ω-Functionalized Lipid Prodrugs of HIV NtRTI Tenofovir with Enhanced Pharmacokinetic Properties

Abstract

Tenofovir (TFV) is the cornerstone nucleotide reverse transcriptase inhibitor (NtRTI) in many combination antiretroviral therapies prescribed to patients living with HIV/AIDS. Due to poor cell permeability and oral bioavailability, TFV is administered as one of two FDA-approved prodrugs, both of which metabolize prematurely in the liver and/or plasma. This premature prodrug processing depletes significant fractions of each oral dose and causes toxicity in kidney, bone, and liver with chronic administration. Although TFV exalidex (TXL), a phospholipid-derived prodrug of TFV, was designed to address this issue, clinical pharmacokinetic studies indicated substantial hepatic extraction, redirecting clinical development of TXL toward HBV. To circumvent this metabolic liability, we synthesized and evaluated ω-functionalized TXL analogues with dramatically improved hepatic stability. This effort led to the identification of compounds 21 and 23, which exhibited substantially longer t_1/2 values than TXL in human liver microsomes, potent anti-HIV activity in vitro, and enhanced pharmacokinetic properties in vivo.

Figure 1.

Once NRTIs and NtRTIs enter cells, they undergo a series of phosphorylation events to generate active NRTI-TP or NtRTI-DP metabolites, respectively. The ensemble of enzymes required for these phosphorylation events includes nucleoside kinases (NKs) that convert NRTIs to NRTI monophosphates (NRTI-MPs), as well as nucleoside monophosphate kinases (NMPKs), which synthesize NRTI diphosphates (NRTI-DPs) and NtRTI monophosphates (NtRTI-MPs). Nucleoside diphosphate kinases (NDPKs) then convert NRTI-DPs and NtRTI-MPs into active metabolites NRTI-TPs and NtRTI-DPs, respectively.

Figure 2.

Structures of TFV, TDF, and TAF and the various FDA-approved cART in which they are incorporated.

Figure 3.

Metabolic fate of TDF and TAF in vivo.

Figure 4.

Design principles of lysoglycerophospholipid-derived TFV prodrugs. While endogenous lysoglycerophospholipids, such as lysophosphatidylcholine, are substrates for phospholipase A (PLA), phospholipase C (PLC), and phospholipase D (PLD), TXL and analogues thereof reported herein are only susceptible to cleavage by PLC.

Figure 5.

Proposed mechanism of antiviral activity. (1) TFV lipid prodrugs (LPDs) first dissociate and diffuse away from HSA (or other plasma proteins) before (2) translocating across the plasma membrane. At the membrane–cytosol interface, (3) TFV LPD is then subject to intracellular cleavage by PLC (or other lipid-metabolizing enzymes such as acid sphingomyelinase), thereby releasing TFV. Two subsequent phosphorylation events, catalyzed by NMPK (4) and NDPK (5), deliver the active TFV-DP metabolite, which (6) inhibits HIV-RT.

Figure 6.

Mouse plasma and liver pharmacokinetic profiles of top ω-functionalized, ether-linked analogues of TXL. Male C57BL/6 mice (n = 3 per time point) were administered a single oral dose (10 mg/kg) of TFV prodrug using 90:10 olive oil:EtOH as a vehicle. Plasma and liver levels of prodrug and TFV were quantified using LC-MS/MS. (a) Plasma concentrations of TFV prodrug (top) and TFV metabolite (bottom) are plotted separately, whereas (b) liver concentrations of TFV prodrug and TFV metabolite are plotted together. Data represent the mean concentration at each time point ± SEM. The figures are generated with GraphPad Prism v9.

Figure 7.

Summary of the design, synthesis, and pharmacological evaluation of a novel series of ω-functionalized lipid prodrugs of HIV NRTI TFV, culminating in the discovery of ω-CF₃ analogue 21.

Scheme 1.. Synthetic Preparation of Preliminary TXL Derivatives^a

^aReagents and conditions: (a) 1-bromohexadecane, TBAB, 50% NaOH (aq), tetrahydrofuran (THF), rt, 93 h, 28%; (b) 20% Pd(OH)₂/C, H₂, EtOAc, rt, 21 h, 91%; (c) trisyl chloride, pyridine, rt, 24–72 h, 14–20%; (d) DCC, Et₃N, DMAP, DMF, 105 °C, 12–24 h, 33–36%; and (e) 1-bromohexadecane, DBU, DMF, 60 °C, 1.5 h, 89%.

Scheme 2.. Synthetic Preparation of ω-Functionalized TXL Derivatives Featuring Methylene Linkers^a

^aReagents and conditions: (a) alkyl halide, n-BuLi, HMPA (or DMPU), THF, −78 °C to rt, 12–24 h, 59–80%; (b) p-TsOH·H₂O, MeOH, rt, 3–8 h, 53–90%; (c) 1,3-diaminopropane, NaH, 55 °C, 12–24 h, 45–64%; (d) TFV, trisyl chloride, pyridine, rt, 24–72 h, 7–53%; (e) TMSCl, n-BuLi, HMPA, THF, rt, 4 h, 63%; (f) PMBCl, NaH, DMF, 50 °C, overnight, 75%; (g) CF₃Si(CH₃)₃, CuI, K₂CO₃, TMEDA, DMF, rt, 48 h, 91%; (h) CAN, MeOH, H₂O, rt, 3 h, 86%; (i) 20% Pd(OH)₂/C, H₂, EtOH, rt, 18 h, 73%; and (j) EDC, DMAP, Et₃N, MeCN, 90 °C, overnight, 22%.

Scheme 3.. Synthetic Preparation of ω-Functionalized TXL Derivatives Featuring Ether Linkers^a

^aReagents and conditions: (a) CBr₄, PPh₃, DCM, rt, 30 min; (b) 1,3-propanediol, NaH, KI, DMF, 95 °C, 3 h, 35% over two steps; (c) TFV, DCC, Et₃N, DMAP, DMF, 95 °C, 17 h, 24%; (d) 2-(3-bromopropoxy)tetrahydropyran or 1-(3-bromopropoxymethyl)-4-methoxy-benzene, NaOH (aq), TBAB, THF, 75 °C, 12–24 h, 41–48%; (e) CF₃Si(CH₃)₃, CuI, K₂CO₃, TMEDA, DMF, rt, 48 h, 69%; (f) CAN, MeOH, H₂O, rt, 3 h, 95%; (g) TFV, trisyl chloride, pyridine, rt, 24–72 h, 19–46%; (h) 20% Pd(OH)₂/C, H₂, EtOAc, rt, 18 h, 78%; (i) TMSCl, n-BuLi, DMPU, THF, −78 °C to rt, overnight, 55%; and (j) p-TsOH·H₂O, MeOH, rt, 3 h, 74%.