Elsevier

Bioorganic & Medicinal Chemistry

Volume 18, Issue 24, 15 December 2010, Pages 8630-8641
Bioorganic & Medicinal Chemistry

Convergent synthesis of fluorescence-labeled probes of Annonaceous acetogenins and visualization of their cell distribution

https://doi.org/10.1016/j.bmc.2010.10.004Get rights and content

Abstract

The convergent synthesis of fluorescence-labeled solamin, an antitumor Annonaceous acetogenin, was accomplished by two asymmetric alkynylations of 2,5-diformyl tetrahydrofuran with an alkyne tagged with fluorescent groups and another alkyne with an α,β-unsaturated γ-lactone. Assay for the growth inhibitory activity against human cancer cell lines revealed that the probe with the fluorescent groups at the end of the hydrocarbon chain may have the same mode of action as natural acetogenins. The merged fluorescence of dansyl-labeled solamin and MitoTracker Red suggests that Annonaceous acetogenins localize in the mitochondria.

Introduction

Over 400 natural polyketides called Annonaceous acetogenins have been isolated from Annona plants growing in tropical and subtropical regions.1 Most acetogenins are white waxy derivatives of long-chain fatty acids (C32 or C34) whose terminal carboxylic acid is combined with a 2-propanol unit at the C-2 position to form a methyl-substituted α,β-unsaturated γ-lactone (Fig. 1). One interesting feature of their chemical structures is the single, adjacent, or nonadjacent 2,5-disubstituted tetrahydrofuran (THF) system with one or two flanking hydroxyl groups(s) at the center of a long hydrocarbon chain. Much attention has also been paid to their broad spectrum of biological activities, including immunosuppressive, antimalarial, insecticidal, and probably the most impressive, antitumor activity. Several strategies for the total synthesis of natural acetogenins2 and their analogues3 have been developed, motivated by their unique structures and attractive biological activities.

It is generally accepted that the mode of action of acetogenins is the inhibition of NADH-ubiquinone oxidoreductase (complex I) in the mitochondria, thereby suppressing ATP production particularly for cancer cells having high metabolic activity, and inducing apoptosis.4, 5, 6 However, the structure–activity relationship for the inhibition of complex I is not completely related to its cytotoxicity. McLaughlin and co-workers suggested that as the mitochondrial assay is cell-free, it does not take into consideration such factors as membrane transport, intracellular transport, and metabolic inactivation.7 Poupon and co-workers synthesized the first fluorescence-labeled acetogenin, dansyl-labeled squamocin, in which γ-lactone was replaced with a fluorescence label, and visualized its cell distribution.8 Yao and co-workers reported that a fluorescence-labeled analogue, in which the THF ring moiety of natural acetogenin was mimicked by ethylene glycol ether unit replacements, showed different distributions in human normal cells and cancer cells.9 Miyoshi and co-workers reported an 125I-labeled acetogenin analogue in which the (trifluoromethyl)phenyldiazirine group served as a substitute for the γ-lactone moiety and a photoreactive group photo-cross-linked to the ND1 subunit of bovine heart mitochondrial complex I with high specificity.10 These reports prompted us to synthesize a novel fluorescence-labeled acetogenin that retains all functionalities,11 because in living cells, the hydrophilic THF moiety in natural acetogenins may be located at the surface of the mitochondrial inner membrane and the γ-lactone moiety may interact with the enzyme active site.12 Miyoshi and co-workers revealed that the hydrocarbon chain on the left side of the THF moiety is not essential for the inhibitory activity against complex I.13 We planned the synthesis of novel fluorescence-labeled acetogenins having a fluorescent group at the left end of the THF moiety. After starting our synthetic study, Sinha and co-workers reported the first synthesis of photoactive asimicin with all functionalities, which has a benzophenone group at the end of the hydrocarbon chain.14 However, the photoactive probe was not evaluated.15 Herein, we describe the full details of the first16 and second-generation17 syntheses of fluorescence-labeled acetogenins, the evaluation of their growth inhibitory activities against human cancer cell lines, and their cell distribution.

Section snippets

First-generation synthesis of fluorescence-labeled solamin

We planned to synthesize fluorescence-labeled acetogenin with our developed stereoselective synthesis of the THF moiety18, 19 and selected solamin,20 which has a comparatively simple structure yet all the functionalities, as the lead compound. The 7-nitrobenzo[c][1,2,5]oxadiazol-4-yl-amino (NBD-NH–) group was employed as a fluorescent tag due to its strong fluorescence with a long wavelength, which is advantageous for the observation of cell distribution.21

The synthesis was started from

Conclusion

We have accomplished the convergent synthesis of fluorescence-labeled solamin, an antitumor Annonaceous acetogenin, by two asymmetric alkynylations of 2,5-diformyl tetrahydrofuran with two kinds of alkynes as the key steps. Examination of their growth inhibitory activity against cancer cells revealed that the introduction of fluorescent groups at the end of the hydrocarbon chain of acetogenins did not interfere with the mode of action of the probes. It was observed that dansyl-labeled solamin 3

Chemistry

Melting points are uncorrected. Optical rotations were measured by using a JASCO DIP-360 digital polarimeter or a JASCO P-1020 digital polarimeter. 1H NMR spectra were recorded in CDCl3 solution with a JEOL JNM-GX-500 spectrometer (500 MHz). 13C NMR spectra were recorded in CDCl3 solution with a JEOL JNM-AL300 spectrometer (75 MHz) or a JEOL JNM-GX-500 spectrometer (125 MHz). All signals are expressed as δ values in ppm downfield from the internal standard tetramethylsilane. The following

Acknowledgments

In vitro antiproliferative activities of fluorescence-labeled solamins and natural solamin against human cancer cell lines were examined by the Screening Committee of New Anticancer Agents supported by a Grant-in-Aid for Scientific Research on Priority Area ‘Cancer’ from The Ministry of Education, Culture, Sports, Science and Technology, Japan. We acknowledge financial support in the form of a Grant-in-Aid for Young Scientists (B) [No. 21790113], a Grant-in-Aid for Scientific Research (B) [No.

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