GB2411353A

GB2411353A - Resveratrol Analogues

Info

Publication number: GB2411353A
Application number: GB0404183A
Authority: GB
Inventors: Richard Michael Hoffman
Original assignee: University of Hertfordshire
Current assignee: University of Hertfordshire
Priority date: 2004-02-25
Filing date: 2004-02-25
Publication date: 2005-08-31
Also published as: GB0404183D0

Abstract

Compounds for use in the treatment of conditions associated with angiogenesis or for diseases associated with cyclooxygenase-2 (COX-2) of the formula: <EMI ID=1.1 HE=36 WI=50 LX=278 LY=762 TI=CF> <PC>wherein when R1 is H or OH, R2 is 3-methylbut-1-enyl, 3-methylbut-2-enyl or similar; and when R1 is OH, R2 is a hydrocarbon chain; and analogues thereof. Thus these compounds may be useful in the treatment of conditions associated with inflammation, angiogenesis and tumour growth.

Description

241 1 353 Resveratrol Analonues

Field of the Invention

The present invention concerns resveratrol analogues obtainable as phytoalexins that have been found to have valuable biological/pharmacological activity.

Background to the Invention

Resveratrol (3, 4', 5-trihydroxy-trans-stilbene - see Figure 1), present in red grapes, is believed to be responsible for the so-called "French paradox", in which consumption of red wine has been shown to be associated with reduced mortality due to heart disease and certain cancers. Many of the beneficial effects of resveratrol have been linked to inhibiting the induction of the pro-inflammatory enzyme cyclooxygenase- 2 (COX2). In addition, resveratrol has anti-tumour, anti angiogenic and pro-apoptotic effects. Thus, resveratrol has potential therapeutic use in a wide range of conditions associated with inflammation, angiogenesis and tumour growth. In this proposal, we have isolated a number of novel resveratrol derivatives, and in preliminary studies, one of these derivatives has been found to have superior properties compared to resveratrol.

Resveratrol is commonly referred to as a phytoalexin (a compound that is induced in a plant in response to stress conditions). In red grapes its production is believed to be stimulated by Botrytis vitiae fungus. Phytoalexins have several advantages as novel pharmaceuticals over normal plant secondary metaboiites.

Firstly, high and reproducible yields can be isolated from commonly occurring plant - .- sources (thus not relying on exotic, and difficult to obtain plants with variable levels of substances). Secondly, phytoalexins are the end result of considerable metabolic input by the plant to produce biologically active (anti-fungal etc) products, thus increasing the likelihood that they will be pharmacologically active. Thirdly plants often produce a family of structurally related substances.

Resveratrol and resveratrol-like compounds/ analogues have been found to exist not only in grapes but also in, amongst others, Polvoonum tinctorium (Chinese Indigo plant), Polvoonum bistorta (Snakeweed) and other PolYqonum species, rhubarb and peanuVgroundnut.

Attempts to synthesise resveratrol analogues for prevention of disease are documented in, for example, POT WO 01/21165, wherein a number of chemically modified resveratrol compounds including dibydroxy, tetrahydroxy and pentahydroxy, dimethoxy, trimethoxy, tetramethoxy and pentamethoxy stilbene derivatives, were prepared and examined for potential cancer preventive activity.

Amongst other findings, that document concluded that 3, 4, 4', 5 tetrahydroxystilbene possesses 10-fold less activity than resveratrol.

As a generality, the publications on research into resveratrol have focused into the compound itself or chemically synthesised analogues and largely ignored the existence of and possible merits of analogues that may be available from natural sources. Hitherto little or no systematic research has been undertaken into the pharmacological effects of phytoalexin analogues of resveratrol.

Summarv of the Invention According to a first aspect of the present invention, there is provided a composition for use as an anti-angiogenic composition and/or as a composition for inhibition of cyclo-oxygenase-2 as a prophylactic or therapy for disease states involving cyclo-oxygenase- 2 comprising a compound having the formula: {1: . t40H:-::: Ho' .. :-. t) Rs

selected from the group consisting of compounds wherein: a) R' = -H or-OH; and R2 = 3-methyl-but-1 enyl; or 3-methyl-but-2 enyl; - or similar; b) R' = -OH; and R2 = a hydrocarbon chain; or c) analogues thereof.

Suitably in (b) the hydrocarbon chain has one or more double bonds and preferably is branched chain.

To particular advantage the compound used may be viewed as a tetrahydroxy resveratrol analogue having a hydrocarbon side chain.

Contrary to expectation, analogues falling within the above formula have been established to have an anti-angiogenic effect and an effect of inhibiting induction of cyclo-oxygenase, and even more surprisingly an effect that is substantially greater than that of resveratrol itself. Furthermore, the compound, although suitably being a tetrahydroxy resveratrol analogue, would appear to have little or no cytotoxicity. In the tetrahydroxy analogue it is speculated that this may be as a result of the hydrocarbon side chain. In the examples the hydrocarbon side chain is branched, being a 3-methyl-but-1-enyl side chain.

The above formula is to be treated as further including giycosylated ' derivatives of compounds of the specified formula, noting that glycosylated..

derivatives of resveratrol are common place in nature and facilitate biological availability of resveratrol for absorption and assimilation. : .'.

Since the compounds of the present invention are obtainable as....

phytoalexins obtained directly from a plant source, there is the potential for high ' reproducible yields through harvesting from commonly occurring plant sources. ..

Brief Descrintion of the Drawinos A preferred embodiment of the present invention will now be more particularly described by way of example with reference to the accompanying drawings wherein: Figure 1 illustrates the chemical formula for resveratrol and Figure 2 the chemical formula of the enhanced activity tetrabydroxystilbene of the present invention (BS2); Figures 3 and 4 illustrate the chemical formulae of two further related phytoalexin compounds isolated from peanut, BS1/1 being a trihydroxystilbene with a 3-methyl-but-1 enyl side chain (Figure 4) and BS1/2 being a trihydroxystilbene having a 3-methyl-but-2-enyl side chain (Figure 3); and Figure 5 is a photomicrograph of a western blot analysis for determining cyclooxygenase-2 induction in mouse macrophage cells for each of resveratrol, and BS2.

Example Protocol for Obtainment of Phvtoalexin Resveratrol Analogues Whole unshelled groundnuts, suitably from America, are first shelled and then sliced to approximately 1 mm thickness and steeped in water incubated at room temperature for between 48 and 72 hours in order for the peanuts to become thoroughly infested with the naturally-occurring microflora present inherently in the peanuts as harvested from their country of origin. Indeed, the shells of the peanuts may be retained with the peanuts or used as a source of phytoalexin in their own ..

right. .... :.

As a result of their incubation with the microfloraNungi the peanuts are stimulated to produce phytoalexins and those phytoalexins are extracted according.. .: to the following protocol. .... . A.

Firstly, the subject peanuts are crushed in a pestle and mortar and mixed and homogenized into an organic solvent such as ethanol and then filtered.

From this point onwards all samples should be protected from light as far as possible. The homogenate may be filtered and evaporated under reduced pressure to a small volume and then partitioned three times against equal volumes of petrol (b.p. 40-60 ) and the petrol fraction discarded. The remaining aqueous fraction may then be partitioned three times against EtOAc and the combined EtOAc phases dried. Samples may then be fractionated on a Silica gel column with petrol (b.p. 40 60 /EtOAc t7:3)) as mobile phase.

Following phytoalexin induction and extraction protocols substantially as described above three different compounds each having the form of an analogue of resveratrol have been isolated from solution and purified by thin layer chromatography. Each has been identified in turn by mass spectrometry. The three compounds, labelled BS1/2, BS2/2 and BS2 are shown in Figures 2 to 4 herein below.

Repeated bioassays have been carried out on each of these using the following protocol. J774 macrophages were plated in 95 well plates, test compounds BS1/2, BS2/2 and BS2 were added and 1 hour later the cells were stimulated with lipopolysaccharide (LPS). After 24 hours, cell extracts were prepared and run on SDS-PAGE. COX-2 induction was analysed by western blot using ECL (enhanced chemiluminescence) detection.

As can be seen, BS2, was found to be a more potent inhibitor of COX2 induction than resveratrol. In addition, this compound has antiangiogenic activity in an in vitro assay for angiogenesis. Interestingly, the compound is apparently not cytotoxic. The compound may be useful as an anti-inflammatory and/or anti angiogenic (and thus anti-tumour) compound with no general toxicity.

Turning to Figure 5, this illustrates a single bioassay experiment to assess induction of cyclooxygenase-2 by the resveratrol analogue and is representative of e..

three separate experiments which were reproducible. :::

Figure 5 is a photomicrograph of a Western blot analysis with antibody to COX-2 unstimulated (lane 2), stimulated with LPS (1,ug/ml) (lane 3) for 24 hours in presence of resveratrol (lane 4 and 5) and novel compound (lane 6 and 7), Lane 1 is COX-2 positive (72kD), Lane 2 is negative control without LPS, Lane 3 is positive control with LPS, Lane 4 is 10,um Resveratrol (1 hour) + 1'ug/ml LPS, Lane 5 is 30'um Resveratrol (1 hour) + 1'ug/ml LPS, Lane 6 is 10,um novel compound BS2 (1 hour) + 1,ug/ml LPS, Lane 7 is 30,um novel compound BS2 (1 hour) + 1,ug/ml LPS The level of inhibition of COX-2 production by J774 macrophages incorporating resveratrol or BS2 was: 10,uM resveratrol 15% +/- 0.06% 30,uM resveratrol 21% ± 0.07% 10 AM BS2 46% ± 0.13%; and hum BS2 54% +/- 0.15% These data are means (+ /- s.e.) of three independent western blot experiments. No cytotoxicity by BS2 was observed. Inhibition of COX-2 production by BS1/1 was similar to resveratrol.

To bioassay for anti-angiogenic activity of the resveratrol analogues, the analogues were tested in the Matrigel In Vitro Assay for angiogenesis. Human umbilical vein endothelial cells (between passages 2-5) were seeded on to Matrigel and tubule formation was observed under a microscope 18 hours later. In example results, 30 hum BS2 completely inhibited tubule formation and 20 hum BS2 partially inhibited tubule formation and with similar results to resveratrol. - ëeee -' e. e e - - ... :--e

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Claims

- CLAIMS

1. A compound for use as an anti-angiogenic composition and/or as a composition for inhibition of cyclo-oxygenase-2 as a prophylactic or therapy for disease states involving cyclo-oxygenase-2 comprising a compound having the formula: Ho 1\ R: selected from the group consisting of compounds wherein: a) R. = -H or -OH; and R2 = 3-methyl-but-1 enyl; or 3-methyl-but-2 enyl; - or 1 0 similar; b) R. = -OH; and R2 = a hydrocarbon chain; or c) analogues thereof.

2. A compound as claimed in Claim 1, wherein in (b) the hydrocarbon chain has one or more double bonds.

3. A compound as claimed in Claim 1 or 2, wherein in (b) the hydrocarbon chain is branched chain.

4. A compound as claimed in Claim 3, wherein the said hydrocarbon chain is a 3-methyl-but-1 enyl chain.

5. A compound as claimed in any preceding claim, wherein R' is -OH and R2 is 3-methyl-but-1 enyl.

6. A composition as claimed in Claim 1, wherein R. is OH and R2 is 3methyl- but-2 enyl.

7. Use of a compound having the formula: R. 1 selected from the group consisting of compounds wherein: a) R' = -H or-OH; and R2 = 3-methyl-but1 enyl; or 3-methyl-but-2 enyl; - or similar; b) R' = -OH; and R2 = a hydrocarbon chain; or c) analogues thereof.

for use in preparation of a medicament as an anti-angiogenic medicament.

8. Use of a compound as claimed in Claim 7, wherein in (b) the hydrocarbon chain has one or more double bonds.

9. Use of a compound as claimed in Claim 7 or Claim 8, wherein in (b) the hydrocarbon chain is branched chain.

10. Use of a compound as claimed in Claim 9, wherein the said hydrocarbon chain is a 3-methyl-but-1 enyl chain.

11. Use of a compound as claimed in any of Claims 7 to 10 inclusive, wherein R. is -OH and R2 is 3-methyl-but-1 enyl.

12. Use of a compound having the formula ,1 selected from the group consisting of compounds wherein: a) R. = -H or -OH; and R2 = 3-methyl-but1 enyl; or 3-methyl-but-2 enyl; - or similar; b) R. = -OH; and R2 = a hydrocarbon chain; or c) analogues thereof.

in preparation of a medicament for prophylaxis or therapy of a diseased state involving cyclo-oxygenase 2.

13. Use of a compound as claimed in Claim 12, wherein in (b) the hydrocarbon chain has one or more double bonds.

14. Use of a compound as claimed in Claim 12 or Claim 13, wherein in (b) the hydrocarbon chain is branched chain.

15. Use of a compound as claimed in Claim 14, wherein the said hydrocarbon chain is a 3-methyl-but-1 enyl chain.

16. Use of a compound as claimed in any of Claims 12 to 15 inclusive, wherein R. is -OH and R2 is 3-methyl-but-1 enyl.