NOVEL COMBINATION OF BENZOQUINAZOLINE ANTIFOLATES AND PROTECTING AGENTS

Abstract: The use of a protecting agent, for example a folate derivative such as folic acid or leucovorin, in combination with a non-competitive folic acid analogue, for example benzoquinazoline derivatives, for use in reducing the side effects associated with the administration of such non-competitive folic acid analogues, and pharmaceutical formulations comprising such combinations are disclosed.

Description: Novel combination of beπzoqulnazol i ne antifolates and protecting agents. The present invention relates to novel combinations of non-competitive folic acid analogues with protecting agents, and to methods of treatment using these combinations. European Patent Application 0505640 provides a method for improving the therapeutic utility of GAR-transformylase inhibitors and other antifolates by co-administering a folate binding protein binding agent to the host undergoing treatment. Thymidvlate synthase is an enzyme catalysing the terminal step in the de novo synthesis of thymidvlate required for DNA synthesis. It has been postulated that inhibitors of this enzyme may be expected to have anti-tumour activity and it has been reported (Jones ej a], J. Med. Chem. 1986, 22, 468) that the in-vivo antitumour activity of N^-propargyl-5,8- dideazafolic acid arises solelv from its inhibitory effect on this enzyme. PCT publication W or a salt thereof, wherein the dotted line represents a single or double bond, R- is Cμ4 alkyl or amino optionally substituted by a C 1.4 alkyl, Cj_5 alkanoyl or benzyl group; R-. RX, R^ and R*^ are the same or different and each is selected from hydrogen, phenyl, halo, nitro. a group S(0) n R^ wherein n is the integer O. 1 or 2 and R° is halo or is C 1.4 alkyl or a group NR9R ** 0 wherein R^ and R- 0 are both hydrogen, a group NR*- - R-- wherein R^ - and R -- are the same or different and each is hydrogen or C j_4 alkyl. a group OR • ' wherein R -* J is hydrogen or C ' 1.4 alkyl optionally substituted by halo; a C ] _4 aliphatic group optionally substituted by a group OR -^ or NR ^R15 wherein R-4 and R** * 5 are the same or different and each is hydrogen or C 1.4 alkyl; or two of R 2 to R* 5 are linked together to form a benzo group. or one of R 2 to R 5 is a group -X-Y-R 16 wherein X is Cl-b, NR 1 7 . CO or S(0) m and m is O, 1 or 2 and R*- 7 is hydrogen or a C1.4 aliphatic group and Y is C b. O, NR** ' * 7 ' or S(0) m ' wherein m' is 0,1 or 2 and R*- 7 ' is hydrogen or a C 1.4 aliphatic group provided that X and Y are only the same when each is CH2. or -X-Y- is a group -0-, -NR^ 7 -, -CH=CH- or -N=N- wherein R- 7 is as hereinbefore defined, R- -- is a C 1.4 aliphatic group or a 5- or 6- membered aromatic ring optionally substituted by a group R- -* at a position at least one carbon atom removed from that linked to Y, the 5- or 6- membered ring being optionally further substituted by a halo atom; and R^ is halo, C \ .4 alkoxy, nitro, nitrile, C1.4 alkyl optionally substituted by halo, halo or a group COR-* --- 1 wherein Rl9 is hydroxy, C \ .4 alkoxy or C 1.5 alkyl optionally substituted by one or two carboxyl groups or l-12 esters thereof or R-9 is a group NR-^^R-** wherein R 2 ^ and R- - are the same or different and each is hydrogen or C 1.4 alkyl optionally substituted by hydroxy or R- ~- is an amino acid group or an ester thereof in which the first nitrogen atom of the amino acid group may be linked to the 5- or 6- membered aromatic ring to form a further 5- or 6- membered heterocyclic ring or R- -- is an C2-3 alkylene group linked to the 5- or 6- membered aromatic ring to form a further 5- or 6- membered ring; R6 and R 7 are the same or different and each is hydrogen. C 1.4 alkyl optionally substituted by hydroxy or C 1.4 alkoxy or together form a benzo group; provided that at least one of R- to R 7 is other than hydrogen and that R4 is not methoxy when R- is hydroxy or methyl is an inhibitor of the enzyme thymidvlate synthase and has anti-tumour activity. By the term halo is meant fluoro. bromo, chloro and iodo. By the term C j .4 aliphatic group is meant a C 1.4 alkyl. alkenyi. or alkynyl group. By the term amino acid group is meant naturally occurring amino acids. Preferred amino acid groups include glycine, glutamic acid and polyglutamic acid groups. When the amino acid group is linked to the 5- or 6- membered aromatic ring, this is via a carbon atom of the aromatic ring adjacent to carbon to which COR 19 is attached. Preferably the dotted line is a double bond. Suitable substituents for the aromatic ring R-6 include halo, C 1.4 alkyl and C1.4 alkoxy each optionally substituted by one to five halo atoms. Most suitably there are one or two substituents selected from fluoro, chloro. methyl, trifluoromethyl and methoxy, and preferably fluoro, or no substituents on the aromatic ring. In one preferred embodiment, -X- Y-R--6 is a group wherein R-° is as hereinbefore defined and preferably a group COR**" as hereinbefore defined and R 22 is hydrogen or fluoro. In a further preferred embodiment X-Y-R**" is a group wherein foNR**^-*-* i -s a* glu-tamic or polyglutamicr acid group and Z is CH2, S or O. Suitably, R^ is an amino group optionally substituted by one or two methyl or ethyl groups or R- is a methyl or ethyl group. Preferably R^ is an amino or methyl group. Suitably, at most only three, and preferably at most only two, of R 2 to R-5 are other than hydrogen and each is independently selected from hydrogen, halo, hydroxy. nitro. C j_3 alkyl optionally substituted by hydroxy or Cj.2 alkoxy, C j .3 alkoxy, amino optionally substituted by one or two methyl or ethyl groups, or a group S(0) n R-* 3 wherein n is 0, 1 or 2 and R 2 *-* is a C1.4 alkyl group or an amino group optionally substituted by one or two methyl or ethyl groups, or one of R 2 to R-- is a group -X-Y-R 2 ^ where R 24 is a group wherein R- °, R- -*--, R- 2 and Z are as hereinbefore defined. In one preferred embodiment R * • •• * • is nitro or a group wherein R*-- 3 , R-" and R 27 are the same or different and each is hydrogen or a C 1.4 alkyl group and t is an integer from 0 to 6. Preferably R-* 5 , R-° and R 27 are hydrogen and t is O. Preferably Z is CFb or S. Preferably one of R 2 to is a group -X- Y-R 24 as hereinbefore defined. Preferably RX is a group -X-Y-R 24 . Suitably R^ and R 7 are the same or different and each is hydrogen, methyl, ethyl or methyl substituted by bromo, hydroxy or methoxy. Preferably R 7 is hydrogen and R° is methyl. Preferably -X-Y- is a group -SO2NRI 7 - or CPfoNRl 7 wherein R^ 7 is as hereinbefore defined. Suitably R^ 7 is hydrogen or a C j _4 alkyl or alkenyl group and preferably R-* 7 is hydrogen or methvl. A further group of c or a salt thereof, wherein R - , R°, R 7 and the dotted line are as hereinbefore defined and R-° to R J are the same or different and each is selected from hydrogen, halo, nitro. a group S(0) n R 8 , a group NR I R 2 , a group OR- 3 , or a C ] _4 aliphatic group optionally substituted by a group OR 14 or NR 14 R 15 wherein R 8 . R n . R 12 , R 1 3 , R 14 and R 1 - 5 are as hereinbefore defined, provided that R 28 to RX - are not all hydrogen and that R 0 is not methoxy wherein R is hydroxy or methyl. A preferred group of compounds of the formula (I) is that of the formula (III): or a salt thereof, wherein R**, R^ and R 7 are as hereinbefore defined and R 32 to R ^ are the same or different and one is a group X-Y-RI6 and the others are the same or different and each is selected from hydrogen, halo, nitro. a group S(O) n R 8 , a group NRU RI 2 . a group OR!*-- or a C1- ..4 aliphatic group optionally substituted by a group OR 4 or NR 4 RI * -\ wherein X.Y,R 8 .R-- ^R 12 ^ 13 ^ 14 ^ 1 5 and R 16 are as hereinbefore defined. Preferably R 33 is a group X-Y-R " as hereinbefore defined. Preferred compounds of the formula (I) include: 3-Amino-9-bromobenzo[f]quinazolin- 1 (2H)-one 3-Amino-9-ethynylbenzo[f]quinazolin-l(2H)-one N-(4-((3-Amino- 1 ,2,5.6-tetrahydro- 1 -oxobenzo[f]quinazolin-9-yl)sulfonamido)benzoyl)-L- glutamic acid N-(4-(( 1 ,2,5,6-tetrahydro-3-methyl- 1 -oxobenzo[f]quinazolin-9-yl)-sulfonamido)benzoyl)-L- glutamic acid N-(4-(( 1 ,2-Dihydro-3-methyl- 1 -oxobenzo[f]quinazolin-9-yl)sulfonamido)benzoyl)-L- glutamic acid N-(4-((( 1 ,2-Dihydro-3-methyl- 1 -oxobenzo[f]quinazolin-9-yl)methyl) amino)-2-fluorobenzoyl)-L-glutamic acid N-(4((( 1 ,2-Dihydro-3-methyl- 1 -oxobenzo[f]quinazolin-9-yl)methyl) amino)benzoyl)-L-glumatic acid (S)-2-(5-((( 1 ,2-Dihydro-3-methyl- 1 -oxobenzo[f]quinazolin-9-yl)methyl)amino- 1 -oxo-2- isoindolinyl)glutaric acid 9-((4-Acetylanilino)methyl)-3-methylbenzo[f]quinazolin- 1 (2H)-one 3-Methyl-9-((4-nitroanilino)methyl)benzo[f]quinazolin- 1 (2H)-one N-(4-(((3 -Amino- 1 ,2-dihydro- 1 -oxobenzo[ ]quinazolin-9-yl)methyl) amino)benzoyl)-L-glutamic acid 3-Amino-9-((4-nitroanilino)methyl)benzo[f]quinazolin- 1 (2H)-one 9-((4-Acetylanilino)methyl)-3-aminobenzo[f quinazolin-l(2H)-one (RS)-2-(2-(4-((( 1 ,2-Dihydro-3-methyl- 1 -oxobenzo[ ]quinazolin-9-yl)methyl) amino )phenyl)-2-oxoethyI)glutaric acid Ethyl-4-(4-((( 1 ,2-dihydro-3-methyl- 1 -oxobenzo[f]quinazolin-9-yl) methyl)amino)phenyl)-4-oxobutyrate 4-(4-((( 1 ,2-Dihydro-3-methyl- 1 -oxobenzo[f]quinazolin-9-yl)methyl) amino)phenyl)-4-oxobutyric acid N-(4-((( 1 ,2-Dihydro-3-methyl- 1 -oxobenzo[fjquinazolin-9-yl)methyl) amino)-2-fluorobenzoyl)glycine Ethyl N-(4-(((l,2-Dihydro-3-methyl-l-oxobenzo[f)quinazolin-9-yl) methyl)amino)-2-fluorobenzoyl)glycinate Certain compounds of the formula (I) contain asymmetric carbon atoms and are. therefore, capable of existing as optical isomers. The individual isomers and mixtures of these are included within the scope of the present invention. It has now been found that the compounds of formula (I) can be used in combination with a protecting agent described hereinafter to prevent or decrease unwanted side effects of these compounds. The present invention relates to the administration of the compounds of formula (I) in combination with a protecting agent which blocks the intestinal toxicity of the compounds of formula (I) without blocking their activity. Suitable protecting agents are folates. for example, folic acid, tetrahydrofolate. 5 -formyltetrahydro folate (leucovorin) or 5- methyltetrahydrofolate. Other protecting agents include thymine or thymidine. The protecting agent may be administered as an oral formulation. It is within the scope of the invention to administer the protecting agent before, during or after the administration of a compound of formula (I). Preferably, the protecting agent will be given slightly before administration of a compound of formula (I), i.e. between 15 minutes and one hour before the compound of formula (I). The present invention also relates to combination therapy comprising the administration of non-competitive folic acid analogues in combination with protecting agents as defined above. This allows the use of the non-competitive folic acid analogues at higher doeses than could be used in the absence of the protecting agent as defined above, and therefore higher, more potent doses can be achieved while minimizing host toxicity. The presence of the protecting agent reduces the toxicity of the non-competitive folic acid analogues at the doses utilized in the absence of the protecting agent (i.e. less toxicity at the same dose in the presence of a protecting agent). Non-competitive folic acid analogues are defined as structural analogues of folic acid (including but not limited to the benzoquinazoline compounds of formula I, e.g., compounds of example 1 or 2) expressing classical non-competitive inhibition kinetics (vs. the folate substrate) on the target enzyme (e.g., thymidvlate synthase. glycineamide ribonucleotide transformylase or dihydrofolate reductase). Alternatively, the non- competitive inhibitors of the present invention are defined as structural analogues of folic acid (as defined above) which retain their tumour cell kill in the presence of the folate protecting agents defined above, on the target enzyme. The non-competitive inhibitor may been given as an intravenous or intraperitoneal bolus or infusion and the protecting agent may be given as an intravenous or intraperitoneal bolus infusion or as an oral formualtion. For a definition of classical non-competitive inhibiton see I.H. Segal ( 1975) Enzyme kinetics. John Wiley and Sons, which is incorporated herein by reference. More specifically, protecting agents selected from folic acid, tetrahydrofolate, 5- formyltetrahydro folate (leucovorin) or 5-methyltetrahydro folate can block the intestinal toxicity of compounds of formula (I) without blocking their anti-tumor activity. Preferably, the non-competitive, folic acid analogue (i.e.. non-competive inhibitor) is a compound of formula (I) and the protecting agent is folic acid or 5-formyltetrahydrofolate. More preferably, the non-competitive, folic acid analogue (non-competitive inhibitor) is a compounds selected from (S)-2-(5-((( l,2-dihydro-3-methyl-l-oxobenzo(f)quinazolin-9- yl)methyl)amino)- 1 -oxo-2-isoindolinyl)glutaric acid. N-(4-((( 1 ,2-dihydro-3-methyl- 1 - oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutamic acid, or N-(4-((l, 2, 5, 6-tetrahydro-3-methyl- 1 -oxobenzo( f)quinazolin-9-yl)sulfonamido)benzoyl )-L-glutamic acid and the protecting agent which is used in combination is folic acid or 5- formyltetrahydrofolate. Most preferbly, the compound (S)-2-(5-(((l,2-dihydro-3-methyl-l- oxoxbenxo(f)quinazolin-9-yl)methyl)amino)-l-oxo-2-isoindolinyl)glutaric acid is utilized in combination with folic acid or 5-formyltetrahydrofolate. The salts of the compounds of the present invention can also be used in combination with the protecting agent or salts thereof. Suitable salts of the compounds of the present invention or of the protecting agents may comprise acid addition salts derived from an amino group or anionic species derived from a compound of formula (I) or protecting agent, for example when this is substituted by a carboxy group, and a cation. In both types of salts, the therapeutic activity resides in the moiety derived from the compounds defined herein and the identity of the other component is of less importance although for therapeutic and prophylactic purposes it is. preferably, pharmaceutically acceptable to the patient. Examples of pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric. nitric and sulphuric acids, and organic acids, such as tartaric. acetic, trifluoroacetic, citric, malic, lactic, fumaric, benzoic, glycollic, gluconic. succinic and methanesulphonic and arylsulphonic. for example p-toluenesulphonic, acids. Examples of salts comprising an anionic species derived from a compound of formula (I) and a cation include ammonium salts, alkali metal salts, such as sodium and potassium salts, alkaline earth salts, such as magnesium and calcium salts, and salts formed with organic bases, for example, amino salts derived from mono-, di- or tri-(lower alkyl) or (lower alkanoDamines. such as triethanolamine and diethylamino- ethylamine. and salts with heterocyclic amines such as piperidine, pyridine. piperazine and morpholine. The pharmaceutically acceptable salts together with the salts which are not thus acceptable have utility in the isolation and/or the purification of the compounds of the invention, and the pharmaceutically unacceptable salts are also useful in being convertible to the pharmaceutically acceptable salts by techniques well known in the art. The route by which the compound or salt of the present invention is administered to the animal may be oral, topical, parenteral (including subcutaneous, intradermal. intramuscular, intravenous or rectal). If the compound or salt is presented in the form of a pharmaceutical formulation, whch is preferred, then the actual formulation employed will of course depend on the route of administration elected by the physician or veterinarian. For example, if oral administration is preferred, then the pharmaceutical formualtion employed is. preferably, one which is suitable for such a route. A therapeutically effective amount of a compound or salt of the present invention will depend upon a number of factors including, for example, the age and weight of the animal, the precise condition requiring treatment and its severity, the nature of the formulation, and the route of administration, and will ultimately be at the discretion of the attendant physician or veterinarian. However, an effective amount of a non-competitive folic acid analogue of the present invention (as described hereinbefore) to be used in combination with a protecting agent (as described hereinbefore) for the treatment of neoplastic growth, for example colon or breast carcinoma will generally be in the range of 0.1 to 100 mg/kg body weight of recipient (mammal) per day and more usually in the range of 1 to 50 mg/kg body weight per day. Thus, for a 70 kg adult mammal, the actual amount per day would usually be from 70 to 3500 mg and this amount may be given in a single dose per day or more usually in a number (such as two. three, four, five or six) of sub-doses per day such that the total daily dose is the same. An effective amount of a salt of the present invention may be determined as a proportion of the effective amount of the compound per ≤e. An effective amount of a compound of the present invention for the treatment of disorders of the immune system (e.g. rheumatoid arthritis and tissue rejection) and related diseases such as psoriasis, will generally be in the range of 0.5-10 mg/kg body weight of recipient (mammal) per day. Thus for a 70kg adult human, the actual amount per day would usually be from about 5-700 mg per day and this amount may be given in a single dose per day or more usually dosing would be intermittent e.g. 12 hourly intervals or weekly. An effective amount of a salt of the present invention may be determined as a proportion of the effective amount of the compound p_≥r ≤e. An effecive amount of a compound of the present invention for the treatment of bacterial and fungal infections is in the range of 0.1 - 100 mg/kg bodyweight of recipient (mammal) per day and preferably in the range of 1 -50 mg/kg body weight per day. Thus, for a 70 kg adult mammal, the actual amount per day is from 70-3500 mg and this amount may be given in a single dose per day or more preferably in a number (such as two, three, four, five or six) of sub-doses per day such that the total daily dose is the same. An effective amount of a salt of the present invention may be determined as a proportion of the effective amount of the compound per se. An effective amount of a protecting agent (e.g., folic acid) of the present invention for use in combination with a non-competitive, folic acid analogue (including but not limited to the compounds of the present invention as described herein) is in the range of 1-300 mg/kg bodyweight of recipient (mammal) per day and preferably in the range of 5-50 mg/kg body weight per day. Thus, for a 70 kg adult mammal, the actual amount per day is from 350- 3500 mg and this amount may be given in a single dose per day or more preferably in a number (such as two, three, four, five or six) of sub-doses per day such that the total daily dose is the same. The treatment of neoplastic growth with a compound of the present invention may at times require the administration to the animal of an antidote or rescue agent, e.g. thymidine. The compounds, pel se, of the present invention which can be used in combination with a protecting agent as described herein include, but are not limited to, those disclosed in PCT publication WO/91/19700. The protecting agents of the present invention are readily available and well known to those skilled in the art. Whilst it is possible for the compounds or salts of the present invention to be administered as the raw chemical, it is preferred to present them in the form of a pharmaceutical formulation. Accordingly, the present invention furthur provides a pharmaceutical formulation, for medicinal application, which comprises a compound of the present invention and a protecting agent or pharmaceutically acceptable salts thereof, as hereinbefore defined, in combination with one or more pharmaceutically acceptable carriers therefor, and optionally one or other therapeutic agredients. The pharmaceutical formulation may optionally contain other therapeutic agents that may usefully be employed in conjunction with the compound or salt of the present invention, for example a pyrimidine nucleoside transport inhibitor that is capable of enhancing the antineoplastic activity of the compounds and salts of the presents invention. The expression "pharmaceutically acceptable" as used herein in relation to the carrier is used in the sense of being compatible with the compound or salt of the invention employed in the formulation and with any other therapeutic agent that may be present, and not being detrimental to the recipient thereof. The carrier itself may constitute one or more excipients conventionally used in the art of pharmacy that enable the compound or salt of the present invention and any other therapeutic agent that may be present, to be formulated as a pharmaceutical formulation. The pharmaceutical formulations of the present invention include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular and intravenous) and rectal administration although the most suitable route will probably depend upon, for example, the condition and identity of the recipient. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods will known in the art of pharmacy. All methods include the step of bringing into association the active ingredient, i.e., the compound or salt and protecting agent of the present invention, with the carrier. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with a liquid carrier or, a finely divided solid carrier or both, and then, if necessary, forming the associated mixture into the desired formulation. The pharmaceutical formulations of the present invention suitable for oral administration may be presented as discrete units, such as a capsule, cachet, tablet, or lozenge, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid such as a syrup, elixir or a draught, or as an oil-in water liquid emulsion or a water-in-oil liquid emulsion. The formulation may also be a bolus, electuary or paste. Generally, a tablet is the most convenient pharmaceutical formulation suitable for oral administration. A tablet may be made by compressing or moulding the active ingredient with the pharmaceutically acceptable carrier. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form, such as a powder or granules, in admixture with, for example, a binding agent, an inert diluent, a lubricating agent, a disintegrating agent and/or a surface active agent. Moulded tablets may be prepared by moulding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient. The pharmaceutical formulations of the present invention suitable for parenteral administration include aqueous and non-aqeuous sterile injection solutions which may contain, for example, an anti-oxidant, a buffer, a bacteriostat and a solution which renders the composition isotonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may contain, for example, a suspending agent and a thickening agent. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. The pharmaceutical formulations of the present invention suitable for rectal administration may be presented as a suppository containing, for example, cocoa butter and polyethylene glycol. Compounds and salts of formula (I) have anti-neoplastic activity in the human colon SW480 adenocarcinoma cell culture cytotoxicity tests in which representative compounds of the present invention have been shown to be active, and in human breast MCF7 adenocarcinoma cell culture. It has thus been established that compounds of the present invention are able to inhibit neoplastic growth. Therefore, compounds and salts of the present invention are of use in medicine and in particular in the treatment of neoplastic growth, including solid tumours such as melanoma, breast and colon tumours in mammals. Accordingly, the present invention yet further provides a method for the treatment of susceptible malignant tumours and leukemia in a animal, e.g.. a mammal, which comprises administering to the animal a therapeutically effective amount of a compound or salt of the present invention in combination with a protecting agent. In the alternative, there is also provided a compound or salt of the present invention in combination with protecting agent. In the alternative, there is also provided a compound or salt of the present invention in combination with a protecting agent for use in medicine and in particular for use in the treatment of a neoplastic growth, e.g.. malignant tumours. The present invention also provides the use of a compound of formula (I) or a salt therof in combination with a protecting agerit for the maufacture of a medicament for the treatment of neoplastic growth. The animal requiring treatment with a compound or salt of the present invention in combination with a protecting agent is usually a mammal, such as a human being. The following examples illustrate the preparation and pharmacological properties of representative compounds which are useful in the present invention and which demonstrate the invention. These examples are illustrations only and should not be construed as limiting or narrowing the scope of the invention. Example 1 (SV2-(5-(((1.2-Dihvdro-3-methyl-l -oxobenzo[f]quinazolin-9-yl)methyl)- amino)- 1 -oxo-2-isoindolinyl)glutaric acid (Compound A) A. Diethyl (S)-2-(4-nitrophthalimido)glutarate Diisopropylethylamine (24 ml, 0.138 mole) (Aldrich) was added to a suspension of 4-nitrophthalic anhydride (25 g, 0.13 mole) (Tokyo Kasei) and L-glutamic acid diethyl ester hydrochloride (35 g, 0.146 mole) (Aldrich) in toluene (130 ml). The reaction mixture was stirred at reflux utilizing a Dean-Stark trap for 2.5 hours. After cooling, the solution was diluted with diethyl ether (300 ml), washed with water (75 ml), saturated NaHCO3 solution (50 ml), dried (MgSO4), and concentrated in vacuo at 70°C to give diethyl (S)-2-(4-nitrophthalimido) glutarate as an oil that solidified to a white solid on standing (35.8 g). M.P. = 65.5-66.5°C. B. Diethvl (S)-2-(4-aminophthalimido)glutarate A suspension of diethyl (S)-2-(4-nitrophthalimido)glutarate (35.6 g, 94.1 mmol) and 10% palladium on carbon (0.5 g) (Aldrich) in ethanol (200 ml) was shaken under a hydrogen atmosphere (40-50 psi) for 26 hours. The solution was filtered through celite and concentrated in vacuo. The residue was purified by chromatography on silica gel (250 g) eluting with diethyl etheπhexane (4: 1) to give diethyl (S)-2-(4- aminophthalimido)glutarate as a viscous yellow oil (29. lg). C. Diethyl (S V2-(5-amino- 1 -oxo-2-isoindolinvnglutarate A solution of diethyl (S)-2-(4-aminophthalimido)glutarate ( 10.5 g, 30.2 mmol) in ethanol ( 150 ml) was cooled in an acetonitrile/C02 bath. Concentrated HC 1 (25 ml) was added followed by 30 mesh granular Zn ( 10.5g. 0.161 mole) (Fisher) when the internal temperature has reached -40°C. The reaction mixure was stirred 1.5 hours at this temperature and a further 1 hour at - 10°C. The excess of Zn was filtered from the solution, 10% palladium on carbon (l .Og) was added, and the solution shaken under hydrogen at (30-50 psi) overnight. The catalyst was removed by filtration through celite and the filtrate concentrated in vacuo. The residue was absorbed onto silica gel ( 15 g) and purified by chromatography on silica gel (440 g) eluting with ethyl acetate:methylene chloride (l :14g). D 9-Bromomethvl-3-methvlbenzo flquinazolin-l(2HVone To a hot solution of 3, 9-dimethylbenzo[f]quinazolin-l(2H)-one (4.00 g, 17.9 mmol) in benzene (2000 ml) under nitrogen was added N-bromo-succinimide (4.00 g, 22.5 mmol). The reaction mixture was stirred just below reflux for 30 minutes, then at a gentle reflux for 30 minutes. The resulting suspension was allowed to cool for 2 hours, the solid filtered and dried at 70°C under reduced pressure to give 9- bromomethyl-3-methylbenzo[fjquinazolin-l(2H)-one (4.32 g, 83% purity by NMR). E. Diethyl (SV2-(5-(((1.2-dihvdro-3-methvl-l -oxobenzo[f]quinazolin-9-yl . methvnamino .-1 -oxo-2-isoindolinvnglutarate A solution of crude 9-bromomethyl-3-methylbenzo[f]quinazolin-l(2H)-one (4.32 g), (S)-diethyl 2-(5-amino-l-oxo-2-isoindolinyl) glutarate (4.0g, 12 mmol), and NaHCO3 (2.0 g, 24 mmol) in DMF (30 ml) was stirred under nitrogen at 105°C for 1.5 hours. After cooling, acetic acid (1 ml, 17 mmol) was added, the reaction mixture transferred to a larger round bottom flask with ethanol. and then concentrated in vacuo onto silica gel (30 g). The absorbed material was purified by chromatography on silica gel eluting with methano methylene chloride (1 :24) and then precipitation of the solid from methylene chloride (~-20ml) with ethyl acetate (~~ 5ml) and methanol (~ 5 ml). The white solid was filtered under nitrogen and dried under high vacuum to give diethyl (S)-2-(5-(((l ,2-dihydro-3-methyl-l- oxobenzo[f]-quinazolin-9-yI)-methyl)amino)- l-oxo-2-isoindolinyl)glutarate (3.27g). F (S)-2-(5-((( 1.2-Dihvdro-3-methyl- 1 -oxobenzo[f]quinazolin-9-v])- methyl .amino')- 1 -oxo-2-isoindolinyl)glutaric acid A solution of diethyl (S)-2-(5-((( 1.2-dihydro-3-methyl-l-oxo-benzo-[f]quinazolin-9- yl)methyl)amino)-l-oxo-2-isoindolinyl)glutarate (3.20g. 5.75 mmol) in 0.2 N NaOH (140 ml) was stirred under nitrogen for 3 hours at room temperature. The solution was then slowly adjusted to pH3 with 1 N HC 1 and the resulting suspension allowed to stir briefly. The white solid was filtered under nitrogen, washed with water, and dried under high vacuum to give (S)-2-(5-(((l,2-dihydro-3-methyl-l- oxobenzo[_]quinazolin-9-y ljmethy l)amino)- 1 -oxo-2-isoindolinyl)glutaric acid (2.85). } H NMR (DMSO-d 6 /D 2 0, 300 MHz) δ: 1.86-2.34 (m, 4H, glu CH 2 , CH 2 ), 2.44 (s. 3H, CH3), 4.25 (very strongly coupled AB pair, 2H, glu NCH 2 Ar), 4.58 (s, 2H. C9-CH ), 4.67-4.75 (m. 1H. glu CH), 6.69-6.77 (m. 2H. Ar), 7.37 (d, J = 8 Hz, 1H Ar), 7.59 (d, J = 9 Hz, 1H, Ar) 7.64 (dd. J = 8.2 Hz. 1H, Ar). 8.01 (d, J= 8 Hz, 1H, Ar), 8.22 (d, J = 9 Hz, 1H, Ar), 9.85 (s, 1H, Ar). Anal Calculated for C27H 2 4N O 6 • 1.6H 2 0: C, 61.27; H, 5.18; R10.58. Found: C 61.29; H, 5.18; N, 10.57. Example - (s)-2-(5-(((3-Amino- 1. 2-dihvdro- 1 -oxobenzorflαuinazolin-9-vl)methvnamino)- 1 - oxo-2-isoindolinvnglutaric acid A. N-(9-Bromomethyl-1. 2-dihydro-l -oxobenzo[f]quinazolin-3-yl .pivalamide N-(9-methyl- 1 ,2-dihydro- 1 -oxobenzo[_]quinazolin-3-y l)pivalamide (15g, 48 mmol) was dissolved in refluxing benzene (4000 ml). The reaction was removed from heat and N-bromosuccinimide (1 1.28g, 64 mmol, Kodak) added. The solution was heated under reflux for 2 hours. Benzene was removed in vacuo, the residue slurried with a small volume of ethanol. filtered and dried under high vacuum to give the bromomethyl derivative. The product was used without further purification. B Diethyl(S)-2-(5-((( 1 ,2-dihydro- 1 -oxo-3-pivalamidobenzo[f]quinazolin-9- ' yl _methyl)amino .-l-oxo-2-isoindolinv0glutarate (S)-diethyl 2-(5-amino-l-oxo-2-isoindolinyl)glutarate (2.8 g) and N-(9- Bromomethyl- 1 ,2-dihydro- 1 -oxobenzo[f]quinazolin-3-yl)pivalamide (2.0g) were heated in dimethylformamide ( 15ml) at 1 15°C for 1.5 hours. The reaction mixture was concentrated onto silica gel. eluting with methylene chloride/methanol (97:3). The fractions containing product were evaporated and stored under high vacuum overnight. The partially crystallized residual oil was suspended in ethyl acetate and filtered. The solid was recrystallized from methanol, filtered and dried under high vacuum to yield the diester (0.21 g) as a white solid. C. (SV2-(5-(((3-Amino- 1.2-dihvdro- 1 -oxobenzoff]quinazolin-9-vπmethyl)amino)- 1 - oxo-2-isoindolinyl)glutaric acid A solution of diethyl (S)-2-(5-(((l,2-dihydro-l-oxo-3-pivalamidobenzo [f]quinazolin-9-yl)methyl)amino)-l-oxo-2-isoindolinyl)glutarate in 0.33 M aqueous sodium hydroxide/tetrahydrofuran ( 12 ml. 1 : 1 ) was heated to reflux. Additional water (6 ml) was added to keep the reaction mixture homogeneous, and the solution was heated under reflux for 2.5 hours. The cooled solution was adjusted to pH 3 with 1M hydrochloric acid, the precipitate filtered off, washed with water and dried under high vacuum. The crude product, a white solid (0.15 g), contained approximately 20% of the 3-pivalamide of the desired diacid by NMR. The solid was redissolved in 0.5 M sodium hydroxide (5ml) and stirred at room temperature for 2 days. The pH of the solution was adjusted to 2, the precipitated solid filtered off. washed with water, and dried at room temperature under high vacuum to yield the diacid as a white solid (0.13 g). - H NMR (DMSO-d 6 , 300 MHz); δ 1.88-2.08 (m. 1H), 2.10-2.30 (m,3H), 2.26 (s, 2H), 4.52 (d, J = 5.6 Hz, 2H). 4.65-4.75 (m. 1H), 6.57 (br s. 2H), 6.67-6.75 (overlapping s and dd, 2H), 7.13 br t, J = 5.6 Hz, 1H), 7.27 (d, J = 8.9 Hz, 1H) 7.35 (d, J = 8.3 Hz, 1H), 7.46 (dd, J = 8.3, 1.1 Hz, 1H ), 7.85 (d, j = 8.2 Hz, 1H), 8.00 (d, J = 8.9 Hz, 1H), 9.68 (s, 1H), 1 1.14 (brs, 1H). 1 1.9- 12.9 (v br s 2H. Anal. Calculated for C 8H23N5O 6 • 2.5H 2 O: C. 57.61 H. 4.93; N, 12.80. Example 3 N-4-(( 1.2.5.6-tetrahvdro-3-methyl- 1 -oxobenzo[f]quinazolin-9-vπ-sulfonamido)benzoyl)-L- glutamic acid A. Diethyl-N-(4-((1.2.5.6-tetrahydro-3-methyl-l-oxobenzoff]quinazolin-9- yl .sulfonamido.benzovl .-L-glutamate N-(4-aminobenzoyl)-L-glutamic acid diethyl ester (6.06g, 0.0188 mole) (Aldrich) and 1.2.5.6-tetrahydro-3-methyl-l-oxobenzo[f]-quinazolin-9-sulfonyl chloride (5.84 g. 0.0188 mole) were dissolved in pyridine (55ml) and the reaction mixture stirred at room temperature for 3.5 hours. The pyridine was removed in vacuo. the residue washed with water, and the pink solid collected by filtration. The crude product was dried under high vacuum, then subjected to chromatography on a Waters Prep 500 instrument (silica Cartridge, elution with methanol: methylene chloride ( 1 :4). The product was recrystallized from ethanol and dried under high vacuum to yield the diethyl ester (5.68g. 51%). B. N-(4-((1.2.5.6-Tetrahydro-3-methyl-l -oxobenzoff]quinazolin-9-ylV sulfonamido)benzoyl)-L-g!utamic acid Diethyl-N-(4-(( 1 ,2,5,6-tetrahydro-3-methyl- 1 -oxober_zo[f]quinazolin-9- yl)sulfonamido) benzoyl)-L-glutamate (4.53 g, 7.6 mmole) was dissolved in N- NaOH (64ml) and the solution stirred at room temperature for 4 hours. The pH of the solution was adjusted to 3.00 with IN HC1, the solid collected by filtration, washed with water and dried under high vacuum to yield the product as an off-white solid (3.94g 96%). 1 HHMR (DMSO-d 6 .300 MHz) δ: 1.84-1.96 (m.lH.glu CH); 2.00-2.10 (m, 1H, glu CH); 2.32(s, 3H, CH 3 , superimposed over t, 2H. glu CH 2 ); 2.71 (m, 2H. Ar CH 2 ); 2.89 (m, 2H, Ar CH 2 ); 4.28-4.36 (m, 1H, glu CH); 7.20 (d, J = 9Hz, 2H, Ar); 7.39 (d, J = 8Hz, 1H, Ar); 7.62 (dd, J =8,2 Hz, 1H, Ar); 7.74 (d, J = 9Hz, 2H, Ar); 8.44 (d, J = 8Hz, 1H, gluNH); 9.21 (d, J = 2Hz, 1H, Ar); 10.73 (s, 1H, S0 2 NH); 12.36 (br s, 2H, CO2H); 12.72 (br s, 1H, NH). Aflϋl Calculated for C 2 5H 2 4N 4 0 8 S. 3/2H 2 0: C.52.91; H,4.79; N,9.87; S,5.65. Found: Q52.98; H.4.78; N,9.87; S,5.58. Example N-(4-(((1.2-Dihvdro-3-methvl-l -oxobenzorf)quinazolin-9-vl .methvlV amino .-2- fluorobenzovl VI .-glutamic acid A. Diethyl N-(4-(((1.2-dihvdro-3-methyl-l-oxobenzo[f]quinazolin- 9-yl) mgthyπaminQ)-2-fluQroben ? c)yl)-t-glW3mat.; To a hot solution of 3,9-dimethylbenzo[f]quinazolin-l(2H)-one (2.0 g, 8.9 mmol) in benzene (1000ml) under nitrogen was added N-bromosuccinimide (NBS) (2.0 g, 11 mmol). The solution was stirred at reflux for 1 hour and then concentrated in vacuo to give crude 9-bromomethyr-3-methylbenzo[f]quinazolin-l(2H)-one. The solid was suspended with diethyl N-(4-amino-2-fluorobenzoyl)-L-glutamate (T. R. Jones et al., UK Patent GB 2175 903 A, 1986)(6.0 g, 18 mmol) in DMF (20 ml) and stirred under 0 nitrogen at 100 C for 30 minutes. The reaction mixture was allowed to cool, N- methylmorpholine ( 1.0 ml, 9.1 mmol) (Aldrich) was added, and the solution concentrated under high vacuum. The residue was purified with silica gel chromatography eluting with methylene chloride:THF (5: 1). Fractions containing product were concentrated in vacuo to a thick paste, the solid suspended in a small volume of diethyl ether, filtered under nitrogen, and dried under high vacuum to give diethyl N-(4-((( 1.2-dihydro-3-methyl- 1 -oxobenzo[f]-quinazolin-9-yl)methyl)amino)- 2- fluorobenzoylVL-glutamate as a white solid (2.3g). B. N-(4-((( 1.2-Dihvdro-3-methyl- 1 -oxobenzof f]quinazolin-9-yl)methyl)- amino .-2-fluorobenzovn-L-glutamic acid A solution of diethyl N-(4-(((l,2-dihydro-3-methyl-l-oxobenzo[f]quinazolin-9-yl) methyl)amino)-2-fluorobenzoyl)-L-glutamate (2.3 g, 4.1 mmol) in ethanol (25 ml) and 0.2 N NaOH (100 ml) was stirred under nitrogen at room temperature for 3 hours. The solution was adjusted to pH 7 with IN HC1 and reduced in volume under vacuum to remove the ethanol. The product was precipitated by acidifying the solution with IN HC1 to pH 3 with stirring under nitrogen. The suspension was stirred 15 minutes, filtered under nitrogen, washed with water, pressed with a sheet of latex to remove excess water, and dried under high vacuum to give N-(4-(((l,2-dihydro-3-methyl-l- oxobenzo[f]quina-zolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutamic acid as a white solid (2.1g). H NMR (DMSO-d 6 ,300 MHz) d: 1.82-2.12 (m, 2H, glu CH 2 ), 2.29 (t, J=7 Hz, 2H, glu CH 2 ), 2.43 (s, 3H. C -CH 3 ), 4.32-4.42 (m, IH, glu CH), 4.57 (d, J = 6 Hz, 2H, C9-CH 2 ), 6.39 (dd, J = 15.2 Hz, IH. Ar), 6.53 (dd, J = 9.2 Hz, IH, Ar), 7.30 (t, J = 6 Hz, IH, ArNH), 7.47 (dd, J = 9,9 Hz, IH. Ar), 7.59(d, J = 9Hz, IH. Ar), 7.61 (dd, J = 8, 2 Hz, IH. Ar), 7.73 (t, J = 7 Hz. IH, glu NH), 8.01 (d, J = 8 Hz. IH, Ar), 8.22 (d, J = 9 Hz, IH. Ar), 9.85 (s, IH, Ar). 12.42 (br s. 2H. C0 2 H's), 12.53 (s, 1H. N 2 H). Δna Calculated for C26H23FN4O6.3/2 H 2 0.1/3 NaCl: C, 56.49; H, 4.74; N, 10.14; Cl, 2.12; Na, 1.37. Found: C. 56.48; H, 4.64; N, 10.20; Cl. 2.01; Na, 1.30. EXAMPLE 5 The thymidvlate synthase inhibitor Compound A (8 mg/kg) was lethal when administered to beagle dogs as an iv bolus for five consecutive days. In contrast Compound A at 20 mg/kg was not lethal when given as an iv bolus 30 minutes after oral administration of folic acid at 50 mg/kg. Similarly, five consecutive days of lOmg/kg of Compound A iv was lethal when given alone. But when the same dose was given 30 minutes after an oral dose of 10, 50, 100 or 200 mg/kg of leucovorin (Wellcovorin) no deaths occurred. EXAMPLE 6 Compound A administered as an ip bolus for five days at 3.2 or 10 mg/kg to nude mice bearing the human tumor GC3TK in the subrenal capsule produced complete inhibition of tumor growth. When 50 mg/kg of folic acid or 200 mg/kg leucovorin was administered orally 30 minutes prior to the ip bolus dose of Compound A complete inhibition of tumor growth was still observed. In another experiment, in the absence of protectant lOmg/kg Compound A produced complete growth inhibition and 3.2 mg/kg proudced 89%) inhibition. When 500 mg/kg folic acid was administered orally 30 min prior to the Compound A, tumor growth inhibition was not significantly different from that observed in the absence of the folic acid. Table : Influence of Folic Acid or leucovorin upon toxicity and antitumor efficacy of Compound A Key: - -: one animal died, one had minimal toxicity m: minimal toxicitv no toxicity

Claims: CLAIMS 1. Use of a protecting agent in the manufacture of a medicament for the reduction of side effects associated with the administration of a non-competitive folic acid analogue. 2. Use according to Claim 1 wherein the protecting agent is a folate derivative. 3. Use according to Claim 2 wherein the folate derivative is selected from folic acid, 5-formyltetrahydrofolate or 5-methyltetrahydrofolate. 4. Use according to Claim 1 wherein the protecting agent is thymine or thymidine. 5. Use according to Claim 1 wherein the non-competitive folic acid analogue is a thymidylate synthase inhibitor. 6. Use according to Claim 5 wherein the thymidylate synthase inhibitor is a benzoquinazoline derivative of formula (I) or a salt thereof, wherein the dotted line represents a single or double bond. R- is Cι_4 alkyl or amino optionally substituted by a Cj_4 alkyl, \.ζ alkanoyl or benzyl group; R 2 , R3, R4 and R*- 5 are the same or different and each is selected from hydrogen, phenyl, halo, nitro, a group S(O) n R-- wherein n is the integer 0, 1 or 2 and R^ is halo or is C 1.4 alkyl or a group NR R 10 wherein R-- and R-® are both hydrogen, a group NR** -- R** 2 wherein R- •* and R^ 2 are the same or different and each is hydrogen or C 1.4 alkyl. a group OR* 0 wherein R-3 is hydrogen or C] _4 alkyl optionally substituted by halo; a C]_4 aliphatic group optionally substituted by a group OR*-^ or NR 4 1 wherein R' and R- -- are the same or different and each is hydrogen or C 1.4 alkyl; or two or R 2 to R* 5 are linked together to form a benzo group, or one of R 2 to R-- is a group -X-Y-R16 wherein X is CH2, NR 17 , CO or S(O) m and m is 0. 1 or 2 and R 17 is hydrogen or a C 1.4 aliphatic group and Y is CH2, NR** 7 ', O, or S(O) m * wherein m' is 0, 1 or 2 and R- 7 ' is hydrogen or C 1.4 aliphatic group provided that X and Y are only the same when each is CH2. or -X-Y- is a group -0-, -NR^ 7 -, -CH=CH- or -N=N- wherein R** 7 is as hereinbefore defined. R**" is a C 1.4 aliphatic group or a 5- or 6- membered aromatic ring optionally substituted by a R^ at a position at least one carbon atom removed from that linked to Y, the 5- or 6- membered being optionally further substituted by a halo atom; and R^ is halo, C1.4 alkoxy, nitro, nitrile, Cι_4 alkyl optionally substituted by halo, halo or a group COR--9 wherein R--9 is hydroxy, Cj^alkoxy, C \. alkyl optionally substituted by one or two carboxyl groups or C \.\2 ester thereof or R-9 is a group NR 2 ^R *^ wherein R ^ and R 2 ^ are the same or different and each is hydrogen or C 1.4 alkyl optionally substituted by hydroxy or R-9 is an amino acid group or an ester thereof in which the first nitrogen atom of the amino acid group may be linked to the 5- or 6- membered heterocyclic ring or R**" is an C2-3 alkylene group linked to the 5- or 6- membered aromatic ring to form a further 5- or 6- membered ring; R and R 7 are the same or different and each is C \ .4 alkyl optionally substituted by hydroxy or C 1.4 alkoxy or together form a benzo group; provided that at least one of R 2 to R 7 is other than hydrogen and that R^ is not methoxy when R- is hydroxy or methyl. 7. Use according to Claim 6 wherein the thymidylate synthase inhibitor is a compound of formula (III) or a salt thereof, wherein R**, R-- and R 7 are as hereinbefore defined and R 32 to R---- are the same or different and one is a group X-Y-R**" and the others are the same or different and each is selected from hydrogen, halo, nitro, a group S(0) n R 8 , a group NR** -R---, a group OR^ or a C 1.4 aliphatic group optionally substituted by a group R 14 or NR14R--5, wherein X, Y, R 8 , R 1 *-, R 12 , R 13 , R 14 , R 15 and R 16 are as hereinbefore defined. Use according to Claim 6 wherein the thymidylate synthase inhibitor is a compound of formula (IV) wherein R-, R^, R 7 and R 32 to R---- are as hereinbefore defined. 9. Use according to Claim 7 wherein the thymidylate synthase inhibitor is selected (S)-2-(5-((( 1 ,2-dihydro-3-methyl- 1 -oxobenzo[fjquinazoIin-9-yl)methyl)amino)- 1 - oxo-2- isoindolinyl) glutaric acid N-(4-((( 1 ,2-dihydro-3-methyl- 1 -oxobenzo[f]quinazolin-9-yl)methyl)amino)-2- fluorobenzoyl)-L-glutamic acid N-(4-(( 1 ,2,5,6-tetrahydro-3-methyl- 1 -oxobenzo[f]quinazolin-9-yl)sulfonamido) benzoyl)-L-glutamic acid. 10. Use of a protecting agent in combination with a non-competitive folic acid analogue for the manufacture of a medicament for the treatment of neoplastic growth. 1 1. Use of a protecting agent in combination with a non-competitive folic acid analogue for the manufacture of a medicament for the treatment of disorders of the immune system and related diseases. 12. Use of a protecting agent in combination with a non-competitive folic acid analogue for the manufacture of a medicament for the treatment of bacterial and fungal infections. 13. A pharmaceutical formulation which comprises a protecting agent and a non- competitive folic acid analogue both as hereinbefore described in combination with one or more pharmaceutically acceptable carriers therefore and optionally one or more other therapeutic ingredients. 14. A method of reducing side effects associated with the administration of a non- competitive folic acid analogue which comprises administering to a mammal in need thereof an effective amount of a protecting agent, either before, concurrently with or after the administration of the non-competitive folic acid analogue. 15. A method for the treatment of a tumor in a mammal comprising administering to said mammal an effective antitumor amount of a non-competitive folic acid analogue in combination with an effective amount of a protecting agent.

Inventors: DUCH DAVID STANLEY, FERONE ROBERT, KOCH ARTHUR, SMITH GARY KEITH, DUCH, DAVID, STANLEY, FERONE, ROBERT, KOCH, ARTHUR, SMITH, GARY, KEITH
Applicants: DUCH DAVID STANLEY, FERONE ROBERT, KOCH ARTHUR, SMITH GARY KEITH, WELLCOME FOUND, DUCH, DAVID, STANLEY, FERONE, ROBERT, KOCH, ARTHUR, SMITH, GARY, KEITH, THE WELLCOME FOUNDATION LIMITED
Application year: 1993
Main classification: A61K31
IPC codes: A61K31, A61K45, A61P31, A61P35, A61P37, A61P43