the important findings in our project.
In various mice models, our collaborators, Grainger et al. have demonstrated that
Tamoxifen inhibited atherosclerosis by suppressing the diet-induced formation of lipid lesions in the aorta.48, 55 Tamoxifen is a potent inducer of autophagy and
autophagy is believed to have beneficial effects for atherosclerosis and plague progression, possibly by degrading damaged intracellular organelles, thereby preventing oxidative injuries and cellular distresses.57 However, Tamoxifen is a well-
known multi-targeting drug and it is not clear which molecular targets of Tamoxifen is/are responsible for the induction of autophagy and the cardioprotective effects of Tamoxifen. In addition, the estrogenic activity of Tamoxifen in the endometrium cells is linked to an increased risk of ulcerous cancers.19 Therefore, the development
of more selective autophagy inducers is needed if they are ever to become medicinally useful in the treatment or prevention of cardiovascular diseases.
We started our project based on the extensive research and findings of Magarian et
al. who had demonstrated that the dichlorocyclopropyl analogues of Tamoxifen were
pure antiestrogens, with very interesting anti-cancer and anti-tubulin properties.75-77,
79, 81-83, 149, 204
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A number of those diaryldichlorocyclopropanes had been synthesized in racemical form and as we attempted to develop a synthetic route for their asymmetric synthesis, we found out the diaryldichlorocyclopropanes were not particularly stable and degraded easily under normal handling conditions. As a result, we abandoned the asymmetric synthesis of diaryldichlorocyclopropane and moved on to try to synthesize the difluorocyclopropyl analogues instead since it was believed that the relative stronger C-F bond may offer greater stability.
Methods for the synthesis of gem-difluorocyclopropanes are extremely limited, with
the vast majority of the reactions reported involving the [2+1] cycloaddition of difluorocarbene to alkenes.86 Additionally, the difluorocarbene is the least reactive
dihalocarbene and thus the synthesis of difluorocyclopropyl analogues of Tamoxifen from unreactive olefins, such as stilbenes, is particularly challenging. Having attempted to generate the difluorocarbene using various methods, we found that TMSCF3 is probably one of the most effective difluorocarbene precursors reported to
date. Not only does TMSCF3 offer many advantages such as low toxicity,
commercial availability and mild reaction conditions, but the catalyst NaI is also inexpensive, reliable and easy to handle. However, the difluorocarbene generated under the various reaction conditions reported by Hu et al.205 is only of moderate
reactivity and does not transform the electron-deficient diarylethenes to their prospective difluorocyclopropanes at all. In our laboratory, we have developed two alternative methods based on Hu et al.’s original finding which provides the desired
diaryldifluorocyclopropanes without the use of highly toxic Cd(CF3)2, the only
reagent ever reported to date to convert cis-stilbene to its difluorocyclopropane
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enhance the reactivity of the difluorocarbene generated using TMSCF3 and allows
rapid transformation of various olefins to their corresponding difluorocyclopropanes in just under 20 minutes. Although the conversion was not particularly impressive for stilbenes (~20%), it was very effective for more reactive and electron-rich olefins, such as styrene, with conversion as high as >95% (as determined by 1H
NMR spectroscopy of the crude reaction mixtures). We also accidently discovered that the iodine ion can attack TMSCF3, giving -CF3 which subsequently decomposes
to :CF2 and reacts with alkenes slowly at room temperature. We observed that large
amount of gases were produced, leading us to believe that -CF3 can deprotonate
CH3CN to give HCF3 as a gaseous by-product as the reactions proceeded. This belief
is supported by the findings of other researchers who reported the extremely basic nature of trifluoromethyl anion and its ability to abstract a proton from CH3CN.135
The loss of trifluoromethyl anions and formation of HCF3 can be limited by
performing the difluorocyclopropanation in sealed pressure tubes. Although slow, the room-temperature difluorocyclopropanation method is particularly suitable for unstable difluorocyclopropanes such as those derived from cis-stilbene in which the
energy barrier for isomerisation is lower than that for difluorocyclopropanation. Any input of energy would cause the cis-stilbene and its analogues to isomerise to their
trans-counterpart, making the stereoselective synthesis of
diaryldifluorocyclopropanes very difficult to achieve at elevated temperatures. Both microwave-assisted and room-temperature difluorocyclopropanation have their own advantages and disadvantages and it would be interesting to try these methods on a wider range of alkenes and determine their efficacies and potential applications.
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A number of derivatives of Tamoxifen have been synthesized and tested for this project, yet only compounds whose structures were similar to selective ligands of AEBS, such as DPPE and PBPE, induced autophagic responses. DPPE and PBPE have been demonstrated to induce autophagy potentially through disruption in cholesterol biosynthesis pathway and induction of sterol accumulation.161 Therefore,
it appears that our drug candidates are also selective ligands of AEBS and the autophagic effect of our compounds is as a result of inhibition of AEBS and cholesterol synthesis on the basis of high structural similarities between our 1,1- diaryldifluorocyclopropyl and 1,1-diarylmethylene compounds with those well- known AEBS ligands.
Furthermore, we found that the presence of an aminoethoxy basic side chain is crucial for induction of autophagic activities and this finding is consistent with the fact that a basic side chain is also important for binding to AEBS.34 We were able to
confirm that DPPE and PBPE were potent autophagic inducers in our independent study, although not analogues with a morpholine moiety attached to the aminoethoxy side chain. The morpholinyl derivatives MBPE should have also induced autophagic activity if they were high-affinity AEBS ligands as reported by Poirot et al.191 and
this inconsistency in our findings deserve further investigation. Moreover, our novel aminoethoxy difluorocyclopropanes were found to be consistently more active than any of the derivatives of diarylmethane in our biological assay. Most importantly, we have demonstrated that our drug candidates stimulate autophagic response through an estrogen receptor independent pathway as they do not bind to the ERs.
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