Late-Stage Lead Diversification Coupled with Quantitative Nuclear Magnetic Resonance Spectroscopy to Identify New Structure–Activity Relationship Vectors at Nanomole-Scale Synthesis: Application to Loratadine, a Human Histamine H₁ Receptor Inverse Agonist

Abstract

An experimental approach is described for late-stage lead diversification of frontrunner drug candidates using nanomole-scale amounts of lead compounds for structure−activity relationship development. The process utilizes C−H bond activation methods to explore chemical space by transforming candidates into newly functionalized leads. A key to success is the utilization of microcryoprobe nuclear magnetic resonance (NMR) spectroscopy, which permits the use of low amounts of lead compounds (1−5 μmol). The approach delivers multiple analogues from a single lead at nanomole-scale amounts as DMSO-d 6 stock solutions with a known structure and concentration for in vitro pharmacology and absorption, distribution, metabolism, and excretion testing. To demonstrate the feasibility of this approach, we have used the antihistamine agent loratadine (1). Twenty-six analogues of loratadine were isolated and fully characterized by NMR. Informative SAR analogues were identified, which display potent affinity for the human histamine H 1 receptor and improved metabolic stability.

Biocatalysis

“…Despite the potential to catalyse remote and unactivated C-H oxidations, which are chemically challenging, enzymes such as cytochrome P450s and other oxygenases are problematic to scale up due to low activity, instability and promiscuity, resulting in a mixture of products 177 . However, these features are well-suited for small-scale, late-stage diversification of biologically active compounds in which the enzyme promiscuity is advantageous to generate new libraries of compounds for evaluation 178,179 . The synthetic utility of the transformation afforded by these enzymes encourages continued efforts to find solutions and realize the potential of these biocatalysts for large-scale manufacture 180 .…”

Enzyme Kits to Facilitate the Integration of Biocatalysis into Organic Chemistry – First Aid for Synthetic Chemists

“…In 2020, Lall et al . demonstrated comprehensive chemo‐ and enzymatic late‐stage functionalisation of the potent antihistamine drug loratadine ( 31 ; Scheme 3A) [125] . On the one hand, the lead compound was exposed to chemical conditions of electrolysis, electrochemical trifluoromethylation, biomimetic metalloporphyrin oxidation (BMO) and alkylation using alkyl sulfinate salts (Baran Diversinates).…”

“… (Chemo‐) enzymatic late‐stage functionalisation of lead drugs ( A ) or natural products ( B , C ) for exploring chemical space of candidate derivatives. A : The drug loratadine ( 31 ) was diversified by several chemical and biocatalytic late‐stage functionalisation techniques via C−H activation [125] . Some of the products ( 32 – 37 ) formed by screening commercial enzyme libraries (i. e., a BM3 mutant kit, microorganism kit, and a kit containing liver microsomes and recombinant liver P450s) are shown.…”

“…In 2020, Lall et al demonstrated comprehensive chemo-and enzymatic late-stage functionalisation of the potent antihistamine drug loratadine (31; Scheme 3A). [125] On the one hand, the lead compound was exposed to chemical conditions of electrolysis, electrochemical trifluoromethylation, biomimetic metalloporphyrin oxidation (BMO) and alkylation using alkyl sulfinate salts (Baran Diversinates). On the other hand, 31 underwent biocatalytic functionalisation by screening three commercially available enzyme kits: P450-BM3 mutants (Codexis), liver microsomes (Corning, Pfizer, and XenoTech), recombinant liver P450s (PanVera), as well as microorganisms (various culture collection facilities, etc.).…”

Future of Biotransformation Science in the Pharmaceutical Industry

“…In addition, analogs 10-13 demonstrated potent and selective inhibition of the kinase and metabolic stability in HLM, which was similar or greater than the corresponding 5-aminooxindole derivatives such as 7. Lead Diversification: An example of how biotransformation studies proved critical in the expansion of the primary pharmacology SAR is evident from the work of Lall et al (2020), where the authors exploited the complex biochemical reactions that can be performed by oxidizing enzymes for diversification of hit/lead chemical series. Original small molecule hits/leads were incubated with a battery of recombinant mammalian and bacterial CYP enzymes, liver microsomes, or microbes to biochemically generate mixtures of products in a single step that would otherwise be very labor-intensive to prepare by traditional synthetic chemistry approaches.…”