Enhanced Plasma Stability and Potency of Aryl/Acyloxy Prodrugs of a BTN3A1 Ligand.

This article is licensed under CC-BY-NC-ND 4.0 pubs.acs.org/acsmedchemlett Letter Enhanced Plasma Stability and Potency of Aryl/Acyloxy Prodrugs of a BTN3A1 Ligand Umed Singh, Girija Pawge, Sarita Rani, Chia-Hung Christine Hsiao, David F. Wiemer, and Andrew J. Wiemer* Cite This: ACS Med. Chem. Lett. 2024, 15, 1771−1777 ACCESS Metrics & More Read Online Article Recommendations sı Supporting Information * ABSTRACT: While ester-based phosphonate prodrugs excel at delivering payloads into cells, their instability in plasma is a hurdle for their advancement. Here, we synthesized new aryl/acyloxy prodrugs of a phosphonate BTN3A1 ligand. We evaluated their phosphoantigen potency by flow cytometry and ELISA and their plasma and cellular metabolism by LC-MS. These compounds displayed low nanomolar to high picomolar potency. Addition of a p-isopropyl group to the phenyl substituent and use of cyclohexyl or p-methoxybenzyl groups as the acyloxy substituent significantly increased human, but not mouse or rat, plasma stability without negatively impacting potency. Combinations of these prodrug moieties further improved stability, with the best combination achieving a half-life of over 12 h in human plasma, a marked improvement on prior compounds. In contrast, oxane analogs improved water solubility and cellular payload delivery but remained unstable in human plasma. The studies suggest that certain ester-based phosphonate prodrugs quickly deliver active payloads inside cells and show substantial stability in human plasma. KEYWORDS: phosphonate, prodrug, butyrophilin, isoprenoid, ligand T he human immune system includes multiple lines of defense against bacterial infection and malignant cell types, including both innate and adaptive responses. While the innate response is rapid and general, the adaptive response is stronger and allows a memory component for more rapid defense against repeated challenges by the same agent. The most abundant facet of the adaptive immune system comprises the αβ T cells, which recognize peptide antigens with the assistance of the major histocompatibility complex (MHC) and immune coreceptors, which are often good therapeutic targets. In contrast, the smaller subset of Vγ9Vδ2 T cells does not rely upon the MHC or recognize peptides.1 Instead, it responds to small organophosphorus compounds, or phosphoantigens, especially the diphosphates produced in the nonmevalonate biosynthesis of isoprenoids found in bacterial but not human metabolism.2 To detect these compounds, Vγ9Vδ2 T cells recognize the co-receptors of the butyrophilin (BTN) family.3 Because Vγ9Vδ2T cells have not been studied exhaustively, their therapeutic manipulation may have untapped potential for treatment of various cancers.4 The most potent natural stimulant of Vγ9Vδ2 T cell proliferation is (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP, 1) (Figure 1),5 which is the final intermediate unique to bacterial biosynthesis of higher isoprenoids.6 To stimulate proliferation, this compound must penetrate the cell membrane to reach its binding site on the butyrophilin receptor BTN3A1,7,8 part of the BTN3/BTN2A1 complex.9−15 That this binding site is within the cell rather than on the cell surface may be the result of an evolutionary response to intracellular bacterial infections. Unfortunately, © 2024 The Authors. Published by American Chemical Society Figure 1. Structures of HMBPP, C-HMBP, and selected prodrug forms. although therapeutic manipulation of Vγ9Vδ2 T cells may have significant potential, HMBPP itself is unlikely to be an attractive drug candidate. The highly charged diphosphate Received: Revised: Accepted: Published: 1771 July 29, 2024 September 12, 2024 September 20, 2024 September 25, 2024 https://doi.org/10.1021/acsmedchemlett.4c00371 ACS Med. Chem. Lett. 2024, 15, 1771−1777 ACS Medicinal Chemistry Letters pubs.acs.org/acsmedchemlett Letter Scheme 1. Combination of p-Isopropylphenyl Ester with Acyloxymethyl Estersa a Reagents and conditions: (a) RCOCH2OCl,34,35 NaI, CH3CN (anhyd), reflux, 24 to 48 h, 18−23%; (b) SeO2, t-BuOOH, 4-hydroxybenzoic acid, CH2Cl2, 0 °C, 3−4 days, 14%; (c) Ac2O, Et3N, rt, overnight, 85−92%. Scheme 2. Synthesis of New Mixed Aryl/Acyloxymethyl Phosphonate Estersa a Reagents and conditions: (a) NaHCO3, Bu4NHSO4, chloromethyl chlorosulfate, CH2Cl2:H2O 1:1, 88−90%; (b) (i) TMSCl, dichloromethane, 0 °C to rt, overnight, 90−95%; (ii) H2O: THF (10:1), 1 h; (iii) Ag2CO3, DMF, 30 min, TBAI, RCOCH2OCl,34,35 acetonitrile, 80 °C, 48 h 18−35%; (c) SeO2, t-BuOOH, 4-hydroxybenzoic acid, CH2Cl2, 0 °C, 3−4 days, 14−17%; (d) Ac2O, Et3N, rt, overnight, 84−92%. functionality does not diffuse readily through the cell membrane16 and has only limited plasma stability.17 To obtain more drug-like compounds, several modifications of the HMBPP ligand have been explored. Incorporation of a C−P bond (2, C-HMBP) in place of the phosphate ester in compound 1 renders C-HMBP phosphatase resistant18 while preserving significant phosphoantigen activity.8,19 Many strategies have been developed to enhance the membrane permeability of charged molecules,20 including phosphates and phosphonates, through the use of prodrugs,21−24 but until our initial report on compound 3 (POM2-C-HMBP) no one had explored a prodrug strategy with phosphoantigens.8 Because it readily crosses the cell membrane and then undergoes hydrolysis to deliver the charged ligand, the prodrug 3 displays significantly higher potency than phosphonate 2.8 Since our first report on the potency of compound 3, several other prodrug forms have been explored with phosphoantigens, including aryl phosphonamidates (e.g., 4),25,26 bisamidates,27,28 mixed aryl/acyloxy diesters (e.g., 5),29,30 and double prodrug forms that included acetate protection of the requisite allylic alcohol.31,32 While several of these compounds have cellular potency in the low nanomolar or even high picomolar range, for improved in vivo capabilities, the ideal prodrug form would have rapid cellular uptake, display high plasma stability, and yield benign fragments upon drug release. Of the possible prodrug forms that we have studied, the dimethyl ester of the parent phosphonate 2 was found to be highly stable but had little or no potency, perhaps because it is too stable to cellular metabolism.8 The bis-pivaloyloxymethyl (POM) compound 3 (POM2-C-HMBP) has good potency in 1772 https://doi.org/10.1021/acsmedchemlett.4c00371 ACS Med. Chem. Lett. 2024, 15, 1771−1777 ACS Medicinal Chemistry Letters pubs.acs.org/acsmedchemlett Letter Scheme 3. Synthesis of the Potential Prodrugs 29a and 29ba a Reagents and conditions: (a) (i) TMSCl, dichloromethane, 0 °C to rt, overnight, 90%; (ii) H2O:THF (10:1), 1 h; (iii) Ag2CO3, DMF, 30 min, TBAI, 19, acetonitrile, 80 °C, 48 h, 19%; (b) SeO2, t-BuOOH, 4-hydroxybenzoic acid, CH2Cl2, 0 °C, 3−4 days, 17%; (c) Ac2O, Et3N, rt, overnight, 86%. bioassays for cell proliferation but rapidly undergoes (...truncated)