GB2296496A

GB2296496A - Pharmaceutical pregnane derivatives

Info

Publication number: GB2296496A
Application number: GB9426080A
Authority: GB
Inventors: Simon Gibbons
Original assignee: Xenova Ltd
Current assignee: Xenova Ltd
Priority date: 1994-12-23
Filing date: 1994-12-23
Publication date: 1996-07-03
Anticipated expiration: 2014-12-23
Also published as: GB2296496B; GB9426080D0

Abstract

Pregnane steroid derivatives obtainable from the roots of the plant Cynanchum otophyllum and their esters are modulators of multi-drug resistance. These compounds are produced by (i) preparing a methanol extract of the roots of Cynanchum otophyllum, (ii) isolating the pregnane derivatives from the fermentation medium, and (iii) if desired, esterifying the pregnane derivatives thus isolated.

Description

PHARMACEUTICAL COMPOUNDS The present invention relates to compounds useful as modulators of multi-drug resistance (MDR), to their preparation and to pharmaceutical and veterinary compositions containing them.

The resistance of tumours to treatment with certain cytotoxic agents is an obstacle to the successful chemotherapeutic treatment of cancer patients. A tumour may acquire resistance to a cytotoxic agent used in a previous treatment. A tumour may also manifest intrinsic resistance, or cross-resistance, to a cytotoxic agent to which it has not previously been exposed, that agent being unrelated by structure or mechanism of action to any agent used in previous treatments of the tumour.

Analogously, certain pathogens may acquire resistance to pharmaceutical agents used in previous treatments of the diseases or disorders to which those pathogens give rise.

Pathogens may also manifest intrinsic resistance, or cross resistance, to pharmaceutical agents to which they have not previously been exposed. Examples of this effect include multi-drug resistant forms of malaria, tuberculosis, leishmaniasis and amoebic dysentery.

The above phenomena are referred to collectively as multi-drug resistance (MDR). As discussed more fully later on, a plasma membrane glycoprotein (P-gp) is implicated in the mechanism which underlies MDR. P-gp has drug binding properties. Certain agents which have the capacity to modulate MDR may therefore also be useful in facilitating the delivery of drugs across the blood brain barrier, and in treating AIDS and AIDS-related complex.

Disadvantages of drugs which have so far been used to modulate MDR, termed resistance modifying agents or RMAs, are that they frequently possess a poor pharmacokinetic profile and/or are toxic at the concentrations required for MDR modulation.

It has now been found that fractionation of a methanol extract of the roots of the plant Cynanchum otophyllum yields a series of pregnane derivatives which, together with their alkanoyl esters, have activity as modulators of multi-drug resistance. The present invention therefore provides the use of a pregnane derivative of formula (A):

wherein Rl is OH, OR wherein R is Cl-C6 alkanoyl, or Ri is a group of formula (a):

wherein n is 0, 1 or 2 and R2 is H or R as defined above; in the manufacture of a medicament for use as a modulator of multi-drug resistance.

The term "the present compounds" will hereinafter be used to refer collectively to the pregnane steroid derivatives of formula (A).

The pregnane steroid derivatives of formula (A) wherein R1 is OH or a group of formula (a) wherein R2 is H and n is 2 are natural products disclosed in Scientia Sinica (series B), vol XXIX NO 3, March 1986, pp 295-301 by Quanzhang et al, where their isolation and anti-epileptic activity are described.

The invention further provides a pregnane derivative of formula (A')

wherein Rl is OR in which R is C1-C6 alkanoyl, or Rl is a group of formula (a'):

wherein n is O or 1 and R2 is H or R as defined above, or n is 2 and R2 is R as defined above, excluding the compounds wherein R1 is OAc or is a group of formula (a') in which n is 2 and R2 is Ac.

A C1-C6 alkanoyl group may be straight or branched. It is, for example, a C1-C4 alkanoyl group such as acetyl, propionyl, t-butanoyl, s-butanoyl or n-butanoyl.

The pregnane steroid derivatives of formula (A) wherein R1 is OH or a group of the above formula (a) wherein R2 is H, both known and novel, are natural products which have been isolated from the roots of the plant Cynanchum otophyllum, a member of the family Asclepiadaceae. Esterification of these natural products gives the corresponding pregnane derivative of formula (A) wherein Rl is OR or a group of the above formula (a) wherein R2 is R.

The present invention therefore provides a process for producing the novel pregnane steroid derivatives of formula (A') which process comprises (i) preparing a methanolic extract of the roots of the plant Cvnanchum otophyllum; (ii) isolating from the said extract a pregnane derivative of formula (A') wherein Rl is a group of formula (a') in which n is 0 or 1 and R2 is H; and/or (iii) if desired to produce a compound of formula (A') wherein Rl is OR excluding OAc or a group of formula (a') in which R2 is the group R, R being as defined above exluding Ac, esterifying either the corresponding pregnane derivative obtained in step (ii) or the corresponding pregnane derivative of formula (A) as defined above wherein R, is OH or a group of formula (a) in which n is 2 and R2 is H.

The following compounds are examples of pregnane derivatives of formula (A) wherein R1 is OH or a group of formula (a) in which R2 is H:

The above compounds 1 to 4 are found in a methanol extract of the roots of Cynanchum otoDhyllum and may be individually recovered and separated using conventional chromatographic fractionations with various chromatographic techniques and solvent systems. The compounds 1 to 4 can thus be obtained in substantially pure form. Compounds 1 to 4 are suitably isolated from the said methanol extract initially by Vacuum Liquid Chromatography (VLC) and then individually separated by High Performance Liquid Chromatography (HPLC).

Typically the methanol extract is subjected to VLC over silica gel eluting first with chloroform in hexane followed by methanol in chloroform, or with hexane and ethyl acetate. The fraction eluted with ethyl acetate is then subjected to gradient preparative HPLC using acetonitrile and water or methanol and water to detect the presence of, and to isolate, the desired compounds 1 to 4. The use of these fractionation techniques will afford purified compositions containing the desired compounds, the presence of which is determined by analysing the various HPLC chromatographic fractions for physicochemical characteristics. The isolation and characterisation of compounds 1 to 4 is described in more detail in the Examples which follow.

Compounds 1 to 4 may be converted into the corresponding esterified pregnane derivatives of formula (A) wherein R1 is OR or a group of formula (a) wherein R2 is R, R being as defined above, by treatment with an appropriate esterifying agent in a suitable solvent. Examples of esterifying agents include acid anhydrides such as acetic anhydride, and Cl-C6 alkanoyl halides such as acetyl chloride. Suitable solvents include pyridine.

Examples of such esterified derivatives include the following compounds 5 to 8 which are the acetyl esters of compounds 1 to 4, respectively:

Compounds 5 to 8 may be prepared from compounds 1 to 4, respectively, for instance by treatment with acetic anhydride in pyridine. Other suitable acetylating agents include acetyl halides such as acetyl chloride.

Cancer cells which exhibit multi-drug resistance, referred to as MDR cells, display a reduction in intracellular drug accumulation compared with the corresponding drugsensitive cells. Studies using in vitro derived MDR cell lines have shown that MDR is often associated with increased expression of a plasma membrane glycoprotein (P-gp) which has drug binding properties. P-gp is thought to function as an efflux pump for many hydrophobic compounds, and transfection studies using cloned P-gp have shown that its overexpression can confer the MDR phenotype on cells: see, for example, Ann.

Rev. Biochem 58 137-171 (1989).

A major function of P-gp in normal tissues is to export intracellular toxins from the cell. There is evidence to suggest that overexpression of P-gp may play a clinical role in multi-drug resistance. Increased levels of P-gp mRNA or protein have been detected in many forms of human cancers leukaemias, lymphomas, sarcomas and carcinomas. Indeed, in some cases P-gp levels have been found to be increased in tumour biopsies obtained after relapse from chemotherapy.

Inhibition of P-gp function in P-gp mediated MDR has been shown to lead to a net accumulation of anti-cancer agent in the cells. For example, Verapamil a known calcium channel blocker was shown to sensitise MDR cells to Vinca alkaloids in vitro and in vivo: Cancer Res., 41, 1967-1972 (1981). The proposed mechanism of action involves competition with the anti-cancer agent for binding to the P-gp. A range of structurally unrelated resistance-modifying agents acting by this mechanism have been described such as tamoxifen (Nolvadex:ICI) and related compounds, and cyclosporin A and derivatives.

Compounds of formula I and their pharmaceutically acceptable salts (hereinafter referred to as "the present compounds") have been found in biological tests to have activity in modulating multi-drug resistance. The results are set out in Example 5 which follows. The present compounds may therefore be used as multi-drug resistance modifying agents, also termed resistance-modifying agents, or RMAs. The present compounds can modulate, e.g. reduce, or eliminate multi-drug resistance.

The present compounds can therefore be used in a method of potentiating the cytotoxicity of an agent which is cytotoxic to a tumour cell. Such a method comprises, for instance, administering one of the present compounds to the tumour cell whilst the tumour cell is exposed to the cytotoxic agent in question. The therapeutic effect of a chemotherapeutic, or antineoplastic, agent may thus be enhanced. The multi-drug resistance of a tumour cell to a cytotoxic agent during chemotherapy may be reduced or eliminated.

The present compounds, both novel and known, have been found in biological tests to have activity in modulating multidrug resistance. The results are set out in Example 6 which follows. The present compounds may therefore be used as multidrug resistance modifying agents, also termed resistancemodifying agents, or RMAs. The present compounds can modulate, e.g. reduce, or eliminate multi-drug resistance. The present compounds can therefore be used in a method of potentiating the cytotoxicity of an agent which is cytotoxic to a tumour cell.

Such a method comprises, for instance, administering one of the present compounds to the tumour cell whilst the tumour cell is exposed to the cytotoxic agent in question. The therapeutic effect of a chemotherapeutic, or antineoplastic, agent may thus be enhanced. The multi-drug resistance of a tumour cell to a cytotoxic agent during chemotherapy may be reduced or eliminated.

The present compounds can also be used in a method of treating a disease in which the pathogen concerned exhibits multi-drug resistance, for instance multi-drug resistant forms of malaria (Plasmodium falciParum), tuberculosis, leishmaniasis and amoebic dysentery. Such a method comprises, for instance, administering one of the present compounds with (separately, simultaneously or sequentially) the drug to which the pathogen concerned exhibits multi-drug resistance. The therapeutic effect of the drug may thus be enhanced.

A human or animal patient harbouring a tumour may be treated for resistance to a chemotherapeutic agent by a method comprising the administration thereto of one of the present compounds. The present compound is administered in an amount effective to potentiate the cytotoxicity of the said chemotherapeutic agent. Examples of chemotherapeutic or antineoplastic agents which are preferred in the context of the present invention include Vinca alkaloids such as vincristine and vinblastine; anthracycline antibiotics such as daunorubicin and doxorubicin; mitoxantrone; actinomycin D; taxanes e.g.

taxol; epipodophyllotoxins e.g. etoposide and plicamycin.

The present compounds can be administered in a variety of dosage forms, for example orally such as in the form of tablets, capsules, sugar- or film-coated tablets, liquid solutions or suspensions or parenterally, for example intramuscularly, intravenously or subcutaneously. The present compounds may therefore be given by injection or infusion.

The dosage depends on a variety of factors including the age, weight and condition of the patient and the route of administration. Typically, however, the dosage adopted for each route of administration when a compound of the invention is administered alone to adult humans is 0.001 to 50 mg/kg, most commonly in the range of 0.01 to 5 mg/kg body weight.

Such a dosage may be given, for example, from 1 to 5 times daily by bolus infusion, infusion over several hours and/or repeated administration.

The present compounds are formulated for use as a pharmaceutical or veterinary composition also comprising a pharmaceutically or veterinarily acceptable carrier or diluent.

The compositions are typically prepared following conventional methods and are administered in a pharmaceutically or veterinarily suitable form.

For example, the solid oral forms may contain, together with the active compound, diluents, such as lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants such as silica, talc, stearic acid, magnesium or calcium stearate and/or polyethylene glycols; binding agents such as starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose, or polyvinyl pyrrolidone; disintegrating agents such as starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dye-stuffs; sweeteners; wetting agents such as lecithin, polysorbates, laurylsulphates. Such preparations may be manufactured in known manner, for example by means of mixing, granulating, tabletting, sugar coating, or film-coating processes.

Liquid dispersions for oral administration may be syrups, emulsions and suspensions. The syrups may contain as carrier, for example, saccharose or saccharose with glycerol and/or mannitol and/or sorbitol. In particular a syrup for diabetic patients can contain as carriers only products, for example sorbitol, which do not metabolise to glucose or which only metabolise a very small amount to glucose. The suspensions and the emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol.

Suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier such as sterile water, olive oil, ethyl oleate, glycols such as propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride. Solutions for intravenous injection or infusion may contain a carrier, for example, sterile water which is generally Water for Injection.

Preferably, however, they may take the form of a sterile, aqueous, isotonic saline solution. Alternatively, the present compound may be encapsulated within liposomes.

The following examples illustrate the invention: Example 1: Extraction and purification of Compound 1 A sample (10g) of a methanol extract of the roots of Cynanchum otophyllum was subjected to VLC, using the technique described in J.Natural Products vol 49, No. 5, 1986, pp. 892900 and 934-936, over TLC grade silica gel eluting with hexane and ethyl acetate. The fraction eluted with ethyl acetate was subjected to gradient preparative HPLC (MeCN:H2O) to afford compound 1 (ref=0.30, 9:1 CHCl3:MeOH) (200 mg) as a white amorphous solid.

Compound 1 eluted after 16.5 mins using the Standard MeCN:H2O analytical HPLC programme on Crib, the details of which are as follows: Time/Min Flow ml/min goo H20 %MeCN Initial 2.0 100 0 2.00 2.0 100 0 15.00 2.0 0 100 20.00 2.0 0 100 25.00 2.0 100 0 29.00 2.0 100 0 30.00 0.0 100 0 Compound 1 had the following characteristics: Molecular formula: C49H78Ol6 Molecular weight: 922 Solubility: CHCl3, MeOH.

UV: Xmax (nm) (E) Solvent 220,282 MeCN/H2O Mass Spec Technique m/z Intensity (W) Electrospray 945(M+Na) 100 480 10 Accurate Mass 945.520227 (C49H78O16Na) Observed 945.518758 (C49H,8O16Na) Calculated I.R. (KBr) : v(cm-1) : 3472 1315 2966 1223 1713 1091 1643 1003 1367 864 1H N.M.R.: 6/ppm in CDCl3: 1.10,1.85(2H m H2-1) 5.47(1H s H-2') 3.20(1H m H-4"') 1.60,1.90 (2H m H2-2) 2.30(1H m H-4') 3.80(1H m H-5"') 3.50(1H m H-3) 1.01(6H d H3-5'/6' 1.16(3H d H3-6"' J=6.8 Hz) J=6.1 Hz) 2.30(2H m H2-4) 2.07(3H s H3-7') 4.45(1H dd H-1"" J=1.6,9.6 Hz) 5.31(1H bs H-6) 4.80(1H bd H-1" 1.45,2.30(2H m J=9.5 Hz) H2-2"") 2.15(2H m H2-7) 1.55,2.05(2H m 3.35(1H m H-3"") H2-2") 1.50(1H m H2-9) 3.75(1H m H-3") 3.15(1H t H-4"" J=8.7 Hz) 1.80(2H m H2-11) 3.20(1H m H-4") 3.25(1H m H-5"") 4.49(1H dd H-12 J=6.7,9.0 3.80 (1H m H-5") 1.27(3H d H-6"" Hz) J=6.1 Hz) 1.90(2H m H2-15) 1.17(3H d H3-6"J= 3.35(3H s H3-7"") 6.1 Hz) 1.80,2.85(2H m H2-16) 3.40(6H s H-7"/H-7'") 1.32(3H s H-18) 4.71(1H bd H-1"'J=8.1 Hz) 1.08(3H s H-19) 1.55, 2.05(2H m H2-2"') 2.12(3H s H-21) 3.75(1H m H-3'") 13C N.M.R.: 6/ppm in CDCl3: 39.8 (C-l) 24.3 (C-11) 27.2 (C-21) 77.1(C-3") 18.3(C-6"') 29.0 (C-2) 71.7 (C-12) 165.9(C-1') 82.6(C-4") 58.3(C-7"') 77.9 (C-3) 57.9 (C-13) 113.1(C-2') 68.6(C-5") 101.5(C-2"") 38.8 (C-4) 88.2 (C-14) 166.6(C-3') 18.3(C-6") 35.5(C-2"") 140.7(C-5) 33.2 (C-15) 38.2 (C-4') 58.1(C-7") 80.7(C-3"") 117.7(C-6) 32.0 (C-16) 20.9 (C-5') 99.8(C-1"') 75.4(C-4"") 34.3 (C-7) 91.6 (C-17) 21.0 (C-6') 35.7(C-2"') 71.7(C-5"") 74.4 (C-8) 9.5 (C-18) 16.6 (C-7') 77.2(C-3"') 18.1(C-6") 43.8 (C-9) 18.6 (C-19) 96.2 (C-l") 82.7(C-4"') 56.3(C-7"") 37.2 (C-10) 208.9(C-20) 35.7 (C-2") 68.4(C-5"') Resonances denoted * may be interchanged.

ExamPle 2: Preparation of Compound 2 A sample (lOg) of a methanol extract of the roots of the plant Cvnanchum otophyllum was subjected to VLC as described in Example 1, over TLC-grade silica gel eluting with hexane and ethyl acetate. The fraction eluted with 90% ethyl acetate in hexane was subjected to gradient preparative HPLC (MeCN:H2O) to afford compound 2 (Rf = 0.40, 9:1 CHCl3:MeOH) (15 mg) as a colourless oil.

The 1H and 13C NMR spectroscopic data obtained for compound 2 were close to those published by Quanzhang et al, 1986 (loc cit). Compound 2 has also previously been isolated from Cynanchum caudatum, as reported by Yamagishi T. and Mitsuhashi H. 1972, Chem. Pharm. Bull. 20 pages 625-626.

Compound 2 therefore had the following characteristics: 1H N.M.R.: b/ppm in CDCl3: 1.06(6H,d,J=6.8 Hz, 2.12(3H,d,J=0.9 Hz, 4.55 (1H, dd, J= H-5'/6') H-7') 5.4, 8.8 Hz, H-12) 1.13 (3H,s, H-18) 2.16 (3H,s, H-21) 5.35 (1H, bt, J=2.8 Hz, H-6) 1.41 (3H,s, H-19) 3.55 (1H,m, H-3) 5.52 (1H, bs, H-2') 13C N.M.R.: 6/ppm in CDCl3: 9.31 C-18 27.02 C-21 38.07 C-4' 71.76 C-12 140.57 C-S 16.41 C-7' 30.75 C-2 38.65 C-1 74.25 C-B 165.87 C-3' 18.55 C-19 31.84 C-16 41.92 C-4 87.97 C-14 166.60 C-1' 20.75 C-5' 33.06 C-15 43.68 C-9 91.44 C-17 208.68 C-20 20.84 C-6' 34.17 C-7 57.84 C-13 112.96 C-2' 24.20 C-11 36.87 C-10 71.55 C-3 117.58 C-6 Example 3:Preparation of Compound 3 A sample (lOg) of a methanol extract of the roots of Cvnanchum otophyllum was subjected to VLC over TLC-grade silica gel eluting with hexane and ethyl acetate. The fraction eluted with ethyl acetate was subjected to gradient preparative HPLC (MeCN:H2O) to afford compound 3 (Rf = 0.57, 9:1 CH2Cl2:MeOH) (30 mg) as a white amorphous solid.

Compound 3 eluted after 15.9 mins using the standard MeCN:H2O analytical HPLC gradient program on C18 described in Example 1.

Compound 3 had the following characteristics: 111 N.M.R.: #/ppm in CDCl3: 1.10,1.87 (2H m H2-1) 1.39 (3H s H-18) 3.85(1H m H-5") 1.55,1.90(2H m H2-2) 1.10 (3H s H-19) 1.20 (3H d H3-6" J=6.8 Hz) 3.55 (1H m H-3) 2.14 (3H s H-21) 3.42 (3H s H-7")* 2.25,2.40(2H m H2-4) 5.49 (1H s H-2') 4.68 (1H bd H-1''' J=9.5 Hz) 5.41 (1H bs H-6) 2.35 (1H m H-4') 1.65,2.25 (2H m H2 2"') 2.15 (2H m H2-7) 1.03 (6H d H3-5'/6' 3.55 (1H m H-3''') J=6.8 Hz) 1.50 (1H m H2-9) 2.09 (3H bs H3-7') 3.15 (1H m H-4''') 1.80 (2H m H2-11) 4.85 (1H bd H-1" 3.55 (1H m H-5''') J=9 .3Hz) 4.52 (1H bt H-12 1.55,2.10 (2H m H2- 1.25 (3H d H3-6''' J=8.8 Hz) 2") J=6.1 Hz) 1.90-2.00(2H m H2- 3.80 (1H m H-3") 3.40 (3H s H-7''')* 15) 1.80,2.85(2H m H2- 3.20 (1H m H-4") 16) Resonances denoted * may be interchanged 13C N.M.R.: #/ppm in CDCl3: 38.8 (C-l) 24.4 (C-11) 27.2 (C-21) 77.3 (C-3") 18.3(C-6''') 29.0 (C-2) 71.7 (C-12) 166.0(C-1') 82.7 (C-4") 58.2(C-7''')* 78.0 (C-3) 58.0 (C-13) 113.2 (C-2') 68.7 (C-5") 38.8 (C-4) 88.3 (C-14) 166.7(C-3') 18.5 (C-6") 140.8 (C-5) 33.2 (C-15) 38.3 (C-4') 57.4 (C-7")* 117.7 (C-6) 32.1 (C-16) 20.9 (C-5') 99.6 (C-1''') 34.4 (C-7) 91.7 (C-17) 21.0 (C-6') 33.9(C-2''') 74.4 (C-8) 9.6 (C-18) 16.6 (C-7') 77.6(C-3''') 43.9 (C-9) 18.7 (C-19) 96.3 (C-1") 72.6 (C-4''') 37.3 (C-10) 208.9(C-20) 35.8 (C-2") 70.8(C-5''') Resonances donated * may be interchanged Example 4: Preparation of Compound 4 A sample (10g) of a methanol extract of the roots of Cvnanchum otophyllum was subjected to VLC over TLC grade silica gel eluting with hexane and ethyl acetate.The fraction eluted with 90% ethyl acetate in hexane was subjected to gradient preparative HPLC (MeCN:H2O) to afford compound 4 (Rf = 0.47, 9:1 CH2C12:MeOH) (15 mg) as a white amorphous solid.

Compound 4 eluted after 15.1 mins using the standard MeCN:H2O analytical HPLC gradient program on C18 described in Example 1.

Compound 4 had the following characteristics: 111 N.M.R.: #/ppm in CDCl3: 1.10,1.87(2H m H2-1) 1.95 (2H m H2-15) 4.79(1H dd H-1" J=1.9, 9.6 Hz) 1.60,1.90 (2H m H2-2) 1.80,2.85(2H m H2- 1.60,2.15(2H m H2-2") 16) 3.55 (1H m H-3) 1.39 (3H s H-18) 3.63 (1H m H-3") 2.25,2.40(2H m H2-4) 1.12 (3H s H-19) 3.20 (1H m H-4") 5.34 (1H bs H-6) 2.15 (3H s H-21) 3.57 (1H m H-5") 2.15 (2H m H2-7) 5.50 (1H s H-2') 1.26 (3H d H3-6" J=6.8 Hz) 1.55 (1H m H2-9) 2.35 (1H m H-4') 3.45 (3H s H-7") 1.80 (2H m H2-11) 1.05 (6H d H3-5'/6' J=6.8 Hz) 4.54 (1H bt H-12 2.10 (3H bs H3-7') J=7.8 Hz) 13C N.M.R.: 6/ppm in CDCl3: 39.0 (C-1) 74.5 (C-8) 33.4 (C-15) 166.1 (C-1') 95.9 (C-1") 29.1 (C-2) 44.0 (C-9) 32.1 (C-16) 113.3(C-2') 34.3 (C-2") 78.0 (C-3) 37.9 (C-10) 91.7 (C-17) 166.8(C-3') 77.7 (C-3") 39.0 (C-4) 24.5 (C-11) 9.6 (C-18) 38.3 (C-4') 72.7 (C-4") 140.9 (C-5) 72.7 (C-12) 18.7 (C-19) 21.0 (C-5') 71.0 (C-5") 117.8(C-6) 58.1 (C-13) 208.9(C-20) 21.1 (C-6') 18.5 (C-6") 34.5 (C-7) 88.3 (C-14) 27.3 (C-21) 16.7 (C-7') 57.4 (C-7") Example 5:Preparation of Compounds 5 to 8 Compounds 5 to 8 were prepared by treating 5mg of each of compounds 1 to 4, respectively, with acetic anhydride (lml) in pyridine (2ml) Compound 5 had the following characteristics: 1H N.M.R.: #/ppm in CDCl3: 1.06 (6H d H3-5'/6' J=6.8 Hz) 4.46 (1H t H-4"" J=9.3 Hz) 1.13 (3H s H-19) 4.78 (1H bd H-1''' J=9.5 Hz) 1.19 (3H d H3-6" J=6.1 Hz) 4.86 (1H bd H-1" J=9.4 Hz) 1.21 (3H d H3-6''' J=6.1 Hz) 5.36 (1H bs H-6) 1.27 (3H d H-6"" J=6.1 Hz) 5.52 (1H s H-2') 1.41 (3H s H-18) 2.09 (3H s acetate CH3) 2.12 (3H s H-7') 2.17 (3H s H-21) 3.32 (3H s H3-7" ") 3.45 (6H s H-7"/H-7''') 4.52 (1H dd H-1" " J=9.7 Hz) 4.56 (1H bt H-12 J=8.7 Hz) Compound 6 had the following characteristics: 1H N.M.R. : #/ppm in CDCl3: 1.07 (6H,d, J=6.8 2.13 (3H, d, J=1.1 5.41 (1H, bt, J=3.1 Hz, H-5'/6') Hz, H-7') Hz, H-6) 1.17 (3H,s, H-18) 2.17 (3H, s, H-21) 5.53 (1H, bs, H-2') 1.41 (3H,s, H-19) 4.63 (1H, m, H-3) 2.03 (3H,s, acetate 4.58 (1H, dd, J=5.4, CH3) 8.3 Hz, H-12) Compound 7 had the following characteristics: 1H N.M.R.: #/ppm in CDCl3: 0.99 (6H d H3-5'/6' J=6.8 Hz) 4.49 (1H dd H-12 J=6.7,8.5 Hz) 1.06 (3H s H-19) 4.72 (1H dd H-1''' J=1.8,9.5Hz) 1.10 (3H d H3-6" J=6.8 Hz) 4.75 (1H dd H-1" J=1.7,9.5 Hz) 1.15 (3H d H3-6"' J=6.1 Hz) 5.28 (1H bs H-6) 1.34 (3H s H-18) 5.44 (1H bs H-2') 2.05 (3H s acetate CH3) 2.06 (3H d H3-7' J=0.9) 2.10 (3H s H-21) 3.32 (3H s H-7")* 3.38 (3H s H-7''')* 4.44 (1H dd H-4''' J=2.9,9.8 Hz) Compound 8 had the following characteristics: 1H N.M.R. ; #/ppm in CDCl3: 1.07 (6H d H3-5'/6' J=6.8 Hz) 4.54 (1H dd H-4" J=2.9,9.6 Hz) 1.14 (3H s H-19) 4.58 (1H dd H-12 J=4.7,8.5 Hz) 1.20 (3H d H3-6" J=6.8 Hz) 4.90 (1H dd H-1" J=2.0,9.2 Hz) 1.42 (2H s H-18) 5.37 (1H s H-6) 2.11 (3H s acetate CH3) 5.41 (1H bs H-2') 2.13 (3H d H3-7'J=0.9 Hz) 2.17 (3H s H-21) 3.42 (3H s H-7") 3.58 (1H m H-3) Example 6: Testing of the Present compounds as modulators of MDR Materials and Methods A multi-drug resistant subline, AR1.0 of the EMT6 mouse mammary carcinoma cell line was cultured in RPMI 1640 medium containing 10% foetal calf serum and 2mM glutamine, at 370C in 5% CO2.

1. Drus accumulation assav AR1.0 cells were seeded into 96-well opaque culture plates (Canberra Packard). The assay medium contained a mixture of tritiated daunorubicin (DNR), a cytotoxic agent, and unlabelled DNR (0.3Ci/ml, 2 M). Compounds 1 to 6 were serially diluted in assay medium over a range of concentrations from 39ng/ml to 20yg/ml. The cells were incubated at 37 C for 1 hr before washing and determination of cell-associated radioactivity. Each assay included a titration of the known resistance modifying agent cyclosporin A as a positive control.

Results were expressed as W maximum accumulation where 100% accumulation is that observed in the presence of the known RMA verapamil at 100 M. Where possible, an IC50 was determined.

The results are shown in Table 1: TABLE 1 Accumulation Compound ICso(M) 1 1 2 30 3 2.5 4 3 5 0.8 6 6 2. Potentiation of doxorubicin toxicity - (Compound 1 onlv) Compound 1 was examined for its ability to potentiate the toxicity of doxorubicin in AR1.0 cells. An initial proliferation assay involved titrating compound 1 against a fixed concentration of doxorubicin (0.86 M) which alone is nontoxic to AR1.0 cells.After a four day incubation, proliferation was measured using the colorimetric sulphorhodamine B assay (Skehan et al;JNCI. 82 pp1107-1112 1990) Compound 1 was shown to sensitise AR1.0 cells to 0.86 M doxorubicin without high innate toxicity: toxicitv (ICso) of 1 toxicitv with 0.86u doxorubicin (IC50) 10 M 0.01 M To measure the extent of this sensitisation a potentiation index was determined for compound 1. Cells were cultured for four days with a titration of doxorubicin @10-11 - 2x10-5M) in the presence of 1yM of compound 1. Proliferation was quantified as described by Skehan et al, loc cit. The ICw (concentration required to reduce proliferation to 50% of the untreated controls) for doxorubicin alone and with compound 1 were derived and used to calculate the potentiation index (PI): IC50 for doxorubicin alone PI= IC50 for doxorubicin plus RMA Potentiation index for compound 1 at 1 M=100 ExamPle 7: Pharmaceutical composition Tablets, each weighing 0.15g and containing 25mg of one of the present compounds can be manufactured as follows: Comnosition for 10.000 tablets Present compound (250g) lactose (800g) corn starch (415g) talc powder (30g) magnesium stearate (5g) The present compound, lactose and half the corn starch are mixed. The mixture is then forced through a sieve 0.5mm mesh size. Corn starch (10g) is suspended in warm water (90ml). The resulting paste is granulated to a powder. The granulate is dried and comminuted on a sieve of 1.4mm mesh size. The remaining quantity of starch, talc and magnesium stearate is added, carefully mixed and processed into tablets.

Claims

1. Use of a pregnane derivative of formula (A):

wherein Rl is OH, a group OR in which R is C1-C6 alkanoyl, or Rl is a group of formula (a):

2. Use according to claim 1 wherein, in formula (I), R1 is OH, OAc or a group of formula (a) wherein R2 is H or Ac.

3. A pregnane derivative of formula (A'):

wherein Rl is a group OR in which R is C1-C6 alkanoyl, or Rl is a group of formula (a'):

wherein n is 0 or 1 and R2 is H or a group R as defined above, or n is 2 and R2 is a group R as defined above, excluding the compounds wherein R1 is OAc or is a group of formula (a') in which n is 2 and R2 is Ac.

4. A compound according to claim 3 wherein, in formula (A'), n is 0 or 1 and R2 is H or Ac.

5. A process for producing a compound as defined in claim 3, which process comprises (i) preparing a methanol extract of the roots of the plant Cvnanchum otophYllum; (ii) isolating from the said extract a pregnane derivative of formula (A') wherein Rl is a group of formula (a') in which n is 0 or 1 and R2 is H; and/or (iii) if desired to produce a compound of formula (A') wherein Rl is OR excluding OAc or a group of formula (a') in which R2 is a group R excluding Ac, esterifying either the corresponding pregnane derivative obtained in step (ii) or the corresponding pregnane derivative of formula (A) as defined in claim 1 wherein Rl is OH or a group of formula (a) in which n is 2 and R2 is H.

6. A pharmaceutical composition comprising a pharmaceutically or veterinarily acceptable carrier or diluent and, as an active principal, a compound as claimed in claim 3 or 4.

7. A compound as defined in claim 3 or 4 for use as a modulator of multi-drug resistance.