Cerebro y aprendizaje

Over the past century nature has provided a rich source of novel bioactive agents which have been developed into efficacious drugs for a multitude of disease indications. Innovative technological advances have provided biochemical techniques by which to screen nature’s tremendous supply of bioactive agents in the hunt for novel PPI modulators. Although still in its infancy, this approach has identified several promising therapeutic candidates, particularly in the area of cancer research.

This approach is exemplified by the discovery of JBIR-22 (4), a 0.2 µM inhibitor of PAC3 homodimerisation identified from a screen of 123,599 natural product extracts via a high- throughput in vitro protein fragment complementation assay (PCA). JBIR-22 (4) is a novel member of the tetramic acid family of natural products, a ubiquitous collection of bioactive agents containing a pyrrolidine-2,4-dione core motif. In particular, JBIR-22 (4) forms part of a small sub-family of tetramic acids defined by a tetramic acid core containing an unnatural 4,4-disubstituted glutamic acid. The absence of a reported total synthesis targeting members of this sub-family is in sharp contrast to their potent activity against therapeutically relevant targets involved in HIV, cancer and microbial pathogenesis.

3.6.1 Harzianic acid – a novel siderophoric plant growth promoting agent

We proposed a general synthetic approach to access the members of this tetramic acid sub-family centred on the development of a masked tetramic acid core which could be modified by the attachment of the broad range of substituents to optimise diversity. A late stage manipulation would then reveal the synthetically challenging tetramic acid moiety. Central to this concept was the design of a short and stereoselective synthesis of 4,4-disubstituted glutamic acids which on cyclisation would provide our AHL biomimetic masked tetramic acid. This was achieved in a three step process involving a diastereoselective tert-butanesulfinamide 38 directed aldol cyclisation followed by an in situ cyclisation to furnish lactone 46. N-methylation of 46 followed by a highly diastereoselective substrate controlled reduction provided the free amine 53 which could be trapped by the appropriate β-ketothioester side chain. To validate this approach as a comprehensive and general protocol to access these tetramic acids we initially focused on the total synthesis of plant growth promoting siderophore harzianic acid (19).

The polyene side chain of harzianic acid (19) was assembled from commerically available trans- hexan-2-ol (59) via oxidation to the corresponding aldehyde 58 and subsequent incorporation of the β-ketothioester functionality by means of a HWE olefination. Optimisation of the silver mediated N-

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acylation protocol developed by Woodward and Ley facilitated the one-pot coupling of 53 and β- ketothioester 56 followed by subsequent cyclisation to afford harzianic acid ethyl ester 64. Microwave assisted hydrolysis of 64 furnished (S,S)-harzianic acid (19) in a separable 3:1 ratio to (R,S)-5’-epiharzianic acid (19b). This stereoselective and convergent route facilitated the first total synthesis of harzianic acid (19) with a LLS of 6 steps and an overall yield of 22% (Scheme 3.48).

Scheme 3.48. First total synthesis of harzianic acid (19).

The efficacy of this synthetic approach is illustrated by the isolation of all four possible diastereomers of harzianic acid (19). These compounds could prove to be highly valuable tools for the investigation of harzianic acid’s anticancer and plant growth promoting activities. In particular, exploration of harzianic acid’s potent siderophoric properties could be vital to unravelling its mode of action. Comparison of the iron binding affinity of (R,S)-5’-epiharzianic acid (19b) and its biological activities, could provide a mechanism by which to explore the proposed correlation between harzianic acid’s iron binding properties and its antifungal and growth promoting functions. Analysis of the biological activity of the harzianic acid enantiomer (19c) could reveal the possibility of a chirally discriminating uptake mechanism or an alternative biological target by which harzianic acid (19) affects its physiological response.

3.6.2 Identification of JBIR-22’s PAC3 binding site

Synthesis of PPI inhibitor JBIR-22 (4) was investigated through two approaches, which were distinguished by the method and stage at which the IMDA cycloaddition to form the decalin ring of JBIR-22 (4) occurred. The first approach involved formation of the tetramic acid core with the attached polyene chain, prior to the IMDA cycloaddition. The second route involved an IMDA cycloaddition of trienal 69 to furnish the decalin ring, prior to its attachment to the masked tetramic acid core. As the absolute configuration of JBIR-22 (4) had not been elucidated, the second route was initially prioritised so the stereochemical configuration of the products could be unambiguously assigned.

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Schreiber ozonolysis of cyclohexene (72), followed by a HWE olefination and acetal deprotection furnished trienal 75. A subsequent chiral imidazolidinone 85 catalysed IMDA cycloaddition afforded the enantiomerically enriched decalins 78a and 78b. Incorporation of S-tert-butyl-thioacetate provided the desired β-ketothioesters 68a and 68b which were coupled to 53 via the end-game strategy utilised in the synthesis of harzianic acid (19). Comparison of spectroscopic data of the pure JBIR-22 diastereomers 4a and 4b with that reported for the isolated JBIR-22 (4) revealed that 4a

contains the correct relative stereochemical configuration. Significant discrepancies between the reported optical rotation value for JBIR-22 (4) and the synthesised sample 4a prevented absolute stereochemical assignment of the natural product. Chiral HPLC or CD analysis of a sample of natural JBIR-22 (4) sample in comparison with that of 4a should enable absolute stereochemical assignment. In summary, this expedient approach facilitated the first total synthesis of JBIR-22 diastereomers 4a

and 4b with a LLS of 10 steps and of overall yield for 4a of 6% (Scheme 3.49).

Scheme 3.49. First total synthesis of JBIR-22 (4a).

The succinct and adaptable nature of the JBIR-22 (4a) synthetic route provides ample opportunity to explore and optimise the activity of this potent PAC3 PPI inhibitor. A particular area of interest is the proposed JBIR-22 binding site. A docking study reported by Izumikawa et al. identified a potential binding site for (2R, 3R, 6S, 11R, 5’R, 7’R)-JBIR-22 diastereomer 4d on the PAC3 homodimerisation interface (Figure 3.18).123 We have identified an alternative binding site on the PAC3 homodimerisation interface using the docking software GOLD (data not shown). Analysis of the biological activity of the JBIR-22 diastereomers in comparison to their proposed binding site may reveal which site is most likely, and facilitate the optimisation of these compounds through a SAR study. Furthermore, it would provide a novel drug binding site for the development of future inhibitors of the proteasomal machinery, a validated anticancer target.

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Figure 3.18. (A) The proposed JBIR-22 binding site on the PAC3 homodimerisation interface.123PAC3 is shown as cartoon surface model with key residues shown in ball and stick model. Carbon atoms are in green. 4d is shown as ball and stick and carbon atoms are in cyan. In all cases, oxygen atoms are in red and nitrogen atoms in blue. (B) Structure of bound JBIR-22 diastereomer 4d.

3.6.3 Probing JBIR-22’s biosynthetic pathway and the role of a “Diels-Alderase”

The synthesis of the JBIR-22 diastereomers 4a and 4b provided pure standards for the development of the late-stage asymmetric IMDA approach. The IMDA precursor 65 was assembled by means of two alternative routes. The initial approach proceeded via a coupling of 53 and the appropriate β- ketothioester 66, followed by subsequent cyclisation to afford 65. In contrast, the alternative approach aimed to circumvent the silver mediated N-acylation by incorporating the polyene side chain via a HWE olefination. This was achieved by N-acylation of amine 53 by the novel Meldrum’s acid derivative 63 to afford the phosphonate substituted masked tetramic acid 96. A tandem one- pot cyclisation-HWE olefination furnished the desired IMDA precursor 65.

IMDA precursor 65 proved to be a very synthetically useful chemical tool, both for the development of an asymmetric IMDA methodology but also as a mechanism to probe the biosynthesis of JBIR-22 (4). JBIR-22 (4) contains a chiral decalin ring similar to that observed in lovastatin (16), equisetin (10) and solanapyrone A (15), which are proposed to be formed by a controversial enzyme-mediated enantioselective [4+2]-cycloaddition. An alternative biosynthesis of the decalin ring of JBIR-22 (4) could involve a non-enzymatic substrate controlled IMDA. To probe this hypothesis, an achiral Lewis acid catalysed cycloaddition of 65 was investigated. This resulted in the formation of the decalin ring as a single diastereomer but as a racemic mixture, thus forming an approximately 1:1 mixture of JBIR-22 ethyl ester diastereomers 77a and 77b. This would suggest that JBIR-22 (4) is formed by a similar biosynthetic pathway to that reported for equisetin (10), involving a “Diels-Alderase” mediated enantioselective IMDA.

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3.6.4 Development of a catalytic asymmetric IMDA protocol

Preliminary investigation of a chiral Lewis acid catalysed IMDA cycloaddition of 65, focused on exploiting the inherent chelating properties of the tetramic acid core. It was proposed that the 1,3- dicarbonyl motif present in the tetramic acid core of 65 could potentially function as a bidentate dienophile. An initial screen identified that two-point binding Cu(II) complexes involving a bisoxazoline 110 or siam ligand 111 catalysed the cycloaddition in high diastereoselectivity and moderate enantioselectivity, affording the diastereomerically enriched 77a cycloadduct (Scheme 3.50). Further insight into the pre-transition state assembly of the catalyst-65 complex may prove to be essential for optimising the diastereoselectivity. Information regarding the effect of the steric environment surrounding the bidentate dienophile on the stereoselectivity of the cycloaddition is limited due to the scarcity of reported IMDA cycloadditions of advanced natural product intermediates. This is compounded by the widespread use of simple oxazolidinone precursors as bidentate dienophiles for the development of catalytic asymmetric IMDA procedures. Therefore, the future optimisation of the catalytic asymmetric IMDA cycloaddition of 65, in addition to providing a concise and highly versatile route to JBIR-22 (4a) and related analogues, would also assist the development of a more general and comprehensive method for the utilisation of catalytic IMDA cycloadditions in the later stages of total synthesis projects.

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