Chapter 2: Synthesis aspects of Ajoene
2.6. Ajoene Analogues with Water-solubility-promoting Substituents
2.6.1 Overview
A library of ajoene analogues were synthesized within our group and tested for their anti-proliferation activity on various cancer lines. We were particularly interested in an oesophageal cancer cell-line called WHCO1. Results of the SAR study are summarised in Table 1, where replacement of R1 and R2 were varied independently. Ajoene derivatives were tested either as separate isomers or as a mixture of isomers depending on the R group used. Anti-proliferation of the WHCO1 cell-line was determined by an MTT assay, which is a colourimetric assay in which live cells appear as purple due to intact mitochondrial reductase enzymes that reduce the MTT dye.
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33 Initially, the R1 substituents (4a- 4g) were varied and it was identified that having an electron-releasing end-group with high lipophilicity provided an increased anti-proliferation activity, with p-methoxybenzyl as the most active substituent on R1. The R2 substituent was also optimised (4j-5) and it was found that p-methoxybenzyl also gave the best anti-proliferation of WHCO1 cells. Interestingly, having a p- fluorobenzyl substituent gave a significantly less active analogue suggesting that having electron- withdrawing groups on the terminus reduces the activity of the central pharmacophore. In cases where separation of isomers was possible it was found that the Z-isomer was slightly more active than the E- isomer by around 30%.42 Hence, replacing both R1 and R2 by p-methoxybenzyl was identified as our most active analogue (see Figure 12), which resulted in an inseparable mixture that showed a twelvefold increase in activity (2.1 M) relative to Z-ajoene (25 M). The IC50 of this analogue is lower than that of the in vitro clinical range for the other chemotherapeutics, cisplatin (9.2 M) and 5- fluororacil (7.9 M) on the same cell-line.35
Figure 12: Advanced lead 5 identified in an SAR study.
Advanced lead 5 was tested for its ability to inhibit tumour growth in a nude-mouse model. However, owing to its lipophilic nature it was not possible to give oral doses and so it was decided to administer it through an intralipid vehicle. The test subjects were each inoculated with 2.50 x 106 WHCO1 cells delivered via subcutaneous injection and tumours were allowed to develop over the duration of the study.
At the end of the study it was found that there was no significant effect on tumour growth in the subjects that received drug treatment compared to the control subjects, and also that the drug had no negative effects on the health of the subjects. Advanced lead 5 that returned very good in vitro activity, was thus ineffective in reducing tumour sizes in an in vivo study. This may have been due to its lipophilic nature resulting in poor drug bio-availability, but it might also have been due to the drug degrading upon administration and thus not reaching the tumours. The latter possibility was further explored when advanced lead 5 was incubated in whole blood for two hours at 37°C and it was found that even after 15 minutes the compound could not be detected by HPLC (see, Figure 13). It may also be postulated that intraperitoneal injection may not have been the best choice for drug delivery. 35, 47
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34 Table 1: Effects of R1 and R2 substitution of WHCO1 cell proliferation.
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35 Figure 13: Metabolic stability of advanced lead 5, as measured by HPLC.
The current project was aimed at addressing the first of these issues by introducing groups at the para- position of the benzyl substituents of 5 that would promote water-solubility. The new target should resemble analogue 5 as closely as possible in order to maximize activity and thus the proposed modified target was conceptualised as depicted in Figure 14. The next section serves to describe in detail the synthesis of ajoenes with substituents that promote an increased degree of aqueous solubility, their characterisation and also their biological evaluation.
Figure 14: Illustration of the target water-soluble ajoene, 6 and 6’.
A second objective was to design the synthesis such that it is possible to place a radioactive 14C-tag on one of the p-methoxy methyl groups in an attempt to track the movement and accumulation in nude mice xenografts using positron emission tomography (PET). In this case, the radiolabed ajoene analogue would still need to resemble 5 as depicted in Figure 15. This was done in collaboration with NTeMBI/ NECSA at the nuclear facilities at Pelindaba in South Africa
Figure 15: Illustration of targeted labelled analogue, 7. -100
1900
0 25 50 75 100 125
5 (pmoles)
Time (mins)
Metabolic Stability of 5 in whole Blood at 37 C
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36 The UCT synthesis was employed to access the two target molecules and so it was decided to start the sequence by synthesis of an advanced intermediate that would allow placement of a radiolabeled tag and also a water-soluble group as the last step based on the availability of the tag. This required one of the aromatic groups to be a free phenol.
The synthesis began with attempts to synthesize ajoene 6’ with a free phenol on the left-hand side of the molecule. A retrosynthetic analysis based on the UCT synthesis is shown in Scheme 11, which indentified synthons 8 and 9 as accessible from methyl p-hydroxybenzoate 12 and p-methoxybenzyl chloride 11 respectively. Reagent 9 had previously been synthesized within our group from the chloride 11.
Scheme 11: Retrosynthetic analysis of target 6’.
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