Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
  • C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
  • C07D217/02—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with only hydrogen atoms or radicals containing only carbon and hydrogen atoms, directly attached to carbon atoms of the nitrogen-containing ring; Alkylene-bis-isoquinolines
  • C07D217/04—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with only hydrogen atoms or radicals containing only carbon and hydrogen atoms, directly attached to carbon atoms of the nitrogen-containing ring; Alkylene-bis-isoquinolines with hydrocarbon or substituted hydrocarbon radicals attached to the ring nitrogen atom
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
  • C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
  • C07D241/10—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
  • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings

Definitions

• the present invention relates to compounds useful as modulators of multi-drug resistance (MDR), to their
• compositions containing them are provided.
• tumours The resistance of tumours to treatment with certain cytotoxic agents is an obstacle to the successful
• 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.
• 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.
• MDR multi-drug resistance
• P-gp plasma membrane glycoprotein
• 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.
• RMAs resistance modifying agents
• the present invention therefore provides a piperazinedione derivative of formula (I):
• R 1 is (i) a group
• p is 0 or 2;
• each of Ra to Re which may be the same or different, is independently selected from hydrogen, C 1 -C 6 alkyl
• halogen atoms C 1 -C 8 alkenyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylthio, halogen, hydroxy, nitro, optionally substituted phenyl, cyano, -CH 2 OH,
• n is 0 or is an integer of from 1 to 6, each of R 11 and R 12 is independently H or C 1 -C 6 alkyl and R 13 is C 1 -C 6 alkyl; or any of Ra and Rb, Rb and Re, Re and Rd or Rd and Re together form a
• R 2 is H, C 1 -C 6 alkyl optionally substituted by a group
• R 11 is as defined above or a phenyl group as defined under (i) above, but is other than H when R 1 is unsubstituted phenyl;
• R 3 and R 4 are hydrogen and the other is a group of formula (A):
• q is an integer of 1 to 4, r is 0 or 1 and R 5 and R 6 , which may be the same or different, are each H or C 1 -C 6 alkoxy, or R 5 and R 6 together form a methylenedioxy group; and - - - - - is a double bond or , when R 1 is as def ined under
• a C 1 -C 6 alkyl group may be linear or branched.
• a C 1 -C 6 alkyl group is typically a C 1 -C 4 alkyl group, for example a methyl, ethyl, propyl, i-propyl, n-butyl, sec-butyl or tert- butyl group.
• a C 3 -C 6 cycloalkyl group may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.
• a halogen is, for example, fluorine, chlorine, bromine or iodine.
• a C 1 - C 8 alkoxy group is typically a C 1 -C 4 alkoxy group, for example a methoxy, ethoxy, propoxy, i-propoxy, n-butoxy, sec-butoxy or tert-butoxy group.
• a C 2 -C 6 alkenyl group is, for example, C 2 -C 4 alkenyl, for example ethenyl, prop-1-enyl or prop-2-enyl.
• a heterocyclic group may be, for example, a pyridine, pyrrole, furan or thiophene group which is linked via any one of its constituent ring atoms. It may be, for instance, a 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-furyl, 3-furyl, 2- thienyl or 3-thienyl group.
• the integer q is from 1 to 4, and is preferably 1 or 2.
• R 5 and R 6 are preferably the same and are preferably C 1 -C 4 alkyl, for instance methyl.
• R 1 is as defined under (i) above, the phenyl group is unsubstituted or is substituted at one or more of
• Ra to Re is other than hydrogen, preferably Rb or Re, especially Re.
• the substituent Ra to Re is preferably selected from a halogen, for instance chlorine, bromine or fluorine; a C 1 -C 6 alkoxy group, for instance OMe; and an acetamido group -NHAc in which Ac denotes acetyl.
• the phenyl group may instead be 2,3-, 2,4-, 2,5-, 2,6-, 3,4- or 3,5- disubstituted, or 2,3,4-, 2,3,5-, 2,3,6- or
• Ra to Re When it is disubstituted, three of Ra to Re are hydrogen and two are other than hydrogen.
• Ra and Rb, or Ra and Re, or Ra and Rd, or Ra and Re, or Rb and Re, or Rb and Rd are other than hydrogen whilst, in each case, the other three of Ra to Re are hydrogen.
• Ra to Re When the phenyl group is trisubstituted, two of Ra to Re are hydrogen and three are other than hydrogen.
• Ra, Rb and Re, or Ra, Rb and Rd, or Ra, Rb and Re, or Rb, Re and Rd are other than hydrogen whilst, in each case, the other two of Ra to Re are hydrogen.
• each of Ra to Re is hydrogen.
• one of Ra to Re is selected from hydroxy, C 1 -C 6 alkoxy, NHCOR 11 , -CO 2 R 11 , -N(R 11 R 12 ), -O(CH 2 ) n N(R 11 R 12 ), -SO 2 R 13 , -CON(R 11 R 12 ) , NO 2 , -SO 2 N(R 11 R 12 ), -SOR 13 , -N(R 11 )COR 12 and halogen and the other four of Ra to Re are H.
• Alkoxy may be, for instance, OMe or OBu n .
• NHCOR 11 is typically -NHAc.
• CO 2 R 11 is typically -COOH or -COOMe.
• N(R 11 R 12 ) is typically NMe 2 .
• -CON(R 11 R 12 ) may be -CONH 2 .
• SO 2 R 13 is typically SO 2 Me,
• SO 2 N(R 11 R 12 ) is for example -SO 2 NMe 2 .
• SOR 13 may be SOMe and -N(R 11 )COR 12 may be -NMeCOBu t .
• Halogen is typically F or Cl.
• Rc is alkoxy, especially OMe or OBu n ; NHCOR 11 , especially -NHAc; -CO 2 R 11 , especially -CO 2 H or -CO 2 Me;
• Ra to Re are all hydrogen, or one or two of Ra to Re are other than hydrogen whilst the others are hydrogen.
• one of Ra, Rb and Rc is other than hydrogen.
• Ra and Rc, or Rb and Rc are other than hydrogen.
• Preferred values for the one or two of Ra to Re which is or are other than hydrogen include C 1 -C 6 alkoxy such as OMe or OBu n , halogen such as Cl or F, hydroxy, -N(R 11 R 12 ),
• Particularly preferred compounds are those wherein Ra, Rb, Rd and Re are each H, and Re is selected from H, OMe -NHAc, -CO 2 H, -CO 2 Me, -CONH 2 , NO 2 , -NMe 2 , SO 2 Me , -SOMe and -SO 2 NMe 2 .
• Ra to Re are preferably each independently selected from H, halogen, hydroxy, C 1 -C 6 alkoxy, nitro, -CH 2 SCOR 13 , -CH 2 SR 11 , -CO 2 R 11 , -OCOR 13 , CF 3 , -O(CH 2 ) n N(R 11 R 12 ), -O(CH 2 ) n CO 2 R 11 ,
• Ra and Rb are independently H, nitro or halogen
• Rc is H, hydroxy, -O(CH 2 ) n N(R 11 R 12 ), -OCOR 13 ,
• Rd is H, halogen, C 1 -C 6 alkoxy, -CH 2 SCOR 13 , -CHzSR 11 or -COjR 11 ; and Re is H, nitro or halogen.
• the benzene ring forms, together with the phenyl group, an optionally substituted naphthalene ring structure.
• R 1 is a phenyl group as defined above which is unsubstituted or mono-substituted at position 2, 3 or 4 by Cl or MeO, or is a pyridyl, furyl or thienyl group
• R 2 is H, CH 3 , cyclopropyl or phenyl
• one of R 3 and R 4 is H and the other is a group of formula (A) wherein q is 2 and each of R 5 and R 6 is a methoxy group.
• R 1 is unsubstituted phenyl
• R 2 is C 1 -C 4 alkyl, preferably methyl, or is phenyl or
• R 3 is H and R 4 is a group of formula (A) wherein q is 2 and each of R 5 and R 6 is MeO.
• R 1 is substituted phenyl as defined above or a furyl, thienyl or pyridyl group
• R 2 is H
• R 3 is H
• R 4 is a group of formula (A) wherein q is 2 and each of R 5 and R 6 is MeO.
• R 1 is substituted phenyl as defined above or a furyl, thienyl or pyridyl group
• R 2 is H
• R 3 is a group of formula (A) wherein q is 2 and each of R 5 and R 6 is MeO
• R 4 is H.
• R 1 is unsubstituted phenyl
• R 2 is C 1 -C 4 alkyl, preferably methyl, phenyl or cyclopropyl
• R 3 is a group of formula (A) wherein q is 2 and each of R 5 and R 6 is MeO, and R 4 is H.
• R 1 is a furyl, thienyl or pyridyl group it is preferably a 3-furyl, 2-thienyl, 3- thienyl or 4-pyridyl group.
• Examples of preferred compounds of the invention are as follows. The compound numbering is adhered to in the rest of the specification.
• hydrochloride (9108) N-(4-(2-(6,7-Dimethoxy-1,2,3,4-tetrahydro-2-isoquinolyl)ethyl)phenyl)-4- ⁇ (3Z,6Z)-6-(4-methoxybenzylidene)-1-methyl-2,5-dioxo-3-piperazinylidene)methylbenzamide, hydrochloride (9109) N-(4-(2-(6,7-Dimethoxy-1,2,3,4-tetrahydro-2-isoquinolyl)ethyl)phenyl)-4-((3Z,6Z)-6-(4-chlorobenzylidene)-1-methyl-2,5-dioxo-3- piperazinylidene)methylbenzamide, hydrochloride (9091)
• R 1 , R 2 and - - - - - are as defined above, with a
• R 7 and R 8 is hydrogen and the other is -CHO, and q, r, R 5 and R 6 are as defined above; in the presence of a base in an organic solvent; and, if desired, converting the resulting compound into a pharmaceutically acceptable salt thereof .
• Suitable bases include caesium carbonate, sodium carbonate, potassium carbonate, sodium hydride, potassium t-butoxide and triethylamine.
• Suitable organic solvents include dimethylformamide
• DMF tetrahydrofuran
• THF tetrahydrofuran
• reaction mixture When sodium hydride or potassium t-butoxide is used as the base the reaction mixture is typically warmed from 0°C to room temperature, or to 40°C. The reaction may be performed for a period of 1 to 4 hours, for example 2 or 3 hours.
• R 1 is as defined above, with an alkylating agent, in an organic solvent in the presence of a base.
• alkylating agent is typically an alkyl halide R 2 -CH 2 X , a methanesulphonate or p-toluenesulphonate ester R 2 CH 2 OSO 2 Me or R 2 CH 2 OSO 2 C 6 H 4 Me , respectively, or a dialkyl sulphate (R 2 CH 2 O) 2 SO 2 , wherein R 2 is as defined above and X is a halogen, for instance Cl Br or I.
• Suitable bases and solvents include sodium hydride in THF or DMF or mixtures thereof, and potassium t-butoxide in t-butanol or THF or DMF or mixtures thereof.
• the reaction mixture is typically warmed from 0°C to room temperature.
• R 1 is as defined under (i) above and R 2 is as defined above with acetic anhydride.
• the reaction is typically performed under reflux, for instance for 1 to 6 hours, typically 3 hours.
• the compound of formula (X) may be prepared by treating a compound of formula (XI):
• the compounds of formula (XI) may be prepared by treating a compound of formula (XII):
• Suitable bases and solvents include triethylamine, caesium carbonate, sodium carbonate, potassium carbonate and sodium hydride in DMF or THF or mixtures thereof, and potassium t-butoxide in t-butanol or DMF or THF or mixtures thereof.
• the temperature of the reaction is typically from 100-140°C, for instance 120- 130°C.
• potassium t-butoxide is used as base the reaction mixture is typically warmed from 0°C to room temperature.
• 1,4-Diacetyl-2,5-piperazinedione may be prepared by the published procedure (S.M. Marcuccio and J.A. Elix, Aust. J. Chem., 1984, 37, 1791).
• the reaction is conducted in an organic solvent either with an excess of the amine of formula (IX), or in the presence of a base such as a tertiary amine, e.g. Et 3 N, or pyridine.
• the organic solvent is an inert organic solvent such as CH 2 Cl 2 .
• the coupling agent used in (a) or (b) with the 3- or 4- formylbenzoic acid, respectively, may be, for instance, 1- cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulphonate or 2-chloro-1-methylpyridinium iodide.
• the activated acid halide or mixed anhydride derivative of 3- or 4-formylbenzoic acid may be produced by
• the mixed anhydride derivative may be prepared by treatment of the carboxylic acid with a halogenating agent, for instance a chlorinating agent such as SOCl 2 , PCl 3 , oxalyl chloride or PCl 5 .
• a halogenating agent for instance a chlorinating agent such as SOCl 2 , PCl 3 , oxalyl chloride or PCl 5 .
• the mixed anhydride derivative may be prepared by treatment of the carboxylic acid with a C 1 -C 6 alkyl haloformate such as iBuOCOCl or EtOCOCl, in the presence of a base such as Et 3 N.
• the reduction step (ii) is typically performed using iron powder and concentrated hydrochloric acid in methanol, usually at a temperature of about 80°C and for a period of 1 to 4 hours, for instance 3 hours. Alternatively it may be carried out by catalytic hydrogenation over a palladium on carbon catalyst in methanolic HCl, isopropanol or acetic acid.
• Suitable salts include salts with pharmaceutically
• inorganic acids include hydrochloric acid, sulphuric acid and orthophosphoric acid.
• organic acids include p-toluenesulphonic acid, methanesulphonic acid, mucic acid and succinic acid.
• MDR cells Cancer cells which exhibit multi-drug resistance, referred to as MDR cells, display a reduction in
• P-gp plasma membrane glycoprotein
• P-gp A major function of P-gp in normal tissues is to export intracellular toxins from the cell.
• 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.
• 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.
• a method of potentiating the cytotoxicity of an agent which is cytotoxic to a tumour cell 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 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.
• 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.
• 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.
• 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.
• a human or animal patient suffering from a disease in which the responsible pathogen exhibits multi-drug resistance may be treated for resistance to a
• therapeutic agent by a method comprising the administration thereto of one of the present compounds.
• Examples of such disease include multi-drug resistant forms of malaria (Plasmodium falciparum), tuberculosis, leishmaniasis and amoebic dysentery.
• MDR modulators also have utility in the delivery of drugs across the blood-brain barrier, and in the treatment of AIDS and AIDS-related complex.
• the present compounds can therefore be used in a method of facilitating the delivery of drugs across the blood brain barrier, and in the
• a human or animal patient in need of such treatment may be treated by a method comprising the administration thereto of one of the present compounds.
• 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.
• a piperazinedione derivative of formula (I) or a pharmaceutically acceptable salt thereof is 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.
• An agent for use as a modulator of multi-drug resistance comprising any one of the present compounds is therefore provided.
• 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; dyestuffs, sweeteners; wetting agents such as lecithin, polysorbates, lauryl sulphates.
• preparations may be manufactured in known manners, 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.
• 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
• lidocaine such as sterile water, olive oil, ethyl oleate, glycols such as propylene glycol, and, if desired, a suitable amount of lidocaine
• hydrochloride Some of the present compounds are insoluble in water. Such compounds may be encapsulated within
• 1,4-diacetyl-2,5-piperazinedione was treated with a series of benzaldehydes R 1 -CHO, where R 1 is as l isted in table 1B , in the presence of potassium t -butoxide in t- butanol-THF (1:1) at 0°C.
• the reaction mixture was allowed to warm to room temperature for the time indicated in the table. Recrystallisation, which was optional, was conducted using the indicated solvent.
• Compound 3.1 was treated with 3.2 in the presence of K 2 CO 3 in DMF, at a temperature of 100°C for 12 hours, to give 3.3 in 78% yield. 3.3 was then reduced with Fe powder in concentrated HCl and MeOH at 80°C for 3 hours to give 3.4 in 51% yield. Alternatively 3.3 was reduced by catalytic hydrogenation at 30psi over a palladium on carbon catalyst in methanolic HCl for 3 hours to give 3.4 in quantitative yield.
• 4-formylbenzoyl chloride was prepared by treating 4-formylbenzoic acid with thionyl chloride in toluene under reflux. It was then treated with compound 3.4, prepared according to Reference Example 5, in CH 2 Cl 2 in the presence of Et 3 N at a temperature of about 0°C and allowed to warm to room temperature, to afford the following compound 4.2 in 53% yield:
• Example 2 The compounds prepared in Example 2 were converted to the corresponding hydrochloride salts by treatment with gaseous HCl in THF.
• Example 4 Selected compounds prepared in Example 4 were converted to the corresponding hydrochloride salts by treatment with gaseous HCl in CH 2 Cl 2 .
• the hydrochloride denoted in Table 5 below by the suffix ".HCl” was in some cases then
• Tablets each weighing 0.15 g and containing 25 mg of a compound of formula (I) or salt thereof can be manufactured as follows: Composition for 10,000 tablets
• the compound of formula (I) or salt thereof, lactose and half of the corn starch are mixed. The mixture is then forced through a sieve 0.5 mm mesh size. Corn starch (10 g) is suspended in warm water (90 ml). The resulting paste is used to granulate the powder. The granulate is dried and broken up into small fragments on a sieve of 1.4 mm mesh size. The remaining quantity of starch, talc and magnesium stearate is added, carefully mixed and processed into tablets.
• the EMT6 mouse mammary carcinoma cell line and the MDR resistant subline AR 1.0 were cultured in RPMI 1640 medium containing 10% foetal calf serum and 2mM glutamine at 37°C in 5% CO 2 .
• Cells were passaged between 1 in 200 and 1 in 2000 in the case of the parental cell line and between 1 in 20 and 1 in 200 in the case of the MDR resistant subline, after trypsinisation (0.25% trypsin, 0.2gl -1 , EDTA).
• Drug accumulation assay AR 1.0 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 (0.3 ⁇ Ci/ml; 2 ⁇ M).
• DNR tritiated Daunorubicin
• cytotoxic agent a cytotoxic agent
• unlabelled DNR 0.3 ⁇ Ci/ml; 2 ⁇ M.
• Compounds of formula I were serially diluted in assay medium over a range of concentrations from 5 nM to 100 ⁇ M.
• the cells were
• results are expressed as % maximum accumulation where 100% accumulation is that observed in the presence of the known RMA verapamil at a concentration of 100 ⁇ M or as an IC 50 .

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