Scaffold manipulation for the synthesis of novel, potentially irreversible Akt inhibitors

Van Der Westhuizen, Leandi (2019-12)

Thesis (PhD)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: Cancer is a leading cause of death and disease worldwide with an increasing number of cases reported annually. There is therefore an ever-present need for improved diagnosis and treatment options. Akt is a protein kinase that belongs to the PI3K/Akt/mTOR signalling pathway and is involved in regulating a number of cellular processes, including proliferation, growth, cellular survival, glucose metabolism, angiogenesis and migration. This kinase has been found to be dysregulated in a variety of diseases, including cancer, with activated Akt linked to reduced patient survival and conferring resistance to conventional cancer treatments. To date, no Akt inhibitors have been approved by the U.S. Food and Drug Administration (FDA) for anticancer therapy and all of the current clinical trial candidates are reversible inhibitors. This suggests an opportunity for further research into this field, especially into irreversible Akt inhibitors. During a previous project, the novel irreversible inhibitor of wild-type Akt1, N-[3-(1-{1-[4-(3-phenylimidazo[1,2-a]pyridin-2-yl)benzyl]piperidin-4-yl}-1H-1,2,3-triazol-4-yl)phenyl]-acrylamide, was synthesised and identified. In order to study the differences in inhibition between irreversible and reversible Akt inhibitors, the first aim of this project was to synthesise the reversible version of this irreversible Akt inhibitor, namely N-[3-(1-{1-[4-(3-phenylimidazo[1,2-a]pyridin-2-yl)benzyl]piperidin-4-yl}-1H-1,2,3-triazol-4-yl)phenyl]-propionamide. After its successful synthesis, evaluation found that both the reversible and irreversible Akt inhibitors bind the targeted allosteric pocket between the kinase and pleckstrin homology (PH) domains, with the irreversible inhibitor targeting both the target cysteine residues, Cys296 and Cys310. As expected, evaluation further revealed the irreversible inhibitor to be a more potent Akt inhibitor than its reversible counterpart. The second aim of this project was to further explore Akt inhibition, with the design and synthesis of a small library of novel target compounds, to be evaluated for their ability as irreversible Akt inhibitors, and thus, as potential anticancer agents. This library included a number of compounds with small alterations from the previously synthesised irreversible Akt inhibitor and also included hybrid compounds between said previously synthesised irreversible Akt inhibitor and an Akt inhibitor of our colleagues, N-(2-oxo-3-{1-[4-(5-oxo-3-phenyl-5,6- dihydro-1,6-naphthyridin-2-yl)benzyl]piperidin-4-yl}-2,3-dihydro-1H-benzo[d]imidazol-5- yl)acrylamide, also called borussertib. This library of compounds was successfully synthesised and evaluated for covalent bond formation to Akt, inhibitory activity against Akt and antiproliferative activity against a number of cancer cell lines. From this library, three compounds were identified as type VI inhibitors of wild-type Akt1, capable of both covalent modification of wild-type Akt1 and of inhibiting its activity. These compounds are N-(6-chloro-2-oxo-3-{1-[4-(3-phenylimidazo[1,2-a]pyridin-2-yl)benzyl]-piperidin-4-yl}-2,3-dihydro-1H-benzo[d]imidazol-5-yl)acrylamide, N-(2-oxo-3-{1-[4-(3-phenylimidazo[1,2-a]pyridin-2-yl)benzyl]piperidin-4-yl}-2,3-dihydro-1H-benzo[d]imidazol-5-yl)acrylamide and N-[3-(1-{1-[4-(5-oxo-3-phenyl-5,6-dihydro-1,6-naphthyridin-2-yl)-benzyl]piperidin-4-yl}-1H-1,2,3-triazol-4-yl)phenyl]acrylamide. The latter of these compounds displayed the best overall rate of covalent bond formation with wild-type Akt1 and also the best Akt inhibitory activity and antiproliferative activity in the biochemical and cellular assays, respectively. This compound has the same eastern scaffold as the previously synthesised irreversible Akt inhibitor and its western scaffold is from the potent Akt inhibitor from our collaborators, borussertib. Ultimately, the reversible counterpart of a previously synthesised irreversible Akt inhibitor was successfully synthesised and characterised and a small library of novel potentially irreversible Akt inhibitors was also designed, synthesised and characterised. These compounds were all evaluated for their inhibition of Akt and the information gained allowed for the further development of the structure-activity relationship (SAR) knowledge of this class of inhibitors. It is hoped that this will be utilised for the design of future generations of irreversible Akt inhibitors with improved properties.

AFRIKAANSE OPSOMMING: Kanker is een die vernaamste oorsake van sterftes en siekte ter wêreld. Met 'n toenemende aantal gevalle wat jaarliks aangemeld word, is daar voortdurend 'n behoefte aan verbeterde diagnose- en behandelingsopsies. Akt is 'n proteïenkinase wat lid is van die PI3K/Akt/mTOR seintransduksiepad en is betrokke by die regulering van 'n aantal sellulêre prosesse, insluitend proliferasie, groei, sellulêre oorlewing, glukosemetabolisme, angiogenese en migrasie. Daar is gevind dat hierdie kinase gedisreguleer is in verskeie siektes, insluitend kanker. Geaktiveerde Akt is gekoppel aan verminderde pasiëntoorlewing en weerstand teen gebruiklike kankerbehandelings. Geen onderdrukkers van Akt is tot op hede deur die Food and Drug Administration van Amerika (FDA) vir kankerbehandeling goedgekeur nie en al die huidige kliniese proefmiddels is omkeerbare onderdrukkers. Dit dui op ‘n geleentheid vir verdere navorsing op hierdie gebied, veral in onomkeerbare onderdrukkers van Akt. ‘n Nuwe onomkeerbare onderdrukker van wilde-tipe Akt1, genaamd N-[3-(1-{1-[4-(3- fenielimidaso[1,2-a]piridin-2-iel)bensiel]piperidin-4-iel}-1H-1,2,3-triasol-4-iel)feniel]- akrielamied, is tydens 'n vorige projek gesintetiseer en geïdentifiseer. Ten einde die verskille in onderdrukking tussen onomkeerbare en omkeerbare onderdrukkers van Akt te bestudeer, was die eerste doel van hierdie projek om die omkeerbare weergawe van bogenoemde onomkeerbare onderdrukker te sintetiseer, naamlik N-[3-(1-{1-[4-(3-fenielimidaso[1,2-a]- piridin-2-iel)bensiel]piperidin-4-iel}-1H-1,2,3-triasol-4-iel)feniel]propionamied. Na die suksesvolle sintese van hierdie verbinding, het evaluering bevind dat beide die omkeerbare en onomkeerbare onderdrukkers Akt bind in die geteikende allosteriese bindingsholte tussen die kinase- en plekstrinhomologie (PH)-domeine. Daar is ook gevind dat die onomkeerbare onderdrukker beide die teikensisteïenresidue, Cys296 en Cys310, teiken. Die evaluering het verder, soos verwag, gedui dat die onomkeerbare onderdrukker 'n sterker onderdrukker van Akt is as sy omkeerbare eweknie. Die tweede doel van hierdie projek was om onderdrukking van Akt verder te ondersoek, met die ontwerp en sintese van 'n klein reeks van nuwe teikenverbindings, ten einde hul vir hul vermoëns as onomkeerbare onderdrukkers van Akt, en dus potensiële antikankermiddels, te evalueer. Hierdie reeks bevat 'n aantal verbindings met klein veranderinge vanaf die voorheen gesintetiseerde onomkeerbare onderdrukker van Akt en ook hibriedverbindings tussen die voorheen gesintetiseerde onomkeerbare onderdrukker van Akt en 'n onderdrukker van ons kollegas, N-(2-okso-3-{1-[4-(5-okso-3-feniel-5,6-dihidro-1,6-naftiridin-2-iel)bensiel] piperidin-4-iel}-2,3-dihidro-1H-benso[d]imidasol-5-iel)akrielamied, ook genoem borussertib. Hierdie reeks van verbindings is suksesvol gesintetiseer en geëvalueer vir die vorming van ‘n kovalente binding met Akt, vir hul onderdrukkende aktiwiteit teen Akt en vir teenprolifererende aktiwiteit teen 'n aantal kankersellyne. Drie verbindings van hierdie reeks is geïdentifiseer as tipe VI-onderdrukkers van wilde-tipe Akt1, wat kovalent aan wilde-tipe Akt1 bind en ook die aktiwiteit daarvan onderdruk. Hierdie verbindings is N-(6-chloro-2-okso-3-{1-[4-(3-fenielimidaso[1,2-a]piridin-2-iel)bensiel]- piperidin-4-iel}-2,3-dihidro-1H-benso[d]imidasol-5-iel)akrielamied, N-(2-okso-3-{1-[4-(3- fenielimidaso[1,2-a]piridin-2-iel)bensiel]piperidin-4-iel}-2,3-dihidro-1H-benso[d]imidasol-5- iel)akrielamied en N-[3-(1-{1-[4-(5-okso-3-feniel-5,6-dihidro-1,6-naftiridin-2-iel)bensiel]- piperidin-4-iel}-1H-1,2,3-triasol-4-iel)pheniel]akrielamied. Van hierdie verbindings, toon laasgenoemde die beste algehele tempo van kovalente bindingsvorming met wilde-tipe Akt1 asook die beste onderdrukkende aktiwiteit teen Akt en teenprolifererende aktiwiteit in die biochemiese en sellulêre toetse onderskeidelik. Hierdie verbinding het dieselfde oostelike gedeelte as die voorheen gesintetiseerde onomkeerbare onderdrukker en sy westelike gedeelte is dié van die sterk onderdrukker van Akt van ons medewerkers, borussertib. Ten einde is die omkeerbare eweknie van 'n voorheen gesintetiseerde onomkeerbare onderdrukker van Akt suksesvol gesintetiseer en gekarakteriseer en 'n klein reeks van nuwe potensiële onomkeerbare onderdrukkers van Akt is ook ontwerp, gesintetiseer en gekarakteriseer. Hierdie verbindings is almal geëvalueer vir hul onderdrukking van Akt en die inligting wat verkry is, het bygedra tot die verdere ontwikkeling van die struktuur-aktiwiteitsverhoudingkennis van hierdie klas onderdrukkers. Daar word gehoop dat dit aangewend sal word vir die ontwerp van toekomstige generasies van onomkeerbare onderdrukkers van Akt met verbeterde eienskappe.

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