NEW ROUTE OF SYNTHESIS FOR TERTIARY ALKYL ESTERS

Abstract: 4-[(2-chloroethyl)-(2-hydroxyethyl)-amino]benzoyl amino acids of formula (IV), wherein X represents a group (a), where P represents hydrogen or a straight or branched chain C1-6 alkyl group, and salts thereof, and processes for their production. The compounds are useful for the production of nitrogen mustard prodrugs.

Description: NEW ROUTE OF SYNTHESIS FOR TERTIARY ALKYL ESTERS THIS INVENTION relates to pro-drugs and is particularly concerned with novel intermediates for the production of enzyme activatable pro-drugs. Over the years, many cytotoxic compounds have been discovered which are of potential use in cancer chemotherap Nitrogen mustards form one important family of such cytotox compounds. The clinical use of cytotoxic compounds in general and nitrogen mustards in particular has been limite because of the poor selectivity in the cytotoxic effect between tumour cells and normal cells. One approach to overcome this problem has involved the development of so-called pro-drugs which are derivative of the cytotoxic drug, often a relatively simple derivative whose cytotoxic properties are considerably reduced compare to those of the Parent drug. Numerous proposals have been made for the administration of such pro-drugs to patients under regimes whereby the pro-drug is only converted to the cytotoxic drug in the region of the intended site of action One particularly promising approach involves the conversion of the nitrogen mustard into a reaction product with an amino acid or oligopeptide to form a'pro-drug which can be converted to the parent nitrogen mustard at the site of intended action under the influence of an enzyme. This approach can be put into practice by the utilization of an antibody/enzyme conjugate in association with a pro-drug. The antibody/enzyme conjugate is one formed from an antibody to tumour associated antigen and an enzyme that will convert the pro-drug to the cytotoxic drug. In clinical practice, the antibody/enzyme conjugate is first administered to the patient and is allowed to localise in the region of the tumour to be treated. The pro-drug is then administered to the patient so that conversion of the pro-drug to the cytotoxic drug is also localised in the region of the tumour to be treated under the influence of the localised enzyme. Such a system is described in International Application PCT/GB88/00181, published as WO88/07378. Specific pro-drugs that can be used in the above- mentioned International Application are those based upon benzoic acid nitrogen mustards. The cytotoxic benzoic acid nitrogen mustard is converted, in accordance with the procedures described in our above-mentioned International Application, into an amide by reaction with an alpha-amino acid, the preferred alpha-amino acid being glutamic acid. In this case, the glutamic acid is linked to the nitrogen mustard through an amide bond formed between the carboxy group of the benzoic acid nitrogen mustard and the alpha- amino group of the glutamic acid. Other pro-drugs can be prepared based on benzoic acid nitrogen mustards where the carboxy group is converted into a derivative with an oligopeptide or other protecting group which is removed .in vivo, under the influence of an enzyme localised in the region of the tumour to be treated. Pro-drugs of the type described in the above- mentioned Application and other pro-drugs embodying the sa principle are administered as pro-drugs where the carboxy groups present in the glutamic acid or analogous residue, example aspartic acid, are in free carboxylic acid form. These pro-drugs are prepared by synthetic methods in which the carboxy group present in the glutamic acid or analogou reactant is protected. One prodrug of particular interest is the compound the formula (I) : 0 CO P II I wherein X represents a group -C-NH-CH-CH 2 CH2-C0 2 P, and where P is a protecting group or hydrogen. The protecting group may be a straight or branched chained C 1-6 alkyl, fo example ethyl or tertiary butyl. The compound of the form (I) can be prepared from a compound of formula (II) : The above mentioned International Application describes th synthesis of compound (I) from compound (II) via an intermediate (III) : by reaction of (III) with ethanesulphonyl chloride in pyridine. However, this reaction results in three major products, since the hydrogens of the two terminal hydroxy groups may each be substituted by a mesyl group and the resulting bis mesyloxy groups may in turn be substituted by chloro group. The three products have to be separated by column chro atography before removal of the protecting groups. Column chromatography is not suitable for large scale preparation of compounds and is therefore a restrictio on the quantity of the compound (I) which may be prepared on a commercially viable scale. It has now been found that the compound of formula (I) may be synthesised in high yield from a novel intermediate of formula (IV) : where X is as defined in formula (I) by reaction of (IV) wit methane sulphonyl chloride in an organic solvent, for examp triethylamine. Since IV is produced as a single main product, it may be purified by recrystallization. Accordingly, the present invention provides 4-[ (2-chloroethyl) (2-hydroxyethyl)-amino]benzoyl amino acid (CHA) for use in the production of 4-[ (2-chloroethyl) (2- mesyloxyethyl)amino]benzoyl amino acids (CMA) . A further embodiment of the invention provides a process for the production of CMA by reaction of CHA with methane sulphonyl chloride. References to CMA and CHA, and precursors thereof, the above and following text, are to be understood to inclu compounds in which the terminal carboxy groups of the amino acid moiety are protected by a group P as defined above. References to these compounds (CHA, CMA and precursors thereof) also include salts thereof. Preferably, these wil be pharmaceutically acceptable salts. Such salts include alkali metal (eg. sodium) , alkaline earth metal (eg. magnesium) and ammonium salts, and acid addition salts suc as the hydrochloride salt. The compound of formula IV may be synthesised from the novel intermediate (V) : Where X is as defined in formula I above by reaction of (V) with ethylene oxide in glacial acetic acid. The compound of formula (V) may be synthesised by the reaction of compound (II) with chloroacetaldehyde in the presence of a borohydride, such as a cyanoborohydride, eg. a metal salt of cyanoborohydride such as sodium cyanoborohydride or in the presence of a transition metal catalyst, eg. palladium or platinum, and hydrogen. The compound of formula II may be made either by reference to the above mentioned International Application or by reference to the Example given below. Thus in accordance with the present invention there is also provided: i) a process for the production of 4-[ (2-chloroethyl) amino]benzoyl amino acids (CA) by the reaction of 4-aminobenzoyl amino acid with chloroacetaldehyde and cyanoborohydride; ii) CA suitable for use in the production of CHA; iii) a process for the production of CHA by reaction of CA with ethylene oxide; iv) a process for the production of CMA which comprises reacting CA with ethylene oxide to produce CHA, and reacting the resulting CHA with methane sulphonyl chloride to obtain CMA; v) a process for the production of CMA which comprises reacting 4-aminobenzoyl amino acids with chloroacetalehyde to produce CA followed by the process described in (iv) above; i) a process for the production of CMA which comprises reacting 4-nitrobenzoyl amino acids with ammonium formate in the presence of a palladium catalyst on charcoal to produce the corresponding 4-aminobenzoyl amino acid, followed by the process described in (v) above, and; vii) the process described in paragraphs (iv) , (v) or (vi above in which the amino acid moieties of the CA, 4-aminobenzoyl amino acids or 4-nitrobenzoyl amino acids are protected by a group P (as defined above) , preferably a tertiary butyl ester group, and the resulting CMA is deprotected by treatment with trifluoroacetric acid or formic acid. During the synthesis of the compound of formula (I) the one or more carboxylic acid groups of the amino acid moiety will be protected. The protecting groups such as ethyl ester groups may be removed by alkaline hydrolysis wi sodium hydroxide, as described in the above mentioned International Application, or where the protecting groups a tertiary butyl ester groups by treatment with trifluoroacetric acid in a substantially non-aqueous medium, or with formic acid. After removal of the protecting group the de-protected prodrug may be recovered by lyophilisation (freeze drying) and stored in the dried state. When necessary, the de-protected prodrug may be transferred into vials and frozen, for example in liquid nitrogen, before freeze-drying. On industrial freeze-dryerε, pre-freezing i however not usually necessary. Lyophilisation may be performed by standard techniques known in the art. The invention is further illustrated by the followi specific reaction scheme and examples: REACTION SCHEME FOR EXAMPLES 1 AND 2 3 duii--tieerrti--buuutiyli glutamic acid Continued/. SUBSTITUTE SHEET REACTION SCHEME FOR EXAMPLES 1 AND 2 CONTINUED SUBSTITUTE SHEET EXAMPLE 1 Synthesis of the Glutamic acid di-t-butylester 10 g (68 mmol) glutamic acid in 290 ml t-butylacetate and 16.6 ml (0.15 mol) of 60 % perchloric acid were shaken for about 15 min until the amino acid and the perchloric acid were dissolved. The solution was kept at room temperature for 5 d. The mixture was cooled to -5"C (ice/NaCl) and extracted wit 0.5 N hydrochloric acid (4x) . The aqueous phase was neutralized with solid sodium carbonate and extracted with ether (6x) . The combined organic phases were washed with saturated aqueous sodium bicarbonate (2x) , dried over magnesium sulphate and evaporated to give 3.5 g (20%) of th glutamic acid di-t-butylester as a pale yellow liquid. SUBSTITUTE SHEET 1 Synthesis of Compound 2 1.1 Synthesis of Compound 1 3.5 ml (25 mmol) of triethylamine was added to a cooled (ice/NaCl) solution of 5.3 g (20 mmol) glutamicacid-d-t- butylester in 70 ml dry dichloromethane. At that temperature 3.7 g (20 mmol) p-nitrobenzoylchloride in 60 ml dry dichloromethane were added dropwise and the solution was stirred overnight at room temperature. The solution was washed with water (5x) , dried over magnesium sulphate and evaporated to form an orange oil. ■fø-NMR (CDCl 3 60 MHz): δ = 1.43 (s, 3CH 3 ) , 1.5 (s, 3CH 3 ) , 1.87 - 2.6 (m, 4H, CH 2 ), 4.4 - 4.83 (m, 1H, N-C-H) , 7.2 - 7.63 (d, broad, 1H, N-H) , 7.8 - 8.33 (m, 4H, arom. H) ppm. 1.2 Synthesis of Compound 2 1.1 g of 10 % palladium on charcoal and 6.6 g (0.105 mol) ammoniumformate were added to the cooled (ice-water) solution of the crude compound 1 in 60 ml dry methanol (exothermic reaction) . The reaction mixture was stirred at room temperature for 1/2 h. SUBSTITUTE SHEET During that time the product precipitated. Dichloromethane was added to dissolve the precipitate and the catalyst was removed by filtration through a celite pad. The filtrate wa evaporated and the residue was taken up in water and dichloromethane. The phases were separated. The organic phase was washed with water, dried over magnesium sulphate and evaporated to give 2. as colourless precipitate. Yield 6.55 g (87 %) of 2, p. 130*C (after recrystallisatio from ethanol/petrolether (40-60 β C). H-NMR (CDCl 3 60 MHZ) : 6 = 1.41 (s, 3CH 3 ) , 1.48 (s, 3CH 3 ) , 2.0 - 2.6 (m, 4H, CH 2 ) , 3.8 - 4.13 (s, broad, 2H exchangeable, NH 2 ) , 4.47 - 4.9 (m, 1H, N-C-H) , 6.4 - 6.87 ( 3H, 1N-H 2 arom. H) 7.6 (d, J = 8Hz, 2H, arom. H) ppm. 2. Synthesis of Compound 4 2.1 Synthesis of the Diester 3 1.5 ml of a 1:1 mixture of 6 N aqueous hydrochloric acid an ethanol, 1.5 ml (10 mmol) chloroacetaldehyde as a 45 % aqueous solution and 0.554 g (9 mmol) sodium cyanoborohydri were added successively to a solution of 3.05 g (8 mmol) dipeptide 2 in 60 ml dry methanol. SUBSTITUTE SHEET The reaction mixture was stirred at room temperature for 5 d, then acidified with some concentrated hydrochloric acid to pH 1 - 2 and evaporated. The residue was taken up in dichloromethane and water. The organic layer was separated and the aqueous layer was extracted with dichloromethane. The combined organic layers were washed with water (2x) and 10% aqueous sodiumbicarbonate solution (lx) , dried over magnesium sulphate and evaporated to give the crude 3.. Another batch was purified by flash chromatography (Rp= 0.44, Si0 2 , ether/petrolether 2 : 1) on silica gel with ether/petrolether (40-60 β C) (2 : 1) as eluant and recrystallization with a little dichloromethane, ether and petrolether (40-60 β C) to afford 3. as colourless crystals (mp. 144.5-144.7*C) . IR (CHC1 3 ): 3356 (broad, N-H), 3010 (C-H), 1712 (C=0) , 1610, 1500, 1437, 1148 cm" 1 . ^H-NMR fCDCl 3 220MHz) : £ = 1.43 (s, 9H, CH 3 ) , 1.50 (s, 9H, CH 3 ), 1.95-2.54 (m, 4H, CH 2 ) , 3.54 (t, J=5.3Hz, 2H, CH 2 ) , 3.7 (t, J-5.3HZ, 2H, CH 2 ), 4.5-4.82 (m, 2H, 1N-H, exchangeable, 1 N-C-H), 6.6 (d, J=8.8Hz, 2H, arom.H), 6.85 (d, J=8.4Hz, IH, 0=C-N-H) , 7.68 (d, J=8.8Hz, 2H, arom.H) ppm. •3^-C-NMR (CDCl3) : δ =-= 27.72 (1CH 2 ), 28.03 (6CH3), 31.70 (1CH 2 ), 42.93 (1CH 2 ), 44.87 (1CH 2 ), 52.62 (1 N-C-H), 80.65 ( 0-C-) , 82.17 (1 0-C-) , 111.96 (2 arom.C-H), 122.70 (1 arom. C-C=0) , 128.90 (2 arom.C-H), 150.10 (1 arom. C-N) , 166.73 (1C=0) , 171.62 (1 0-C=0) , 172.5 (1 0-C=0) ppm. Ms: m/e = 440 (M+) , 182 (100%). microanalysis: found C 59.98%, H 7.67%, N 6.30%, calculated for C 22 H 33 C1N 2 0 5 C 59.92%, H 7.54%, N 6.35%. 2.2 Synthesis of Dipeptide 4. Gaseous ethylene oxide was passed through a solution of the crude 3. in 50 ml glacial acetic acid at room temperature for 3/4 h. The solution was stirred in a stoppered flask at roo temperature for 2 d. The solution was diluted with 60 ml of water and extracted with dichloromethane (3x) . The combined organic phases were washed with water, dried over magnesium sulphate and evaporated in vacuo. The residue was purified by flash chromatography on silica gel with ether as eluant (Rp=0.22, Si0 2 ether) to afford 2.537 g (65%) 4. as colourless precipitate. SUBSTITUTE SHEET 759 mg 4. were recrystallized from a little dichloromethane, ether and petrolether (40-60 β C) to give 483 mg 4. as colourless crystals (mp. 97-99°C). IR (CHC1 3 ): 3429 (broad, NH, OH) , 3009 (C-H) , 1719 (C=0) , 1607, 1498, 1437, 1150cm" "1 . •3--H-NMR (CDCl3 220MHz) : δ = 1.42 (s, 9H, CH 3 ) , 1.49 (s, 9H, CH 3 ), 1.92-2.52 (m, 4H, CH 2 ) , 2.65 (S, broad, IH, exchangeable, OH), 3.54-3.72 (m, 4H, CH 2 ) , 3.72-3.86 (m, 4H, CH 2 ), 4.60-4.80 (m, IH, N-C-H), 6.68 (d, 2H, J=8.8Hz, arom.H), 6.88 (d, IH, J=8.4Hz, N-H), 7.68 (d, 2H, J=8.8Hz, arom.H) ppm. KS: m/e = 484 (M + ) , 448 (M+-HC1) , 190 (100%) microanalysis: found C 59.32%, H 7.71%, N 5.65%, calculated for C 24 H 37 C1N 2 0 6 C 59.43%, H 7.69%, N 5.78%. 3. Synthesis of Compound Σ> 1.5 ml of triethylamine and 0.5 ml (6.5 mmol) methanesulfonylchloride were added to 2.605 g (5.67 mmol) 4 . in 50 ml dichloromethane at 5"C. After stirring lh at 5'C the reaction mixture was poured into 300 ml of water. The phases were separated and the aqueous phase extracted with SUBSTITUTESHEET dichloromethane (2x) . The combined ' organic phases were washed with water (3x) , dried over magnesium sulphate and evaporated. The crude product 5 was crystallized from ether/petrolether (40-60 β C) to give 2.336 g (75%) as colourless crystals (mp. 74.5-75.5*C) . IR (CHC1 3 ) : 3430 (N-H), 3009 (C-H), 1722 (C=0) , 1648, 1608, 1496, 1368 (S0 2 -0) , 1175, 1153 cm" 1 . ^H-NMR (CDCI 3 . 220MHZ): δ = 1.44 (S, 9H, CH 3 ) , 1.51 (s, 9H, CH 3 ), 1.94-2.52 (m, 4H, CH 2 ) , 2.95 (s, 3H, CH 3 ) , 3.68 (t, J=6.2Hz, 2H, CH 2 ), 3.76-3.9 (m, 4H, CH 2 ) , 4.39 (t, J-5.5HZ, 2H, CH 2 ), 4.63-4.74 (m, IH, N-C-H), 6.71 (d, J=8.8Hz, 2H, arom. H) , 6.82 (d, J=7.5Hz, IH, N-H), 7.76 (d, J=8.8Hz, 2H, arom. H) ppm. EXAMPLE 2 The di-t-butyl ester 5 (3.00g, 5.33 mmol) produced in Example 1 is stirred in formic acid (98%, 600 ml) at 10*C for 48 h. It is then transferred into vials and frozen in liquid nitrogen, prior to lyophilisation on a freeze dryer. When all the acid has been removed, the vials are capped while still under vacuum on the freeze dryer. The deprotection is quantitative and gives the dicarboxylate 6 as a white powder as final product (2.40g, 100%) NMR. (Me 2 SO-d 6 ) δ 1.98 (m,2H, CH 2 CH 2 C0 2 H) , 2.34(t,2H, J = 7.3 HZ, CH 2 CH 2 C0 2 H) , 3.16 (s,3H, CH3SO3) , 3.77 (s, 4H, C1CH 2 CH 2 ), 3.83 (t, 2H, J = 5.4 Hz, CH 3 S0 3 CH 2 CH 2 ) , 4.33 (m,3H, CH 3 S0 3 CH 2 CH 2 & CH) , 6.82 (ABq, 2H, J = 8.9 Hz, arom H- 3,5), 7.77 (ABq, 2H, arom H-2,6), 8.27 (d, IH, J - 7.8Hz, NH) mass spectrum FAB m/z 451([M+H + ] ,17%) , 401(M-C1CH 2 ,7%) , 304(M-NHCH(CO 2 H)CH 2 CH 2 CO 2 H, 100%) Anal. C 17 H 23 N 2 O 8 ClS-0.2H 2 O) Expected Found C 44.92 44.89 H 5.19 5.41 N 6.17 5.78 Cl 7.79 7.83 7.05 6.97 SUBSTITUTE SHEET EXAMPLE 3 The di-t-butyl ester 5 (92 g, 163 mmol) produced in Example 1 is stirred in formic acid (98%, 18.4 1) at 10*C f 48 hours. It is then transferred into vials and placed in LSL-secfroid FCFV600 freeze dryer (Life Sciences Labs) The solution is frozen in situ prior to lyophilisation when all the acid has been removed, the vial are capped while still under vacuum on the freeze dryer. T dicarboxylate 6 is obtained as a white powder (68 g, 92%) . Anal. C 17 H 23 N 2 0 8 C1S Expected Found C 45.28 45.09 H 5.14 5.41 N 6.21 6.41 Cl 7.86 7.76 S 7.11 7.40 SUBSTITUTE SHEET

Claims: CLAIMS 1. A 4-[ (2-chloroethyl)-(2-hydroxyethyl)- amino]benzoyl amino acid of formula (IV) 0 CO P II ' I wherein X represents a group -C-NH-CH-CH 2 CH 2 -C0 2 P where P represent hydrogen or a straight or branched chain Cι_g alkyl group, and salts thereof. 2. A compound according to claim 1 wherein P is an ethyl or tertiary butyl group. 3. A process for the production of a 4-[(2- chloroethyl)-(2-hydroxyethyl)-amino]benzoyl amino acid of formula (IV) 0 C0 P II I wherein X represents a group -C-NH-CH-CH 2 CH 2 -C0 2 P where P represent hydrogen or a straight or branched chain C-^g alkyl group, and salts thereof, which comprises reaction of a - 27 - compound of formula (V) where X is as defined above, with ethyle -ne oxide. 4. A process according to claim 3 wherein the compound of formula (V) is obtained by reaction of a compoun of formula (II) or salts thereof, where X is as defined in claim 3, with chloroacetaldehyde in the presence of a borohydride or a transition metal catalyst and hydrogen. 5. A process according to claim 4 wherein the compound of formula (II) is obtained by reaction of a compound of formula (Il-a) wherein X is as defined in claim 3, with ammonium formate i the presence of a palladium catalyst. 6. A process according to any one of claims 3 to SUBSTITUTE SHEET which further includes converting the compound of formula (IV) or a salt thereof obtained into a compound of formula (I) or a salt thereof wherein X is as defined in claim 3, by reaction with methane sulphonyl chloride. 7. A process according to any one of claims 3 to 6 wherein in the moiety X, the groups P are both ethyl. 8. A process according to any one of claims 3 to 6 wherein in the moiety X, the groups P are both t-butyl. 9. A process according to claim 8 which further includes removal from the moiety X of both t-butyl groups by deprotection in the presence of formic acid. 10. A 4-[ (2-chloroethyl) mino]benzoyl amino acid of formula (V) 0 C0 P • II I wherein X represents a group -C-NH-CH-CH 2 CH 2 -C0 2 P SUBSTITUTE SHEET where P represents hydrogen or a straight or branched chain C-^g alkyl group, or a salt thereof. 11. A process for the production of a compound of formula (I-a) or a salt thereof which comprises deprotection of a compound of formula (I-b) with formic acid.

Inventors: MANN JOHN, SPRINGER CAROLINE, MANN, JOHN, SPRINGER, CAROLINE
Applicants: MANN JOHN, SPRINGER CAROLINE, MANN, JOHN, SPRINGER, CAROLINE
Application year: 1990
Main classification: C07C237
IPC codes: C07B61, C07C231, C07C237, C07C303, C07C309