Source: https://patents.google.com/patent/US5587161
Timestamp: 2018-02-19 23:57:09
Document Index: 428034399

Matched Legal Cases: ['Art 9385', 'Art 9385', 'Art. 9385', 'Art 9385', 'Art 9385', 'Art 9385', 'Art 9385']

US5587161A - Prodrugs for antibody directed enzyme prodrug therapy - Google Patents
Prodrugs for antibody directed enzyme prodrug therapy
US5587161A
US5587161A US08361424 US36142494A US5587161A US 5587161 A US5587161 A US 5587161A US 08361424 US08361424 US 08361424 US 36142494 A US36142494 A US 36142494A US 5587161 A US5587161 A US 5587161A
US08361424
Robert I. Dowell
Cancer Research Campaign Technology Ltd
A61K47/6899—Antibody-Directed Enzyme Prodrug Therapy [ADEPT]
C07C275/40—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by nitrogen atoms not being part of nitro or nitroso groups
C07C311/51—Y being a hydrogen or a carbon atom
Where Pr1 and/or Pr2 represents a benzyloxycarbonyl group, deprotection is preferably effected by hydrogenation. Such hydrogenation may be effected by any convenient means such as in the presence of platinum or Raney nickel, but is preferably effected by the use of palladium in the presence of carbon. The hydrogenation is advantageously effected in the presence of an inert solvent, preferably a non-protonic solvent, especially ethyl acetate, tetrahydrofuran or polar aprotic solvent such as dimethylformamide; preferably at a temperature of 0°-100° C., more preferably at a temperature of 15°-50° C. and especially at ambient temperature; and preferably for 1-24 h.
Where Pr1 and/or Pr2 represents a t-butyloxycarbonyl group, the deprotection reaction may advantageously be effected in the presence of an acid, advantageously a strong acid such as trifluoroacetic acid HCl, HBr, HI or formic acid. Where it is desired to use a solvent, inert non protonic solvents such as CH2 Cl2 or diethyl ether are preferred. The reaction is conveniently effected at a temperature of 0°-100° C., more conveniently at 0°-30° C. and especially at ambient temperature.
According to a further feature of the present invention there is provided a process for the preparation of compounds of formula Ia, wherein X is O and Y is O, and the salts thereof which process comprises reacting a compound of the formula: ##STR3## (wherein R1, R2, R1a, R2a, R3, R4, R5a, R5b, R5c, and R5d are as hereinbefore defined X is O, Y is O and L represents a leaving atom or group) with a compound of the formula: ##STR4## (wherein Pr1 and Z1 are as hereinbefore defined) whereby to form a compound of formula Ia, and, if desired, converting the compound of formula Ia to a salt thereof. Advantageously L is Cl, Br, I, 4-nitrophenoxy or pentafluorophenoxy. Conveniently the reaction is effected in the presence of a solvent at a temperature of -10° C. to 100° C. and preferably at 20°-50° C.
Preferred reaction conditions include effecting the reaction in the presence of an organic solvent (especially chloroform, ethyl acetate, toluene, dimethylformamide and CH2 Cl2) preferably at 5°-50° C. preferably for 1-24 h. Compounds of formula (II) constitute a further feature of the present invention which compounds may for example be prepared by reacting a corresponding phenol containing an anilino mustard group either with an aryl chloroformate, for example nitrophenyl chloroformate (especially 4-nitrophenylchloroformate) or with phosgene whereby to form a compound of formula (II).
Preferred reaction conditions include effecting the reaction in the presence of an organic solvent (especially ethyl acetate or chloroform), preferably at 15°-50° C. (especially at ambient temperature), preferably for 1-10 h.
Compounds of formula (III) wherein W represents group (3) as herein defined may be prepared from compounds of formula (III) wherein W represents group (2) as herein defined using standard conditions. Preferable standard conditions include reaction with nitrogen nucleophiles (especially ammonia or a primary or secondary amine) preferably at 25°-50° C. preferably for about 24 h.
Compounds of formula (III) wherein W represents group (9) as herein defined may be prepared by reduction of compounds of formula (XV) as herein defined to a corresponding primary alcohol using standard conditions (especially using diborane, or a mixed anhydride reaction followed by sodium borohydride reduction). The corresponding primary alcohol is converted by standard methods for example using methane sulphonyl chloride and trimethylamine at 0° C. in the presence of CH2 Cl2 into a leaving group such as Br, I or mesylate; which leaving group is displaced by a group of formula:
According to a further feature of the present invention there is provided a process for the preparation of compounds of formula (IV) and the salts thereof, which process comprises reacting a compound of formula (V) (wherein R1, R2, R1a, R2a, R3, R4, and R5a-d are as hereinbefore defined) with a compound of formula L1 --(C═O)--L2 (wherein L1 and L2 represent leaving groups) whereby to form a compound of formula (IV). Values for L1 and L2 include Cl, CCl3, imidazolyl and aryloxy (especially phenoxy). Preferred reaction conditions include effecting the reaction in the presence of an organic solvent (preferably polar aprotic, especially ethyl acetate) at 5°-25° C. for about 15 min. ##STR7##
According to a further feature of the present invention there is provided a process for the preparation of compounds of formula (Ia) as hereinbefore defined, wherein X is --CH2 -- and Y is O, and the salts thereof which process comprises: reacting a compound of formula (II) or a salt thereof, as hereinbefore defined, with the provisos that X is --CH2 --, Y is O and L is advantageously pentafluorophenoxy, Cl, --O--(CO)--C1-6 alkyl preferably branched alkyl, especially C1-4 alkyl and the product of a corresponding phenyl acetic acid containing an anilino mustard reacted with a carbodiimide (especially dicyclohexylcarbodiimide); with a compound of formula (III) as hereinbefore defined using standard reaction conditions (for example effecting the reaction in the presence of polar aprotic solvents such as dimethylformamide, ethyl acetate or tetrahydrofuran for 1-24 h at 20°-50° C.) whereby to form a compound of formula (Ia), and, if desired, converting the compound of formula (Ia) to a salt thereof. Compounds of formula (II) may be prepared using standard methods from corresponding phenyl acetic acids containing an anilino mustard group.
According to a further aspect of the present invention there is provided a process for the preparation of a compound of formula (Ia) as hereinbefore defined, wherein W represents group (8) (that is --CONH--SO2 R11) as hereinbefore defined or a salt thereof which process comprises: reacting a compound of formula (III) as hereinbefore defined with the proviso that W only represents group (8); with a compound of formula (II) as hereinbefore defined using reaction conditions known per se whereby to form a compound of formula (Ia) wherein W represents group (8), and, if desired, converting the compound of formula (Ia) into a salt thereof. Preferred reaction conditions include effecting the reaction in the presence of an organic solvent (preferably a polar aprotic solvent, especially ethyl acetate or dichloromethane), preferably at 5°-50° C. (especially ambient temperature), preferably for 1-5 h.
According to a further feature of the present invention there is provided a process for the preparation of compounds of formula (Ia) hereinbefore defined, wherein X is N or O, Y is O, R1 and R2 are Cl, Br, I or OSO2 Me (especially Cl), R1a is H, R2a is H, R3 is H and R4 is H, and the salts thereof which process comprises reacting a compound of formula: ##STR11## (wherein R5a-d, Pr1 and Z1 are as hereinbefore defined and X is O or NH) with either: a phosphorous halogenating agent (especially phosphorus pentachloride) or thionyl chloride in the presence of an organic solvent (preferably non-polar aprotic, especially CH2 Cl2), preferably heated at reflux for 1-2 h (especially 90 min); or methyl sulphonyl chloride in the presence of an organic solvent (preferably polar aprotic, especially pyridine) whereby to form a compound of Formula (Ia) wherein X is O or NH, Y is O, R1 and R2 are Br, I, OSO2 Me or Cl, R1a is H, R2a is H, R3 is H and R4 is H, and, if desired converting the compounds of formula (Ia) into a salt thereof. When methyl sulphonyl chloride is used, hydroxy groups in the compound of formula (XIX) may be converted into either Cl and/or OSO2 Me depending on the temperature used for the reaction. The dichloro compounds (that is R1 ═R2 ═Cl) may conveniently be obtained by effecting the reaction at 70° C. for 15 min; and the corresponding compound where R1 ═Cl and R2 ═OSO2 Me may conveniently be obtained by effecting the reaction at 50° C. for 10 min.
When R1 and R2 are Br and R1 and R2 are I, methane sulphonyl anhydride is preferably substituted for methane sulphonyl chloride because this removes any problems of competing halogen in the reaction. The compound of formula XIX (0.002M) was dissolved in CHCl3 (30 ml). Triethylamine (1.12 ml) and methysulphonyl anhydride (0.008M) were added at ambient temperature, the mixture stirred for 2 hours and then washed with water. The product so obtained was dried over MgSO4, filtered and evaporated to an oil. The oil was dissolved in dry DMF and lithium iodide (or bromide; 0.005M) added, stirred at 80° C. for 2 hours, cooled, poured into water and extracted with ether. The product so obtained was dried over MgSO4, filtered, evaporated to dryness and purified by flash column chromatography.
A process for the preparation of compounds of formula (I), wherein W represents a tetrazol-5-yl group, comprises reacting a compound of formula: ##STR14## (wherein Z111 represents Z as hereinbefore defined with the proviso that W represents a tetrazol-5-yl group; with compounds of formula (II) as hereinbefore defined under standard conditions; whereby to form a compound of formula I wherein W represents a tetrazol-5-yl group and, if desired, converting the compound of formula I to a salt thereof. The standard conditions include reaction in a polar aprotic solvent (especially DMF) in the presence of a base (preferably dimethylamino pyridine, especially triethylamine) for at least 2 h (preferably 20 h) at a temperature of 20°-50° C. (especially 25° C.).
The in vitro cytotoxic potency of prodrugs, prodrugs plus enzyme and drugs was measured in a cytotoxicity assay similar to that described by Skehan et al (J. Natl. Cancer Inst 82, 1107-1112, 1990). LoVo cells (ECACC No: 87060101) were diluted in DMEM media (containing 10% FCS, 1% glutamine and 0.2% gentamycin) plated out in 96 well microtitre plates at a density of 2,500 cells/well and incubated overnight at 37° C. in 5% CO2. Various concentrations of prodrug, corresponding drug as control or prodrug plus enzyme (1U CPG2 activity/well--one unit of enzyme being the amount required to hydrolyse 1 μmole of methotrexate/min/ml at 37°) were added to these cells and following either a 1 hr or 24 hr incubation period the cells were washed, fresh medium added and the cells incubated at 37° C. in 5% CO2. Three days after addition of compound, TCA was added to the wells (16% final concentration) and the amount of cellular protein adhering to the plates was assessed by the addition of SRB dye (Skehan et al). The optical density at 540 nm was measured and expressed as a percentage of the OD540 in control wells which received no compound. The potency was expressed as the concentration required to inhibit cell growth by 50% (IC50). Prodrug on its own should generally possess low activity in the test relative to prodrug in the presence of CPG2 (that is CPG2 enzyme is necessary for activation of prodrug to drug). Direct addition of chemically synthesised drug (not needing CPG2 activation) acts as a control in the assay.
ii) operations were carried out at room temperature, that is in the range 18°-25° C. and under an atmosphere of an inert gas such as argon;
A solution of dibenzyl N-(4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl)-L-glutamate (see (2) in scheme 1) (6 g) in ethyl acetate (100 ml) was hydrogenated over 30% palladium on carbon (0.6 g) for 2 h. When the theoretical amount of hydrogen had been taken up the catalyst was removed by filtration and the filtrate evaporated to dryness. The residue was taken up into hot ether (25 ml) and hexane added until cloudy. On cooling N-(4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl)-L-glutamic acid (see (3) in Scheme 1) was obtained as a white crystalline solid (3.4 g) 79% yield m.p. 87°-89°. NMR 7.0 (d) 2H; 6.6 (d) 2H; 6.2 (d) 1H; 4.4 (m) 1H; 3.5-3.7 (m) 8H; 2.0-2.6 (m) 4H.
The titled compound was also prepared in a different polymorphic form, not soluble in ether, with a melting point of 128°-130° C.
A solution of 4-nitrophenyl-chloroformate (1.43 g) in chloroform (15 ml) was added to a mixture of 4-[bis(2-chloroethyl)amino]phenol hydrochloride (Biochem. Pharmacol 17 893 (1968)) (1.93 g), triethylamine (2 ml) and chloroform (20 ml). After 2 h at ambient temperature the mixture was evaporated to dryness and the residue chromotographed on Merck silica gel Art 9385. On elution with hexane/ethyl acetate and recrystallisation from benzene:petroleum ether (3:1) the product 0-(4-[N,N-bis(2-chloroethyl)amino]phenyl)-0'-(4-nitrophenyl)carbonate (see (1) in Scheme 1) was obtained as a yellowish solid (1.4 g) (50%) mp=66°-7°.
Triethylamine (3.8 ml) was added to 5.5 g of the product so obtained in chloroform (40 ml), followed by addition of L-Glutamic acid dibenzyl ester tosylate (13.75 g). The mixture was stirred and heated at 60° for 4 h and evaporated to dryness. The residue was chromatographed on silica gel (Merck Art 9385) and eluted with 3% ethyl acetate in chloroform to obtain the required starting material dibenzyl-2-([bis(2-chloroethyl)amino]phenoxycarbonyl)glutamate (see (2) in Scheme 1) 6.2 g (77% yield) as a white solid m.p. 85°-7°.
The process described in Example 1 was repeated using dibenzyl N-(4-[N,N-bis(2-chloroethyl)amino]-3-methyl-phenoxycarbonyl)-L-glutamate in place of dibenzyl N-(4-[N,N-bis(2-chlorethyl)amino]phenoxycarbonyl)-L-glutamate to obtain N-(4-[N,N-bis(2chloroethyl)amino]-3-methyl-phenoxycarbonyl)-L-glutamic acid as a white solid, m.p. 160°-162° C.
(b) Benzyl bromide (4.24 g) was added to a mixture of the product so obtained (6 g), potassium hydroxide (1.6 g) and ethanol (40 ml). The mixture was stirred and heated at reflux for 2 h, cooled and concentrated by evaporation. The residue was poured onto water (100 ml), extracted twice with ethyl acetate, dried and evaporated to obtain 2,2'-(4-benzyloxy-2-toluidino)diethanol as a solid (7.2 g) mp=70°-72° C.
(c) Phosphorous pentachloride (11.4 g) was added in portions to the product so obtained (7 g) in chloroform (50 ml) at 10°-20° C. The mixture was then heated at reflux for 90 min, cooled and poured onto water. The organic phase was separated and washed with aqueous sodium bicarbonate solution, water and evaporated to dryness. The residue was chromatographed on silica gel. After elution with hexane/ethyl acetate (2:1) 4-benzyloxy-3-methyl-N,N-bis(2-chloroethyl)-aniline (2.2 g) was obtained as an oil.
An alternative reaction for use in lieu of the reaction immediately above is as follows. Methane sulphonyl chloride (2.5 ml) was added at 0°-5° C. to a solution of the product obtained in step (b) (2.6 g) in pyridine (8 ml). The mixture was then heated at 70° C. for 15 min, cooled and poured onto dilute citric acid solution (100 ml). The mixture was extracted twice with ethyl acetate, dried and evaporated to obtain 4-benzyloxy-3-methyl-N,N-bis(2-chloroethyl)-aniline as an oil 2.6 g (84%).
A saturated solution of hydrogen chloride in ether (120 ml) was added to a solution of ditertbutyl N-(-4-[N,N-bis(2-chloroethyl)amino]-phenylcarbamoyl)-L-glutamate (4.4 g) in ethyl acetate (20 ml). After 1 hour at ambient temperature the mixture was evaporated to a solid. This solid was triturated with ether to obtain N-(4-[N,N-bis(2-chloroethyl)amino]phenylcarbamoyl)-L-glutamic acid hydrochloride (3.5 g) as a grey solid m.p=148°-150° C. (see Scheme 3)
A mixture of p-fluoronitrobenzene (14.1 g) and diethanolamine (30 ml) was stirred and heated at 130° C. for 2 h. The mixture was cooled to about 60° C. and poured onto 1L of water containing 10 ml of 48% caustic soda solution. After cooling to 15° C. a precipitate was filtered off and dried to obtain 2,2'-(4-nitroanilino)diethanol (20.7 g) (92%) m.p.=102°-104° C.
Thionyl chloride (30 ml) was added, with cooling, to a mixture of the product so obtained (20 g), dichloromethane (200 ml) and pyridine (7 ml). After the addition the mixture was heated at reflux for 1 hour. After cooling the mixture was diluted with an equal volume of dichloromethane and carefully washed twice with water, dried and evaporated to obtain [N,N-bis(2-chloroethyl)]-4-nitro-aniline as a solid 21 g m.p.=81°-3° C.
To a solution of the product so obtained (0.53 g) in redistilled tetrahydrofuran (20 ml) was added 30% palladium on carbon catalyst (100 mg). The mixture was stirred under an atmosphere of hydrogen for 2 h and the catalyst then removed by filtration. The filtrate was evaporated to dryness and the residue redissolved in ether (20 ml) and a solution of hydrogen chloride gas in ether added to slight excess. The resulting 4-[N,N-bis(2-chloroethyl)amino]anilinium chloride was obtained as a solid and dried. Yield=0.5 g m.p. 238°-40° C. (d).
To a solution of triphosgene (Aldrich) 200 mg in chloroform (10 ml) at 0°-5° C., was added the product so obtained (539 mg) followed by triethylamine (0.83 ml). After 15 min at room temperature a solution of L-glutamic acid ditertbutyl ester (0.31 g) in chloroform (5 ml) was added. The mixture was allowed to stand at ambient temperature for 18 h, washed with water, dried and evaporated to dryness. The residue was chromatographed on Merck silica gel and eluted with hexane--ethyl acetate (3:1) to obtain the desired starting material ditertbutyl 4[bis-2-(chloroethylamino)]phenylcarbamoyl-L-glutamate 0.44 g as an oil (see Scheme 3).
A solution of dibenzyl N-(4-[N,N-bis-(2-chloroethyl)amino]phenylcarbamoyl)-L-glutamate (1.138 g) in DMF (15 ml) was hydrogenated over 10% Pd/C for 16 h. After filtration and evaporation in vacuo, the residue was dissolved in CHCl3 (20 ml). After 18 h the crystalline precipitate was filtered off and dried in vacuo to obtain N-(4-[N,N-bis-(2-chloroethyl)amino]phenylcarbamoyl)-L-glutamic acid. Yield, 730 mg (93%). After recrystallization from acetone/CHCl3 microscopic rods formed m.p. 116°-118°. NMR (CD3 COCD3): δ8.0(s)1H; 7.2(d)2H; 6.6(d)2H; 6.2(d)2H NH; 4.4(m)1H; 3.6(m)8H; 2.5-1.9(m)4H.
To a solution of dibenzyl N-(4-[N,N-bis(2-chloroethyl)-amine]-3-fluorophenyl-carbamoyl) L-glutamate (0.4 g) in ethyl acetate (10 ml) was added 30% palladium on carbon (50% moist) (160 mg) and the mixture stirred under an atmosphere of hydrogen for 1 hour. After filtration of the catalyst the filtrate was evaporated to dryness. N-(4-[N,N-bis(2-chloroethyl)-amine]3-fluorophenylcarbamoyl L-glutamic acid was obtained after trituration of the oily residue with ethyl acetate/hexane as a white powder (210 mg, mp 111°-114° C.). The starting material dibenzyl N-(4-[N,N-bis (2-chloroethyl)amino]-3-fluorophenylcarbamoyl)L-glutamic acid was obtained as follows:
A suspension of 4-[N,N-bis-(2-chloroethyl)amino]-3-fluoroanilinium oxalate (3.5 g) in anhydrous ethyl acetate (200 ml) and potassium carbonate (5.5 g) was cooled under argon to 5° C. To this mixture was added a solution of phosgene in toluene (1.9M; 5.5 ml). After the addition the mixture was stirred at room temperature for 10 min, filtered and the filtrate dried over magnesium sulphate. The dried filtrate obtained was added in one portion to a mixture of dibenzyl glutamate p toluenesulphonate (5 g), potassium carbonate (2 g) and ethyl acetate (100 ml). Triethylamine (2 ml) was added and the mixture stirred for 20 min at ambient temperature. The mixture was filtered and the filtrate evaporated to dryness. The residue was chromatographed on silica gel eluting with ethyl acetate/hexane (1:2) to obtain the desired starting material as an oil, which crystalized. Yield=5.5 g m.p. 81°-84° C.
4-[N,N-bis(2-chloromethyl)amino]-3-fluoroanilium oxalate was prepared as described in Example 3 except that 3,4 difluoronitrobenzene was used as starting material in place of p-fluoronitrobenzene to obtain 4-[N,N-bis-(2-hydroxyethyl)amino]fluoronitrobenzene m.p.=99°-101° C.
The product so obtained was treated with thionyl chloride as described in Example 3 to obtain 4-[N,N-bis(2-chloroethyl)-3-fluoronitrobenzene. m.p. 66°-8° C.
The product so obtained was hydrogenated as described in Example 3 using ethyl acetate as solvent. The mixture was filtered and the filtrate evaporated to low volume and redissolved in ether. A saturated solution of oxalic acid in ether was added to excess and the desired product 4-[N,N-bis(2-chloromethyl)amino]-3-fluoroanilinium oxalate collected. m.p=146°-8° C.
The product so obtained was hydrogenated using ethyl acetate as solvent as described in Example 3. The catalyst was removed by filtration and the filtrate evaporated to low volume, redissolved in ether and saturated ethereal oxalic acid added to excess. The oxalate salt was obtained by filtration m.p.=118°-21° C.
A solution of the product so obtained (2) (5.01 g) in acetic acid (30 ml) was hydrogenated over 10% palladium on carbon for three days. After filtering, the solution was cooled and ethylene oxide (5 ml) added, and left at ambient temperature for 22 hours. Solvent was evaporated and the residue was partitioned between ethyl aceate and water. The organic phase was separated, washed with water, dried (Na2 SO4) and evaporated to dryness. The residue was chromatographed on silica gel; eluted with ethyl acetate in chloroform (2:1) to obtain 4-[bis(2-hydroxyethyl)amino]phenyloxycarbonyl-L-glutamic acid di-t-butyl ester (4) (3.93 g) 69% m.p. 91°-93° C.
A solution of the product so obtained (4) (0.86 g) in pyridine (3 ml) was stirred with methanesulphonyl chloride (0.6 ml) at 2° C. for 20 min, followed by 50° C. for 10 min. The reaction mixture was partitioned between ethyl acetate and water. The organic phase was separated, washed with water, dried (Na2 SO4) and evaporated to dryness. The residue was chromatographed on silica gel; eluted with ethyl acetate in dichloromethane (1:9) to obtain 4-[(2-chloroethyl)[2-(mesyloxy)ethyl]aminophenyloxycarbonyl-L-glutamic acid-di-t-butyl ester (6) as an oil (0.44 g) 43%.
TABLE 1__________________________________________________________________________ ##STR15##Ex NoR.sup.5a-b   mpt   ClCH.sub.2 CH.sub.2 M                           Aromatics                                   αCH                                         CH.sub.2 CH.sub.2                                                 Other__________________________________________________________________________ 9   CH.sub.3 (R.sup.5a)             160-2° C.                   3.34-3.54(m)8H                           6.9-7.22(m)3H;                                   4.1(m)1H                                         2.0-2.5(m)4H                                                 2.27(s)3H CH.sub.310   Pr.sup.i (R.sup.5a)             156-8° C.                   3.32-3.55(m)8H                           6.95-7.28(m)3H;                                   4.1(m)1H                                         2.0-2.49(m)4H                                                 3.7(m)1H CH                                                 1.14(d)2H(CH.sub.3).                                                 sub.211   CH.sub.3 (R.sup.5b)             124-6° C.                   3.7(m)8H                           6.5-6.9(m)3H                                   4.1(m)1H                                         1.9-2.3(m)4H                                                 2.1(s)3H CH.sub.312   F (R.sup.5a)       3.6(m)8H                           6.8-7.2(m)3H                                   4.1(m)1H                                         2.0-2.49(m)4H13   CHCHCHCH           3.6(m)8H                           6.6-8.0(m)6H                                   4.2(m)1H                                         2.1-2.5(m)4H(R.sup.5a-b)14   Cl (R.sup.5b)             106-8° C.                   3.6(m)8H                           6.7-7.1(m)3H                                   4.1(m)1H                                         1.9-2.4(m)4H15   Cl (R.sup.5a)             148-150° C.                   3.4-3.54(m)8H                           7.0-7.4(m)3H)                                   4.1(m)1H                                         1.9-2.36(m)4H__________________________________________________________________________
TABLE 2______________________________________ ##STR16##R5.sup.a or .sup.b           m.p. or NMR data                         Reference______________________________________Me (R5.sup.a)   176-9° C.                         AldrichPr.sup.i (R5.sup.a)           172-5° C.Cl (R5.sub.a)   6.5-6.7(m)3H  J. Chem. Soc.           Aromatics;    (1928), 2703           8.8(s)1H OH;           4.6(br s)2H NH.sub.2 ;           159.5° C.Me (R5.sup.b)   174-6° C.                         AldrichCl (R5.sub.b)   146-8° C.F (R5.sub.a)    6.6-6.9(m)3H           Aromatics;           9.4(s)1H OH;           4.4(m)2H NH.sub.2DiCH.sub.3      260-2° C.(R5.sup.a = R5.sup.b = CH.sub.3)           (as hydrochloride ex Aldrich)(R5.sup.a and R5.sup.b           273° C.together represent           (as hydrochoride ex Aldrich)CHCHCHCH)______________________________________
TABLE 3__________________________________________________________________________ ##STR17##R5.sup.a-b   HOCH.sub.2 CH.sub.2 N                 Aromatics                         Other__________________________________________________________________________Me (R5.sup.a)        2.8-3.3(m)8H                 6.4-6.9(m)3H                         2.03(s)3H CH.sub.3Pr.sup.i (R5.sup.a)        3.0-3.4(m)8H                 6.6-7.1(m)3H                         3.6(m)1H; 1.1(d)6H Pr.sup.iCl (R5.sup.a)        3.15-3.54(m)8H                 6.7-7.2(m)3HCH.sub.3 (R5.sup.b)        3.3-3.5(m)8H                 6.4-6.6(m)3H                         2.06(s)3H CH.sub.3Cl (R5.sup.b)        3.2-3.6(m)8H                 6.5-6.8(m)3HF (R5.sup.a) 3.1-3.4(m)8H                 6.5-6.9(m)3Hdi CH.sub.3  2.9-3.4(m)8H                 6.6-7.0(m)3H                         2.0(s)3H; 2.2(s)3H CH.sub.3(R5.sup.a = R5.sup.b = CH.sub.3)(R5.sup.a and R5.sup.b        3.2-3.4(m)8H                 6.8-8.3(m)11Htogether representCHCHCHCH__________________________________________________________________________
TABLE 4__________________________________________________________________________ ##STR18##R5.sup.a-b    HOCH.sub.2 CH.sub.2 N                  Aromatics                         CH.sub.2                              Other__________________________________________________________________________Me (R5.sup.a) 3.0-3.2(m)8H                  6.8-7.5(m)8H                         5.0(s)2H                              2.2(s)3H; CH.sub.3Pr.sup.i (R5.sup.a)         2.9-3.3(m)8H                  6.8-7.5(m)8H                         5.0(s)2H                              3.6(m)1H; 1.1(d)6H Pr.sup.iCl (R5.sup.a) 3.05-3.35(m)8H                  6.9-7.5(m)8H                         5.1(s)2H                              2.2(s)3H CH.sub.3CH.sub.3 (R5.sup.b)         3.1-3.4(m)8H                  6.9-7.3(m)8H                         5.0(s)2HCl (R5.sup.b) 3.3-3.5(m)8H                  6.8-7.4(m)8H                         5.0(s)2HF (R5.sup.a)  3.2-3.4(m)8H                  6.8-7.5(m)8H                         5.0(s)2HDi CH.sub.3   2.9-3.3(m)8H                  6.8-7.4(m)8H                         5.0(s)2H                              2.2(s)3H; 2.1(s)3H CH.sub.3(R5.sup.a = R5.sup.b = CH.sub.3)(R5.sup.a and R5.sup.b         3.2-3.4(m)8H                  6.9-8.3(m)6H                         5.3(s)2Htogether representCHCHCHCH)__________________________________________________________________________
TABLE 5__________________________________________________________________________ ##STR19##R5.sup.a-b   R.sup.1          R.sup.2            CH.sub.2 CH.sub.2 N                    CH.sub.2                         Aromatics                                 Other__________________________________________________________________________Me (R5.sup.a)        Cl          Cl            3.3-3.5(m)8H                    5.08(s)2H                         6.8-7.4(m)8H                                 2.3(s)3H MePr.sup.i (R5.sup.a)        Cl          Cl            3.2-3.5(m)8H                    5.04(s)2H                         6.8-7.4(m)8H                                 3.7(m)1H; 1.1(d)6HCl (R5.sup.a)        Cl          Cl            3.4-3.6(m)8H                    5.1(s)2H                         6.9-7.4(m)8HCH.sub.3 (R5.sup.b)        Cl          Cl            3.3-3.6(m)8H                    5.1(s)2H                         6.9-7.4(m)8HCl (R5.sup.b)        Cl          Cl            3.6(m)8H                    5.1(s)2H                         6.8-7.4(m)8HF (R5.sup.b) Cl          Cl            3.4-3.6(m)8H                    5.06(s)2H                         6.8-7.4(m)8HDi CH.sub.3  Cl          Cl            3.2-3.5(m)8H                    5.06(s)2H                         6.8-7.4(m)7H                                 2.2(s)3H; 2.1(s)3HCH.sub.3(R5.sup.a = R5.sup.b = CH.sub.3(R5.sup.a and R5.sup.b        Cl          Cl            3.4-3.7(m)8H                    5.3(s)2H                         7.1-8.3(m)11Htogether representCHCHCHCH__________________________________________________________________________
TABLE 6______________________________________ ##STR20##              m.p. of HCl saltR5.sup.a-b         (°C.)______________________________________Pr.sup.i (R5.sup.a)              124-7Me (R5.sup.a)      164-7Me (R5b)           122-4Cl (R5.sup.b)      156-8F (R5.sup.a)       123-5(R5.sup.a = R5.sup.b = CH.sub.3) di-CH.sub.3              144-6(R.sup.5 and R5.sup.b              180-4together representCHCHCHCHCl (R.sup.5a)       119-121______________________________________
TABLE 7______________________________________ ##STR21##R.sup.5a-b   ClCH.sub.2 CH.sub.2 N                aromatics   other______________________________________CH.sub.3 (R5.sup.a)   3.57(m)4H;   7.2-8.4(m)7H                            2.32(s)3HCH.sub.3   3.35(m)4HPr.sup.i (R.sup.5a)   3.5(m)4H;    7.2-8.4(m)7H                            3.7(m)1HCH;   3.34(m)4H                1.1(d)2H(CH.sub.3)CH.sub.3 (R.sup.5b)   3.7(m)4H;    6.7-8.4(m)7H                            2.2(s)3HCH.sub.3   3.32(m)4H3F      3.5-3.7(m)8H 7.2-8.1(m)7H1,4 Naphth   3.5-3.5(m)8H 7.2-8.4(m)10H2Cl     3.75(m)8H    6.7-8.4m(7H)______________________________________
TABLE 8__________________________________________________________________________ ##STR22##R.sup.5a-b  ClCH.sub.2 CH.sub.2 N           Aromatics                    ArCH.sub.2 O                         αCH                              CH.sub.2 CH.sub.2                                     other__________________________________________________________________________CH.sub.3 (R.sup.5a)  3.42(m)8H           7.35-6.9(m)13H                    5.2(s)2H                         4.4(m)1H                              2.5-2.1(m)4H                                     2.3(s).sup.3H CH.sub.3                    5.1(s)2HPr.sup.i (R.sup.5a)  3.5(m) 3.3(m)8H           7.35-6.7(m)13H                    5.2(s)2H                         4.3(m)1H                              2.5-2.1(m)4H                                     3.6(m)1HCH                    5.1(s)2H         1.1(d6H                                     2 × CH.sub.3CH.sub.3 (R.sup.5b)  3.7(m) 3.3(m)8H           6.6-7.4(m)13H                    5.14(s)2H                         4.2(m)1H                              2.5-2.1(m)4H                                     2.1(s)3H                    5.09(s)2H        CH.sub.33F     3.60(m)8H           6.7-7.4(m)13H                    5.13(s)2H                         4.2(m)1H                              2.5-2.1(m)4H                    5.08(s)2H1,4 Naphthyl    7.1-8.4(m)16H                    5.1(s)2H                         4.2(m)1H                              2.5-2.1(m)4H                    5.16(s)2H2Cl    3.7(m)8H 6.7-7.4(m)13H                    5.09(s)2H                         4.2(m)1H                              2.5-2.1(m)4H                    5.15(s)2HCl (R5.sup.a)  3.45-3.62(m)8H           6.98-7.36(m)13H                    5.09(s)2H                         4.2(m)1H                              2.1-2.5(m)4H                    5.16(s)2H__________________________________________________________________________
A suspension of α-benzyl 4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-glutamic acid-γ-anilide (2.0 g) in ethyl acetate (50 ml) was hydrogenated over 10% palladium on carbon (0.15 g) for 4 h. The catalyst was removed by filtration and the filtrate was evaporated to dryness under reduced pressure at 35°. The product, 4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-glutamic acid-γ-anilide (1 in Scheme 6) was obtained as a white crystalline solid, 1.4 g (83%) m.p. 110°.
A solution of triethylamine (10 mls) in dichloromethane (10 mls) was added to a mixture of 4-nitrophenyl-chloroformate (7.25 g) and 4-(N,N-bis(2-chloroethyl)amino)phenol hydrochloride (10 g) in dichloromethane (100 mls) over a period of two hours. After stirring at room temperature for 16 h the solvents were removed under reduced pressure and the residue was chromatographed on silica gel (Merck Art. 9385). Elution with dichloromethane and evaporation of the eluates yielded the product as a red oil. Trituration with hexane gave a yellow solid which was recrystallised from benzene/hexane to give 0-(4-[N,N-bis(2-chloroethyl)amino)phenol-0'-(4-nitrophenyl)carbonate as orange crystals, 10.4 g (71%). m.p. 68°.
N-t-BOC-L-glutamic acid α-benzyl ester (10 g) and dicyclohexylcarbodiimide (6.1 g) were dissolved in dichloromethane (120 mls) and stirred at room temperature for 10 minutes. Aniline (2.8 mls) was added and the mixture stirred at room temperature for 16 hours. The mixture was filtered and the precipitate washed with dichloromethane (2×15 mls). The filtrate was washed successively with saturated NaHCO3 solution (2×100 mls) and water (100 mls) then evaporated. The resulting solid was recrystallised from EtOAc/hexane to give colourless plates, 8.2 g (67%). The γ-anilide (12.0 g) and p-toluenesulphonic acid (5.4 g) in benzene (300 mls) were refluxed for 40 minutes and allowed to cool overnight. The precipitate was filtered, dried at the pump and recrystallised from EtOAc/MeOH to give colourless plates of tosyl-α-benzyl-L-glutamic acid γ-anilide, 8.2 g (58%).
which was recrystallised from ethyl acetate/hexane to give colourless crystals, m.p. 129°.
A solution of di-t-butyl 4-[N,N-bis(2-chloroethyl)amino]-3-methylphenylcarbamoyl-L-glutamate (0.6 g) in dichloromethane (6 ml) was cooled to 0° C., trifluoroacetic acid (15 ml) was added. This solution was then left at 0° C. for 3 days. The solution was then evaporated to dryness to yield (4-[N,N-bis(2-chloroethyl)amino]-3-methylphenylcarbamoyl-L-glutamic acid as an oil. Yield 0.49 g. NMR: 8.46(s)1H); 7.15(m)3H; 6.4(d)1H; 4.18(m)1H; 3.55(m)4H; 3.35(m)4H; 2.3(m)2H; 2.23(s)3H, 2.0(m)2H.
Potassium carbonate (27.5 g) was added to a solution of 3-methyl-4-nitroaniline (Journal of Organic Chemistry 33, 3498 (1968) (7.6 g) in ethyl acetate (150 ml), followed by dropwise addition of a 1.9M solution of phosgene in toluene (27.5 ml) maintaining the temperature below 30° C. The mixture was then stirred at ambient temperature for 1 hour. L-glutamic acid di-t-butyl ester (13 g) was added to the mixture which was then stirred at ambient temperature overnight. The solution was then filtered, washed with water, and the organic layer dried over MgSO4 and evaporated to an oil. This was then chromatographed on silica, eluted with Hexane: ethyl acetate 3:1 to give di t-butyl(3-methyl-4-nitrophenylcarbamoyl-L -glutamate as an oil. Yield=11.19 g 51%). NMR: 9.16(s)1H; 8.0(d)1H; 7.43(m)2H; 6.70(d)1H; 4.1(m)1H, 2.51(s)3H; 2.25(m)2H; 1.8(m)2H; 1.41(d)8H.
Methanesulfonyl chloride (3.8 ml) was added dropwise to a solution of di t-butyl 4-[N,N-bis(2-hydroxyethyl)amino]-3-methylphenylcarbamoyl-L-glutamate (4 g) in pyridine (60 ml) under an argon atmosphere maintaining the temperature below 30° C. After addition, the solution was stirred at 80° C. for 2 hours. The solution was cooled and poured onto 10% citric acid (500 ml), extracted with ethyl acetate, washed with water, the organic layer dried over MgSO4 and then evaporated to a brown oil. This oil was chromatographed on silica eluted using 5:1 hexane:ethyl acetate to give di t-butyl 4-[N,N-bis(2-chloroethyl)amino[-3-methylphenylcarbamoyl-L-glutamate as an oil. Yield=7.23 g. 28%. NMR: 8.42(s)1H; 7.1(m)3H; 6.36(d)1H; 4.13(m)1H; 3.50(m)4H; 3.31(m)4H); 2.3(m)2H; 2.23(s)3H; 1.9(m)2H; 1.4(s)9H; 1.35(s)9H.
Methanesulfonyl chloride (2.91 ml) was added dropwise to a solution of di t-butyl 4-[N,N-bis(2-hydroxyethyl)amino]benzylcarbonyl-L-glutamate (2.88 g) in pyridine (45 ml) under an argon atmosphere, maintaining the temperature below 25° C. After the addition the solution was stirred at 80° C. for 1 hour. The solution was then cooled and poured onto 10% citric acid (500 ml) extracted into ether, washed with water, the organic layer was dried over MgSO4 and then evaporated to a brown oil. This oil was chromatogaphed on silica eluting with hexane: ethyl acetate 2:1 to give di t-butyl 4-[N,N-bis(2-chloroethyl)amino]benzylcarbonyl-L-glutamate as an oil (1.3 g) (42%). NMR: 8.19(d)1H; 7.10(d)2H; 6.66(d)2H; 4.10(m)1H; 3.69(s)8H; 3.32(s)2H; 2.21(m)2H; 1.9(m)2H; 1.37(d)18H. Yield 1.32 g 42%.
A solution of di t-butyl 4-[N,N-bis-(2-chloroethyl)-amino]phenylcarbamoyl-L-glutamate (prepared as described in Example 3) (500 mg) in 98% formic acid (10 ml) was allowed to stand at ambient temperature for 24 h. The solution was evaporated to dryness and the residue chromatographed on Merck silica gel Art 9385 in dichloromethane/ethyl acetate/formic acid (7:2:1) to give the title compound as an oil which crystallized. m.p.=117°-9° C. EXAMPLE 21
TABLE 9______________________________________ ##STR23##Ex No       X     R             M.pt______________________________________22          Cl    F             111-114° C.23          Cl    Cl            oil24          Cl    CN            105-7° C.______________________________________
TABLE 10______________________________________ ##STR24##   R            m.p______________________________________   F            99-101   Cl           Oil   CN           151-4______________________________________ *NMR(DMSOd.sub.6) 8.15(d)1H: 8.05(q)1H; 7.3(d)1H; 4.65(t)2H(OH); 3.5(m)8H
TABLE 11______________________________________ ##STR25##X              R           m.p.______________________________________Cl             F           66-8° C.Cl             Cl          Oil*Cl             CN          106-9Br             F           66-68° C.______________________________________ *NMR(CDCl.sub.3): 8.3(d)1H; 8.1(q)1H; 7.25(d)1H; 3.75(t)4H; 3.6(t)4H.
TABLE 12______________________________________ ##STR26##                    m.p.X            R           (of oxalate salt)______________________________________Cl           F           146-8° C.Cl           Cl          118-21° C.Cl           CN          112-6° C.Br           F           134-6° C.______________________________________
TABLE 13______________________________________ ##STR27##X              R           m.p.______________________________________Cl             F           81-4° C.Cl             Cl          Oil*Cl             CN          Oil**Br             F           Oil***______________________________________ *NMR(CDCl.sub.3): 7.4(m)1H; 7.3(m)10H; 7.1(m)2H; 5.2(s)2H; 5.05(s)2H; 4.6(m)1H; 3.5-3.4(m)8H; 2.6-2.0(m)4H. **NMR(CDCl.sub.3): 8.25(s)1H; 7.6(m)2H); 7.4-7.2(m)10H); 7.1(d)1H; 6.2(d)1H(NH); 5.2(s)2H); 5.1(s)2H; 4.6(m)1H; 3.7-3.5(m)8H; 2.7-2.2(m)4H. ***NMR: 7.4-6.8(m)13H; 5.7(d)1H; 5.2(s)2H; 5.1(s)2H; 4.6(m)1H; 3.7-3.3(m)8H; 2.6-1.9(m)4H.
TABLE 15__________________________________________________________________________Ex No    ClCH.sub.2 CH.sub.2 N--       Aromatics               αCH                     CH.sub.2 --X                             Other__________________________________________________________________________25  3.69(m)8H       6.7-6.9(m)4H               3.97(m)1H                     1.79-1.99(m)2H                             2.2(t)2H                             6.7(bs)1H                             7.27(bs)1H                             7.89(bs)1H26  3.70(s)8H       6.70-6.93(-)4H               4.0(m)1H                     1.85-2.2(m)2H                             2.42(m)2H                             3.19(s)3H                             7.90(d)1H27  3.71(s)8H       6.69-7.38(m)8H               4.04(m)1H                     1.9-2.1(m)2H                             2.45(m)2H                             2.26(s)3H                             7.89(d)1H                             9.83(s)1H28  3.73(s)8H       6.7-6.9(m)4H               4.08(m)1H                     2.1-2.27(m)2H                             3.03(t)2H                             8.03(d)1H                             12.79(bs)1H29  3.69(s)8H       6.72-6.93(m)4H               4.38(m)1H                     3.46-3.8(m)2H                             8.12(d)1H30  3.70(s)8H       6.6-7.6(m)8H               4.05(m)1H                     1.8-2.3(m)2H                             2.45(m)2H                             3.50(s)2H                             7.9(d)1H                             9.9(s)1H                             12.5(broad s) 31*    3.67(s)8H       6.5-8.5(m)8H               4.10(m)1H                     1.8-2.3(m)2H                             2.5(m)2H32  3.70(s)8H       6.22-7.21(m)               4.03(m)1H                     1.8-2.2(m)2H                             2.50(m)2H                             7.79(d)1H                             9.52(s)1H__________________________________________________________________________ *NMR run with CD.sub.3 CO.sub.2 D added
The compound of Example 28 was prepared as described in Example 1 by hydrogenation of benzyl 4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-γ-(5-tetrazolyl)-.alpha.-amino-L-butyrate. The compound of Example 28 was also prepared by a second method as set out immediately hereinafter. A process analogous to that described in Example 1 was followed but using (S)-2-amino-4-(1 H-1,2,3,4-tetrazol-5-yl)butyric acid (Z. Grzonka et al. Tetrahedron, 33: 2299-2302, 1977) instead of L-glutamic acid dibenzyl ester in the reaction with 0-(4-[N,N-bis(2-chloroethyl)amino]-phenyl)-01 -(4-nitrophenyl)-carbonate. The reaction was carried out in dry DMF with 2 equivalents of triethylamine for 20 hours at 25° C. After evaporation to dryness, the residue was dissolved in ethyl acetate, washed with dilute citric acid, dried and evaporated to dryness. The product crystallised slowly from ethyl acetate. The residue was recrystallised from ethyl acetate to give the compound of Example 28 (m.p. 173°-5° C.).
N-Boc-α-benzyl-L-glutamate [E. Klieger et al, Ann. 673. 196-207, 1964] (10 g) in 50 ml dry dichloromethane was treated with dimethylaminopyridine (DMAP) (3.9 g) and dicyclohexylcarbodiimide (6.73 g). Methane sulphonamide (3.04 g) was then introduced to the reaction flask and the reaction continued at 25° C. for 20 hrs. Dichloromethane was evaporated and the residue redissolved in ethyl acetate. Ethyl acetate solution was then washed with 0.25M citric acid, followed by water and then dried over Na2 SO4 (anhydrous). Evaporation of the ethyl acetate extract gave a residue which was purified on silica using flash column chromatography using as eluent methylene chloride, then 5% methanol/methylene chloride and then 10% methanol/methylene chloride to give the Boc-protected acyl sulphonamide: ##STR37##
3.6 g of the Boc-protected acyl sulphonamide was suspended in 50 ml ethyl acetate followed by the addition of 8 equivalents of HCl saturated ethyl acetate (22.4 ml of 3.1M solution). The starting material sulphonamide went into solution and the reaction was allowed to continue for 20 hrs, under argon, at 25° C. α-Benzyl γ-mesyl-L-glutamine hydrochloride of the formula: ##STR38## was produced as a white solid which was filtered off and washed with anhydrous ether before drying in a dessicator. NMR: δ2.08(m)2H; 2.52(m)2H; 3.20(s)3H; 4.09(t)1H; 5.15-5.35(dd)2H, 7.39-7.50(m)5H, about 9 (broad) 3H.
α-Benzyl- 4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-glutamate was prepared as described in Example 1 from 0-(4-[N,N-bis(2-chloroethyl)amino]-phenyl)-0'-(4-nitrophenyl)carbonate but substituting α-benzyl-L-glutamate (Ref. C. Coutsogeorgopoulos et al J. Am. Chem. Soc. 83, 1885, 1961) for L-glutamic acid dibenzyl ester. Moreover the reaction was performed in dry DMF at 25° C. for 2 hrs. The product was purified by column chromatography (silica, Merck Art 9385) using ethyl acetate/hexane mixtures in the range 70/30 respectively to 100% ethyl acetate. The NMR data for the product is set out in Table 17.
α-Benzyl 4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-glutamate (300 mg) in dry tetrahydrofuran (5 ml) was treated with 1.1 equivalents of triethylamine. Isobutylchloroformate (0.08 ml, 1.1 equiv) in dry THF (10 ml) was added slowly to the reaction mixture at -25° C. After 15 minutes, 1.1 equivalents (0.08 ml) m-toluidine in dry tetrahydrofuran (5 ml) was added. The mixture was allowed to warm up to 25° C. and was then continuously stirred for 18 hrs. The reaction mixture was then filtered and the filtrate evaporated to give a residue which was purified by flash column chromatography using mixtures of ethyl acetate/hexane as eluent. Evaporation of appropriate fractions yielded α-benzyl 4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-γ-(3-methylphenyl)-L-glutamine as a white crystalline solid. The NMR data from this product is set out in Table 17.
1-amino-1-carboxy-2-(5-thiotetrazole)ethane was prepared by adding β-chloroalanine (18.50 g, 115.6 mmol) (Sigma Chemical Co) and 5-thiotetrazole (11.79 g, 115.6 mmol (prepared as described in European Patent Publication No. 33965) to 230 mls of a 2M sodium hydroxide solution with stirring. The reaction mixture was then heated from room temperature to 90° C. for 11/2 hours. The reaction mixture was then allowed to cool to room temperature and then cooled further with ice/methanol bath cooling, the mixture was acidified to pH4.0 with concentrated hydrochloric acid and stirring was maintained for 1/2 hour. The resulting precipitate was filtered off, washed with cold water and then with ether and filtered dry with suction to give 6.9 g. The mother liquor's pH was re-checked and found to have risen to about 5.5. The above procedure was repeated and a further 2.3 g of product obtained. This was dried under high vacuum.
NMR: δ3.30-3.50 ppm(m)2H; 4.16-4.22 ppm(q)1H; 7.47 ppm (broad) H2 O exchanging with NH2
Elemental Analysis--Expected C=23.2, H=4.4; N=33.8 (+1 mol of H2 O) Found C=23.1; H=4.4; N=33.7 (9.6% H2 O)
1-{4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonylamino}-1-benzyloxycarbonyl-2-(5-thiotetrazole)-ethane was prepared as described in Example 1 from 0-(4-[N,N-bis(2-chloroethyl)amino]-phenyl)-0'-(4-nitrophenyl)carbonate, but substituting γ-(5-thiotetrazolyl)-α-benzyloxycarbonyl-amino-L-butyric acid (Ref. Z Grzonka et al Tetrahedron Letters 33 2399-2302, 1977) for L-glutamic acid dibenzyl ester. Dry dimethylformamide was used as solvent and the reaction was carried out at 25° C. The reaction mixture worked up after 2 hours and the product purified by flash column chromatography on silica and eluting with a mixture made up of 2% formic acid in ethyl acetate/methylene chloride (3:1 by volume). The NMR data for this product is set out in Table 16.
3-amino-phenylacetic acid benzyl ester p-toluene sulphonic acid was prepared by adding 3-aminophenylacetic acid (10 g) and p-toluene sulphonic acid monohydrate (13.2 g) to benzyl alcohol (27.2 ml) in toluene (30 ml). The mixture was heated under reflux and the water formed collected in a Dean-Stark receiver. When all the water had been distilled off the mixture was allowed to cool to 25° C. before diluting with diethyl ester and placing in an ice-bath for 1 hr. The crystalline p-toluene sulphonate was filtered off and dried in a dissicator (23.5 g) NMR δ2.31(s)3H, 3.82(s)2H, 5.11(s)2H, 7.11(d)2H, 7.25-7.45(m)8H, 7.52(d)2H.
α-Benzyl 4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-γ-[3-(5-tetrazolyl)phenyl]-L-glutamine, for use in Example 31 was prepared as described in Example 27, but substituting 3-(tetrazol-5-yl)-aniline for m-toluidine. The 3-(tetrazol-5-yl)aniline required as a starting material was prepared as follows. To a solution of 5-(3-nitrophenyl) tetrazole (Finnegan W. G.; Henry R. A. and Lolquist R. J.A.C.S. 80:3908 (1958)) (52 g) in ethanol (2.51) was added 10% palladium on carbon (5 g) and the mixture stirred under an atmosphere of hydrogen for 16h. The catalyst was removed by filtration and the filtrate evaporated to give the desired starting material (40.5 g; m.p.=188°-9° C.).
3-(benzoyloxycarbonylamino)aniline used as an intermediate in preparation of Example 32, was prepared as follows. To a cold solution (0°) of m-phenylene diamine (10 g) in ethyl acetate (200 ml) was added aq KHCO3 (9 g in 300 ml water) with stirring. Benzyl chloroformate (13.2 ml) in ethyl acetate (100 ml) was added dropwise over ten minutes, stirred at 0° C. for one hour and then made acid (pH2) by addition of M.HCl (aq). The product was extracted into ethyl acetate (250 ml), washed with brine, dried over MgSO4 and reduced in "vacuo" to give on oil. The oil was chromatographed on Merck silica gel Art 9385 and eluted with hexane/ethyl acetate (7:3) to give 8 g of the intermediate as a low melting point solid (36% yield).
TABLE 16__________________________________________________________________________X         ClCH.sub.2 CH.sub.2 N--             Aromatics                      ArCH.sub.2 O                           αCH                                 CH.sub.2 --X                                         Other__________________________________________________________________________CH.sub.2 CO.sub.2 H     3.63(m)8H             6.62-7.31(m)9H                      5.11(s)2H                           4.14(m)1H                                 1.8-2.05(m)2H                                         7.89(d)1H                                         2.35(m)2HCH.sub.2 CONH.sub.2     3.71(m)8H             6.7-7.3(m)9H                      5.15(s)2H                           4.13(m)1H                                 1.8-2.03(m)2H                                         2.23(t)2H                                         6.77(s)1H                                         7.28(s)1H                                         8.08(d)1HCH.sub.2 CONHSO.sub.2 Me     3.71(m)8H             6.7-7.37(m)9H                      5.15(s)2H                           4.15(m)1H                                 1.8-2.1(m)2H                                         2.49(m)2H                                         3.19(s)3H                                         11.7(bs)1H                                         8.1(d)1HCH.sub.2 CONH--     3.71(m)8H             6.69-7.42(m)13H                      5.18(s)1H                           4.19(m)1H                                 1.9-2.2(m)2H                                         2.49(m)2H                                         2.25(s)3H                                         8.11(d)1H                                         9.83(s)1HCH.sub.2 --     3.71(m)8H             6.7-7.37(m)9H                      5.17(s)2H                           4.23(m)1H                                 2.1-2.3(m)2H                                         8.23(m)2H                                         3.03(t)2HCH.sub.2 CONH--     3.70(m)8H             6.6-7.6(m)18H                      5.10(s)2H                           4.19(m)1H                                 1.9-2.3(m)2H                                         2.45(m)2H                      5.17(s)2H          3.67(s)2H                                         8.13(d)1H                                         9.90(s)1HCH.sub.2 CONH--     3.70(s)8H             6.6-8.5(m)13H                      5.15(s)2H                           4.2(m)1H                                 1.9-2.3(m)2H                                         2.15(m)2H                                         8.15(d)1H                                         10.2(s)1H__________________________________________________________________________
a) A solution of the product bis(2-mesyloxyethyl)amino]phenoxycarbonyl-L-glutamic acid di-t-butyl ester (1.0 g) (See Scheme 9-compound 11) in acetonitrile (50 ml) was stirred with sodium iodide (1.0 g) at 70° C. for 20 h. The reaction mixture was filtered and the filtrate concentrated under vacuum. The residue was chromatographed on silica gel; eluted with ethyl acetate in cyclohexane (1:5) to obtain 4-[N,N-bis(2-iodoethyl)amino]phenoxycarbonyl-L-glutamic acid di-t-butyl ester (see Scheme 9-Compound 14) as an oil (0.75 g) 68% yield. NMR: 1.41 (s) 9H; 1.43 (s) 9H; 1.81-1.95 (m) 1H; 2.34 (m) 2H; 3.31 (t) 4H; 3.72 (t) 4H; 4.00 (m) 1H; 6.67 (d) 2H; 6.93 (d) 2H; 7.91 (d) 1H.
A solution of 4-[N,N-bis(2-hydroxyethyl)amino]phenoxycarbonyl-L-glutamic acid di-t-butyl ester (2.53 g) (obtained as described in Example 8-see also Scheme 4) in pyridine (9 ml) was stirred with methanesulphonyl chloride (1.8 ml) at 2° C. for 20 min followed by 80° C. for 11 min. The reaction mixture was partitioned between ethyl acetate and citric acid/water (10%). The organic phase was separated, washed with water, dried (Na2 SO4) and evaporated to dryness. The residue was chromatographed on silica gel; eluted with ethyl acetate in dichloromethane (1:9) to obtain 4-[N,N-bis(2-mesyloxyethyl)amino]phenoxycarbonyl-L-glutamic acid di-t-butyl ester (Compound 11 in Scheme 9) as an oil (0.95 g) 28% yield. NMR: 1.41 (s) 9H; 1.43 (s) 9H; 1.8-1.99 (m) 1H; 2.34 (m) 2H; 3.16 (s) 6H; 3.72 (t) 4H; 3.9 (m) 1H; 4.31 (t) 4H; 6.78 (d) 2H; 6.92 (d) 2H; 7.9 (d) 1H.
A solution of bis(2-mesyloxyethyl)amino]phenoxy-carbonyl-L-glutamic acid di-t-butyl ester (0.48 g) (compound 11 in Scheme 9-obtained as described in Example 8-see also Scheme 4) in acetonitrile (30 ml) was stirred with lithium bromide (0.26 g) at 70° C. for 22 h. The reaction mixture was filtered and the filtrate concentrated under vacuum. The residue was chromatographed on silica gel; eluted with ethyl acetate in dichloromethane (1:5) to obtain 4-[N,N-bis(2-bromoethyl)amino]phenoxycarbonyl-L-glutamic acid di-t-butyl ester (compound 12 in Scheme 9) as an oil (0.37 g) 83% yield. NMR: 1.41 (s) 9H; 1.43 (s) 9H; 1.8-1.98 (m) 1H; 2.34 (m) 2H; 3.58 (t) 4H; 3.76 (t) 4H; 3.97 (m) 1H; 6.72 (d) 2H; 6.93 (d) 2H; 7.93 (d) 1H.
Ethylene oxide (6.6 g) was added to 3-fluoro-4-nitroaniline (1.3 g) in glacial acetic acid (30 ml) and the reaction mixture was kept in a closed flask at laboratory temperature for 72 h. The solution was evaporated under reduced pressure to half of its original volume, diluted with saturated aqueous sodium chloride solution and extracted three times with ethyl acetate. The ethyl acetate extracts were combined, washed with saturated aqueous sodium bicarbonate, evaporated, and the residue was purified by flash column chromatography on silica gel. After elution with hexane containing 50% (v/v) ethyl acetate to remove unchanged starting material and monosubstituted product. Elution with ethyl acetate gave the product 2',2'-(3-fluoro-4-nitroanilino)diethanol (m.pt. 99°-101° C.).
The product so obtained (280 mg) was dissolved in dichloromethane (7.5 ml), pyridine (0.1 ml) was added and the solution was cooled in an ice/water bath. Thionyl chloride (0.25 ml) was added dropwise, with stirring. When the addition was complete the reaction mixture was heated under reflux for 1 hour and then left at laboratory temperature for 20 hours. The solution was diluted with dichloromethane (10 ml) and washed three times with water, dried over sodium sulphate, evaporated, and the residue was recrystallised from methanol to give the product [N,N-bis(2-chloroethyl]-3-fluoro-4-nitroaniline (m.pt 97°-98° C.).
The product so obtained (200 mg) in tetrahydrofuran (7.5 ml) was stirred for 16 hours in an atmosphere of hydrogen in the presence of palladium/charcoal (20 mg of 5% w/w). The catalyst was removed by filtration and the solvent was evaporated. The resulting residue was dissolved in the minimum volume of methanol and the crude product was precipitated by addition of excess diethyl ether saturated with hydrogen chloride. Recrystallisation from methanol/diethyl ether gave the product 4-[N,N-bis(2-chloroethyl)amino-2-fluoro-anilinium chloride (m.pt. 195°-200° C.,d).
TABLE 17__________________________________________________________________________ ##STR39##               % Expected                         % FoundEx. No.W              C  H  N   C  H  N__________________________________________________________________________36   CONH-p-C.sub.6 H.sub.5 COOH               51.5                  5.26                     7.83                         51.7                            5.10                               7.5037   CONH-n-C.sub.4 H.sub.9               51.9                  6.32                     9.09                         52.0                            6.38                               8.9738   CONH-sec-C.sub.4 H.sub.9               51.9                  6.32                     9.09                         52.2                            6.34                               9.0939   CONH-n-C.sub.3 H.sub.7               50.9                  6.07                     9.37                         50.6                            6.35                               8.9540   CONH-i-C.sub.3 H7               50.9                  6.07                     9.37                         51.1                            6.07                               9.4041   CONHC.sub.6 H.sub.11               54.1                  6.40                     8.60                         54.3                            6.73                               8.7242   COHNCH.sub.2 C.sub.6 H.sub.5               55.6                  5.48                     8.46                         56.0                            5.52                               8.2143   CONH-p-C.sub.6 H.sub.5 OH               53.0                  5.06                     8.43                         53.0                            5.24                               7.90__________________________________________________________________________
A solution of the intermediate dibenzyl 4-[N,N-bis(2-chloroethyl)amino]-phenylcarbamoyl-L-glutamate (1.138 g) in DMF (15 ml) was hydrogenated over 10% Pd/C for 16 hours. After filtration and evaporation in vacuo, the residue was dissolved in CHCl3 (20 ml). After 18 hours the crystalline precipitate was filtered off and dried in vacuo to obtain 4-[N,N-bis-(2-chloroethyl)amino]phenylcarbamoyl-L-glutamic acid. Yield, 730 mg (93%). After recrystallisation from acetone/CHCl3 microscopic rods formed m.p. 116°-118° C. NMR (CD3 COCD3): δ8.0(s)1H; 7.2(d)2H; 6.6(d)2H; 6.2(d)2H NH; 4.4(m)1H; 3.6(m)8H; 2.5-1.9(m)4H.
Dibenzyl glutamate p-tolunesulphonate (available Bachem U.K.; 0.25 g) was dissolved in dry methylene chloride (10 ml)-under argon and cooled to 0° C. Pyridine (0.162 ml) was added, followed rapidly by phosgene in toluene (1.93M, 0.311 ml). The solution was stirred at 0° C. for 2 hours, pyridine (0.05 ml) added and followed by 4-[N,N-bis (chloroethyl)amino]anilinium chloride in one portion. The mixture was stirred for 10 minutes at 0° C. and then for 18 hours at room temperature. The reaction mixture was then diluted with ethyl acetate and water. The organic layer was then washed in turn with dilute citric acid (2×), water and saturated brine; dried and evaporated to give the desired dibenzyl intermediate as a solid.
To a solution of triphosgene (1 g) in chloroform (80 ml) was added at 10° C. 4-[N,N-bis-(2-chloroethyl)amino]anilinium chloride (2.7 g). Whilst keeping the temperature at 10° C., triethylamine (4.15 ml) was added and the mixture was stirred and allowed to warm to ambient temperature for 15 minutes. To this mixture was added in one portion dibenzyl glutamate tosylate and triethylamine (1.7 ml). After 1.5 hours at ambient temperature the mixture was diluted with chloroform (100 ml), washed twice with water, dried and evaporated to dryness. The residue was chromatographed on Merck silica gell Art 9385, eluting with ethyl acetate hexane to give dibenzyl 4[N,N-bis(2-chloroethyl)amino]-phenylcarbamoyl-L-glutamate (2.5 g). m.p. 119°-22° C.
(a) 3-amino-phenylacetic acid benzyl ester p-toluene sulphonic acid was prepared by adding 3-aminophenylacetic acid (10 g) p-toluene sulphonic acid monohydrate (13.2 g) to benzyl alcohol (27.2 ml) in toluene (30 ml). The mixture was heated under reflux and the water formed collected in a Dean-Stark receiver. When all the water had been distilled off the mixture was allowed to cool to 25° C., before diluting with diethyl ether and placing in an ice-bath for 1 hour. The crystalline p-toluene sulphonate was filtered off and the product dried in a dessicator (23.5 g) NRM δ 2.31(s)3H, 3.82(s)2H, 5.11(s)2H, 7.11(d)2H, 7.25-7.45(m)8H, 7.52(d)2H.
(b) N Boc α benzyl L glutamate, (5 g) in 20 ml) dry DMF at 5° C. was heated with (1.1 eq; 2.45 g) hydroxybenzothiazole (HOBT) and the reaction mixture stirred at this temperature under argon for 10 minutes. (DCCI) Dicyclohexylcarbodimide (1.1 eq; 3.37 g) was then added and the reaction stirred for a further 10 minutes at 5° C. before allowing to warm to 25° C. and stirring for a further 45 minutes. 3 amino phenylacetic acid benzyl ester p-toluene sulphonic acid (the product obtained in (a)) (1.1 eq; 6 g) together with 1.1 eq triethylamine (2.23 ml) in 10 ml dry DMF was then added and the reaction mixture stirred at 25° C. for a further 20 hours. The precipitate of dicyclohexylurea was then filtered off, and the DMF filtrates evaporated to dryness. The residue was then redissolved in EtoAc. The ethyl acetate solution was then washed with NaHCO3 (aq), then brine and then dried over Na2 SO4 (anhydrous). Evaporation of the ethylacetate extract gave a residue which was then purified by flash column chromatography using 30,40 and 50% EtoAc/hexane mixtures as eluent. Evaporation of appropriate fractions yielded 5 g of product (compound 4 in scheme 11).
(c) The product obtained in (b) (5 g) was suspended in 10 ml ether and 5 ml dichloromethane added (to aid solubility) followed by 8 eq of saturated etheral/HCl. The reaction mixture was then allowed to stir for 20 hours at 25° C. The product was at this stage an immiscible oil. The ether was then evaporated and the residue azeotroped twice with toluene before adding ether and evaporating down to yield 5 g of product (compound 5 in scheme 11) as a yellow foam.
(d) 1.1 eq (0.66 g) of 1.1 carbonyldiimidazole in 20 ml dry THF at 5° C. was treated with a solution containing 1 g of 4[N.N bis(2-chloroethyl)amino]anilinium chloride. The reaction mixture was then stirred at 5° C. for 15 minutes before adding 1 eq (1.84 g) of the product obtained in (c) with 1.1 eq (0.56 ml) triethylamine in 10 ml dry THF, and stirred for a further 2 hours at 25° C. The triethylamine hydrochloride ppt was then removed by filtration and the THF filtrates evaporated and the residue redissolved in EtoAc. The ethyl acetate solution was then washed with water, followed by 0.25M citric acid, then brine and dried over Na2 SO4 (anhydrous). Evaporation of the ethyl acetate gave a residue which was purified by flash column chromatography using 30,40 and 50% ethylacetate/hexane mixture as eluent. Evaporation of appropriate fractions yielded the desired intermediate (compound 6 in scheme 11).
The process described in Example 16 was repeated using α-benzyl 4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-glutamic acid-γ-(3,5-dicarboxybenzyl)anilide in place of α-benzyl 4-(N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-glutamic acid-γ-anilide to obtain 4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-glutamic acid-γ-(3,5-dicarboxy) anilide as colourless crystals (m.p. 167°-170° C.).
1. A two component system, each component for use in association with the other, which comprises: (i) a first component that is an antibody or fragment thereof capable of binding a given antigen, the antibody or fragment thereof being conjugated to a carboxypeptidase G enzyme capable of converting a compound of Formula I or physiologically acceptable salt thereof into a cytotoxic drug; and
(ii) a second component that is a compound of Formula I or a physiologically acceptable salt thereof convertible under the influence of the carboxypeptidase G enzyme to the cytotoxic drug,
said compound of Formula I having the structure: ##STR41## wherein R1 and R2 each independently represents chlorine, bromine, iodine, OSO2 Me, or OSO2 phenyl (wherein phenyl is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from C1-4 alkyl, halogen, --CN and --NO2);
R5a, R5b, R5c and R5d each independently represents hydrogen, C1-4 alkyl optionally containing one double bond or one triple bond, C1-4 alkoxy, halogen, cyano, --NH2, --CONR7 R8 (wherein R7 and R8 are as defined below), --NH(C1-4 -alkyl), --N(C1-4 -alkyl)2 and C2-5 alkanoyl; or R5a and R5b together represent
c) --CH═CH--CH═CH--, --CH═CH--CH2 or --CH2 --CH═CH-- each optionally substituted with 1, 2, 3 or 4 substituents said substituents each independently selected from the group consisting of C1-4 alkyl, C1-4 alkoxy, halogen, cyano, nitro, C2-5 alkanoyl and --CONR7 R8 (wherein R7 and R8 are as defined below);
X represents O or NH;
Z represents --V--W where V is --CH2 --T-- in which T is --CH2 --, --O--, --S--, --(SO)--, or --(SO2)-- (provided that when V has sulphur or oxygen as its second atom, W is other than --COOH) and said group V optionally further carrying one or two substituents Q1 and/or Q2 on carbon; wherein Q1 and Q2 each independently represents C1-4 alkyl or halogen, or, when Q1 and Q2 are bonded to adjacent carbon atoms, Q1 and Q2 together may additionally represent a C3 -C4 alkylene radical optionally substituted with 1, 2, 3 or 4 substituents independently selected from the group consisting of C1-4 alkyl and halogen; and
(2) --(C═O)--O--R6 wherein R6 represents C1-6 alkyl, C3-6 cycloalkyl or aryl (as defined in 3 below) group;
(3) --(C═O)--NR7 R8 wherein R7 and R8 each independently represent hydrogen or a C1-6 alkyl, C3-6 cycloalkyl, aryl, heteroaryl linked to N via carbon or C7-9 aralkyl group wherein aryl is phenyl; heteroaryl is a 5 or 6 membered ring containing 1 to 3 heteroatoms selected from the group consisting of nitrogen and sulphur; the aryl moiety per se, the heteroaryl moiety and the aryl moiety of the aralkyl group may be substituted on carbon with 1-4 substituents selected from the group consisting of --COOH, --OH, --NH2, --CH2 --NH2, --(CH2)1-4 --COOH, tetrazol-5-yl and --SO3 H and the alkyl moiety may optionally carry a methyl group;
(6) PO3 R10 R10 (wherein the R10 radicals, which may be the same or different, are as herein defined)
(7) a tetrazol-5-yl group
(b) C1-6 -alkyl optionally substituted with substituents selected from the group consisting of aryl as defined below, C1-4 alkyl, CF3 or halogen; and
(c) perfluoro-C1-6 alkyl; wherein aryl is phenyl or phenyl having 1-5 substituents wherein the substituents are selected from the group consisting of halogen, --NO2, --CF3, C1-4 alkyl, C1-4 alkoxy, --NH2, --NHCOCH3, --CONH2, --OCH2 COOH, --NH(C1-4 alkyl), --N(C1-4 alkyl)2, --NHCOOC1-4 alkyl, --OH, --COOH, --CN and --COOC1-4 alkyl; and
(9) --M--Het wherein M represents S, SO or SO2 and Het represents a 5 or 6 membered heterocyclic aromatic ring linked to M via a carbon atom of the aromatic ring, said aromatic ring containing 1, 2, 3 or 4 heteroatoms selected from the group consisting of 0, N and S said aromatic ring optionally being substituted on carbon atoms of the ring with 1, 2, 3 or 4 substituents selected from the group consisting of --OH, --SH, --CN, --CF3, NH2 and halogen; or
salts of said compound of formula I.
2. The system according to claim 1 wherein R1 and R2 independently represent I, Br, Cl, OSO2 Me and OSO2 phenyl wherein phenyl is substituted with 1 or 2 substituents in the 2 and/or 4 positions.
12. The system according to claim 1 wherein the compound of Formula I is:
N-(4-[ N, N-bis(2-chloroethyl)amino]phenoxycarbonyl)- L-glutamic acid or salts thereof.
13. The system according to claim 1 wherein the compound of Formula I is:
N-(4-[ N, N-bis(2-iodoethyl)amino]phenoxycarbonyl)- L-glutamic acid or salts thereof.
14. The system according to claim 1 wherein the compound of Formula I is selected from:
a) ( S)-2(4-[ N, N-bis(2-chloroethyl)amino]phenoxycarbonylamino)-4-(1H-1,2,3,4-tetrazol-5-yl)butyric acid and salts thereof;
b) N-(4-[ N, N-bis(2-chloroethyl)amino]-3-fluorophenylcarbamoyl)- L-glutamic acid and salts thereof;
c) N-(4-[ N, N-bis(2-chloroethyl)amino]phenylcarbamoyl)- L-glutamic acid and salts thereof;
d) N-(4-[ N, N-bis(2-iodoethyl)amino]phenoxycarbonyl)- L-glutamic acid and salts thereof; and
e) N-(4-[ N, N-bis(2-chloroethyl)amino]phenoxycarbonyl)-L-glutamic acid-γ-(3,5-dicarboxy)anilide or salts thereof.
15. The system according to claim 1 wherein the compound of Formula I is:
N-(4-[ N, N-bis(2-chloroethyl)amino]phenoxycarbonyl)- L-glutamic acid-γ-(3,5-dicarboxy)anilide or salts thereof.
16. The system according to claim 1, wherein Het of W(9) is selected from the group consisting of pyridyl, pyrrolyl, 1,2,3-triazinyl and 1,2,4-triazinyl.
20. The system according to claim 10 wherein said enzyme is carboxypeptidase G2 (CPG2) and said compound of Formula I is:
21. The system according to claim 10 wherein said enzyme is carboxypeptidase G2 (CPG2) and said compound of Formula I is:
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