Synthesis of phthalates of betulinic acid and betulin with cytotoxic activity

doi:10.1016/j.bmc.2005.03.006

Bioorganic & Medicinal Chemistry

Volume 13, Issue 10, 16 May 2005, Pages 3447-3454

https://doi.org/10.1016/j.bmc.2005.03.006 Get rights and content

Abstract

Synthesis of 3β-O-phthalic esters from betulinic acid and its esters and synthesis of phthalic esters from betulin and its monoacetates using classical acylation procedure with phthalic anhydride. The evaluation of cytotoxicity of the prepared compounds was using numbers of tumor cell lines in MTT test. It was discovered that hemiphthalic esters had better cytotoxicity than starting compounds as betulinic acid or quite inactive betulin.

Graphical abstract

Synthesis of 3β-O-phthalic esters from betulinic acid and its esters and synthesis of phthalic esters from betulin and its monoacetates using classical acylation procedure with phthalic anhydride. The evaluation of cytotoxicity of the prepared compounds was using number of tumor cell lines in MTT test. It was discovered that hemiphthalic esters had better cytotoxicity than starting compounds as betulinic acid or quite inactive betulin.

Introduction

Betulinic acid (1) (3β-hydroxy-lup-20(29)-en-28-oic acid), pentacyclic lupane triterpene, is a known natural compound with various biological effects.1, 2 Cytotoxic activity against various malignant versus nonmalignant cell lines belongs to most intensively studied³ effects of these compounds. Betulinic acid (1) demonstrated selective anticancer activity against neuroectodermal tumors,⁴ including human melanoma, neuroblastoma, and Ewing sarcomas. Therefore a lot of effort is focused on modification5, 6 of betulinic acid in order to increase and broader its biological activity against tumor cells of various histogenetic origin despite the fact that its mechanism of effect is still being studied⁷ and is not clear enough. In contrast to betulinic acid (1), betulin (5) (lup-20(29)-en-3β,28-diol) has no significant cytotoxic activities. However, based on structural similarity with betulinic acid (1), one could expect that appropriate derivatization could increase its cytotoxic potency.

In this paper we performed synthesis of new triterpenic compounds, particularly those derivatized on hydroxy groups (mainly 3β- and 28-hydroxy groups).⁸ Since derivatization of hydroxy group of betulinc acid (1) with lipophylic groups (e.g., acetates) lead to decrease of biologic activity,⁹ right choice of appropriate substitution is critical for improvement of pharmacological properties of new compounds. In this respect we have mainly paid attention to the synthesis of hemiesters of lower diacids using competent anhydride.¹⁰ This procedure can lead to acquirement of large groups of new polar derivatives with increased cytotoxic activity. Here we report our results on synthesis of betulinic acid and betulin phthalate esters and the effect of this substitution on biological activity of new compounds. Methyl- and ethyl-phthalates were also prepared within the structure–activity relationship study.

Section snippets

Chemistry

Betulinic acid (1) was isolated from bark of plane trees Platanus acerifolius using extraction with MeOH according to literature.⁵ Treatment of acid 1 with etherical solution of diazomethane, resp. diazoethane, gave methyl 2, resp. ethyl ester 3, in quantitative yield. Reaction of acid 1 with benzyl bromide in the presence of DBU afforded benzyl ester 4 in the yield 85%.

Betulin (5) was isolated from the birch bark (Betula pendula) using the known extraction procedure.¹¹ Monoacetate 6 was

Results and discussion

Our preliminary investigation showed that betulinic acid (1) and betulin (5) derivatives are potential lead compounds for new anti-tumor agents. Majority of hemiphthalates showed increased cytotoxic activity judged against starting compounds. The lowest effect of hemiphthalates was found in the case of bishemiphthalate 5a, since activity of those substances increased only slightly compared with starting betulin (5). On the other side, derivative 6a was substantially better than the parent

Conclusion

Within the worldwide research of betulinic acid (1) and betulin (5) in the field of anti-tumor agents, number of structural modifications, and derivatizations were studied. Our study demonstrates that derivatization of 3β-hydroxy group of these triterpenoids with lower diacids can result in compounds with higher cytotoxic activity against cell lines of different histogenetic origin, including drug resistant tumors. Even derivatization of quite inactive betulin (1) can lead to compounds with

Experimental

Melting points were determined using a Kofler block and are uncorrected. Optical rotations were measured using CHCl₃ solutions (unless otherwise stated) on an Autopol III (Rudolph Research, Flanders, NJ) polarimeter, with an accuracy of ±2°. NMR spectra were recorded on a Varian UNITY INOVA 400 instrument (¹H NMR spectra at 399.95 MHz) using CDCl₃ solutions (unless otherwise stated), with SiMe₄ as an internal standard. Skeletal signals in the region approx. 0–2 ppm were not recorded, except

Chemicals

Phthalic anhydride, DMAP, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and SDS were purchased from Sigma–Aldrich, s.r.o., Czech Republic.

Cell lines

Cell lines CEM, HT29, K562, PC-3, and SK MEL2 were purchased from the American Tissue Culture Collection (ATTC). Paclitaxel-resistant subline of K562 cells (K562-tax) was prepared and characterized in our laboratories.¹⁵

MTT cytotoxicity assay

Cell suspensions were prepared and diluted according to the particular cell type and the expected target cell density

Acknowledgements

This study was supported in part by the Ministry of Education of the Czech Republic (MSM 113100001, 151100001), which paid for instrumental equipment, by the Czech Science Foundation (203/03/D152), from which the chemicals were paid and by MPO project (FT-TA/027) from which HPLC column, silica gel and all other material support were paid. Biological testing was supported by the Czech Science Foundation (301/03/1570). We are grateful to Iva Tislerova for measurement of NMR spectra. Special

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Synthesis of phthalates of betulinic acid and betulin with cytotoxic activity

Abstract

Graphical abstract

Introduction

Section snippets

Chemistry

Results and discussion

Conclusion

Experimental

Chemicals

Cell lines

MTT cytotoxicity assay

Acknowledgements

Cancer Lett.

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem.

Nat. Med.

Med. Res. Rev.