An efficient and concise synthesis of a selective small molecule non-peptide inhibitor of cathepsin L: KGP94

Highlights

• New routes include the synthesis of a key phenolic-protected, functionalized benzophenone intermediate.

• An alternative approach enables access to the new analogue.

• Inexpensive starting materials, mild conditions, and excellent overall yield.

• Reasonable, reliable, and suitable method for both bench and industrial scales.

Abstract

KGP94 is a potent, selective, and competitive inhibitor of the lysosomal endopeptidase enzyme (Cathepsin L) currently in preclinical trials for the treatment of metastatic cancer, which is a leading cause of cancer-associated death. Herein, we report two new synthetic routes for synthesizing the target compound through four consecutive steps, using a Weinreb amide approach starting from a common 3-bromobenzoyl chloride. A key step in the approach is a coupling reaction of a readily available Grignard reagent with amide 4 to produce 6, a previously unreported coupling pattern. These new strategies offer an efficient and alternative approach to synthesis of target compound with an excellent overall yield.

Introduction

Cathepsin L is a group of lysosomal cysteine protease enzyme, which hydrolysed the peptide of endocytosed foreign proteins [1], [2], [3], [4]. In extracellular space, this active enzyme promotes the degradation of extracellular proteins such as collagen, fibronectin, and laminin, and participate in numerous physiological processes including angiogenesis, apoptosis, hyperproliferation, tumor progression and, metastasis by malignant cells [5], [6], [7], [8], [9], [10], [11], [12]. The enzyme is also involved in several pathological conditions such as atherosclerosis, rheumatoid arthritis, diabetes, inflammatory status, immune, liver fibrosis, kidney, viral infection, invasion, and metastasis of tumors and other diseases [13], [14], [15], [16], [17], [18], [19], [20]. Recent studies have shown that this vital enzyme is also linked with the entry of respiratory syndrome coronavirus 2 (SARS-CoV-2) into the human cells through the alternative endolysosomal pathway [20]. Thus, Cathepsin L is a special interest as a target for the development of novel therapeutic agents. Over the years, several classes of small molecule inhibitors have been discovered targeting human cathepsin L and synthesized incorporating different electrophilic moieties include the aldehyde of the N-(I-naphthalenylsulfonyl) peptide derivative, the cyclic carbonyl in azepanone, the carbonyl of thiocarbazate, epoxide in Clik 148, the nitrile in the purine analogue, the nitrile in the triazine analogue and the α,β-unsaturated amide of gallinamide A, that can interact with the catalytic site residue Cys-25 thiolate of cathepsin L.[20]

In past decade, a novel inhibitor (referred to as KGP94) was discovered and developed as an inhibitor of human cathepsin L with high activity (IC₅₀ = 131.4 nM) and lower cytotoxicity (GI₅₀ = 26.9 µM) against various human cell lines (Fig. 1) [21], [22], [23]. KGP94 is a selective inhibitor of the cathepsin L toward human type I collagen that is currently in preclinical development for the treatment of metastatic cancer.[25] Previous studies have reported that the lead compound also has a potential application in the treatment of Chagas’ disease (also known as American Trypanosomiasis).[40] The title compound 1 is an attractive lead, structurally comprises a benzophenone thiosemicarbazone system substituted with one m-bromo and hydroxyl group on two opposite aromatic rings [21], [22], [23], [24], [25], [26], [27], [28], [29]. Mechanistically, the thiol group of this inhibitor forms an important reversible and transient covalent bond with the thiolate moiety of the enzyme active site Cys25, and it has been proved by molecular modelling studies[27], [30], [31], [32]. As a result of their potent inhibition, lower cytotoxicity, and attentive mechanism of action, the compound has attracted considerable attention in preclinical studies.[41]

Thus, to support further preclinical studies with this candidate, two synthetic methods have been reported [21], [22]. The previously used synthetic routes of KGP94 are shown in Scheme 1, Scheme 2. The first one, developed in a medicinal chemistry background, led to the milligram-scale, and allowed the in-vitro assessment of KGP94 in inhibitory activity. This initial route consisted of 6 steps from two commercially available materials and resulted in an overall yield of 14.6%. Inconveniently, the product obtained by this method resulted in the inseparable trace impurities (replacement of bromo with hydrogen atom in halogen-metal exchange reaction), due to the use of excess amount of magnesium metal in Grignard preparation. (Scheme 1). To reduce the impurities and enhance the yield as well as purity of the lead compound, it was later optimized by Parker et al. in 2017 (Scheme 2) consisting of 5 steps with an overall yield of 50.0%. The method was also disclosed in a patented process by the same research group [23]. The two synthetic routes described the cross-coupling as the key step. After oxidation, condensation, and deprotection, KGP94 (I) was finally obtained.

However, the major limitations were encountered with these two routes, including synthesis of coupling materials, usage of hazardous reagents, long sequence of steps, uncontrolled impurities, tedious synthetic procedures, lack of an alternative route and poor overall yields.

These problems forced us to redesign the synthesis to provide an optimal route, to lead compound. Herein, we present our attempts for the development of an efficient synthetic route with concise steps and increased overall yield.