CZ20003688A3 - Method of inhibiting MRP1 - Google Patents

Abstract

The present invention relates to a compound of formula I which is useful for inhibiting resistant tumors in which the resistance is due in part or in whole to MRP-1.

Description

Method of MRP1 inhibition

Field of the invention

The present invention relates to compounds of formula I which are useful for inhibiting resistant tumors in which the resistance is due in part or in full to MRP-1.

State of the art

Chemotherapy, along with surgery and radiotherapy, is an effective treatment for many cancers. Indeed, there are currently several types of cancer that are considered curable with chemotherapy, including Hodgkin's disease, large cell lymphoma, acute lymphocytic leukemia, testicular tumors, and early stages of breast cancer. Other cancers, such as ovarian cancer, small cell lung cancer, and advanced breast cancer, are not yet curable, but respond positively to combination chemotherapy.

One of the most significant unsolved problems in anticancer therapy is drug resistance. After selection for resistance to a single cytotoxic drug, cells can become cross-resistant to a wide range of drugs with different structures and cellular targets, such as alkylating agents, antimetabolites, hormones, platinum drugs, and natural compounds. This phenomenon is referred to as multidrug resistance (MDR). In some cell types, resistance is an intrinsic feature of the cells, while in others, such as small cell lung cancer, it is usually an acquired trait.

Such resistance is known to be multifactorial and is caused by at least two proteins: the 170 kDa P-glycoprotein (MDR-1) and the later identified 190 kDa multidrug resistance protein.

4

4

Λ • 4 4 « • 4 · 4

4 4 4 4

4 «

4 «4 drug resistance (MRP-1). Although both MDR1 and MRP1 belong to the ATP-binding transport protein superfamily, they are structurally very different molecules and have less than 15% amino acid homology. Despite the structural differences between these two proteins, no significant differences in the resistance pattern of MDR1 and MRP1 cell lines were known in 1994. However, the association - or lack thereof - of MRP1 and resistance to specific oncolytics is known. See Cole et al., Pharm. Characterization of Multidrug Resistant MRP1-transfected Human Tumor Cells, Cancer Research 54: 5902-5910, 1994. Doxorubicin, daunorubicin, epirubicin, vincristine, and etoposide are substrates of MRP1, i.e. MRP1 can bind to these cytostatics and redistribute them away from their site of action, out of the nucleus, and out of the cell. Id. and Marquard, D. and Center, M.S. Cancer Research 52: 3157, 1992.

Doxorubicin, daunorubicin and epirubicin are anthracyclines. They are isolates of various strains of Streptomyces and act by inhibiting nucleic acid synthesis.

These agents are useful in the treatment of tumors of the bone, ovary, bladder, thyroid, and especially breast. They are also useful for the treatment of acute lymphoblastic and myeloblastic leukemia, Wilms' tumor, neuroblastoma, soft tissue sarcomas, Hodgkin's and non-Hodgkin's lymphomas, and bronchogenic carcinoma.

Vincristine, a member of the vinca-alkaloid class, is isolated from the common plant, periwinkle (Vinca rosea Linn). The mechanism of action of vincristine is still being investigated, but is related to the inhibition of microtubule formation in the mitotic spindle. Vincristine is useful in the treatment of acute leukemia, Hodgkin's disease, non-Hodgkin's malignant lymphomas, rhabdomyosarcoma, neuroblastoma, and Wilms' tumor.

• ♦ · · * 9»

Etoposide, a member of the epipodophyllotoxin class of cytostatics, is a semisynthetic derivative of podophyllotoxin. Etoposide acts as a topoisomerase inhibitor and is useful for the treatment of testicular and lung tumors.

It is currently unknown what causes a cell line to develop resistance through the MDR1 or MRP1 mechanism. Given the tissue specificity of these transporters and/or in cases where one of the mechanisms predominates, it would be advantageous to have a selective inhibitor for one of these proteins. Furthermore, when administering a drug or drugs that are substrates for one of the proteins, it would be advantageous to co-administer an agent that is a selective inhibitor of that protein. Therefore, the development of compounds that are selective inhibitors of MDR1 or MRP1 is desirable.

The essence of the invention

The present invention relates to compounds of formula I:

where:

m is an integer from 1 to 6;

R is selected from the following groups: COR ^3- , amino, NH-Pg or NHCOR ² ;

R ¹ is selected from the following groups: hydroxy, C 1 -C 4 , alkoxy or NR ³ R ⁴ ;

Pg is a protecting group for the amino group;

• ·

R ² is selected from the following groups: C 1 -C 4 alkyl, substituted C 1 -C 4 alkyl, aryl, substituted aryl, (CH ₂ ) 4 -heterocycle, (CH 2 ) 4 -substituted heterocycle;

R ³ is independently hydrogen or C 1 -C 4 alkyl;

R ⁴ is selected from the following groups: C _x -C _e alkyl, norbornan-2-yl, aryl, substituted aryl, CH ₂ CH(CH ₃ )phenyl, (CH ₂ )-heterocycle, (CH ₂ )-substituted heterocycle; and n is 0, 1 or 2; or pharmaceutically acceptable salts or solvates thereof.

The present invention further relates to a method of inhibiting MRP1 in a mammal comprising administering an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt or solvate thereof, to a mammal in need of such treatment.

In another embodiment, the present invention relates to a method for inhibiting resistant tumors or tumors that could develop resistance, comprising administering to a mammal in need of such treatment an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt or solvate thereof, in combination with an effective amount of a cytostatic agent,

The present invention also relates to a pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, in combination with one or more cytostatic agents, pharmaceutical carriers, diluents and/or additives.

The present invention consists in the discovery that a selected group of compounds - compounds of formula I - are selective inhibitors of the multidrug resistance protein (MRP1) and are therefore useful for the treatment of multidrug resistance (MDR) caused by MRP1 in resistant tumors and in tumors in which it could be • · · · 4 4 • · · · · 4

4 444 9 4 4 • · · · · « · * · * ·

resistance arises.

The term inhibition, as used in connection with MRP1, and MRP1 inhibition, refers to preventing, mitigating, stopping, limiting, slowing or reversing the progression or reducing the ability of MRP1 to redistribute a cytostatic agent from its site of action, most commonly from the nucleus of a tumor cell and outside the cell.

The term "effective amount" of a compound of formula I, as used herein, refers to an amount of a compound of the present invention that is capable of inhibiting MRP1. The term "effective amount" of a cytostatic agent refers to an amount of a cytostatic agent effective for inhibiting tumor, resistance, and the like.

The term inhibition of a resistant tumor or a tumor that could develop resistance refers to preventing, mitigating, stopping, limiting, slowing or reversing the progression, slowing the growth or killing of a resistant tumor or a tumor that could develop resistance.

The term resistant tumor refers to a tumor that is resistant to chemotherapy, where this resistance is partially or fully due to MRP1. Such tumors include, without limitation, tumors of the bladder, bone, breast, lung (small cell), testicular, and thyroid, as well as more specific tumor types such as acute lymphoblastic or myeloblastic leukemia, Wilms tumor, neuroblastoma, soft tissue sarcoma, Hodgkin and non-Hodgkin lymphoma, and bronchogenic carcinoma.

A tumor that could develop resistance is a tumor that does not have inherent resistance and is not currently present, but may develop resistance as a result of the action of MRP1 after initiation of chemotherapy. Accordingly, the methods of the present invention include prophylactic and therapeutic administration of compounds of formula I.

The term chemotherapy refers to the use of one or more cytostatics, where at least one cytostatic is a substrate for MRP1. A substrate for MRP1 is a cytostatic that binds to MRP1 and is redistributed from its site of action (the cell nucleus) outside the cell, causing less efficacy of the therapy.

The terms treatment or cure have their usual meanings, which include preventing, inhibiting, alleviating, ameliorating, halting, limiting, slowing or reversing the progression or reducing the severity of drug resistance caused by MRP1 in multidrug resistant tumors.

In the general formulas of the present invention, the general chemical terms have their usual meanings. For example, the term C^-C^alkyl refers to methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, cyclobutyl, s-butyl and t-butyl. The term C _^ -C _^ alkyl refers to monovalent, straight, branched or cyclic saturated hydrocarbon chains containing 5 or 6 carbon atoms and includes the groups defined by the term CC alkyl. Furthermore, the term C^-C^alkyl also includes, for example, cyclopentyl, pentyl, hexyl, cyclohexyl and the like.

The term aryl refers to phenyl, benzyl and naphthyl.

The term heterocycle refers to a monovalent, saturated, unsaturated or aromatic monocyclic or fused ring system containing a total of 5 to 7 or 8 to 10 atoms, respectively, which contains 1 to 3 heteroatoms selected independently from the group consisting of oxygen, sulfur and nitrogen.

Examples of heterocyclic groups are furanyl, indolyl, thiophenyl, isoxazolyl, all their partially or fully saturated analogues, for example tetrahydrofuran and the like, where the site of attachment to the heterocycle is at any position of the ring available for substitution.

The term substituted heterocycle refers to a heterocyclic ring substituted 1-2 times independently with groups such as C-C _alkyl , halogen, benzyl, phenyl, trifluoromethyl or oxo-.

The term substituted aryl refers to a phenyl, benzyl or naphthyl group, respectively, substituted 1 to 5 times independently with halogen, hydroxy, trifluoromethyl, N(R ^X ) 2 , NH-Pg , C 1 -C 4 alkoxy, benzyloxy, CO 2 R ^X , SO 2 NH ₂ , trifluoromethoxy or nitro.

The term substituted C1-C4 alkyl refers to a C1-C4 alkyl group in which 1 to 3 hydrogens have been replaced by the same halogen, such as trifluoromethyl.

The terms halogen or halide refer to fluorine, chlorine, bromine or iodine.

The term C ₁ -C ₄ alkoxy refers to a C ₁ -C ₄ alkyl group attached through an oxygen atom.

The term amino protecting group, as used herein, refers to an amino substituent commonly used to block or protect an amino group during the reaction of other functional groups of a compound. Examples of such amino protecting groups are formyl, trityl, phthalimide, acetyl, trichloroacetyl, chloroacetyl, bromoacetyl, and iodoacetyl, urethane-type blocking groups such as benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl (FMOC), and the like; and similar amino protecting groups. The type of amino protecting group is not critical as long as the derivatized amino group is stable under the conditions of subsequent reactions at other positions of the molecule and can be removed at a convenient time without disturbing the remainder of the molecule. Similar amino protecting groups used in cephalosporins, penicillins, and peptides are also included within this term. Additional examples of groups encompassed by the above terms are described in T.W. Greene, Protective Groups in Organic Synthesis, John Wiley and Sons, New York, N.Y. 1991, Chapter 7, which is hereby incorporated by reference by reference to Greene. A preferred protecting group for the amino group is t-butylcarbonyl.

The term activating group for a carbonyl refers to a substituent on the carbonyl that renders the carbonyl susceptible to nucleophilic addition. Suitable activating groups are those that cause the removal of an electron from the carbonyl.

Such groups include, for example, alkoxy, aryloxy, aromatic nitrogen-containing heterocycles or amino groups such as oxybenzotriazole, imidazolyl, nitrophenoxy, pentachlorophenoxy,

N-oxysukeinimide, N,N'-dicyclohexylisoure-O-yl,

N-hydroxy-N-methoxyamino and the like; acetates, formates, sulfonates such as methanesulfonate, ethanesulfonate, benzenesulfonate or p-toluenylsulfonate and the like; halides, especially chloride, bromide or iodide.

In general, the term pharmaceutical, when used as an adjective, means substantially nontoxic to a living organism. For example, the term pharmaceutical salt, as used herein, • * refers to a salt of a compound of formula I that is substantially nontoxic to a living organism. See, for example, Berge, S.M., Bighley, L.D. and Monkhouse, D.C., Pharmaceutical Salts, J. Pharm. Sci. 66: 1, 1977. Typical pharmaceutical salts include salts prepared by reacting compounds of formula I with inorganic or organic acids. Such salts are known as acid or base addition salts. These pharmaceutical salts often have increased solubility compared to the parent compounds and are therefore often more suitable for the preparation of liquid formulations or emulsions.

Examples of pharmaceutical acid addition salts of the compound of formula I are sulfate, pyrosulfate, hydrosulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, ethanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelic acid, and the like.

Examples of pharmaceutical base addition salts of compounds of formula I are ammonium, lithium, potassium, sodium, calcium, magnesium, methylamine, diethylamine, ethylenediamine, cyclohexylamine and ethanolamine salts and the like.

The term solvate refers to an aggregate that comprises one or more molecules of a solute, such as a compound of formula I, with one or more molecules of a solvent.

The term suitable solvent refers to a solvent that is inert to the reactions taking place and that dissolves the reactants sufficiently for the desired reaction to occur. Examples of suitable solvents are dichloromethane, chloroform,

1.2- dichloroethane, diethyl ether, acetonitrile, ethyl acetate,

1,3-dimethyl-2-imidazolidone, tetrahydrofuran, dimethylformamide, toluene, chlorobenzene, dimethyl sulfoxide, mixtures thereof, and the like.

The term carbonyl activating agent refers to an agent that changes the carbonyl of a carboxylic acid to a group more susceptible to nucleophilic addition and includes, for example, the agents listed in The Peptides, Gross and Meienhofer, Eds., Academie Press (1979), Chapter 2, and M. Bodanzsky, Principles of Peptide Synthesis, 2nd Edition, Springer-Verlag Berlin Heidelberg, 1993, hereinafter referred to as The Peptides and Peptide Synthesis, respectively. Specifically, carbonyl activating agents include nucleophilic halogen sources such as thionyl bromide, thionyl chloride, oxalyl chloride, and the like; alcohols such as nitrophenol, pentachlorophenol, and the like; amines such as N-hydroxy-N-methoxamine, and the like; acid halides such as acetic, formic, methanesulfonic, ethanesulfonic, benzenesulfonic, or p-toluenesulfonic halides, and the like; and compounds such as 1,

1'-carbonyldiimidazole, benztriazole, imidazole,

N-hydroxysuccinimide, dicyclohexylcarbodiimide and the like.

The term suitable thermodynamic base refers to a base that acts as a scavenger for any protons that may be produced as by-products of the reaction, or a base that allows reversible deprotonation of the acidic substrate and is sufficiently reactive to effect the desired reaction without any undesirable reaction. Examples of thermodynamic bases include, for example, carbonates, bicarbonates and hydroxides (e.g., lithium, sodium or potassium carbonate, bicarbonate or hydroxide), tri(C _x -C ₄ alkyl)amines or aromatic nitrogen-containing heterocycles (e.g., pyridine).

Although all compounds of the present invention are useful, some compounds are more preferred than others. The following list sets forth several groups of preferred compounds, compositions, and methods. It should be understood that each item in the list may be combined with other items in the list to form further groups of preferred embodiments:

a) m is an integer from 2 to 6;

b) R is an amino group;

c) R is t-butyloxycarbonylamino;

d) R is trifluoroacetylamino;

e) R is 3,4,5-trimethoxybenzoylamino;

f) R is a carboxy group;

g) R is 3,4,5-trimethoxyanilinylcarboxy-;

h) R is 3,4,5-trimethoxybenzylaminylcarboxy-;

i) the compound is selected from the Examples;

j) the compound is a pharmaceutical salt;

k) the compound is a hydrochloride salt;

l) a method in which the mammal is a human;

m) a method in which the cytostatic agent is doxorubicin, daunorubicin, epirubicin, vincristine and etoposide;

n) a method in which the tumor is Wilms' tumor, bladder, bone, breast, lung (small cell), testicular, or thyroid tumor, or is acute lymphoblastic or myeloblastic leukemia, neuroblastoma, soft tissue sarcoma, Hodgkin's and non-Hodgkin's lymphoma, or bronchogenic carcinoma; and

o) a composition in which the cytostatic agent is doxorubicin, daunorubicin, epirubicin, vincristine and etoposide.

·· ·♦ • ..··-♦

• ·

The compounds of the present invention can be prepared by a variety of procedures, some of which are illustrated in the following schemes. The particular order of steps required to prepare compounds of formula I depends on the particular compound being synthesized, the starting compound, and the relative lability of the substituted groups.

Compounds of formula I can be prepared from compounds of formula II by the method described in Scheme 1, in which R and m are as defined above.

Compounds of formula I can be prepared by dissolving or suspending a compound of formula II in a suitable solvent and adding a suitable thermodynamic base. A common and preferred solvent is dimethylformamide. A common and preferred thermodynamic base is sodium hydroxide added as a 2N solution in methanol. The reactants are usually mixed at ambient temperature and the resulting solution is usually allowed to react for a period of from 30 minutes to 18 hours. Preferably, the mixture is mixed for about 1 to 10 hours and most preferably, it is mixed for 3.5 to 7 hours. The base is usually used in a large molar excess, usually in a 4- to 8-fold molar excess relative to the compound of formula II. Preferably, about 5- to 7-fold molar excess is used.

• · ··

Some intermediates of the compounds of formula I, described below, can also be prepared by the method described above.

Any amino or hydroxy protecting groups contained in the cyclized compound of formula I may optionally be removed as described in Green to provide compounds of formula I containing a free amino or hydroxy group. Preferred protecting groups and methods for their removal are provided in the Examples.

Compounds of formula I, where R is COR ¹ , can also be prepared from compounds of formula I(a) as described in Scheme 2, where R ^s is an activating group for a carboxyl group, R ^s is C ₁ -C ₅ alkoxy or NR ³ R ⁴ and m is as defined above.

Compounds of formula I(a), prepared by the method of Scheme I, can be converted to other compounds of the present invention. For example, acids of formula I(a) can be activated to form activated carboxylic acids of formula III using techniques well known in the art. See The Peptides,

Peptide Synthesis and Examples of the Invention.

Compounds of formula I(b) can then be prepared by dissolving or suspending a compound of formula III in a suitable solvent, optionally in the presence of a suitable thermodynamic base, and adding an amine of formula IV. A common and preferred solvent is dichloromethane. Preferred bases are triethylamine and piperidinylmethyl polystyrene resin. The amine is usually used in a large molar excess, for example in 1,

5- to 3-fold molar excess relative to the compound of formula III. Preferably, about 1.8- to 2.2-fold molar excess is used. The reaction is usually carried out at a temperature in the range of 0 °C to the reflux temperature of the solvent for a reaction time of 10 minutes to 18 hours. Preferably, the reaction is carried out at a temperature in the range of 15 °C to 40 °C for a reaction time of 5 minutes to 2.5 hours.

Alternatively, the compound of Example I(a) can be activated and the addition of the compound of Formula IV can be carried out in one pot as described in Examples 26 and 38.

Compounds of formula I(b) wherein R ⁵ is C ₁ -C ₆ alkoxy, i.e. esters, can be prepared by methods well known in the art. For a description of the conversion of activated carboxylic acids to esters, see Larock, Comprehensive Organic Transformations, pp. 978-979,

VCH Publishers, New York, N.Y. 1989, hereinafter referred to as Larock. Alternatively, these compounds of formula I(b) may be prepared directly from the acids of formula I(a) by the method set forth in Larock at pages 966-972.

Compounds of formula I, where R is amino- or NHCOR ² , can be prepared from compounds of formula I(c) as described in scheme φ ·

Ϊ5 φφφ

3, where m, Pg, ^R2 and ^R5 are as defined above.

Compounds of formula I(c) can be converted into other compounds of the present invention. For example, a compound of formula I(c) can be deprotected as described in Greene's or the Examples to give a compound of formula I(d). This compound of formula I(d) can also be converted into another compound of the present invention. For example, a compound of formula I(d) dissolved or suspended in a suitable solvent and optionally in the presence of a thermodynamic base can be reacted with a compound of formula VI to give a compound of formula I(e). A conventional and suitable solvent is dimethylformamide or a mixture of dichloromethane and dimethylformamide. If a base is used, triethylamine or N-methylmorpholine is usually preferred. Furthermore, if a base is used, the base and the compound of formula VI are usually used in stoichiometric amounts or in large molar ratios. For example, a 1.0- to 4-fold molar ratio is used.

4 4 • 4

4 4

4

4

W «v 4* • -4- · · 4 · • · « · 4 4

444444 4 • · 4 4 4 ·· ·· 44

If no base is used, an excess of the compound I(d) is usually used, so the compound of formula VI is usually used in a relatively large stoichiometric excess. It is preferred to carry out the reaction in the presence of a base with about 1.8 to 2.2 equivalents of the compound of formula VI. The reaction is usually carried out at a temperature in the range of about 0 °C to about the reflux temperature of the solvent for 10 minutes to 24 hours. Preferably the reaction is carried out at a temperature of about 15-40 °C for 4 to 18 hours.

Pharmaceutical salts of the compounds of the present invention are typically prepared by reacting a compound of formula I with an equimolar amount or excess of an acid. The reagents are typically combined in a common solvent such as diethyl ether, tetrahydrofuran, methanol, ethanol, isopropanol, benzene, and the like for acid addition salts, or water, alcohol, or a chlorine solvent such as dichloromethane for base addition salts. The salts typically precipitate from solution within about one hour to about ten days and can be isolated by filtration or other conventional methods.

Acids commonly used to prepare pharmaceutical acid addition salts are inorganic acids such as hydrochloric, hydrobromic, hydroiodic, sulfuric, phosphoric, and the like, and organic acids such as p-toluenesulfonic, methanesulfonic, ethanesulfonic, oxalic, p-bromophenylsulfonic, carbonic, succinic, citric, tartaric, benzoic, acetic, and the like. Preferred pharmaceutical acid addition salts are salts formed with inorganic acids such as hydrochloric, hydrobromic, and sulfuric acids, and salts formed with organic acids such as maleic, tartaric, and methanesulfonic acids.

Bases commonly used for the preparation of pharmaceutical base addition salts are inorganic bases such as ammonium hydroxides or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates and the like. Such bases suitable for the preparation of salts according to the present invention include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate and the like. Potassium and sodium salts are particularly preferred.

It should be appreciated that the particular counterion forming part of a salt of the present invention is not critical, as long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not cause undesirable qualities of the salt as a whole.

The starting materials for the methods of the present invention can be obtained in a variety of ways. For example, compounds of formula II, I(a) and I(c) can be prepared by the method of Scheme 4, where m and R are as defined above.

Scheme 4

Compounds of formula II may be prepared by dissolving or suspending a compound of formula VIII and a suitable thermodynamic base. A common and suitable solvent is dichloromethane. A suitable thermodynamic base is usually triethylamine. This

• · »9

The ηδ amide-forming reaction is usually carried out in the presence of dimethylaminopyridine (DMAP). The compound of formula VIII is usually and preferably used in an equimolar amount relative to the compound of formula VII, but a small excess (about 0.05 to about 0.15-fold molar excess) is acceptable. The thermodynamic base is usually used in a slight molar excess. For example, about 1.01- to 1.2-fold molar excess relative to the compound of formula VII is used. A 1.05- to 1.15-fold molar excess is preferred. DMAP is used catalytically. For example, it is used in an amount of about 5-15 mole percent relative to the compound of formula VIII. A 10 mole percent is preferred.

Although the transformations described in Schemes 2 and 3 can be carried out before the cyclization described in Scheme 1, it is advantageous to carry out these reactions after the cyclization to prepare compounds of Formula VII with a fully functional R substituent. Therefore, the preferred materials for the reactions of Schemes 1 and 4 are compounds of Formula II and VII, where R is NH-Pg or COR ¹ , where R ¹ is Cx-Ce alkoxy. Furthermore, when the reaction of Scheme 1 is carried out with a compound of Formula II, where R is COR ¹ , where R ¹ is Cx- _Cg alkoxy, under the conditions described for the cyclization, the ester is cleaved to the acid, i.e., compounds of Formula I(a) are formed.

Compounds of formulae IV, V, VI, VII and VIII are known in the art and, if not commercially available, can be readily synthesized by standard procedures used in the art.

The optimum reaction time of Schemes 1-4 can be determined by monitoring the reaction progress using conventional chromatographic techniques. Furthermore, it is advantageous to carry out the reactions of the present invention in an inert atmosphere, such as argon, or more preferably nitrogen. The choice of solvent is usually not critical if • 4 • * 4' • · 4 • 444 « • >

• 4 · 4 4 • * • 4 • 4 .·» . 4 « 4 · 4 « · 4

4 4 • ♦ 4 the solvent used is inert to the reactions taking place and sufficiently dissolves the reactants to allow the desired reaction to take place. The compounds of formula I(d) and II are preferably isolated and purified before their use in the next reaction.

These compounds may be crystallized from the reaction solution during their formation and then recovered by filtration, or the reaction solvent may be removed by extraction, evaporation or decantation. These intermediates and final compounds of formula I may be further purified, if desired, by conventional techniques such as recrystallization or chromatography on solid supports such as silica gel or alumina.

The following Preparations and Examples are provided to better illustrate the embodiments of the present invention and should not be construed as limiting the scope of the invention. It will be apparent to those skilled in the art that various modifications may be made without departing from the scope or spirit of the present invention. All publications cited in the application are understood by those skilled in the art to which the invention relates. The terms and abbreviations used have their usual meanings unless otherwise indicated. For example, °C,

N, mmol, g, ml, M, IR, MS(FD), and MS(IS) refer to degrees Celsius, normal or normality, millimole, gram, milliliter, molarity, infrared spectrometry, field desorption mass spectrometry, and spray ionization mass spectrometry. Furthermore, the absorption maxima listed for IR spectra are only selected maxima and not all of the maxima observed.

Examples of embodiments of the invention

Preparations

Preparation 1: N-t-butyloxycarbonyl-N'-(3-[2-chloro-6-fluorophenyl]-5-methylisoxazol-4-oyl)-1,2-diaminoethane

Nt-Butyloxycarbonyl-1,2-diaminoethane (355 mg, 2.22 mmol), dimethylaminopyridine (27 mg, 0.222 mmol) and triethylamine (247 mg, 2.44 mmol) were mixed in 5 mL of dry dichloromethane. 3-(2-chloro-6-fluorophenyl)-5-methylisoxazol-4-oyl chloride (607 mg, 2.22 mmol) was added slowly. The reaction mixture was stirred for 3 h at ambient temperature under a nitrogen atmosphere. The reaction mixture was diluted with ethyl acetate and water. The ethyl acetate was separated and washed with 3 x 25 mL each of 1N aqueous hydrochloric acid and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was treated with hexanes to crystallize 794 mg of the title compound (90%). EA -calculated for: C _ie H _2x N ₃ O ₄ ClF: C, 54.34; H, 5.32; N, 10.56. Found: C, 54.30; H, 5.36; N, 10.44. MS (IS) m/z 398 (M+).

Preparation 2: N-t-butyloxycarbonyl-N'-(3-[2-chloro-6-fluorophenyl]-5-methylisoxazol-4-oyl)-1,3-diaminopropane

Nt-Butyloxycarbonyl-1,3-diaminopropane (574 mg, 3.29 mmol) and 3-(2-chloro-6-fluorophenyl)-5-methylisoxazol-4-oyl chloride (903 mg, 3.29 mmol) were converted to 1.36 g of the title compound according to Preparation 1 (100%). EA calculated for: C _{x 5} H ₂₃ N ₃ O ₄ C1F: C, 55.41; H, 5.63; N, 10.20. Found: C, 55.17; H, 5.71; N, 9.99. MS (IS) m/z 412 (M+).

Preparation 3: N-t-butyloxycarbonyl-N'-(3-[2-chloro-6-fluorophenyl]-5-methylisoxazol-4-oyl)-1,4-diaminobutane

Nt-Butyloxycarbonyl-1,4-diaminobutane (985 mg, 5.23 mmol) and 3-(2-chloro-6-fluorophenyl)-5-methylisoxazol-4-oyl chloride (1.43 g, 5.23 mmol) were converted to 2.23 g of the title compound by the procedure of Preparation 1, except changing the reaction time to 18 hours (100%). EA calculated for: C ₂₀ H ₂₅ N ₃ 0 ₄ ClF: C, 56.41; H, 5.92; N, 9.87. Found: C, 56.12; H, 5.75; N, 9.67. MS (IS) m/z φφφφ φ · · · ' · · • · · · I» · · · · · φφφ* · · · ·· • »····* · · · · · φφ φφ ·Φ φφφ ··

426 (M+).

Preparation 4: N-t-butyloxycarbonyl-N'-(3-[2-chloro-6-fluorophenyl]-5-methylisoxazol-4-oyl)-1,5-diaminopentane

N-t-Butyloxycarbonyl-1,5-diaminopentane (1.04 g, 5.14 mmol) and 3-(2-chloro-6-fluorophenyl)-5-methylisoxazol-4-oyl chloride (1.41 g, 5.14 mmol) were converted to 2.14 g of the title compound according to Preparation 1 (95%). MS (IS) m/z 412 (M+).

IR(CHCl ₃ ) 3496, 3012, 2936, 1708, 1663, 1611, 1510 cm ¹ .

Preparation 5: N-t-butyloxycarbonyl-N'-(3-[2-chloro-6-fluorophenyl]-5-methylisoxazol-4-oyl)-1,6-diaminohexane

Nt-Butyloxycarbonyl-1,6-diaminohexane, hydrochloride salt (1.02 g, 4.03 mmol) and 3-(2-chloro-6-fluorophenyl)-5-methylisoxazol-4-oyl chloride (1.11 g, 4.03 mmol) were converted to 1.84 g of the title compound according to Preparation 1 (100%). EA calculated for: C ₂₂ H ₂₉ N ₃ O ₄ C1F: C, 58.21; H, 6.44; N, 9.26. Found: C, 57.97; H, 6.20; N, 9.43. MS (IS) m/z 454 (M+).

Preparation 6: Methyl 4-aminobutanoate, hydrochloride

Nt-Butyloxycarbonyl-4-aminobutanoic acid (1 g, 4.92 mmol) was dissolved in 10 mL of freshly prepared hydrochloric acid-methanol mixture (2.5 mL of acetyl chloride in 35 mL of methanol). The reaction mixture was stirred for 2 hours and then distilled. The residue was taken up in ethyl acetate, washed three times with aqueous sodium bicarbonate solution and three times with brine, dried over sodium sulfate, filtered and concentrated to give 708 mg of the title compound (94%). EA calculated for: C ₅ H _i2 C1NO ₂ : C, 39.10; H, 7.87; N, 9.12. Found: C, 38.89;

H, 7.65; N, 8.94. MS (FD) m/z 118 (M+ for free amine).

Preparation 7: Methyl 5-aminopentanoate, hydrochloride

Nt-Butyloxycarbonyl-5-aminopentanoic acid (1 g, 4.60 mmol) was converted to the title compound according to Preparation 6 in 705 mg (91%). EA calculated for C ₆ H ₁₄ ClNO ₂ : C, 42.99; H, 8.42; N, 8.36. Found: C, 43.02; H, 8.15; N, 8.18. MS (FD) m/z 132 (M+ for free amine).

Preparation 8: Methyl 6-aminohexanoate, hydrochloride

Nt-Butyloxycarbonyl-6-aminohexanoic acid (500 mg, 2.16 mmol) was converted to the title compound according to Preparation 6 to yield 357 mg (91%). EA calculated for: C ₇ H ₁₅ ClNO ₂ : C, 46.28; H, 8.88; N, 7.71. Found: C, 46.04; H, 8.88; N,

7.51. MS (FD) m/z 146 (M+ for free amine).

Preparation 9: Methyl 7-aminoheptanoate, hydrochloride

N-t-Butyloxycarbonyl-7-aminoheptanoic acid (1 g, 4.08 mmol) was converted to the title compound according to Preparation 6 to yield 742 mg (93%). EA calculated for C12H14ClNO3: C, 49.10; H, 9.27; N, 7.16. Found: C, 49.10; H, 9.18; N,

7.03. MS (FD) m/z 160 (M+ for free amine).

Preparation 10: Methyl N-(3-[2-chloro-6-fluorophenyl]-5-methylisoxazol-4-oyl)-4-aminobutanoate

Methyl 4-aminobutanoate hydrochloride (668 mg, 4.34 mmol) and 3-(2-chloro-6-fluorophenyl)-5-methylisoxazol-4-oyl chloride (1.19 g, 4.35 mmol) were converted to 1.60 g of the title compound by the procedure of Preparation 1 except that the residue was not treated with hexanes (100%). EA calculated for: C 12 H 12 N 12 O 12 ClF: C, 54.17; H, 4.55; N, 7.90. Found: C, 54.41; H, 4.58; N, 7.78.

MS (FD) m/z 355 (M+).

Preparation 11: Methyl N-(3-[2-chloro-6-fluorophenyl]-5-methylisoxazol-4-oyl)-5-aminopentanoate

Methyl 5-aminopentanoate hydrochloride (694 mg, 4.14 mmol) and 3-(2-chloro-6-fluorophenyl)-5-methylisoxazol-4-oyl chloride (1.24 g, _4.52 mmol) were converted to 1.36 g of the title compound according to Preparation 10 (100%). EA calculated for: _{C17H18N2O4C1F} : C, 55.37; H, _{4.92 ;} N, 7.60. Found: C, 55.44; H, 5.00; N, 7.52. MS (FD) m/z 368 (M+).

Preparation 12: Methyl N-(3-[2-chloro-6-fluorophenyl]-5-methylisoxazol-4-oyl)-6-aminohexanoate

Methyl 6-aminohexanoate hydrochloride (349 mg, 1.92 mmol) and 3-(2-chloro-6-fluorophenyl)-5-methylisoxazol-4-oyl chloride (527 mg, 1.92 mmol) were converted to 2.23 g of the title compound according to Preparation 10 (100%). EA calculated for:

C H N O C1F: C, 56.48; H, 5.27; N, 7.32. Found: C, 56.66;

IQ 20 2 4

H, 5.20; N, 7.07. MS (FD) m/z 382.2 (M+).

Preparation 13: Methyl N-(3-[2-chloro-6-fluorophenyl]-5-methylisoxazol-4-oyl)-7-aminoheptanoate

Methyl 7-aminoheptanoate hydrochloride (733 mg, 3.75 mmol) and 3-(2-chloro-6-fluorophenyl)-5-methylisoxazol-4-oyl chloride (1.03 g, 3.75 mmol) were converted to 1.48 g of the title compound according to Preparation 10 (100%). EA calculated for:

C H N O C1F: C, 57.51; H, 5.59; N, 7.06. Found: C, 57.78;

ZL 9 2 2 2 4

H, 5.70; N, 6.80. MS (FD) m/z 396.1 (M+).

Examples

Example 1: 1-(N-t-butyloxycarbonyl-2-aminoethyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one

Nt-Butyloxycarbonyl-N'-(3-(2-chloro-6-fluorophenyl)-5-methylisoxazol-4-oyl)ethylene-1,2-diamine (772 mg, 1.94 mmol) was dissolved in 10 mL of dry dimethylformamide and stirred under nitrogen at ambient temperature. Aqueous sodium hydroxide (5 mL) was added and the resulting solution was stirred for 4 hours. The reaction mixture was partitioned between 50 mL of 1N aqueous hydrochloric acid and ethyl acetate. The ethyl acetate was washed three times with 25 mL of brine and dried over sodium sulfate. The solution was concentrated in vacuo to give 721 mg of the title compound (98%). EA calculated for: ^C 1SH ₂₀ C1N ₃ 0 ₄ : C, 57.22; H, 5.34; N, 11.12. Found: C, 57.21; H, 5.23; N, 10.97. MS (IS) m/z 376 (MH).

Example 2: 1-(N-t-butyloxycarbonyl-3-aminopropyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one

N-t-Butyloxycarbonyl-N'-(3-(2-chloro-6-fluorophenyl)-5-methylisoxazol-4-oyl)-1,3-diaminopropane (1.31 g, 3.18 mmol) was converted to 1.22 g of the title compound by the procedure of Example 1 except that the reaction time was 6 hours. EA calculated for: C H ClN O : C, 58.24; H, 5.66; N, 10.72. Found: C, 58.48;

IL 9 2 2 3

H, 5.52; N, 10.73. MS (IS) m/z 392 (M-H).

Example 3: 1-(N-t-butyloxycarbonyl-4-aminobutyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one

Nt-Butyloxycarbonyl-N'-(3-(2-chloro-6-fluorophenyl)-5-methylisoxazol-4-oyl)-1,4-diaminobutane (2.23 g, 5.23 mmol) was converted to 2.11 g of the title compound by the procedure of Example • · • 9 • · · · 9 • •99 · • · · · · · * · · · · 99 except that after concentration the residue was azeotroped with xylenes to remove residual dimethylformamide. EA calculated for: C_ _n H TIN O: C, 59.19; H, 5.96; N, 10.35. Found: C, 58.71; H, 5.92; N, 9.96. MS (FD) m/z 405 (MH).

Example 4: 1-(N-t-butyloxycarbonyl-5-aminopentyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one

Nt-Butyloxycarbonyl-N'-(3-(2-chloro-6-fluorophenyl)-5-methylisoxazol-4-oyl)-1,5-diaminopentane (2.11 g, 4.80 mmol) was converted to 1.13 g of the title compound by the procedure of Example 1 except that the reaction time was 6 hours and after concentration the residue was recrystallized from dichloromethane/hexanes. (56%). EA calculated for: C _ai H _2e ClN ₃ O ₄ : C, 60.07; H, 6.24; N, 10.01. Found: C, 60.07; H, 6.30; N, 10.11. MS (IS) m/z 420 (MH).

Example 5: 1-(N-t-butyloxycarbonyl-6-aminohexyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one

N-t-Butyloxycarbonyl-N'-(3-(2-chloro-6-fluorophenyl)-5-methylisoxazol-4-oyl)-1,6-diaminohexane (1.84 g, 4.05 mmol) was converted to 1.45 g of the title compound according to the procedure of Example 3. (82%). EA calculated for: C H C IN O: C, 60.89; H, 6.50;

2 2 8 3

N, 9.86. Found: C, 61.19; H, 6.33; N, 9.51. MS (IS) m/z

434.6 (M-H).

Example 6: 1-(2-aminoethyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one, trifluoroacetate

1-(N-t-Butyloxycarbonyl-2-aminoethyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one (687 mg, 1.82 mmol) was dissolved in 10 mL of dichloromethane and trifluoroacetic acid (3 mL) was added. The mixture was stirred under nitrogen at

at ambient temperature for 2 hours. The solvents were removed and the residue was precipitated with ethyl acetate/hexanes to give 830 mg of the title compound. (100%). ES(FD) m/z 277 (M+ for free amine). IR(KBr) 3145, 1778, 1665, 1646, 1626 cm ^-1 .

Example 7: 1-(3-aminopropyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one, trifluoroacetate

1-(Nt-Butyloxycarbonyl-3-aminopropyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one (922 mg, 2.35 mmol) was converted to 837 mg of the title compound by the procedure of Example 6 except that the reaction time was 3 hours. (88%). EA calculated for: C _xe H _is ClF ₃ N ₃ O ₄ : C, 47.36; H, 3.73; N, 10.36. Found: C, 47.44; H, 3.70; N, 10.34. MS (IS) m/z 292 (M+ for free amine).

Example 8: 1-(4-Aminobutyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one, trifluoroacetate

1-(N-t-Butyloxycarbonyl-4-aminobutyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one (3.14 g, 7.75 mmol) was converted to 2.90 g of the title compound according to the procedure of Example

7. (89%). EA calculated for: Ο ₃₇ Η ₁₇ Ο1Ρ ₃ Ν ₃ Ο ₄ : C, 48.64; H, 4.08; N, 10.01. Found: C, 48.49; H, 3.95; N, 9.91. MS (IS) m/z 306 (M+ for free amine).

Example 9: 1-(5-aminopentyl)-isoxazoloΪ3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one, trifluoroacetate

1-(N-t-Butyloxycarbonyl-5-aminopentyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one (1.08 g, 2.57 mmol) was converted to 1.01 g of the title compound according to the procedure of Example

7. (89%). EA calculated for: C ₁₈ H ₁₉ C _{1 F 3} N ₃ O ₄ : C, 49.84; H, 4.41;

N, 9.69. Found: C, 50.08; H, 4.23; N, 9.69. MS (IS) m/z 320 (M+ for free amine).

Example 10: 1-(6-aminohexyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one, trifluoroacetate

1-(Nt-Butyloxycarbonyl-6-aminohexyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one (1.40 g, 3.23 mmol ⁾ was converted to 1.30 g of the title compound according to the procedure of Example 7. (90%). EA calculated for: _C19H21ClF3N3 (\ ^: _C , 50.96; H, 4.73;

N, 9.38. Found: C, 51.20; H, 4.62; N, 9.31. MS (IS) m/z 334 (M+ for free amine).

Examples 11 and 12: 1-(N-trifluoroacetate-2-aminoethyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one and 1-(N-3,4,5-trimethoxybenzoyl-2-aminoethyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one

1-(2-Aminoethyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one, trifluoroacetate (50 mg, 0.128 mmol) was suspended in 3 mL of dry dichloromethane and N-ethylmorpholine (12.9 mg, 0.128 mmol) and 1 mL of dry dimethylformamide was added. The resulting solution was stirred under nitrogen at ambient temperature and 3,4,5-trimethoxybenzoyl chloride (59 mg, 0.255 mmol) was added and the resulting mixture was stirred for approximately 18 hours. The reaction was continued by addition of ethyl acetate and water. The ethyl acetate was separated and washed three times with 1N aqueous hydrochloric acid, sodium bicarbonate and brine. The ethyl acetate was dried over sodium sulfate and filtered. The ethyl acetate was removed and the residue was chromatographed (silica gel, 1:1 ethyl acetate:hexanes) to give 21 mg of the title compounds as the trifluoroacetate amides and 12 mg of the title compounds as the benzoyl amides. (20%) Trifluoroacetate amide: MS(IS) m/z 372 (M-H). Benzoyl amide: MS(IS) m/z 472 (M+).

Examples 13 and 14: 1-(N-trifluoroacetate-3-aminopropyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one and 1-(N-3,4,5-trimethoxybenzoyl-3-aminopropyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one

1-(3-Aminopropyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one, trifluoroacetate (50 mg, 0.128 mmol) and 3,4,

5-Trimethoxybenzoyl chloride (0.9 equivalents) was converted to the title compounds according to the procedure of Examples 11 and 12 to give 6 mg of the title compounds as trifluoroacetate amides and 48 mg of the title compounds as benzoyl amides. (65%)

Trifluoroacetate amide: MS(IS) m/z 388 (M-H). Benzoyl-amide:

MS(FD) m/z 485 (M+). IR(KBr) 3284, 1673, 1634, 1585, 1339 cm ¹ .

Example 15: 1-(N-3,4,5-trimethoxybenzoyl-4-aminobutyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one

1-(4-Aminobutyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one, trifluoroacetate (75 mg, 0.179 mmol) was dissolved in 2 mL of dry dimethylformamide and stirred under nitrogen at ambient temperature. Triethylamine (54 mg, 0.537 mmol) was added and the solution turned purple. Then

3,4,5-trimethoxybenzoyl chloride (62 mg, 0.268 mmol) and the resulting mixture was stirred for approximately 18 hours. The reaction mixture was diluted with ethyl acetate and washed three times with IN aqueous hydrochloric acid, aqueous sodium bicarbonate and brine, dried over sodium sulfate, filtered and concentrated. The residue was chromatographed (silica gel, ethyl acetate) and the appropriate fractions were recrystallized from dichloromethane ·· ·· ·· , · ·· c ··..· · · · · · · · ·

J*~·· ······· · · • ······ · · · · 9 • · ·· ·· ·· · · · · · to give 40 mg of the title compound. (45%). MS(IS) m/z 500 (M+). IR (KBr) 1674, 1634, 1597, 1585, 1501 cm“ ³ -.

Examples 16 and 17: 1-(N-trifluoroacetate-5-aminopentyl)-isoxazolo(3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one and 1-(N-3,4,5-trimethoxybenzoyl-5-aminopentyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one

1-(5-aminopentyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one, trifluoroacetate and

3,4,5-Trimethoxybenzoyl chloride (0.9 equivalents) was converted to the title compounds according to the procedure of Examples 11 and 12 to give 0.7 mg of the title compounds as trifluoroacetate amides and 41 mg of the title compounds as benzoyl amides. (51%)

Trifluoroacetate amide: MS(IS) m/z 416 (M-H). Benzoyl-amide:

MS(FD) m/z 513 (M+). IR(KBr) 1667, 1629, 1597, 1580, 1500 cm” ¹ .

Examples 18 and 19: 1-(N-trifluoroacetate-6-aminohexyl)-isoxazolo(3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one and 1-(N-3,4,5-trimethoxybenzoyl-6-aminohexyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one

1-(6-Aminohexyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one, trifluoroacetate and

3,4,5-Trimethoxybenzoyl chloride (0.9 equivalents) was converted to the title compounds according to the procedure of Examples 11 and 12 to yield

10.7 mg of the title compounds as trifluoroacetate-amides and 36 mg of the title compounds as benzoyl-amides. (45%)

Trifluoroacetate amide: EA calculated for: C _{x 5} H _is C1F ₃ N ₃ O ₃ : C, 53.09; H, 4.46; N, 9.78. Found: C, 53.34; H, 4.37; N,

9.72. MS (IS) m/z 430 (M+).

Benzoyl amide: EA calculated for: C _2v H _3Q ClF ₃ N ₃ O _g : C, 61.42; H, 5.73; N, 7.96. Found: C, 61.25; H, 5.79; N, 7.69. MS (FD) • ·φ ·· • φ · · « • ΦΦΦΦ· « m/z 528 (Μ+).

Example 20: 1-(3-Carboxypropyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one

Methyl N-(3-[2-chloro-6-fluorophenyl]-5-methylisoxazol-4-oyl)-4-aminobutanoate (1.54 g, 4.35 mmol) was converted to 1.15 g of the title compound by the procedure of Example 1 except that the reaction time was 3 hours. (82%). MS (FD) m/z 320 (M+). IR(KBr) 3145, 2925, 1740, 1732, 1651, 1627, 1598 cm- ¹ .

Example 21: 1-(4-Carboxybutyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one

Methyl N-(3-[2-chloro-6-fluorophenyl]-5-methylisoxazol-4-oyl)-5-aminopentanoate (200 mg, 0.542 mmol) was converted to 180 mg of the title compound according to the procedure of Example 20 (100%).

EA calculated for: C _{x 6} H ₁₅ C 1 N ₂ O ₄ : C, 57.41; H, 4.52; N, 8.37. Found: C, 57.43; H, 4.53; N, 8.08. MS (FD) m/z 334.1 (M+).

Example 22: 1-(5-Carboxypentyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one

Methyl N-(3-[2-chloro-6-fluorophenyl]-5-methylisoxazol-4-oyl)-6-aminohexanoate (735 mg, 1.92 mmol) was converted to 499 mg of the title compound according to the procedure of Example 21 (74%).

EA calcd for C 12 H 13 ClN 14 O 14 : C, 58.54; H, 4.91; N, 8.03. Found: C, 58.51; H, 4.99; N, 7.89. MS (FD) m/z 348 (M+).

Example 23: 1-(6-Carboxyhexyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one

Methyl-N-(3-[2-chloro-6-fluorophenyl]-5-methylisoxazol-4-oyl)*· ·· · ·« • · · · · ·· · ·· • ··· ··· · « · · « • ···» ··· ·· ·· ·· ··· ·· ···

-7-aminoheptanoate (1.49 g, 3.75 mmol) was converted to 1.07 g of the title compound according to the procedure of Example 21. (79%).

EA calculated for ^C18H19ClN2O4 : C, _59.59 ; H, 5.28; N, 7.72. Found: _C , 59.66; _H , 5.49; N, 7.64. MS (FD) m/z 362 (M+).

Example 24: 1-(4-oxo-4-(3,4,5-trimethoxyphenylamino)butyl)isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one

5-(Isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one-1-)butanoic acid and 3,4,5-trimethoxyaniline were converted to the title compound according to the procedure of Example 26 to yield _{67 mg. (88%). EA calculated for C24H24C1N3O6 : C} , _59.32 ;

H, 4.98; N, 5.12. Found: C, 59.52; H, 5.12; N, 8.41. MS (FD) m/z 484.9 (M-H).

Example 25: 1-(4-oxo-4-(3,4,5-trimethoxybenzylamino)butyl)isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6chloroquinolin-2-one

5-(Isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one-1-)butanoic acid and 3,4,5-trimethoxybenzylamine were converted to the title compound according to the procedure of Example 26 to yield _{37 mg. (47%). EA calculated for C24H24C1N3O6 : C} , _60.06 ;

H, 5.24. Found: C, 60.20; H, 5.49. MS (FD) m/z 498.9 (M-H).

Example 26: 1-(5-oxo-5-(3,4,5-trimethoxyphenylamino)pentyl)isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6chloroquinolin-2-one

1-(4-Carboxybutyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one (46 mg, 0.137 mmol) was dissolved in 3 mL of dry dichloromethane and stirred at ambient temperature under a nitrogen atmosphere. 1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDCI, 26 mg, 0.137 mmol) was added followed by 3,4,5-trimethoxyaniline (25 mg, 0.137 mmol) and the resulting mixture was stirred for approximately 18 hours. TLC (ethyl acetate) showed that the reaction was not completely complete, so a catalytic amount of dimethylaminopyridine was added and the reaction mixture was stirred for an additional 18 hours. The reaction mixture was diluted with ethyl acetate and washed three times each with 1N aqueous hydrochloric acid, sodium bicarbonate, brine, dried over sodium sulfate, filtered and the ethyl acetate was removed to give 41 mg of the title compound (60%). EA calculated for:

C H TIN O: C, 60.06; H, 5.24; N, 8.41. Found: C, 59.94;

S 2 6 3 6

H, 5.47; N, 8.14. MS (FD) m/z 498.9 (M-H).

Example 27: 1-(5-oxo-5-(3,4,5-trimethoxybenzylamino)pentyl)isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6chloroquinolin-2-one

1-(4-Carboxybutyl)-isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one (40 mg, 0.119 mmol) was suspended in 3 mL of dry dichloromethane and stirred at ambient temperature under nitrogen. EDCI (23 mg, 0.119 mmol) was added and the reaction mixture became a solution. Then 3,4,

5-trimethoxybenzylamine (23.6 mg, 0.119 mmol) and a catalytic amount of DMAP were added and the resulting mixture was stirred for approximately 18 hours. TLC (ethyl acetate) indicated that the reaction was complete. The reaction mixture was diluted with ethyl acetate and washed three times with each of 1N aqueous hydrochloric acid, sodium bicarbonate, brine, dried over sodium sulfate, filtered, and the ethyl acetate was removed to give 43 mg of the title compound (70%). EA calculated for: C ₂₆ H ₂₈ ClN ₃ O _e : C, 60.76; H, 5.49; N, 8.18. Found: C, 60.89; H, 5.66; N, 7.93. MS (FD) m/z 512.8 (MH).

·· ·· 44 4 ·»

4 4' 4 » · ·· « *· • · · · · · * «· • 44···« 4 4 · 4 4 • · ···· · » ·· 4· 44 ··· *4 4

Example 28: 1-(6-OXΟ-6-(3,4,5-trimethoxyphenylamino)hexyl)isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6chloroquinolin-2-one

5-(Isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-on-1-yl)hexanoic acid and 3,4,5-trimethoxyaniline were converted to the title compound by reaction according to Example 26 to yield 56 mg. (77%). EA calculated for: ^C 26 ^H 2S ^C1N 3° _S : C, 60.76; H, 5.49; N, 8.17. Found: C, 60.18; H, 5.34; N, 7.82. MS (FD) m/z 513.0 (MH).

Example 29: 1-(6-oxo-6-(3,4,5-trimethoxybenzylamino)hexyl)isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6chloroquinolin-2-one

5-(Isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-on-1-yl)hexanoic acid and 3,4,5-trimethoxyaniline were converted to the title compound by reaction according to Example 26 to yield 63 mg. (83%). EA calculated for: C ₂₇ H ₃₀ ClN ₃ O _g : C, 61.42; H, 5.73; N, 7.96. Found: C, 61.32; H, 5.67; N, 7.68. MS (FD) m/z 527.0 (MH).

Example 30: 1-(7-oxo-7-(3,4,5-trimethoxyphenylamino)heptyl)isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one

5-(Isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-on-1-yl)heptanoic acid and 3,4,5-trimethoxyaniline were converted to the title compound by reaction according to Example 26 to yield 52 mg. (71%). EA calculated for: C ₂₇ H ₃₀ ClN ₃ O _g : C, 61.42; H, 5.73; N, 7.96. Found: C, 61.34; H, 5.82; N, 7.68. MS (FD) m/z 526.9 (MH).

*· » · · • 9 ·· ·* *· • * · » »44 • 944 44 • · ··· » 9 · • · 9 * · »· ·· ·« «

Example 31: 1-(7-oxo-7-(3,4,5-trimethoxybenzylamino)heptyl)isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one

5-(Isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-on-1-yl)heptanoic acid and 3,4,5-trimethoxybenzylamine were converted to the title compound by reaction according to Example 26 to yield 69 mg. (92%). EA calculated for: C ₂₆ H ₃₂ ClN ₃ O _s : C, 62.05; H, 5.95; N, 7.75. Found: C, 61.78; H, 5.82; N, 7.49. MS (FD) m/z 541 (MH).

The compounds of the present invention are inhibitors of MRP1. Therefore, the compounds of the present invention can be used to inhibit any tumor having inherent or acquired resistance to cytostatics caused in part or in whole by MRP1. In other words, treatment of such tumors with an effective amount of a compound of the present invention will cause the tumor to be more sensitive to chemotherapy that is less effective due to the action of MRP1.

Vincristine, epirubicin, daunorubicin, doxorubicin and etoposide are cytotoxic drugs that are substrates of MRP1. See Cole et al., Pharmacological Characterization of Multidrug Resistant MRP-transfected Human Tumor Cells, Cancer Research 54: 5902-5910, 1994. Since MRP1 is ubiquitous in mammals, especially in humans (Nooter, K. et al., Expression of the

Multidrug-Resistance Associated Protein (MRP) Gene in Human Cancers, Clin. Can. Res. 1: 1301-1310 (1995), the efficacy of chemotherapy used to inhibit tumors using any of these agents may be reduced by the action of MRP1. Thus, tumors of the bladder, bone, breast, lung (small cell), testicular, and thyroid, as well as more specific types of tumors such as acute lymphoblastic or myeloblastic leukemia, may be

Wilms' tumor, neuroblastoma, soft tissue sarcoma, Hodgkin's and non-Hodgkin's lymphoma and bronchogenic carcinoma, inhibited by a combination of one or more of the above cytostatics and a compound of the present invention.

The biological activity of the compounds of the present invention was evaluated using an initial screening assay that rapidly and accurately measures the activity of the test compound in inhibiting MRP1 or MDR1. Assays suitable for assessing this ability are well known in the art. See, for example, T. McGrath et al., Biochemical Pharmacology, 38: 3611, 1989; D. Marquardt and M.S. Center, Cancer Research 52: 3157, 1992; D. Marquardt et al., Cancer Research 50: 1426, 1990; and Cole et al., Cancer Research 54: 5902-5910, 1994.

Assay for inhibition of MRP-1-mediated resistance to doxorubicin and MDR-1-mediated resistance to vincristine.

HL60/ADR and HL60/VCR are continuous cell lines that are selected for resistance to doxorubicin and vincristine, respectively, by culturing HL60, a human acute myeloblastic leukemia cell line, in increasing concentrations of doxorubicin and vincristine until highly resistant variants are obtained.

HL60/ADR and HL60/VCR cells were cultured in RPMI 1640 (Gibco) containing 10% fetal bovine serum (FBS) and 250 µg/ml GENTAMYCIN™ (Sigma); cells were harvested; washed with assay medium (same as culture medium); counted and diluted to 2 x 10 ⁵ cells/ml in assay medium. 50 µl aliquots of cells were plated into wells of a 96-well tissue culture plate. One column of each 96-well plate served as a negative control and was

• · • » * · • • · · · « • · · · • · β tf • · · W · · φ • · · · • Λ • · « l> ·· » · · · · • · ·

culture medium containing no cells is placed.

Test compounds and reference compounds were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 5 mM. Samples were diluted to 20 µM in assay medium and 25 µl of each test compound was added to 6 wells. Standards for the assay were run in 4 replicates. 25 µl of 0.4% DMSO was added to 4 wells as a control (solvent). Assay medium was added to all wells to achieve a final volume of 100 µl per well.

The plates were incubated at 37°C for 72 hours in a humidified incubator with an atmosphere of 5% carbon dioxide. Cell viability and vitality were measured by oxidation of a tetrazolium salt under appropriate conditions. The plates were incubated for 3 hours at 37°C. Absorbance was determined at 490 nm using a microplate reader.

The ability of the test compound to inhibit the resistance of HL60/ADR and HL60/VCR cells to doxorubicin was determined by comparing the absorbance of wells containing the test compound and the cytostatic (doxorubicin) with the absorbance of wells containing the cytostatic without the test compound. Controls were used to eliminate background and to verify that the results were not artificial. The results of this assay are expressed as a percentage of cell growth inhibition. The cytostatic alone does not usually inhibit the growth of HL60/ADR and HL60/VCR cells at the concentrations used.

Representative compounds of formula I showed significant activity in inhibiting MRP1 multidrug resistance. Many compounds showed statistically significant increase in activity when combined with a cytostatic compared to the activity of the cytostatic alone. In addition, most of the tested compounds showed

O i » · · · » tftf tf • tf · · · • tf · • tf · ·' • tf • tf • » • tf significant degree of selective inhibition of the HL60/ADR cell line compared to the HL60/VCR cell line.

When administering a cytostatic in the method of the present invention, the dose of the cytostatic may vary. It should be understood that the actual dose of the cytostatic administered will be determined by the physician, depending on relevant circumstances such as the type of disease being treated, the selected route of administration, the type of cytostatic administered, the age, weight and response of the particular patient (mammal) and the severity of the patient's symptoms. The amount of cytostatic administered is determined by the physician and is monitored by the physician. A useful resource for determining the dose of the cytostatic is The Physician's Desk Reference, published by the Medical Economics Company in Montvale, NJ 07645-1742, which is updated annually.

Preferred compositions - and methods of the present invention utilizing such compositions - are those that do not contain a cytostatic agent. It is therefore preferred to administer the compounds of the present invention separately from the cytostatic agent. The cytostatic agents disclosed in this application are commercially available and may be obtained in pre-formulated forms suitable for the methods of the present invention.

The compounds of formula I alone or in combination with a cytostatic agent are usually administered in the form of a pharmaceutical composition. These compositions can be administered by various routes, including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular and intranasal. Such compositions are prepared by methods well known in the art of pharmacy and contain at least one active compound of formula I.

The present invention also includes methods of using pharmaceutical compositions comprising, as an active ingredient,

a compound of formula I, and optionally a cytostatic agent, in association with pharmaceutical carriers. In the manufacture of the compositions of the present invention, the active ingredient is usually mixed with an additive, diluted with an additive or enclosed in a carrier, which may be in the form of a capsule, powder, paper or other container. When the additive serves as a diluent, it may be a solid, semi-solid or liquid material which serves as a vehicle, carrier or medium for the active ingredient. The compositions may thus be in the form of tablets, pills, powders, cachets, powders, wafers, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solid compositions or in a liquid medium), ointments containing, for example, up to 10% by weight of the active ingredient, soft or hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.

In preparing a composition, it may be necessary to grind the active compound to achieve a suitable particle size before mixing with the other ingredients. If the active compound is substantially insoluble, it is usually ground to a particle size of less than 200 mesh. If the active ingredient is water soluble, the particle size is adjusted by grinding to achieve uniform distribution in the composition, for example to a size of 40 mesh.

Examples of suitable excipients are lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methylcellulose. The compositions may further comprise: glidants such as talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl and propyl hydroxybenzoates; sweeteners; and flavoring agents. The compositions of the present invention may be formulated to provide rapid, delayed or • · ·· · · f ·· * • · · · *·*·*· ( · c·

S* · · · · · i*· • ··· · · · I · · · · ♦ · ·· ·· ·· ·· ·· *·· delayed release of the active ingredient after administration to the patient, using procedures well known in the art.

The compositions are usually prepared in dosage unit form, each dosage containing from about 5 to about 100 mg, preferably from about 10 to about 30 mg, of each active ingredient. The term dosage unit refers to a physically discrete unit suitable for single administration to a human subject and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect and suitable pharmaceutical additives.

The compounds of formula I are effective over a wide range of doses. For example, the daily dose is usually in the range of about 0.5 to about 30 mg/kg of body weight. In the treatment of adults, a dose in the range of about 1 to about 15 mg/kg/day, in a single dose or divided into several doses, is preferred. However, the actual amount administered will be determined by the physician, according to relevant circumstances, such as the type of disease being treated, the chosen route of administration, the type of compound administered, the age, weight and response of the particular patient and the severity of the symptoms in the patient, and therefore the stated dose range does not limit the scope of the present invention. In some circumstances, an adequate dose may be lower than the stated range, while in other circumstances higher doses may be used without causing adverse effects, provided that such higher doses are first divided into several smaller doses for administration during the day.

For the preparation of solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical excipient to form a material containing a homogeneous mixture of the compound of the present invention. The term homogeneous, as used herein, means that the active ingredient is uniformly present in the material so that the material can be divided into equally effective dosage units such as tablets, pills and capsules. This solid material is then divided into dosage units of the above type containing from 0.1 to 500 mg of the active ingredient of the present invention.

The tablets or pills of the present invention may be coated or otherwise processed to provide a dosage form having a prolonged action. For example, a tablet or pill may comprise an inner dose component and an outer dose component, the outer component forming a shell for the inner component. The two components may be separated by an enteric layer which prevents disintegration in the stomach and allows the inner component to pass intact into the duodenum, or delay its release. Various materials may be used for such enteric layers or coatings, including many polymeric acids and mixtures of polymeric acids with suitable materials, such as shellac, cetyl alcohol and cellulose acetate.

Novel compositions in liquid forms for oral or injectable administration include aqueous solutions, suitably flavored syrups, aqueous or oily suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutical aqueous or organic solvents, or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharmaceutical excipients as described above. Preferably, the compositions are administered orally or nasally to the respiratory tract to achieve local or systemic effect. Compositions in preferred pharmaceutical solvents may be nebulized using inert gases. Nebulized solutions may be inhaled directly from a nebulizing device, or the nebulizing device may be connected to a face mask, tent, or intermittent positive pressure breathing apparatus. Compositions in the form of solutions, suspensions, or powders may be administered, preferably orally or nasally, from devices delivering the composition in a suitable manner.

The following examples of compositions are illustrative only and do not limit the scope of the invention. The term active ingredient refers to a compound of formula I or a pharmaceutical salt or solvate thereof, optionally with one or more cytostatics.

Example of resource 1

A hard gelatin capsule containing the following ingredients is prepared:

Component

Active ingredient Starch

Magnesium stearate

Quantity (mg/capsule) 30.0

305.0

5.0

The above ingredients are mixed and filled into hard gelatin capsules in an amount of 340 mg.

Example of resource 2

Tablets are prepared using the following ingredients:

Component

Quantity (mg/capsule)

42 -'44 4· * · · 4 4 • 44 4 4 44 44 4 »44444 4 4 44 4 44 444« 4 · 44 44 44 444 4« 4 Active ingredient 25.0 Cellulose, microcrystalline 200.0 Colloidal silicon dioxide 10.0 Stearic acid 5.0

The ingredients are mixed and compressed to form tablets weighing 240 mg.

Example of resource 3

A dry powder inhalation agent is prepared using the following ingredients:

Component Weight (%)

Active ingredient 5

Lactose ⁹⁵

The active ingredient is mixed with lactose and the mixture is added to a dry powder inhalation device.

Example of resource 4

Tablets, each containing 30 mg of the active ingredient, are prepared as follows:

Amount

Ingredients (mg/tablet)

Active ingredient 30.0 mg Starch 45.0 mg Microcrystalline cellulose 35.0 mg Polyvinylpyrrolidone {as a 10% solution in water) 4.0 mg Sodium carboxymethyl starch 4.5 mg Magnesium stearate 0.5 mg Talc 1.0 mg

• · tf * 9 9

9 9

9 9 9.

9 tf

Total

120 mg

The active ingredient, starch and cellulose are sieved through a U.S. No. 20 mesh sieve and mixed thoroughly. The polyvinylpyrrolidone solution is mixed with the obtained powder and pressed through a U.S. No. 16 mesh sieve. The granules thus prepared are dried at 50-60°C and sieved through a U.S. No. 16 mesh sieve. Sodium carboxymethylcellulose, magnesium stearate and talc, which have been previously sieved through a U.S. No. 30 mesh sieve, are then added to the granules and, after mixing, the granules are compressed on a tabletting machine to obtain tablets weighing 120 mg.

Example of resource 5

Capsules, each containing 40 mg of the active ingredient, are prepared as follows:

Folder Quantity (mg/capsule) Active ingredient 40.0 mg Starch 109.0 mg Magnesium stearate 1.0 mg

Total 150.0 mg

The active ingredient, cellulose, starch and magnesium stearate are mixed, sieved through a No. 20 mesh U.S. sieve and filled into hard gelatin capsules in amounts of 150 mg.

Example of resource 6

Suppositories, each containing 25 mg of the active ingredient, are prepared as follows:

* · ·· · · · «· • · · · · · ·· · • <99999 9 · · · 9 * · ···· · 9 • 9 · · · * 9 » Λ 9 9 ·

Ingredient Quantity

Active ingredient 25 mg

Glycerides of saturated fatty acids up to 2000 mg

The active ingredient is sieved through a No. 60 mesh U.S. sieve and suspended in glycerides of saturated fatty acids which have been previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal capacity 2.0 g and allowed to cool.

Example of resource 7

Suspensions, each containing 50 mg of active ingredient per 5.0 ml, are prepared as follows:

Ingredient Quantity

Active ingredient 50.0 mg Xanthan gum 4.0 mg Carboxymethyl cellulose sodium (11%)/ microcrystalline cellulose (89%) 50.0 mg Sucrose 1.75 mg Sodium benzoate 10.0 mg Flavor and coloring q.s. Purified water to 5.0ml

The active ingredient, sucrose and xanthan gum are mixed, sieved through a U.S. No. 10 mesh sieve and then mixed with a previously prepared solution of microcrystalline cellulose and sodium carboxymethylcellulose in water. Sodium benzoate, flavoring and coloring are diluted with a small amount of water and added with constant mixing. Water is then added to obtain a sufficient volume.

444494 4 Φ · 4 4

4 9 4 4 9 4

9 9 4 4 4 4 4 4 4

Example of resource 8

Capsules, each containing 15 mg, as follows:

Component

Active ingredient

Starch

Magnesium stearate

Total

The active ingredient, cellulose, starch, is sieved through a U.S. No. 20 mesh gelatin sieve in an amount of 425 mg.

active ingredients, are prepared

Quantity (mg/capsule) 15.0 mg

407.0 mg 3.0 mg

425.0 mg and magnesium stearate are mixed and filled into hard capsules

Example of resource 9

The intravenous preparation is prepared as follows:

Component

Active ingredient

Isotonic saline solution

Quantity 250.0 mg

1000ml

Example of resource 10

The local remedy is

Component

Active ingredient

Emulsifying wax

Liquid paraffin White soft paraffin is prepared as follows:

Quantity 1-10 g g

grams to 100 grams

4ο •4 44 4« 4 44 • 4'4 4 · 4 4 4 * 44 • 444 44 4 44 • *44444 4 4 · 4 4 • 4 444* 44

44 44 444 44 4

White soft paraffin is heated until melted. Liquid paraffin and emulsifying wax are added with mixing and mixing is continued until dissolved. The active ingredient is added and mixing is continued until a dispersion is formed. The mixture is then cooled to a temperature at which it solidifies.

Example of resource ll

Sublingual or buccal tablets, each containing 10 mg of active ingredient, may be prepared as follows:

Ingredient Quantity per tablet

Active ingredient 10.0 mg Glycerol 210.5 mg Water 143.0 mg Sodium citrate 4.5 mg Polyvinyl alcohol 26.5 mg Polyvinylpyrrolidone 15.5 mg Total 410.0 mg

Glycerol, water, sodium citrate, polyvinyl alcohol and polyvinyl pyrrolidone are mixed together with continuous stirring and maintaining the temperature above about 90°C. Once the polymers have gone into solution, the solution is cooled to about 50-55°C and the active ingredient is slowly mixed in. The homogeneous mixture is poured into molds made of inert material to form a diffusion matrix containing the drug with a thickness of about 2-4 mm. This diffusion matrix is then cut to obtain individual tablets of suitable size.

Another preferred device used in the methods of the present invention is a transdermal device (patch).

Such transdermal patches can be used to allow continuous or discontinuous flow of a compound of the present invention into the body in controlled amounts. The preparation and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, for example, U.S. Patent No. 5,023,252, issued June 11, 1991, which is incorporated herein by reference. Such patches can be prepared for continuous, pulsed, or other selected delivery of pharmaceutical agents.

It is often desirable or necessary to deliver a pharmaceutical agent to the brain, either directly or indirectly. Direct techniques typically require the placement of a drug delivery catheter into the ventricular system of an individual to cross the blood-brain barrier. One such implantable delivery system used to transport biological factors to specific anatomical regions of the body is described in U.S. Patent 5,011,472, issued April 30, 1991, which is incorporated herein by reference.

Indirect techniques, which are usually more advantageous, usually involve the preparation of formulations that allow drug permeation by converting hydrophilic drugs to lipid-soluble drugs or prodrugs. This is usually achieved by blocking hydroxyl, carbonyl, sulfate, and primary amine groups present on the drug, which increases the lipid solubility of the drug and allows its transport across the blood-brain barrier. Alternatively, the penetration of hydrophilic drugs can be enhanced by intra-arterial infusion of hypertonic solutions, which can temporarily open the blood-brain barrier.

Claims (13)

3? ten fc o nároky claims «9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9,999,999 99 99 «9« 99 99 999 A compound of formula I wherein: m is an integer from 1 to 6; R is selected from the following groups: COR ¹ , amino, ΝΗ-Pg or NHCOR ² ; R ¹ is selected from the following groups: hydroxy, C 1 -C 8 alkoxy or NR ³ R ⁴ ; Pg is an amino protecting group; R ² is selected from the following groups: C, -C _e alkyl substituted with CC alkyl, aryl, substituted aryl, _{(CH2) n} heterocycle, (CH _z) -subs t ituovaný heterocycle; R ³ is independently hydrogen or C 1 -C 6 alkyl; R ⁴ is selected from the following groups: C 1 -C 4 alkyl, norboran-2-yl, aryl, substituted aryl, CH ₂ CH (CH ₃ ) phenyl, (CH ₂ ) _n -heterocycle, (CH ₂ ) substituted heterocycle; and n is 0, 1 or 2; or a pharmaceutically acceptable salt or solvate thereof. A compound according to claim 1, wherein m is an integer from 2 to 6 and R is selected from the following groups: t-butyloxycarbonylamino, trifluoroacetylamino, 3,4,5-trimethoxybenzylamino, 3,4,5-trimethoxyanilinecarboxy, 3,4,5-trimethoxybenzylaminylcarboxy; or a pharmaceutically acceptable salt or solvate thereof. 3. A method of inhibiting MRP1 in a mammal comprising administering to a mammal in need of such treatment an effective amount of a compound of Formula I: where: m is an integer from 1 to 6; R is selected from the following groups: COR ^X , amino, NH-Pg or NHCOR ² ; R ³ - is selected from the following groups: hydroxy, C ₁ -C ₆ alkoxy or NR ³ R ⁴ ; Pg is an amino protecting group; R ² is selected from the following groups: C _x -C _e alkyl substituted with CC alkyl, aryl, substituted aryl, _{(CH2) n} heterocycle, (CH ₂₎ -substituted heterocycle; R ³ is independently hydrogen _or a Cl-C ~ _alkyl; R ⁴ is selected from the following groups: _x C ~ C _alkyl, norbornane-2-yl, aryl, substituted aryl, CH ₂ CH (CH ₃₎ phenyl, (CH) ^ - het, (CH ₂₎ substituted heterocycle; and n is 0, 1 or 2; or a pharmaceutically acceptable salt or solvate thereof. 4. The method of claim 3 wherein the mammal is a human. A method according to claim 4, wherein the compound of formula I is a compound wherein m is an integer from 2 to 6 and R is selected from the following groups: t-butyloxycarbonylamino, trifluoroacetylamino, 3,4, 5-trimethoxybenzylamino, 3,4,5-trimethoxyanilinecarboxy, 3,4,5-trimethoxybenzylaminylcarboxy; or a pharmaceutically acceptable salt or solvate thereof. 6. A method of inhibiting resistant or resistant tumors in a mammal comprising administering to a mammal in need of such treatment an effective amount of a compound of Formula I: where: m is an integer from 1 to 6; R is selected from the following groups: COR ^X , amino, NH-Pg or NHCOR ² ; ^R1 is selected from the following groups: hydroxy, C _x -C _alkoxy or NR ³ R ^4; Pg is an amino protecting group; R ² is selected from the following groups: C ₁ -C ₆ alkyl, substituted C ₁ -C ₄ alkyl, aryl, substituted aryl, (CH ₂ ) heterocycle, (CH ₂ ) _n -substituted heterocycle; R ³ is independently hydrogen or C ₁ -C ₆ alkyl; R ⁴ is selected from the following groups: C 1 -C 4 alkyl, norboran-2-yl, aryl, substituted aryl, CH ₂ CH (CH ₃ ) phenyl, (CH) -heterocycle, (CH) -substituted heterocycle; and n is 0, 1 or 2; or a pharmaceutically acceptable salt or solvate thereof; 5 η * · 9 · 9 9 99 · ·· -L ···· · · 9 9 9 9,999,999 9 9,99 9 9 9 9 9 9 9 99 99 99 999 99 999 in combination with an effective amount of one or more cytostatic agents. 7. The method of claim 1 wherein the mammal is a human. The method of claim 7, wherein the compound of formula I is a compound wherein m is an integer from 2 to 6 and R is selected from the following groups: t-butyloxycarbonylamino, trifluoroacetylamino, 3,4, 5-trimethoxybenzylamino, 3,4,5-trimethoxyanilinecarboxy, 3,4,5-trimethoxybenzylaminylcarboxy; or a pharmaceutically acceptable salt or solvate thereof. The method of claim 7, wherein the cytostatic is selected from the group: doxorubicin, daunorubicin, epirubicin, vincristine, and etoposide. The method of claim 7, wherein the tumor is selected from Wilms' tumor, bladder, bone, breast, lung (small cell), testes or thyroid, or is associated with acute lymphoblastic and myeloblastic leukemia, neuroblastoma, soft tissue sarcoma, Hodgkin's and non-Hodgkin's lymphoma or bronchogenic carcinoma. 11. A pharmaceutical composition comprising a compound of formula I: £ 0 Φ Φ - - - - - - - - - - 44 44 44 44 44 44 44 44 44 44 44 44 44 44 44 44 444 44 4 where: m is an integer from 1 to 6; R is selected from the following groups: COR ¹ , amino, NH-Pg or NHCOR ² ; R ¹ is selected from the following groups: hydroxy, C _x C _g alkoxy or -NR ³ R ^4; Pg is an amino protecting group; R ² is selected from the following groups: C _x -C _alkyl, substituted C, -C ₄ alkyl, aryl, substituted aryl, _(CH2) ^ - het, (CH) -substituted heterocycle; R ³ is independently hydrogen or _C -C _alkyl; R ⁴ is selected from the following groups: _C, -C _g alkyl, hydroxy, norbornane-2-yl, aryl, substituted aryl, CH ₂ CH (CH ₃₎ phenyl, (CH) _n heterocycle, (CH ₂₎ substituted heterocycle; and n is 0, 1 or 2; or a pharmaceutically acceptable salt or solvate thereof; in combination with one or more pharmaceutical carriers, diluents or excipients. 12. A pharmaceutical composition comprising: (a) a compound of formula I wherein: m is an integer from 1 to 6; R is selected from the following groups: COR ¹ , amino, NH-Pg or NHCOR ² ; 9 9 9 9 9 9 99 ·· ··· · · · ······ R ¹ is selected from the following groups: hydroxy, C 1 -C 6 alkoxy or NR ³ R ⁴ ; Pg is an amino protecting group; R ² is selected from the following groups: C 1 -C 6 alkyl, substituted C 1 -C 6 alkyl, aryl, substituted aryl, (CH ₂ ) 4 -heterocycle, (CH ₂ ) substituted heterocycle; R ³ is independently hydrogen or C 1-6 alkyl; R ⁴ is selected from the following groups: C 1 -C 6 alkyl, norboran-2-yl, aryl, substituted aryl, CH ₂ CH (CH ₃ ) phenyl, (CH) _n -heterocycle, (CH ₂ ) substituted heterocycle; and n is 0, 1 or 2; or a pharmaceutically acceptable salt or solvate thereof; (b) one or more cytostatic agents; and (c) one or more pharmaceutical carriers, diluents or excipients. The composition of claim 12, wherein the cytostatic is selected from the group: doxorubicin, daunorubicin, epirubicin, vincristine, and etoposide.