Original article
6-Substituted 2-oxo-2H-1-benzopyran-3-carboxylic acid derivatives in a new approach of the treatment of cancer cell invasion and metastasis

https://doi.org/10.1016/j.ejmech.2008.01.024Get rights and content

Abstract

Novel 6-substituted 2-oxo-2H-1-benzopyran-3-carboxylic acid derivatives were synthesized and their potency in reducing the invasive behaviour of HT 1080 fibrosarcoma cells was evaluated. Structure–activity relationships were deduced from biological results and will be used in further design of new active compounds. In particular, the acetoxymethyl substituent found at the 6-position of previously described active compounds can be replaced by an acetamidomethyl substituent without loss of potency; while the presence of an aryl ester function at the 3-position was preferred to a thioester or an amide function to induce marked biological activity.

This work confirms the interest of aryl esters of 6-substituted coumarin-3-carboxylic acids as potential new anti-cancer agents.

Introduction

One of the most difficult problems arising during cancer therapy is the occurrence of cancer cell invasion responsible for the spread of tumour cells throughout the body. Such an event leading to metastases is an important cause of the bad prognosis of numerous cancers. Although anti-cancer chemotherapy mainly consists in the use of cytotoxic agents interfering with cell division, another research field has recently emerged with the design and identification of agents able to inhibit or limit the metastatic process. Indeed, many efforts are now focussed on the search of compounds interfering with the cancer cell invasion process and expressing anti-angiogenic properties [1]. Examples of drugs, among which monoclonal antibodies and small molecules, are currently under clinical investigation and some of them have recently been approved for use in man in the treatment of several cancers associated or not with conventional chemotherapies or radiation therapies [2], [3], [4], [5], [6], [7], [8], [9]. These drugs comprise vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) inhibitors (monoclonal antibodies) as well as VEGF and EGF receptor tyrosine kinase inhibitors (small-molecule inhibitors), but also matrix metalloproteinase (MMP), urokinase (uPA), cycloogygenase-2 (COX-2) or methionine aminopeptidase inhibitors [1].

We recently reported that two synthetic 6-substituted coumarin-3-carboxylic acid derivatives, 3-chlorophenyl 6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylate (1) and 3-bromophenyl 6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylate (2) (Fig. 1), expressed a marked potency in inhibiting cancer cell invasion in vitro and tumour growth in vivo [10]. Simple natural and synthetic coumarins such as esculetin (3), osthole (4) and the synthetic 6-nitro-substituted coumarin (5) (Fig. 1) are also known for a long time to exert anti-cancer properties, but, for most of them, by an unknown mechanism [11], [12], [13], [14]. Recently reported coumarin derivatives such as G8935 (6) (Fig. 1), structurally related to 1 and 2 and identified as mitogen activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) kinase inhibitors (MEK1 inhibitors), raise the possibility that the coumarin-3-carboxylates 1 and 2 may exert their anti-cancer activity through inhibition of MEK1 [15]. Another recent work showed that coumarin-3-carboxamides (7, Fig. 1) tightly related to the chemical structure of 1 and 2 selectively inhibited the tumour cells that have a high level of epidermal growth factor receptor (EGRF or erbB1) and erbB2 (HER2) receptor expression [16].

Interestingly, the chemical structure of the active coumarins 1 and 2 may be compared to that of linomide (roquinimex) (8, Fig. 2), a quinolinone isostere of coumarins known to be an immunomodulator, but also expressing anti-angiogenic activity in vitro and in vivo [17], [18], [19]. Moreover, the preferred conformation adopted by linomide, as found in the crystal [20] (8′; Fig. 2), can be superimposed to the preferential conformation adopted, in the solid state and probably in solution, by aryl esters of coumarin-3-carboxylic acid derivatives (9′; Fig. 2) in accordance to recent published crystallographic and molecular modelling studies [21], [22]. In contrast to aryl esters of 6-substituted coumarin-3-carboxylic acids, the corresponding aryl amides 10 (Fig. 2) are not able to adopt such conformation because a strong intra-molecular hydrogen bound is established between the hydrogen atom of the exocyclic NH group and the lactonic carbonyl group [21].

Using coumarins 1 and 2 as lead compounds, the present work aimed at describing new examples of coumarin-3-carboxylic acid derivatives in order to better define structure–activity relationships that could explain their anti-invasive activity. Particular attention was paid to the replacement of the oxygen atom included in the exocyclic ester function in the 3-position by a sulphur atom (thioesters) or by a nitrogen atom (amides) with or without a methyl group. The importance of the nature and the size of the substituent in the 6-position was also examined. Among them, the replacement of the metabolically labile ester function (acetoxymethyl moiety) in the 6-position by a more stable amide function (acetamidomethyl moiety) was investigated. In these series, the presence of a halogen atom in the meta position of the phenyl ring in the 3-position (preferably a chlorine or a bromine atom) was maintained like in the structure of the lead compounds 1 and 2.

Section snippets

Chemistry

Scheme 1 illustrates the synthetic pathway giving access to the coumarinic derivatives characterised by the introduction of a thioester function or an amide function in the 3-position.

Compound 12 obtained from 11 by acetylation was converted into the corresponding acid chloride 13 as previously described [21], [23]. The acyl chloride intermediate 13 then reacted with appropriate thiophenols and anilines to provide the thioesters 14, the secondary amides 15 and the tertiary amides 16 (Scheme 1).

Results and discussion

The new synthetic coumarins were examined in a ‘Boyden chamber’ invasion assay using HT 1080 fibrosarcoma cells in order to determine their potency in reducing the invasive behaviour of tumour cells [10]. The assay consists in measuring the ability of cells treated or not with the coumarinic derivatives tested at different concentrations to pass through type IV collagen-coated Transwell cell culture inserts (chemoinvasion assay). Cell invasion in the absence of coumarin derivative was

Conclusion

The present work aimed at describing new examples of coumarin-3-carboxylic acid derivatives structurally related to lead coumarins 1 and 2 expressing anti-invasive activity in vitro and anti-tumoral effect in vivo. Among the different chemical modifications examined, the replacement of the ester function in the 6-position of 1 and 2 by an amide function was allowed without loss of potency in the ‘Boyden chamber’ chemoinvasion assay with HT 1080 fibrosarcoma cells. By contrast, an aryl ester

Chemistry

Melting points were determined with a Büchi-Tottoli capillary apparatus and are uncorrected. IR spectra were recorded as KBr pellets on a Perkin–Elmer 1000 FTIR spectrophotometer. The 1H NMR spectra were recorded on a Bruker AW-80 (80 MHz) or a Bruker Avance (500 MHz) instrument using d6-DMSO as the solvent with TMS as an internal standard; chemical shifts are reported in δ values (ppm) relative to that of internal TMS. The abbreviations s = singlet, d = doublet, t = triplet, q = quadruplet, m = multiplet,

Acknowledgements

This study was supported by grants from the National Fund for Scientific Research (F.N.R.S., Belgium) from which P. de Tullio is Research Associate. The technical assistance of Y. Abrassart and F. Olivier is gratefully acknowledged.

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