Expanding the toolbox of metabolically stable lipid prodrug strategies

Nucleoside- and nucleotide-based therapeutics are indispensable treatment options for patients suffering from malignant and viral diseases. These agents are most commonly administered to patients as prodrugs to maximize bioavailability and efficacy. While the literature provides a practical prodrug playbook to facilitate the delivery of nucleoside and nucleotide therapeutics, small context-dependent amendments to these popular prodrug strategies can drive dramatic improvements in pharmacokinetic (PK) profiles. Herein we offer a brief overview of current prodrug strategies, as well as a case study involving the fine-tuning of lipid prodrugs of acyclic nucleoside phosphonate tenofovir (TFV), an approved nucleotide HIV reverse transcriptase inhibitor (NtRTI) and the cornerstone of combination antiretroviral therapy (cART). Installation of novel lipid terminal motifs significantly reduced fatty acid hepatic ω-oxidation while maintaining potent antiviral activity. This work contributes important insights to the expanding repertoire of lipid prodrug strategies in general, but particularly for the delivery and distribution of acyclic nucleoside phosphonates

Design and Synthesis of potent benzimidazolone HIV Non-nucleoside reverse transcriptase inhibitors

Thesis (PhD)--Stellenbosch University, 2018.ENGLISH ABSTRACT: Since the 1980’s, HIV has plagued the population on a global scale, with millions of newly infected individuals reported every year. However, with the introduction of combination therapy, which can significantly suppress viremia to almost undetectable levels in the infected populace, the disease can be managed to a point where the infected population can live almost normal lives. Unfortunately, although able to improve quality of life and prevent the onset of AIDS, combination therapy is not curative as issues related to drug resistance and adherence can lead to the re-emergence of high viremia, AIDS and, inevitably, death. Consequently, there remains a need for the continued development of new and superior ARVs that are effective against wild-type and resistant strains of HIV and are well tolerated for chronic use. In an effort to address this need, our group has focused on the design and synthesis of new NNRTIs. In the clinic, NNRTIs are an important part of first-line regimens employed in the treatment of HIV. In particular, our group focused on the synthesis of a series of small benzimidazolone-containing NNRTIs which were initially designed to address lability issues exhibited by a series of potent indole-based NNRTIs. These first-generation benzimidazolones were readily synthesized over five steps and, following evaluation in an HIV whole cell assay, were found to be potent inhibitors of HIV RT, but were susceptible to clinically relevant resistant strains such as K103N and Y181C. As a result, we synthesized a series of second-generation benzimidazolone NNRTIs which were designed to overcome, specifically, the Y181C resistant strain. Starting from 2-amino-3-nitrophenol, the benzimidazolone precursor for these compounds was synthesized over six steps. This precursor was then coupled to various aryl or heteroaryl halides by way of an Ullmann reaction or SNAr. Of this small library, one compound in particular was found to be potent (with low nanomolar activity), not only against wildtype, but also against Y181C, Y188C and the double mutant K103N/Y181C. Furthermore, this compound, 3-chloro-5-((3-ethyl-2-oxo-1-((2-trimethylsilyl)ethoxy)methyl)-2,3-dihydro-1H-benzo[d]imidazol-4- yl)oxy)benzonitrile, exhibited only low levels of susceptibility against the most problematic K103N resistant strain. We envisaged that by introducing additional electrostatic interactions between our potent lead compound and the NNIBP we would succeed in optimizing the efficacy of our compound against wild-type and resistant strains of HIV. In order to achieve these additional interactions we adopted two different approaches. The first approach focused on targeting a lysine residue located at the top of a narrow hydrophobic chimney towards the back of the NNIBP. To this end, we installed a cyanovinyl substituent onto our lead compound which, based on docking studies, would protrude into the chimney and form a hydrogen bond with the targeted lysine. Installation of the cyanovinyl substituent was achieved using the well-established Heck coupling reaction. Although this compound, (E)-3-(2-cyanovinyl)-5-((3-ethyl-2-oxo-2,3-dihydro-1Hbenzo[ d]imidazol-4-yl)oxy)benzonitrile, was also a potent inhibitor of HIV RT, it was unfortunately not significantly more potent than our existing lead compound. The second approach employed a molecular hybridization technique to form a combination of our lead compound and efavirenz, in order to achieve additional hydrogen bonding to the backbone of Lys101. This new hybrid compound, 3-chloro-5-((4,4-dimethyl-2-oxo-1,4-dihydro-2H-benzo[d][1,3]oxazin-5- yl)oxy)benzonitrile, was successfully synthesized over seven steps and found to be slightly more potent than our lead compound with an improved selectivity index.AFRIKAANSE OPSOMMING: Sedert die 1980’s het MIV in ‘n globale pandemie geraak waar miljoene mense jaarliks gediagnoseer word. Die gebruik van kombinasie terapie het gelei tot grootskaalse onderdrukking van viremie in so ‘n mate dat dit gevolglik onopspoorbaar is, en dus sorg dat mense met HIV amper ‘n normale lewe kan lei. Alhoewel die behandeling van mense met HIV hulle lewens gehalte verbeter het en die aanvang van VIGS verhoed, is hedendaagse behandeling nie genesend nie, en as gevolg van dwelm-bestandhied moontlik kan lei to die herverskyning van ‘n hoë viralelading, VIGS en dan dood. As gevolg van hierdie dilemma, is daar tans ‘n groot nood vir navorsing en onwikkeling vir nuwe ARV medisyne wat effektief is teen wilde-tipe en dwelm-bestande MIV stamme, tesame met minimale newe effekte wat ‘n resultaat is van daaglikse gebruik. Om die tekort aan nuwe en effektiewe antiretrovirale medisyne aan te spreek, het ons groep gefokus op om nuwe nie-nukleosied-omgekeerde transkriptase-inhibeerders (NNRTI’s) te ontwerp en te sintetiseer, sedert NNRTI’s beskou word as ‘n belangrike gedeelte vir die behandeling van MIV. Ons groep het onder andere gefokus op die sintesise van ‘n klein reeks molekules wat ‘n bensimidasoloon kern bevat. Die reeks was aanvanklik gesintetiseer om probleme rakend die chemiese stabiliteit van ‘n voorheen gesintetiseerde reeks indool NNRTI’s, ook deur ons groep ontwikkel, aan te spreek. Die eerste generasie reeks bensimidasoloon molekules was maklik geskep oor vyf stappe, en heel sel toetse teenoor MIV omgekeerde transkriptase het getoon dat hulle kragtige inhibeerders van die ensiem was, maar was ook onder andere vatbaar vir relevante MIV stamme soos K103N en Y181C. As gevolg daarvan het ons ‘n tweede generasie reeks bensimidasoloon NNRTI’s ontwerp om hierdie tekortkoming teenoor K103N en spesifiek die Y181C weerstandige stam te verbeter. Deur te begin met 2- amino-3-nitro fenol was die bensimidasoloon voorloper gesintetiseer in ses stappe. Die voorloper was dan gekoppel met menigte aromatiese en heteroaromatiese haliede deur middel van of die Ullmann-koppel reaksie of SNAr. Een molekule uit hierdie reeks, 3-chloro-5-((3-ethyl-2-oxo-1-((2- trimethylsilyl)ethoxy)methyl)-2,3-dihydro-1H-benzo[d]imidazol-4-yl)oxy)benzonitrile, was aktief gewees met lae nanomolaar aktiwiteit teenoor die wilde-tipe MIV maar ook teenoor Y181C, Y188C en die dubbel mutant K103N/Y181C, met net ‘n klein hoeveelheid weerstand teenoor die problematiese K103N MIV stam. Deur addisionele elektrostatiese interaksies by te voeg tussen ons aktiefste molekule en die NNIBP, het ons probeer bevestig of ons die aktiwitiet teenoor die wilde tipe MIV stam sal kan verbeter. Om hierdie idee van addisionele interaksies te laat realiseer het ons twee verskillende metodes benader. In die eerste metode het ons daarop gefokus om ‘n lisien residu te teiken wat in die boonste gedeelte van die smal hidrofobiese skoorsteen in die aktiewe setel van die NNIBP geleë is. Ons het ‘n nitriel-viniel groep geïnstalleer op ons mees aktiefste molekule wat, deur middle van dokstudies, getoon het dat die nitrielviniel groep moontlik sal inbeweeg in die skoorsteen gedeelte en ‘n waterstof-binding vorm met die lisien residu. Inkorporering van die nitriel-viniel substituent was gedoen deur die uitvoering van ‘n Heck-koppel reaksie. Alhoewel die nitriel-viniel molecule, (E)-3-(2-cyanovinyl)-5-((3-ethyl-2-oxo-2,3-dihydro-1Hbenzo[ d]imidazol-4-yl)oxy)benzonitrile, goeie aktiwiteit getoon het teenoor MIV omgekeerde transkriptase, was dit nie moontlik om die oorspronlike aktiewe molekuul se aktiwiteit drasties te verbeter nie. Die tweede benadering het ‘n molekulêre hibridisasie tegniek ingesluit waar ons ons aktiewe molekuul en efavirenz gebaster het sodat daar moontlik ‘n addisionele waterstof-binding gemaak kan word met ‘n Lys101 residu in die aktiewe setel. Hierdie hibried molekuul, 3-chloro-5-((4,4-dimethyl-2-oxo-1,4-dihydro- 2H-benzo[d][1,3]oxazin-5-yl)oxy)benzonitrile, was suksesvol gesintetiseer oor sewe stappe, en was meer aktief as die oorspronglike aktiewe molekuul, maar het ook ‘n verbeterde selektiwiteits indeks getoon in vergelyking met die oorspronklike aktiewe molekuul

The design and synthesis of novel HIV-1 non-nucleoside reverse transcriptase inhibitors

Thesis (MSc)--Stellenbosch University, 2015.ENGLISH ABSTRACT: Since its discovery in the 1980’s, HIV has affected the lives of millions of individuals around the globe. Despite obvious need and an enormous amount of research a cure has remained elusive due to the rapid onset of mutated forms of the virus. However, there has been considerable success in reducing viral levels of infected individuals through the use of highly active antiretroviral therapy (HAART). The first-line regimen HAART mainly targets reverse transcriptase (RT) through the employment of two nucleoside RT inhibitors (NRTIs) and a nonnucleoside RT inhibitor (NNRTI). NNRTIs target an allosteric pocket situated about 10 Å from the catalytic site and cause a conformational change in the enzyme upon binding, leading to the inhibition of viral replication. There are currently 5 FDA approved NNRTIs on the market which successfully inhibit viral replication, but the use of these drugs is becoming limited due to the onset of drug resistant strains of the virus. In light of this need for the development of novel NNRTIs, we set out to explore new territory in NNRTI drug design with a goal of maintaining efficacy in the presence of both wild-type and mutated forms of HIV-1. To this end we designed three different NNRTI scaffolds along three different research thrusts. The first of these focused on the synthesis of 15 novel flexible triazole containing compounds. With these compounds we sought to achieve π-π stacking interactions with conserved amino acid residue Trp229 in the hope that we would be able to maintain efficacy in the presence of mutated forms of the virus. An additional feature included hydrogen bonding interactions to the backbone of Lys103. However, despite having thoroughly explored the triazole ring with multiple substitution arrangements, these compounds had very poor to no activity against whole cell HIV-1. Secondly we focused on the synthesis of a 4-hydroxyindole scaffold as a potential NNRTI. The focus here was to achieve interactions to Trp229 and simultaneously achieve hydrogen bonding interactions to the backbone of Lys101 at the entrance of the pocket. This was a novel concept in this class of compounds. We were able to successfully synthesize the indole core as a proofof-concept using the Knoevenagel-Hemetsberger method however; this compound had no activity against HIV-1. Lastly, in our quest to synthesize a novel NNRTI that could maintain efficacy against HIV-1 we decided to attempt to improve upon the stability of a lead indole-based compound synthesized previously within our research group. The lead compound was found to be potent with an IC50 of 1 nM but was unstable in acidic media due to the presence of a methoxy functionality situated at the 3-position on the indole. We sought to overcome this issue by introducing a substituted aryl amine functionality at this position. We were successful in synthesizing our desired compound but unfortunately it was significantly less active against whole cell HIV-1 than the lead compound. However, we were not completely deterred as there are a number of unexplored bioiososteres as possibilities to improve upon the stability of the lead compound while maintaining its excellent activity profile.AFRIKAANSE OPSOMMING: Sedert die ontdekking van die menslike immuniteitsvirus (MIV) in die 1980’s, het die virus al die lewens van miljoene mense wêreldwyd geaffekteer. Ten spyte van die ooglopende behoefte aan ‘n geneesmiddel sowel as meer navorsing, bly ‘n keermiddel sover onbekombaar as gevolg van die verskillende mutasies wat binne die virus gebeur. Ten spyte hiervan, was daar al heelwat sukses in terme van ‘n verlaging van die virale vlakke in besmette individue deur die gebruik van hoogsaktiewe antiretrovirale terapie (HAART). As ‘n eerste behandeling, teiken HAART meestal trutranskriptase (RT) deur die inspanning van twee nukleosied trutranskriptase inhibeerders (NRTIs) en ‘n nie-nukleosied trutranskriptase inhibeerder (NNRTI). NNRTIs teiken ‘n allosteriese leemte wat ongeveer 10 Å weg van die katalitiese posisie is en veroorsaak dan ‘n konformasie verandering in die ensiem tydens die bindingsproses, wat dan lei tot die inhibisie van die virus se replikasie. Daar is tans 5 FDA goedgekeurde NNRTIs op die mark wat virale replikasie inhibeer, maar die gebruik van hierdie middels word alhoemeer belemmer as gevolg van die onwikkeling van weerstandige stamme van die virus. Met die oog op hierdie nood aan die ontwikkeling van nuwe NNRTIs, het ons gepoog om new gebiede te ondersoek in terme van die ontwerp van NNRTIs, met die doel om die effektiwiteit teen beide die wilde-tipe sowel as die gemuteerde vorme van HIV-1 te behou. Vir hierdie doeleindes het ons drie verskillende NNRTI steiers ontwerp, wat drie navorsingsdoeleindes na streef. Die eerste van hierdie doeleindes was die sintese van 15 nuwe buigsame triasool-bevattende middels. Met hierdie middels het on gepoog om π-π pakkingsinteraksies te behaal met aminosuur residu, Trp229, en sodoende die effektiwiteit van die NNRTIs in die gemuteerde vorm van die virus te behou. ‘n Additionele eienskap wat bygevoeg is, is ‘n waterstofbindingsinteraksie met die ruggraat van Lys103. Ten spyte van pogings om verskeie substitusie patrone om die triasool-ring te ondersoek, het hierdie middels baie swak tot geen aktiwiteit teen heel sel HIV-1 getoon nie. Tweedens, was die fokus op die sintese van ‘n 4-hidroksieindool steier as ‘n potensiele NNRTI. Die fokus hier was om ‘n interaksie met Trp229 te kry terselfdetyd as ‘n waterstofbindingsinteraksie met die ruggraat van Lys101, wat by die opening van die bindingssak is. Hierdie was ‘n nuwe konsep vir hierdie klas van middele. Ons het die indool-kern van hierdie molekules suksesvol gesintetiseer deur middel van ‘n Knoevenagel-Hemetsberger metode, maar ongelukkig het hulle geen aktiwiteit teen HIV-1 getoon nie. Laastens het ons gepoog om ‘n nuwe NNRTI te sintetiseer wat effiktiwiteit teen HIV-1 behou, deur te probeer om vorderings te maak op die stabiliteit van ‘n indool-gebaseerde hoof-middel wat al voorheen deur ons navorsingsgroep geraporteer is. Hierdie hoof-middel het ‘n IC50 waarde van 1 nM gelewer, maar was onstabiel in suur medium as gevolg van die teenwoordigheid van ‘n metoksie-groep in die 3-posisie van die indool. Ons het gepoog om hierdie probleem te oorkom deur ‘n gesubtitueerde arielamien in hierdie posisie te plaas. Ons was suksesvol hierin, maar ongelukkig was die middel heelwat minder aktief teen die heel sel HIV-1 as die metoksie-weergawe. Ten spyte hiervan, is ons optimisties dat ons hierdie probleem kan oorkom, aangesien daar verskeie bioisostere is wat die stabilitiet van middel kan verbeter terwyl dit moontlik die effektiwiteit kan behou

Design and Optimization of Novel Competitive, Non-peptidic, SARS-CoV‑2 M^pro Inhibitors

The SARS-CoV-2 main protease (Mpro) has been proven to be a highly effective target for therapeutic intervention, yet only one drug currently holds FDA approval status for this target. We were inspired by a series of publications emanating from the Jorgensen and Anderson groups describing the design of potent, non-peptidic, competitive SARS-CoV-2 Mpro inhibitors, and we saw an opportunity to make several design modifications to improve the overall pharmacokinetic profile of these compounds without losing potency. To this end, we created a focused virtual library using reaction-based enumeration tools in the Schrödinger suite. These compounds were docked into the Mpro active site and subsequently prioritized for synthesis based upon relative binding affinity values calculated by FEP+. Fourteen compounds were selected, synthesized, and evaluated both biochemically and in cell culture. Several of the synthesized compounds proved to be potent, competitive Mpro inhibitors with improved metabolic stability profiles