The Searcey lab has a great interest in using nature as an inspiration for the design and synthesis of drug molecules. The first natural product inhibitor of the p53/MDM2 interaction, chlorofusin (figure1.33), was of particular interest to the Searcey group due to its unnatural cyclic peptide and chromophore moietites.118
N O O Cl O O O OH HN O HN O NH OH HN O N H O O NH2 O NH NH2 O NH O HN O HO O N H O (CH2)6CH3 R S R L-Asn 3 D-Asn 4 D-Leu 5 L-Thr 6 D-Leu 7 D-ADA 8 L-Orn 9 L-Thr 1 L-Ala 2
Figure 1.33: Structure of chlorofusin, indicating the amino acids and stereochemistry
Chlorofusin was first isolated in 2001 by Duncan et al from the Fusarium sp. Mi- crodocium caespitosum, a type of marine sponge.119 Initial structural work was carried out with a combination of ESI mass spectroscopy,1H-NMR,13C-NMR, COSY, NOESY and ROESY. ESI+ revealed masses corresponding to the (M+H)+ and (M+Na)+, re- sulting in masses of 1363.7 Da and 1385.7 Da respectively.118 There was also evidence of a chlorine atom due to isotopes present on the ESI spectra (a 3:1 ratio which cor- responded to 35Cl and 37Cl). The 1H-NMR data, 13C-NMR data and 2D experiments
permitted the assignment of the amino acid macrocycle, revealing an unnatural amino acid at position 8, aminodecanoic acid. The chromophore was assigned using a combina- tion of13C-NMR and1H-NMR, revealing 8 quarternary carbons and a complex splitting pattern towards the aliphatic end of the spectrum. The initial screening was done using a DELFIA-modified ELISA assay, producing a Kd 4.7 µM and IC50 of 4.6 µM.
A follow-up paper was published by Duncan et al in 2002, detailing the modality of binding of chlorofusin to MDM2.119 The binding of MDM2 to the N-terminus was stud-
ied using surface plasmon resonance. The MDM2 protein was immobilised onto the carboxymethlated dextran surface of the sensor chip through covalent bonding. MDM2
quantification was done using the Bradford method, Chlorofusin was passed over the surface of the chip containing either MDM2 or a ubiquitin control at varying concentra- tions. The data suggested that that chlorofusin initially binds to MDM2 rapidly, leading to a conformational change in the protein, after which there is a second, slower binding step. At present, this is the limit of our knowledge of the mode of chlorofusin binding to MDM2, as at present there are no crystal structures published.
In 2003, two papers were published on the chlorofusin peptide, one involving the first synthesis of the chlorofusin peptide (and diastereomers in an attempt to determine stereochemistry) on solid phase by Searcey et al120 and one on the assignment of the asparagine stereochemistry by Boger et al completed using solution-phase chemistry.121 The synthesis published by Searcey et al utilised Fmoc solid phase peptide synthesis with side-chain immobilisation on Rink Amide MBHA resin, followed by head-to-tail synthesis, followed by cyclisation.120 The starting amino acid was Fmoc-Asp-ODMab, as the DMab group could be easily removed in the presence of hydrazine to uncover the free carboxylic acid, which could then cyclise with the terminal amine of asparagine in the presence of HOBt and DIC. All amino acids were purchased enantiomerically pure with the exception of 2-aminodecanoic acid. 2-Aminodecanoic acid was synthesised initially as a racemate and the enantiomers of the peptide sequence were separated by semi-preparative reverse phase HPLC, as the structural conformation at the time was unclear.
The 2003 paper published by Boger et al explored the total synthesis of chlorofusin and assignment of the asparagines at positions 3 and 4 in solution phase. Instead of synthesising the peptide in solid phase as per Searceys protocol, Boger synthesised the peptide in fragments using Boc, Fmoc, benzyl, SES and CBZ protection. The fragments were coupled together using HOAt and EDCI until the full cyclic peptide was produced. Four separate variants were synthesised by this method, each containing either 3-L-Asn or 3-D-Asn and 4-L-Asn or 4-D-Asn. 1H-NMR and 13C-NMR analyses compared to the natural product permitted the absolute stereochemical configuration of 3-L-Asn and 4-D-Asn.
In 2007, Yao et al examined the stereochemical assignment of the azaphilone chro- mophore of chlorofusin.122 Retrosynthesis of the chlorofusin natural product initially
separated the ornithine side chain from an isochromene. The isochromene was further analysed by ring opening of the furan side chain, hydrolysis of the butyl ester and ring opening to produce a Sonogashira precursor.
A variety of different azaphilone analogues were synthesised and condensed onto the ornithine side chain, each synthesised as a racemate. The compounds were separated by chiral HPLC and analysed by X-ray single crystal analysis. The analysis suggested
that the stereochemical configuration of the azaphilone was (4S,8R,9S). This was then challenged by Boger et al in the same year, who also analysed the different diastere- omers against the natural product peptide.123 Again, analysis was done by 1H-NMR,
13C-NMR, COSY, ROESY, HMQC, HMBC and it was shown that the stereochemical
assignment of (4R,8S,9R) was a near-identical match to the natural product, whereas the assignment proposed by Yao et al showed distinct comparisons with the natural product.
In 2007, the first analogues of chlorofusin were synthesised by Searcey et al.77 Using the methodology detailed in his 2003 paper, the cyclic peptide portion was analysed. This time, however, it was possible to synthesise enantiomerically pure Fmoc-Ada-OH using diethylacetoamidomalonate and 1-bromooctane, followed by hydrolysis and enzy- matic resolution. Analogues of the peptide were synthesised as well as simple aromatic substitution in place of the natural azaphilone. ELISA assay was undertaken, but no hits were determined within the assay. Interestingly, although the stereochemistry was unimportant for activity, the whole molecule was required in order for binding to occur. In conclusion, the structure of chlorofusin and its activity has been finalised and at present no analogues based on the whole chlorofusin molecule have been shown to be ac- tive against the p53/MDM2 interaction. Also, evidence for chlorofusins binding modality in the hydrophobic pocket of MDM2 is limited and at present there are no published crystal structures of this binding. Although inhibitors have already been explored, there is still scope for a wider variety of inhibitors to be investigated that have yet to be mentioned within the literature, which is one of the main purposes of this thesis.