumors associated with Nectin-4 expression.

Heteroalicyclic carboxamidines as inhibitors of inducible nitric oxide synthase; the identification of (2R)-2-pyrrolidinecarboxamidine as a potent and selective haem-co-ordinating inhibitor.

Heteroalicyclic carboxamidines were synthesised and evaluated as inhibitors of nitric oxide synthases. (2R)-2-Pyrrolidinecarboxamidine, in particular, was shown to be a highly potent in vitro (IC(50)=0.12 µM) and selective iNOS inhibitor (>100-fold vs both eNOS and nNOS), with probable binding to the key anchoring glutamate residue and co-ordination to the haem iron.

Synthesis and biological activity of a series of tetrasubstituted-imidazoles as P2X(7) antagonists.

A series of analogues of the pyrazole lead 1 were synthesized in which the heterocyclic core was replaced with an imidazole. A number of potent antagonists were identified and structure-activity relationships (SAR) were investigated both with respect to activity at the P2X(7) receptor and in vitro metabolic stability. Compound 10 was identified as a potent P2X(7) antagonist with reduced in vitro metabolism and high solubility.

Structure-activity relationships and in vivo activity of (1H-pyrazol-4-yl)acetamide antagonists of the P2X(7) receptor.

Structure-activity relationships (SAR) of analogues of lead compound 1 were investigated and compound 16 was selected for further study in animal models of pain. Compound 16 was shown to be a potent antihyperalgesic agent in both the rat acute complete Freund's adjuvant (CFA) model of inflammatory pain [Iadarola, M. J.; Douglass, J.; Civelli, O.; Naranjo, J. R. rain Res.1988, 455, 205] and the knee joint model of chronic inflammatory pain [Wilson, A. W.; Medhurst, S. J.; Dixon, C. I.; Bontoft, N. C.; Winyard, L. A.; Brackenborough, K. T.; De Alba, J.; Clarke, C. J.; Gunthorpe, M. J.; Hicks, G. A.; Bountra, C.; McQueen, D. S.; Chessell, I. P. Eur. J. Pain2006, 10, 537].

Synthesis and structure-activity relationships of a series of (1H-pyrazol-4-yl)acetamide antagonists of the P2X7 receptor.

High-throughput screening identified compound 1 as a potent P2X(7) receptor antagonist suitable for lead optimisation. Structure-activity relationships (SAR) of a series of (1H-pyrazol-4-yl)acetamides were investigated and compound 32 was identified as a potent P2X(7) antagonist with enhanced potency and favourable physicochemical and pharmacokinetic properties.

Synthesis and evaluation of 3-amino-6-aryl-pyridazines as selective CB(2) agonists for the treatment of inflammatory pain.

A series of 3-amino-6-aryl-pyridazines have been identified as CB(2) agonists with high efficacy and selectivity against the CB(1) receptor. Details of the investigation of structure-activity relationships (SAR) are disclosed, which led to the identification of pyridazine analogue 35, a compound with high potency in an in vivo model of inflammatory pain.

Identification of 2-oxo-N-(phenylmethyl)-4-imidazolidinecarboxamide antagonists of the P2X(7) receptor.

A backup molecule to compound 2 was sought by targeting the most likely metabolically vulnerable site in this molecule. Compound 18 was subsequently identified as a potent P2X(7) antagonist with very low in vivo clearance and high oral bioavailability in all species examined. Some evidence to support the role of P2X(7) in the etiology of pain is also presented.

Discovery and structure-activity relationships of a series of pyroglutamic acid amide antagonists of the P2X7 receptor.

A computational lead-hopping exercise identified compound 4 as a structurally distinct P2X(7) receptor antagonist. Structure-activity relationships (SAR) of a series of pyroglutamic acid amide analogues of 4 were investigated and compound 31 was identified as a potent P2X(7) antagonist with excellent in vivo activity in animal models of pain, and a profile suitable for progression to clinical studies.

2-Amino-5-aryl-pyridines as selective CB2 agonists: synthesis and investigation of structure-activity relationships.

2-Amino-5-aryl-pyridines, exemplified by compound 1, had been identified as a synthetically tractable series of CB(2) agonists from a high-throughput screen of the GlaxoSmithKline compound collection. Described herein are the results of an investigation of the structure-activity relationships (SAR) which led to the identification a number of potent and selective agonists.

Identification and optimisation of a novel series of pyrimidine based cyclooxygenase-2 (COX-2) inhibitors. Utilisation of a biotransformation approach.

Many years of work have been invested in the identification of potent and selective COX-2 inhibitors for the treatment of chronic inflammatory pain. One issue faced by workers is the balance between the lipophilicity required for potent enzyme inhibition and the physical properties necessary for drug absorption and distribution in vivo. Frequently approaches to reduce lipophilicity through introduction of polar functionality is hampered by highly challenging chemistry to prepare key molecules. We have complemented traditional synthetic chemistry with a biotransformations approach which efficiently provided access to an array of key target molecules.

Identification of [4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)-2-pyrimidinyl] amines and ethers as potent and selective cyclooxygenase-2 inhibitors.

A novel series of [4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)-2-pyrimidine-based cyclooxygenase-2 (COX-2) inhibitors, which have a different arrangement of substituents compared to the more common 1,2-diarylheterocycle based molecules, have been discovered. For example, 2-(butyloxy)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)pyrimidine (47), a member of the 2-pyrimidinyl ether series, has been shown to be a potent and selective inhibitor with a favourable pharmacokinetic profile, high brain penetration and good efficacy in rat models of hypersensitivity.

The role of the cylooxygenase pathway in nociception and pain.

Cycloxygenase (COX) pathways have long been targeted for the treatment of inflammatory pain, initially through the use of NSAIDs. With the demonstration of two major COX isoforms, COX-1 and COX-2, involved in the production of prostanoids, but with different distribution and regulation, selective COX-2 inhibitors have been developed. This review covers factors influencing COX enzyme activity, the role of their products in the development and maintenance of pain and discusses recent safety concerns of COX-2 inhibitors.

The cyclooxygenase-2 inhibitor GW406381X [2-(4-ethoxyphenyl)-3-[4-(methylsulfonyl)phenyl]-pyrazolo[1,5-b]pyridazine] is effective in animal models of neuropathic pain and central sensitization.

The pathogenic form of the cyclooxygenase (COX) enzyme, COX-2, is also constitutively present in the spinal cord and has been implicated in chronic pain states in rat and man. A number of COX-2 inhibitors, including celecoxib and rofecoxib, are already used in man for the treatment of inflammatory pain. Preclinically, the dual-acting COX-2 inhibitor, GW406381X [2-(4-ethoxyphenyl)-3-[4-(methylsulfonyl)phenyl]-pyrazolo[1,5-b]pyridazine, where X denotes the free base], is as effective as rofecoxib and celecoxib in the rat established Freund's Complete Adjuvant model with an ED(50) of 1.5 mg/kg p.o. compared with 1.0 mg/kg p.o. for rofecoxib and 6.6 mg/kg p.o. for celecoxib. However, in contrast to celecoxib (5 mg/kg p.o. b.i.d.) and rofecoxib (5 mg/kg p.o. b.i.d.), which were without significant effect, GW406381X (5 mg/kg p.o. b.i.d.) fully reversed mechanical allodynia in the chronic constriction injury model and reversed thermal hyperalgesia in the mouse partial ligation model, both models of neuropathic pain. GW406381X, was also effective in a rat model of capsaicin-induced central sensitization, when given intrathecally (ED(50) = 0.07 mug) and after chronic but not acute oral dosing. Celecoxib and rofecoxib had no effect in this model. Several hypotheses have been proposed to try to explain these differences in efficacy, including central nervous system penetration, enzyme kinetics, and potency. The novel finding of effectiveness of GW406381X in these models of neuropathic pain/central sensitization, in addition to activity in inflammatory pain models and together with its central efficacy, suggests dual activity of GW406381X compared with celecoxib and rofecoxib, which may translate into greater efficacy in a broader spectrum of pain states in the clinic.