WO2001060827A1

WO2001060827A1 - Novel p-gp-inhibiting compounds

Info

Publication number: WO2001060827A1
Application number: PCT/GB2001/000689
Authority: WO
Inventors: Mark Gumbleton; Abedel-Nasser Abulrob; Allan Denver Russell; Claire Simons
Original assignee: University College Cardiff Consultants Limited
Priority date: 2000-02-17
Filing date: 2001-02-19
Publication date: 2001-08-23
Also published as: AU2001237533A1; GB0003685D0

Abstract

A compound of general formula (I), or a pharmaceutically acceptable salt or prodrug thereof wherein ∫u⊃a∫/u⊃ , ∫u⊃b∫/u⊃ , ∫u⊃c∫/u⊃ , ∫u⊃d⊃/u⊃ , ∫u⊃e∫/u⊃ and ∫u⊃f⊃/u⊃ may be absent or may independently represent the presence of a single bond; X is selected from the group consisting of CH, CH2, NH, O and S; Y is selected from the group consisting of H or O; whereby: in the case where Y represents O: ∫u⊃a∫/u⊃ represents a single bond so as to complete a double bond to O as represented by Y; ∫u⊃b∫/u⊃ is absent; and X is selected from the group consisting of CH2, NH, O and S; or in the case where Y represents H: ∫u⊃a∫/u⊃ is absent; ∫u⊃b∫/u⊃ may be absent or may represent a single bond so as to complete a double bond to X; whereby, in the absence of ∫u⊃b∫/u⊃ , X is selected from the group consisting of CH2, NH, O and S; or in the case where ∫u⊃b∫/u⊃ represents a single bond so as to complete a double bond to X, X represents CH; R1 is selected from the group consisting of H, C¿1-6? alkyl, NH2, NH(C1-6 alkyl), N(C1-6alkyl)2 and OR?5; R2, R3 and R4¿ are independently selected from the group consisting of H, OH, C¿1-6? alkoxy and OR?5, or R3 and R4¿ can together form the residue of a 5 or 6 membered heterocyclic ring, with the proviso that at least one of R?2, R3 and R4¿ represents OR5; wherein R5 is selected from the group consisting of C¿5-20? alkyl, C5-20 alkenyl, C5-20 alkylene(C3-6 cycloalkyl), C5-20 alkenylene(C3-6cycloalkyl), C5-20 alkylene(heterocycle) and C5-20 alkenylene(heterocycle), where heterocycle represents a 3 to 5 membered heterocyclic ring containing at least one oxygen heteroatom and where said cycloalkyl or heterocycle can be substituted with one or more C1-4 alkyl groups.

Description

NOVEL P-GP-INHIBITING COMPOUNDS

The present invention is concerned with compounds for lowering the resistance of target cells to selected therapeutic agents, formulations containing the same and pharmaceutical uses thereof. In particular, the present invention is ^' concerned with sensitisation of mammalian cells to certain drugs and the increase of drug transport into mammalian cells, and for enhancing susceptibility of microorganisms to antimicrobial agents.

Chemotherapy is a primary form of conventional cancer treatment today. However, a major problem associated with cancer chemotherapy is the ability of tumour cells to develop resistance to the cytotoxic effects of anti-cancer drugs during the course of treatment. It has been observed that tumour cells can become simultaneously resistant to several chemotherapeutic drugs with unrelated chemical structures and mechanisms of action. This phenomenon is referred to as multidrug resistance. Many tumours intrinsically have multidrug resistance (for example, adenocarcinomas of the colon and kidney) while other tumours acquire multidrug resistance during the course of therapy (for example, neuroblastomas and childhood leukemias) .

Tsuruo and coworkers showed that a number of different agents inhibit multidrug resistance, rendering cells sensitive to chemotherapy. Included in this category are the calcium channel blockers verapamil and SR33557 (Tsuruo T, et al; Cancer Res . (1982) 42:4730-3 and Jaffrezou J P, et al; J. Biol . Chem. (1991) 266:19858-64), antiarrhythmic agents like quinidine (Sehested M, et al ; Br. J. Cancer

(1989) 60:809-14), the immunosuppressant cyclosporin A

(Hait N, et al ; Cancer Commun . (1989) 1:35-43, and Nooter K, et al; Int . J. Cancer (1990) 45:263-8), and the anticancer drug tamoxifen (Chatterjee M and Harris A L; Br.

J. Cancer (1990) 62:712-7). Most of the known multidrug resistance reversing drugs used in clinical trials have strong, deleterious side effects at physiologically- achievable concentrations. For example, calcium channel blockage and cardiovascular side effects are associated with continuous, high-dose intravenous verapamil therapy, and dose-limiting or immunosuppression often occur when using cyclosporine A. These drawbacks restrict the clinically-achievable dose of such agents and ultimately, their usefulness.

It is therefore an object of the present invention to provide a more effective agent which substantially reverses multidrug resistance in mammalian cells without any deleterious side effects.

The best documented and clinically relevant mechanism for multidrug resistance in tumour cells is correlated with the expression of P-glycoprotein, the product of the MDR1 gene. P-glycoprotein (P-gp) is a member of the ATP-binding cassette (ABC) superfamily of membrane transport proteins that serve to transport a variety of molecules, ranging from ions to proteins, across cell membranes. P-gp is a broad specificity efflux pump located in the cell membrane, and functions by decreasing the intracellular accumulation of many lipophilic drugs, including some widely used anticancer agents such as anthracyclines , vinca alkaloids, epipodophyllotoxins, actinomycin D and taxol, thereby rendering cells resistant to these drugs (Pastan I and Gottesman M M; Annu . Rev. Med . (1991) 42:277-86, and Schinkel A H and Borst P; Semin . Cancer Biol . (1991) 2:213- 26) .

Human P-glycoprotein is also expressed in several types of normal epithelial and endothelial tissues. The highest levels of MDRl expression are usually found in tumours derived from MDRl-expressing normal tissues, for example, renal, adrenocortical or colorectal carcinomas (Cordon-

Cardo C, et al ; J Histochem . Cytochem . (1990) 38:1277-87). In other types of solid tumours and leukemias, MDRl expression prior to treatment is usually relatively low or undetectable, but a substantial fraction of such malignancies express high levels of MDRl after exposure to chemotherapy (Goldstein J, et al ; J. Natl . Cancer Inεt . (1989) 81:116-24) .

The discovery of P-gp and its occurrence in a variety of tumour types has stimulated the search for compounds that are capable of blocking its function and consequently, of reversing resistance. These investigations have resulted in identification of a large number of so-called chemosensitisers or reversing agents. Some of these compounds act by inhibiting the pumping action of P-gp while the mechanism of action of others is still undetermined. In addition to inhibiting the efflux of various drugs from tumour cells, multidrug resistance modulators that interact with P-gp should also enhance the oral bioavailability of nutrients or drugs (that are affected by the action of P- gp) through the gastrointestinal tract. The modulators are believed to work by interfering with the efflux pump action of P-gp.

P-gp is present in the luminal sides of the endothelial cell layer of the capillary tube of the brain. It is this capillary tube that functions physiologically as the blood- brain barrier. The blood brain barrier is believed to restrict the entry of many different types of compounds (including drugs whose site of action is within the brain) from entering the brain. Multidrug resistance modulators that interact with P-gp also can function to enhance bioavailability of a drug to the brain by interacting with P-gp and thus interfering with the drug efflux pump action of P-gp on the treatment drug. This interference permits more of the treatment drug to cross the blood-brain barrier into the brain and remain there (Cordon-Cardo C, et al ; Proc . Na tl . Acad . Sci . USA . (1989) 86:695-8) .

It is therefore a further object of the present invention to provide a more effective agent which increases bioavailability of materials (such as a pharmaceutically active material or a nutrient) to mammalian cells and in particular to increase bioavailability of a pharmaceutically active material to the brain across the blood-brain barrier. A further aspect of the present invention is concerned with enhancing the susceptibility of microorganisms to antimicrobial agents, such as antibiotics. The constant use of antibiotics in the hospital environment has selected bacterial populations that are resistant to many antibiotics. These populations include opportunistic pathogens that may not be strongly virulent, but that are resistant to a number of antibiotics, thereby producing a serious problem in medical microbiology.

Bacteria have developed several different mechanisms to overcome the action of antibiotics and biocides. The same general concepts also apply to microorganisms other than bacteria. One major mechanism is where access of the antibiotic to the target is prevented or reduced by decreasing the transport of the antibiotic into the bacterium, or by increasing the efflux of the drug from the bacterium to the outside medium. In either case, the concentration of drug at the target site is reduced, thereby allowing bacterial survival in the presence of one or more antibiotics which would otherwise inhibit or kill the bacterial cells. Some bacteria utilize both a low permeability of the cell wall (including membranes) mechanism and an active efflux pump to overcome the action of antimicrobial agents (Nikaido H; Sci ence (1994) 15;264:382-8) .

Efflux pumps are widely involved in antibiotic and biocide resistance. Once in the cytoplasm or periplasm a drug can be transported back to the outer medium, thus removing a significant fraction of the antibiotic molecules which manage to enter the bacterium, thereby maintaining a very low intracellular antibiotic concentration. This transport is mediated by efflux pumps comprising protein assemblies which export substrate molecules from the cytoplasm or periplasm of a cell, in an energy dependent fashion.

Different efflux pumps can efflux specifically a particular drug or group of drugs, such as the NorA system that transports quinolones, or Tet A that transports tetracyclines; or they can efflux a large variety of molecules, such as certain efflux pumps of Pseudomonas aeruginosa, or MsrA efflux pump in Staphylococcus aureus (Neyfakh A A, et al . ; Antimicro- . Agents Chemother. (1993) 37:128-9) .

An "efflux pump inhibitor" can be defined as a potentiator which specifically interferes with the ability of an efflux pump to export antibiotics, but might also interfere with exporting normal (physiological) substrates of that pump. The inhibitor may have intrinsic antimicrobial (for example, antibacterial) activity of its own, but at least a significant portion of the relevant activity is due to the efflux pump inhibiting activity. The mechanism of action/regulation of the pump by an efflux pump inhibitor could arise by inhibiting the normal function of the pump, the normal expression of the pump, or a combination of both. Therefore, it has been found that when an antibacterial agent is administered in conjunction with the efflux pump inhibitor, the antibacterial agent, which would otherwise be maintained at a very low intracellular concentration by the export process, can accumulate to a concentration which will inhibit the growth of the bacterial cells. In addition, if a potentiator reduces the Minimum Inhibitory Concentration (MIC) of an antibacterial agent (where the MIC is the minimum concentration of an antibacterial agent which will completely inhibit growth) in a resistant strain, then such treatment may enable a reduction in the amount of antibacterial agent administered. This could reduce side effects of an antibiotic, and/or decrease the frequency of administration.

NorA is an example of a membrane-associated multidrug efflux protein that can decrease susceptibility to fluoroquinolones in Staphylococcus aureuε . NorA inhibition can increase fluoroqumolone killing activity and also the post-antibiotic effect following removal of the drug. However, studies with the alkaloid reserpme (the first identified inhibitor of NorA) indicate that reserpme is toxic to humans at the concentrations required to inhibit NorA (Schmitz F J, et al . ; J^". Antimicrob . Che-i.ot.her. (1998) 42:807-10). More durable and potent NorA inhibitor compounds are needed that can improve killing activity and prevent resistance.

It is therefore a further object of the present invention to provide a more effective agent which enhances the susceptibility of microorganisms (particularly bacteria) to antimicrobial agents to which these microorganisms have acquired resistance.

According to the present invention, therefore, there s provided a compound of general formula (I) , or a pharmaceutically acceptable salt or prodrug thereof

wherein (I)

_ a _ , _ b _ , _ c _ , _ d _ , _ e _ and _ f _ may be absent or may independently represent the presence of a single bond;

X is selected from the group consisting of CH, CH₂, NH, 0 and S;

Y is selected from the group consisting of H or 0;

whereby: in the case where Y represents O:-

_ a _ represents a single bond so as to complete a double bond to 0 as represented by Y;

_ b _ is absent; and

X is selected from the group consisting of CH₂, NH, 0 and S; or

in the case where Y represents H:- _ a _ is absent;

_ b _ may be absent or may represent a single bond so as to complete a double bond to X;

whereby,

in the absence of _ b _ , X is selected from the group consisting of CH₂, NH, O and S; or

in the case where _ b _ represents a single bond so as to complete a double bond to X, X represents CH;

R¹ is selected from the group consisting of H_, C₁_₆ alkyl, NH₂, NH(C₁-₆ alkyl) , N(C₁_₆ alkyl )₂ and OR⁵;

R², R³ and R⁴ are independently selected form the group consisting of H, OH, C__.₆ alkoxy and OR⁵, or R³ and R⁴ can together form the residue of a 5 or 6 membered heterocyclic ring, with the proviso that at least one of R², R³ and R⁴ represents OR⁵;

wherein R⁵ is selected from the group consisting of C₅. ₂₀alkyl, C_s.₂₀ alkenyl, C₅.₂₀alkylene (C₃_₆ cycloalkyl), C₃- ₂₀alkenylene (C₃_₆ cycloalkyl), C₅_₂₀alkylene (heterocycle) and C₅_₂₀alkenylene (heterocycle) , where heterocycle represents a 3 to 5 membered heterocyclic ring containing at least one oxygen heteroatom and where said cycloalkyl or heterocycle can be substituted with one or more C₁_₄alkyl groups .

According to a particular aspect of the present invention, it may be preferred that in compounds of formula (I) Y represents H and a is absent. Typically, it may be further preferred according to this particular aspect of the present invention that in compounds of formula (I) _ b _ , _ c _ , _ d _ , _ e _ and _ f _ independently represent single bonds and X represents CH. It will of course be appreciated that in the case where _ b _ , _ c _ , _ d _ , _ e _ and _ f _ represent single bonds substantially as described above, then these single bonds are present to complete double bonds between respective carbon atoms of compounds of formula (I) as described above. According to the above described aspect of the present invention, there is, therefore, provided a subgroup of compounds according to the present invention as represented by formula (IA)

(IA)

where R¹, R², R³ and R⁴ are substantially as hereinbefore described.

According to an alternative aspect of the present invention there are provided compounds of formula (I) wherein Y represents 0, _ a _ represents a single bond so as to complete a double bond to O, _ b _ is absent and X is selected from the group consisting of CH₂, NH, 0 and S. According to this alternative aspect of the present invention it may be preferred that _ c _ represents a single bond. It will of course be appreciated that in this case _ c _ completes a double bond between respective carbon atoms of compounds of formula (I) substantially as hereinbefore described. It may be further preferred that _ c _ , _ d_ , _ e _ and _ f _ each independently represent single bonds and again it will be appreciated that _ c _, _ d _ , _ e _ and _ f_. __ complete double bonds between respective carbon atoms of compounds of formula (I) substantially as hereinbefore described.

It is often further preferred in the above described alternative aspect of the present invention that X represents 0 and as such- there is further provided a subgroup of compounds according to the present invention of formula (IB)

(IB)

where R¹, R², R³ and R⁴ are substantially as hereinbefore described with reference to compounds of formula (I) .

Suitably in compounds of formula (I) according to the present invention R⁵ is selected from the group consisting of C₅_₂₀ alkenyl, C₅_₂₀alkenylene (C₃_₆ cycloalkyl), C₅.₂₀alkylene (heterocycle) and C₅_₂₀alkenylene (heterocycle) , wherein heterocycle represents a 3 to 5 membered heterocyclic ring containing at least one oxygen heteroatom and where said cycloalkyl or heterocycle can be substituted by one or more C₁.₄alkyl groups.

More suitably R⁵ is selected from the group consisting of

-CH₂[C_a(H_a)H _;_!_= C_b(H_b)CH₃(CH₂)₂]_n CH=C (CH₃) _2> where n is 1 or 2, _ g _ represents an optional single bond to complete a double bond between C_a and C_b and when _ g _ is present to represent a single bond as above then H_a and H_b are absent;

- ( CH₂ ) _mCH=CH₂ , where m is 4 to 8 ;

-CH₂ [CH=CCH₃ ( CH₂ ) ₂] _P cyclopropyl , where P is 1 or 2 ;

- ( CH₂ ) _qCR_aR_b ( CH₂ ) _r epoxy , where q is 2 to 4 , r is 2 to 4 , R_a and R_b can independently represent H or methyl ; and

-CH₂ [CH=CCH₃ ( CH₂ ) ₂ ] _S epoxy , where S is 1 or 2 ; where cyclopropyl and epoxy can either be unsubstituted or substituted by one or more methyl groups.

Preferably in compounds of formula (I) according to the present invention, R⁵ is selected from the group consisting of CH₂CH = CCH₃ (CH₂) ₂CH=C (CH₃) ₂, CH₂CH₂CHCH₃ ( CH₂ ) ₂CH = C ( CH₃ ) _{2 ,}

CH₂CH=CCH₃ (CH₂) ₂CH=CCH₃ (CH₂) ₂CH=C (CH₃) _2, (CH₂) ₆CH=CH_2,

CH₂CH=CCH_: (CH₂ 2/ 2

; CH₂ ) ₂CHCH₃ ( CH₂ ) ( CH₂ )

^•

More preferably, in compounds of formula (I) according to the present invention, R⁵ is either CH₂CH=CCH₃ (CH₂) ₂CH=C (CH₃) ₂ or

_A It may be particularly preferred according to the present invention that R², R³ and R⁴ are independently selected from the group consisting of H, OH,

and OR⁵ where R5 is substantially as hereinbefore described, with the proviso that at least one of R², R³ and R⁴ represents OR⁵. It may be even more particularly preferred that R², R³ and R⁴ are independently selected form the group consisting of H and OR⁵ where R⁵ is substantially as hereinbefore described, with the proviso that at least one of R², R³ and R⁴ represents OR⁵.

A further subgroup of compounds according to the present invention can be represented by following formula (1C) , or a pharmaceutically acceptable salt or prodrug thereof

(IC)

where either of R² or R⁴ represents OR⁵.

In the case where R² represents OR⁵, R³ and R⁴ may preferably together form the residue of a 5 or 6 membered heterocyclic ring. Suitably, R³ and R⁴ together form the residue of a 5 membered heterocyclic ring containing an oxygen heteroatom, such as oxapentene or oxapentadiene .

Alternatively, in the case where R⁴ represents OR⁵, R² and R³ may preferably independently represent H.

Suitably in compounds of formula (I) according to the present invention R¹ is selected from the group consisting of H, C _₄ alkyl, N(Cj_._₄ alkyl)₂ and OR⁵, wherein R⁵ is substantially as hereinbefore described, and more preferably R¹ is selected from the group consisting of H, methyl, ethyl, propyl and OR⁵, wherein R⁵ is substantially as hereinbefore described. Preferably R¹ is H.

It is often preferred for compounds according to the present invention that in compounds of formula (IC) substantially as hereinbefore described R³ does not represent CH₂CH=C(CH₃)CH₂CH₂CH=C(CH₃)₂ when R¹, R² and R⁶ represent H and R⁴ and R⁵ form the residue of an oxapentene or oxapentadiene ring although this preference might not be required for uses of the present invention substantially as herein before described. According to the present invention, there is further provided a compound of general formula ( ID) , or a pharmaceut ically acceptable salt or prodrug thereof

( ID)

where R¹ , R² and R⁶ each is H or C₁-₄alkyl ; one of R³ and R⁵ is

where X and Y together are -O- or complete a double bond; R⁴ and R⁵, or R³ and R⁴, optionally together represent

CH=CH—O

I I so as to complete an oxapentene or oxapentadiene ring; and when any of R³, R⁴ and R⁵ is not as defined above, it is H or C₁-₄alkyl.

When any of R¹, R², R³, R⁴, R⁵ and R^δ is alkyl, it is preferably methyl. However, it is most preferred that R¹, R² and R⁶ are all H.

It is preferred that there is provided by the present invention one or more compounds selected from compound A, compound B and compound C (compound B and C being the most preferred) :

Compound A

Compound B

Compound C

The above compounds as provided by the present invention may be used alone or in combination for substantially lowering resistance by target cells to one or more therapeutic agents. In particular, compounds as provided by the present invention may be used as inhibitors of P- glycoprotein in mammalian cells and/or to lower antimicrobial resistance in at least one microorganism. Compounds according to the present invention substantially as hereinbefore described, and m particular compounds A, B and C substantially as hereinbefore described, may be made synthetically substantially as hereinafter described or it may be possible to isolate these compounds from citrus fruits. In this latter embodiment, compounds according to the invention may preferably be isolated in substantially pure form.

The present invention preferably further comprises a pharmaceutical formulation comprising at least one compound according to the present invention substantially as hereinbefore described, together with a pharmaceutically acceptable carrier, diluent or exc pient therefor. The formulation is suitable for use in lowering resistance by target cells to one or more therapeutic agents. In particular, formulations according to the present invention are suitable for use in inhibiting P-glycoprotein m mammalian cells and/or to reduce antimicrobial resistance in at least one microorganism.

The pharmaceutical formulations according to the present invention can preferably be administered to mammals (preferably humans) to inhibit cell P-glycoprotein and/or to reduce antimicrobial resistance m at least one microorganism present in the mammal .

According to the present invention, the preferred route of administration of the formulations is oral. The formulation may be m the form of a tablet, capsule, or in the form of a powder or the like to be taken in water.

Alternatively, pharmaceutical formulations according to the present invention may be in a form suitable for intravenous injection, or in slow release form.

There is further provided by the present invention in combination a compound substantially as hereinbefore described and at least one therapeutic agent, for simultaneous, separate or sequential use in therapy. Typically, the therapeutic agent is such as to be susceptible to resistance thereto by target cells to which the agent is to be administered.

Where the pharmaceutical formulation is for use to reduce antimicrobial resistance in at least one microorganism present in the mammal, it is preferable that the pharmaceutical formulation is administered in conjunction with the antimicrobial agent to which the microorganism has become resistant to.

According to a preferred aspect of the present invention, therefore, the therapeutic agent may comprise at least one antimicrobial agent (preferably an antibiotic) . The antimicrobial agent may be selected from the group consisting of beta lactams, glycopeptides, macrolides, quinolones, tetracyclines, aminoglycosides and biocides .

As mentioned above, inhibition of mammalian cell P- glycoprotein substantially reverses multidrug resistance in mammalian tumour cells. It is therefore a preferred feature of the present invention that a therapeutic agent substantially as hereinbefore described comprises at least one oncolytic agent or anti -cancer chemotherapeutic agent. There is, therefore, provided in combination a compound substantially as hereinbefore described and at least one oncolytic agent, for simultaneous, separate or sequential use in the treatment of cancer. The oncolytic or chemotherapeutic agent may be selected from the group consisting of taxol, vinblastine, vincristine, daunorubicin and doxorubicin administered at a therapeutically effective amount .

Inhibition of mammalian cell P-glycoprotein increases bioavailability of materials (such as a pharmaceutically active material or a nutrient) to mammalian cells and increases bioavailability of a pharmaceutically active material to the brain across the blood-brain barrier. It is therefore a further preferred feature of the present invention, to provide a pharmaceutical formulation according to the present invention together with a suitable material .

According to a further embodiment of the present invention, there is provided a method of treating a patient having target cells susceptible to, or exhibiting resistance to, one or more therapeutic agents, which method comprises administering to the patient a compound according to the present invention substantially as hereinbefore described in an amount effective to substantially alleviate the resistance. The method may further comprise administering to the patient a therapeutically effective amount of one or more therapeutic agents susceptible to resistance by target cells substantially as hereinbefore described. Substantially as herein before described the therapeutic agent typically comprises an antimicrobial agent substantially as hereinbefore described or an oncolytic agent substantially as hereinbefore described.

There is further provided by the present invention a method of inhibiting P-glycoprotein in mammalian cells and/or of reducing antimicrobial resistance in at least one microorganism, which method comprises administration of any of the above compounds or pharmaceutical formulations according to the present invention.

It is preferred that a method according to the present invention is used on mammals (preferably humans) to inhibit P-glycoprotein in mammalian cells and/or reduce antimicrobial resistance in at least one microorganism present in the mammal .

It is preferred that a method of reducing antimicrobial resistance in at least one microorganism present in a mammal comprises administration of the antimicrobial agent to which the microorganism has become resistant to together with any of the compounds or pharmaceutical formulations according to the present invention.

According to a further embodiment of the present invention, there is provided a method of substantially reversing multidrug resistance in mammalian tumour cells, which method comprises administration of any of the above compounds or pharmaceutical formulations according to the present invention. It is preferred that the compounds or pharmaceutical formulations are administered with at least one oncolytic agent or anti-cancer chemotherapeutic agent.

According to a further embodiment of the present invention there is provided a method of increasing bioavailability of materials to mammalian cells, which method comprises administration of any of the above compounds or pharmaceutical formulation according to the present invention. It is preferred that the compounds or pharmaceutical formulations are administered with a suitable material, such as a pharmaceutically active material or a nutrient.

There is further provided by the present invention a process of preparing a compound substantially as hereinbefore described, which process comprises reacting a compound of formula (II)

(H)

wherein

at least one of R^2a, R^3a and R^4a represents -OX, where X is a leaving group, and R^2a, R^3a or R^4a that do not represent -OX, respectively represent R², R³ or R⁴;

whereby OX in formula (II) can be reacted with a compound of formula R⁵Y, where Y is a leaving group, to yield -OR⁵ in compounds of formula (I) .

The present invention further provides a process of preparing a compound according to the present invention, whereby a first compound of formula (I) is interconverted to a second compound of formula (I) .

The present invention will now be further illustrated by the following Examples which do not limit the scope of the invention in any way.

Examples

Isolation of active components from Grapefruit oil

Isolation and purification of the components from grapefruit oil was achieved by column chromatography using silica gel 60 (230-400 mesh, Merck) . The column was monitored by TLC on precoated silical plates (Kielsel gel 60 F _2S4, BDH) . Visualisation of the TLC plates was by UN, and by charring with vanilin stain. Grapefruit oil (26.7g) was loaded on a silica column packed with petroleum ether. Flushing the column with copious amounts of petroleum ether eluted the bulk of the material which would appear to be the ^x oil' components which was not visible under UN light and appeared to be responsible for the grapefruit scent . Further washing with petroleum ether-ethyl acetate 9:1 v/v eluted first another oil like component (R_£ 0.72, blue colouration on staining with vanilin) followed by 7-{ [ (2E) - 3 , 7-Dimethyl-2 , 6-octadienyl] oxy} -2H-2-chromenone (compound A, R_f 0.65, highly fluorescent under UN light, light blue colouration on staining with vanilin, C₁₉H₂₂0₃ MW : 298.381) . On increasing the polarity of the mobile phase to petroleum ether-ethyl acetate 4:1 v/v, a further oil like component GF3 was eluted (R_f 0.38, blue/purple colouration on staining with vanilin) . GF3 was an inseparable 2:1 mixture of 4- { [ (E) -5- (3 , 3-Dimethyl-2- oxiranyl) -3-methyl-2-pentenyl] oxy} -7H-furo [3 , 2-g] chromen- 7-one as the major component (compound B, Bergamottin epoxide C₂₁H₂₂0₅ MW : 354.4018) and 7- { [ (E) -5- (3 , 3- Dimethyl -2 -oxiranyl) -3-methyl-2-pentenyl] oxy} -2H-2 - chromenone as the minor component (compound C, coumarin epoxide C₁₉H₂₂0₄ MW : 314.3804) . The quantity of compound A (a pale yellow waxy solid) obtained was 254 mg, and 288mg of GF3 (comprising compound B and C) a yellow syrup component was also obtained.

Confirmation of GF3 Structures

The individual components of GF3 , compound B and compound C, were identified by ¹H and ¹³ C NMR and by mass spectroscopy and confirmed by comparison with the synthesised compounds.

Preparation of compound B (4- { [ (E) -5- (3 , 3-Dimethyl-2- oxiranyl) -3-methyl-2-pentenyl1 oxy} -7H-furo T3 , 2 -ql chromen- 7-one)

To a solution of bergamottin (15mg, 0.044 mmol) in dry dichloromethane (1ml) was added meta-chloro-peroxybenzoic acid (8.2mg, 0.047mmol) and the reaction stirred at room temperature under nitrogen overnight. The reaction mixture was diluted with dichloromethane (2ml) , washed with aqueous

NaHC0₃ (2ml) , then the dichloromethane solution dried

(MgS0₄) and concentrated under reduced pressure. Purification was undertaken by column chromatography.

Preparation of compound C (7- {[ (E) -5- (3 , 3 -Dimethyl-2- oxiranyl) -3 -methyl -2 -pentenyll oxy} -2H-2-chromenone)

To a solution of compound A (50mg, 0.17mmol) in dry dichloromethane (2ml) was added meta-chloro-peroxybenzoic acid (31mg, O.lδmmol) and the reaction stirred at room temperature under nitrogen for 2 hours . The reaction mixture was diluted with dichloromethane (3ml) , washed with aqueous NaHC0₃, then the dichloromethane solution dried

(MgS0₄) and concentrated under reduced pressure to give a pale yellow waxy solid (the crude yield was 53mg) .

Purification was undertaken by column chromatography

(petroleum ether-ethyl acetate 4:1 v/v) , and collection and evaporation of the fractions containing pure material, gave the product compound C as a pale yellow solid (yield was 28mg (53%) ) .

Monitoring susceptibility of microorganism to anti- microbial agents

Staphylococcus aureus is a clinically significant member of the Gram-positive group of bacterial pathogens. It gives rise to serious infections, and may produce bacteremia, endocarditis, and meninngitis. Methicillin-resistant Staphyloccus aureus strains were chosen for evaluation because they are considered a significant medical problem, in view of the fact that methicillin is the drug of choice for treatment of S . aureus infection in the common penicillin-resistant strains.

Minimum Inhibitory Concentration (MIC) determination

The term 'MIC as used herein denotes the lowest concentration of an antimicrobial agent which prevented visible microorganism groth. MIC determinations were undertaken for an antimicrobial agent alone and an antimicrobial agent in the presence of one or more test compounds. The test compounds included grapefruit (GF) oil, compound A according to the present invention, GF3 (a mixture of compound B + C according to the present invention) , compound C according to the present invention and carbonyl cyanide m-chlorophenylhydrazone (CCCP) a de- energiser which blocks the energy dependent Nor A efflux pump. The MIC determinations were carried out by employing in each Example as hereinafter described a selected fixed concentration of each test compound. The variation then seen for the MIC determinations for the antimicrobial agents investigated in the presence of the test compounds provided an indication of the effectiveness of the test compound in facilitating a low concentration of antimicrobial agent to be employed. In other words, the lower the MIC observed for an antimicrobial agent in the presence of a selected test compound, the more effective the test compound in facilitating inhibition of microorganism growth by an antimicrobial agent. The MICs were determined on nutrient agar plates. Test compounds were dissolved in ethanol , where the final concentration of ethanol didn't exceed 0.5% and had no deleterious effect on bacterial growth. An aliquot of 0.1 ml of the stock solution was added to 20 ml of agar media to make the final concentration of the test compounds as shown in the tables. Aliquots of exponentially growing cultures of the specified bacteria was then inoculated into each nutrient agar plate. After incubating for 24 hours at 37°C, the agar plates were evaluated for visible growth. The MIC was determined as the lowest concentration of the antimicrobial agent which prevented visible microorganism growth in the culture. The relative MICs were measured in relation to the MIC of cultures incubated with the antimicrobial agent alone. The results are shown in Table 1 to 6.

Table 1 Susceptibility (MICs) of methicillin sensitive Staphylocccus aureus (MSSA) to ethidium bromide in the presence and absence of test compounds .

The results of Table 1 show that none of the compounds tested reduced the MICs against MSSA. Table 2 Susceptibility (MICs) of methicillin resistant Staphylocccus aureus (MRSA) strain 9543 to ethidium bromide in the presence and absence of the test compounds .

The results of Table 2 show that GF3 extract can reduce the MIC in MRSA strain 9543 by 6-fold compared to CCCP which reduce it by 25-fold.

Table 3 Susceptibility (MICs) of methicillin resistant Staphylocccus aureus (MRSA) strain 7 to ethidium bromide in the presence and absence of the test compounds .

The results of Table 3 show that GF3 extract can reduce the MIC in MRSA strain 7 by 6-fold compared to CCCP which reduce it by 25-fold.

Table 4 Susceptibility (MICs) of methicillin resistant Staphylocccus aureus (MRSA) strain 50325 clinical isolate to ethidium bromide in the presence and absence of the test compounds .

The results of Table 4 show that GF3 extract can reduce the MIC in MRSA by 6-fold and achieve similar activity as the de-energizing agent CCCP.

Table 5 Susceptibility (MICs) of methicillin sensitive Staphylocccus aureus (MSSA) strains LST, OX and S3 to norfloxacin in the presence and absence of the test compounds .

The results of Table 5 show that none of the compounds tested reduced the MICs in the MSSA stains tested.

Table 6 Susceptibility (MICs) of methicillin resistant Staphylocccus aureus (MRSA) strains 16565, 9543, 5 and 7 to norfloxacin in the presence and absence of the test compounds .

The results of Table 6 show that GF3 extract and compound A can reduce the MICs in the MRSA strains tested by around 20-fold. This example demonstrates that the MIC of norfloxacin could be significantly reduced by the addition of GF3 extract or compound A.

Analysis of inhibition of P-glycoprotein mediated transport in human cell lines

The human breast carcinoma cell lines MCF-7 and MCF-7/ADR were employed in the following examples. As hereinbefore described P-glycoprotein (P-gp) is the product of the MDRl gene. MCF-7 cells do not express P-gp, in the MCF-7/ADR cell line however, the MDRl gene is expressed and the MCF- 7/ADR cells express P-gp. Furthermore, MCF-7/ADR cells are known to be highly resistant to the oncolytic agent doxorubicin.

In the present example MCF-7 and MCR-7/ADR cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% FBS, 100 μg/ml streptomycin and 100 U/ml penicillin G. To maintain the MDRl phenotype, MCF-7/ADR cells were cultured in the presence of 0. lμg/ml doxorubicin. The cells were cultured in a humidified, 5% C0₂ atmosphere tissue culture incubator and subcultured every 3-4 days by trypsin/EDTA disaggregation to maintain logarithmic growth. Validation of MDRl gene expression in MCF-7/ADR cells was confirmed by MRK16 and reverse transcriptase-polymerase chain reaction (RT-PCR) using specific primers. It was confirmed that P- gp was expressed by MCF-7 cells by flow cytometric immunofluorescence labelling using monoclonal antibody against P-gp.

The accumulation and retention of rhodamine 123 and doxorubicin in MCF-7 and/or MCF-7/ADR cells was studied using the method hereinafter described.

Rhodamine 123 is a P-gp substrate which can be used as a fluorescent probe to provide a quantitative, functional assay of P-gp activity. Rhodamine 123 will be pumped out of the cell due to the action of P-gp leading to a reduced amount of rhodamine 123 fluorescent accumulated and retained within the cell. Incubation of the cell with a P- gp inhibitor will cause accumulation and retention of fluorescent rhodamine 123 within the cell. The degree of accumulation and retention of fluorescent rhodamine 123 in MCG-7/ADR cells seen in the presence of test compounds compared to accumulation and retention of fluorescent rhodamine 123 in the absence of test compounds provided an indication of the effectiveness of the test compound in inhibiting P-gp function. Doxorubicin is an antibiotic compound which can also be used as a fluorescent probe. Doxorubicin hydrochloride exhibits cytotoxic activity and is used as a drug in the treatment of a number of different cancers.

The accumulation and retention of doxorubicin in MCF-7/ADR cells was measured in the presence or absence of test compounds in the Example as hereinafter described. The degree of accumulation and retention of doxorubicin in MCF- 7/ADR cells in the presence of test compounds compared to accumulation and retention of fluorescent rhodamine 123 in the absence of test compounds provided an indication of the effectiveness of the test compound in facilitating accumulation and retention of doxorubicin in MCF-7/ADR cells, thus increasing the time doxorubicin ass retained within the cells before being pumped out . In other words the greater the accumulation and retention of doxorubicin in the MCF-7/ADR cells observed in the presence of the selected test compound, the more effective the test compound in facilitating the effectiveness of doxorubicin as a therapeutic agent .

The test compounds included grapefruit (GF) oil, compound A according to the present invention, GF3 (a mixture of compound B and C according to the present invention) and verapamil, a known P-gp inhibitor. P-glycoprotein mediated transport of rhodamine 123 and doxorubicin in human cell lines

The method used was similar to the approach described by Lee et al (Mol . Pharmacol . (1994) 46:627-638). Briefly, control cells and those pretreated with the test compounds were suspended (5 xlO⁵ cells /ml) in culture medium containing either rhodamine 123 (0.2 μg /ml) or doxorubicin (1.3 μg /ml) (plus co-incubation with respective test compounds if appropriate) and incubated in the dark for 60 min at 37°C. Following this, the cells were pelleted (x 600 g centrifugation for 5 min) , washed in PBS, 50% of the cells were then aliquoted for flow cytometric analysis representative of the accumulation phase of either rhodamine 123 or doxorubicin.

The remaining 50% of the cells were used to study cellular retention of either rhodamine 123 or doxorubicin. The remaining cells were resuspended in culture medium free of either rhodamine 123 or doxorubicin but containing, where appropriate, the respective test compounds. Efflux was conducted over 120 min at 37°C, after which the cells were pelleted, washed in ice-cold PBS and analysed by flow cytometry.

Flow cytometry was performed on a FACScan flow cytometer

(minimum of 4000 events for each sample) and gated populations (excluding cellular debris and large cellular aggregates) analysed for cell associated fluorescence (FL-1 for rhodamine-123 and FL-2 for doxorubicin, respectively) . Each treatment was represented by at least 4 replicates, and each experiment repeated at least twice. The fluorescence of gated cell populations was analysed using validated analysis software, WinMDI . The results are illustrated in Figures 1 and 2 as hereinafter disclosed.

To confirm that none of the compounds tested contribute to the fluorescence. MCF-7/ADR cells were incubated with the tested compounds alone (i.e. without the fluorescent probe) at different excitation and emission spectrum (FL1 and FL2) . The results are shown in table 7.

Table 7 Fluorescence (MFI) in the FL1 and FL2 spectrum of MCF-7/ADR cells incubated with the test compounds.

Table 7 shows that none of the test compounds show substantial fluorescence when tested alone.

The present invention will now be illustrated with reference to the following figures which are by way of example only.

Figure 1 illustrates the accumulation and retention of doxorubicin (1.3μg/ml) in MCF-7/ADR cells (which cells express P-gp and are doxorubicin resistant) measured by flow cytometry. The P-gp inhibitory activity of grapefruit oil (0.1%), compound A (20μg/ml)and GF3 (20μg/ml) were compared with verapamil (40μg/ml) .

Figure 2a illustrates the accumulation and retention of rhodamine 123 in non P-gp expressing MCF-7 cells. Figure 2b illustrates the accumulation and retention of rhodamine 123 in P-gp expressing MCF-7/ADR cells. The control used in this example was the cells (MCF-7 and MCF-7/ADR cells respectively) incubated with rhodamine 123 alone.

Figure 2b shows that at a concentration of 20μg/ml, GF3 prevented P-gp from pumping out rhodamine 123 in both the accumulation and retention phase in the MCF-7/ADR cells. Figure 2a shows that GF3 had substantially no effect on the accumulation or retention phases in the non P-gp expressing MCF-7 cells.

Claims

Claims _

A compound of general formula (I) , or a pharmaceutically acceptable salt or prodrug thereof

wherein (I)

_ a _ , _ b _, _ c _, _ d _, _ e _ and _ f _ may be absent or may independently represent the presence of a single bond;

X is selected from the group consisting of CH, CH₂, NH, 0 and S;

Y is selected from the group consisting of H or 0;

whereby : in the case where Y represents O:-

_ b _ is absent; and

X is selected from the group consisting of CH₂, NH,

O and S; or

in the case where Y represents H:- _ a _ is absent;

whereby, in the absence of _ b _, X is selected from the group consisting of CH₂, NH, O and S; or

R¹ is selected from the group consisting of H_ C_-₆ alkyl, NH₂, NH(C_!_₆ alkyl) , N (c__.₆ alkyl) ₂ and OR⁵;

R², R³ and R⁴ are independently selected form the group consisting of H, OH, C .₆ alkoxy and OR⁵, or R³ and R⁴ can together form the residue of a 5 or 6 membered heterocyclic ring, with the proviso that at least one of R², R³ and R⁴ represents OR⁵;

wherein R⁵ is selected from the group consisting of C₅_ ₂₀alkyl , C₃-₂₀ alkenyl , C₅_₂₀alkylene (C₃_₆ cycloalkyl) , C₅_ ₂₀alkenylene (C₃-₆ cycloalkyl) , C_s.₂₀alkylene (heterocycle) and C₅-₂₀alkenylene (heterocycle) , where heterocycle represents a 3 to 5 membered heterocyclic ring containing at least one oxygen heteroatom and where said cycloalkyl or heterocycle can be substituted with one or more C₁-₄alkyl groups .

2 . A compound according to claim 1 , wherein Y represents H and _ a _ is absent .

3. A compound according to claim 2 , wherein _ b _ , _ c _ , _ d _ , _ e _ and _ f _ independently represent single bonds and X represents CH.

4. A compound according to claim 1, wherein Y represents O, _ a _ represents a single bond so as to complete a double bond to O, _ b _ is absent and X is selected from the group consisting of CH₂, NH, O and S.

5. A compound according to claim 4, wherein _ c _ represents a single bond.

6. A compound according to claim 5, wherein _ c _, _ d _ , _ e _ and ______ f _ independently represent single bonds .

7. A compound according to claim 4, wherein X represents O.

8. A compound according to any of claims 1 to 7, wherein R⁵ is selected from the group consisting of C₅.₂₀ alkenyl, C₅- ₂₀alkenylene (C₃__s cycloalkyl), C₅-₂₀alkylene (heterocycle) and C₅_₂₀alkenylene (heterocycle) , wherein heterocycle represents a 3 to 5 membered heterocyclic ring containing at least one oxygen heteroatom and where said cycloalkyl or heterocycle can be substituted by one or more C₁_₄alkyl groups .

9. A compound according to claim 8, wherein R⁵ is selected from the group consisting of

-CH₂[C_a(H_a)H₌^_: C_b(H_b)CH₃(CH₂)₂]_n CH=C(CH₃)₂ , where n is 1 or 2 , _ 2 _ represents an optional single bond to complete a double bond between C_a and C_b and when _ g _ is present to represent a single bond as above then H_a and H_b are absent,-

(CH₂)_mCH=CH₂ where m is 4 to 8;

-CH₂ [CH=CCH₃ ( CH₂ ) ₂] _P cyclopropyl , where P is 1 or 2 ; - (CH₂)_qCR_aR_b(CH₂)_r epoxy, where q is 2 to 4, r is 2 to 4, R_a and R_b can independently represent H or methyl ; and

-CH₂ [CH=CCH₃ (CH₂) ₂] _s epoxy, where S is 1 or 2, and where cyclopropyl and epoxy can either be unsubstituted or substituted by one or more methyl groups.

10 A compound according to claim 9, wherein R⁵ is selected from the group consisting of CH₂CH=CCH₃ (CH₂) ₂CH=C (CH₃) _2ι CH₂CH₂CHCH₃ (CH₂) ₂CH=C (CH₃) _2,CH₂CH=CCH₃ (CH₂) ₂CH=CCH₃ (CH₂) ₂CH=C (C

(CH₂)₂CHCH₃(CH₂

11. A compound according to claim 10, wherein R⁵ is either CH₂CH=CCH₃(CH₂)₂CH=C(CH₃)₂ or

12. A compound according to any of claims 1 to 11, of following formula (IC) , or a pharmaceutically acceptable salt or

(IC) where either or R² or R⁴ represents OR⁵

13. A compound according to claim 12, wherein R² represents OR⁵ and R³ and R⁴ together form the residue of a 5 or 6 membered heterocyclic ring.

14. A compound according to claim 12, wherein R⁴ represents OR⁵ and R² and R³ independently represent H.

15. A compound according to any of claims 1 to 14, wherein R¹ is selected from the group consisting of H, C^ alkyl, N(Ci.₄ alkyl) ₂ and OR⁵, wherein R⁵ is as defined in claim 1.

16. A compound according to claim 15, wherein R¹ is selected from the group consisting of H, methyl, ethyl, propyl and OR⁵, wherein R⁵ is as defined in claim 10.

17. A compound according to claim 16, wherein R¹ is H.

18. A compound according to any of claims 1 to 17, wherein R³ and R⁴ together form the residue of a 5 membered heterocyclic ring containing an oxygen heteroatom.

19. A compound according to claim 18, wherein said heterocyclic ring is oxapentene or oxapentadiene.

20. A compound of general formula (ID) , or a pharmaceutically acceptable salt or prodrug thereof,

[ID) where R¹, R² and R⁶ each is H or C^alkyl; one of R³ and R⁵ is

where X and Y together are -O- or complete a double bond; R⁴ and R⁵ , or R³ and R⁴, optionally together represent

CH=CH—O I I so as to complete an oxapentene or an oxapentadiene ring; and when any of R³, R⁴ and R⁵ is not as defined above, it is H or C_halky1.

21. A compound aaccording to claim 20, when any of R¹, R², R³, R⁴, R⁵ and R⁶ is C^ alkyl, it is methyl.

22. A compound according to claim 20 or 21, where R¹, R² and R⁶ are independently H.

23. At least one compound selected from compound A, compound B and compound C_, or a pharmaceutically acceptable salt or prodrug thereof :

Compound A

Compound B

Compound C

24. At least one compound selected from compound B and C or a pharmaceutically acceptable salt or prodrug thereof

Compound B

Compound C

25. A pharmaceutical formulation comprising at least one compound according to any of claims 1 to 24, together with a pharmaceutically acceptable carrier, diluent or excipient therefor.

26. In combination, at least one compound according to any of claims 1 to 24 and at least one therapeutic agent, for simultaneous, separate or sequential use in therapy.

27. In combination according to claim 26, wherein said therapeutic agent is susceptible to resistance thereto by target cells to which said agent is administered.

28. In combination according to claim 26 or 27, wherein said therapeutic agent comprises at least one oncolytic agent.

29. In combination according to claim 28, wherein said oncolytic agent is selected from the group consisting of taxol, vinblastine, vincristine, daunorubicin and doxorubicin.

30. In combination according to clam 26 or 27, wherein said therapeutic agent comprises at least one antimicrobial agent.

31. In combination according to claim 30, wherein said antimicrobial agent is selected from the group consisting of beta lactams, glycopeptides, macrolides, quinolones, tetracyclines, aminoglycosides and biocides.

32. A compound according to any of claims 1 to 24, for use in therapy .

33. A compound according to any of claims 1 to 24, for use in the manufacture of a medicament for substantially lowering resistance to one or more therapeutic agents.

34. A compound according to any of claims 1 to 24, for use in the manufacture of an oncolytic medicament.

35. A compound according to any of claims 1 to 24, for use in the manufacture of an antimicrobial medicament .

36. A method of treating a patient having target cells susceptible to, or exhibiting resistance to, one or more therapeutic agents, which method comprising administering to said patient a compound according to any of claims 1 to 24 in an amount effective to substantially alleviate said resistance.

37. A method according to claim 36, which further comprises administering to said patient a therapeutically effective amount of said one or more therapeutic agents susceptible to resistance by said target cells.

38. A method according to claim 37, wherein said therapeutic agent comprises at least one oncolytic agent.

39. A method according to claim 38, wherein said oncolytic agent is selected from the group consisting of taxol, vinblastine, vincristine, dauhrubicin and daunorubicin.

40. A method according to claim 37, wherein said therapeutic agent comprises at least one antimicrobial agent.

41. A method according to claim 40, wherein said antimicrobial agent is selected from the group consisting of beta lactams, glycopeptides, macrolides, quinolones, tetracyclines, aminoglycosides and biocides .

42. A process of preparing a compound according to any of claims 1 to 24, which process comprises substantially isolating said compound from grapefruit oil.

43. A process of preparing a compound according to any of claims 1 to 24, which process comprises reacting a compound of formula (II)

(π)

wherein at least one of R^2a, R^3a and R^a represents -OX, where X is a leaving group, and R^2a, R^3a or R^a that do not represent -OX, respectively represent R², R³ or R⁴;

whereby OX in formula (II) can be reacted with a compound of formula R⁵Y, where Y is a leaving group, to yield -OR⁵

44. A process of preparing a compound according to any of claims 1 to 24, which process comprises interconverting a first compound as defined in any of claims 1 to 24 to a second compound as defined in any of claims 1 to 24.