Intermediates for difluoroglutamic acid conjugates with folates and anti-folates for the treatment of neoplastic diseases

Abstract: This invention relates to intermediates for the preparation of certain conjugates of folates and antifolates with difluoroglutamic acid which are useful in the treatment of patients suffering from certain neoplastic diseases including leukemia, melanomas, carcinomas, sarcomas and mixed neoplasias.

Description: This invention relates to novel intermediates for the preparation of novel difluoroglutamic acid conjugates with folates and anti-folates which are useful in the treatment of neoplastic diseases. Folates and classical antifolates such as MTX¹ are converted intracellularly to poly(γ-glutamyl) metabolites by the enzyme folylpolyglutamate synthetase. PteGlu n + ATP + L-Glu → PteGlu n+1 + ADP + P i Since it is now known that folylpolyglutamates are essential to the proper functioning of folate metabolism, and antifolylpolyglutamates are implicated in the cytotoxic action of classical antifolates such as MTX, folylpolyglutamate synthetase has become an important enzyme for study in folate biochemistry and biochemical pharmacology. In this regard, the specificity of this enzyme for pteroyl and L-glutamate substrates has been extensively investigated. For pteroyl substrates, the structure of the heterocyclic component can vary considerably, but a terminal L-glutamate residue has been shown to be absolutely required for substrate activity in all reports to this time. Specificity for the incoming amino acid is strict, but not absolute. L-homocysteic acid and D,L- erythro - or D,L- threo -4-fluoroglutamate can all serve as efficient alternate substrates, but in each case incorporation causes chain termination. Chain termination by the 4-fluoroglutamate diastereomers demonstrates the stringent specificity for L-glutamate at the γ-glutamyl acceptor site. F₂Glu of the formula 3A is a potent, concentration-dependent inhibitor of poly(γ-glutamylation) using [³H]Glu and either methotrexate (4-NH₂-10-CH₃PteGlu) or tetrahydrofolate as substrates. Applicants have determined that F₂Glu acts as an alternate substrate, but in contrast to the previously characterized alternate substrate 4-fluoroglutamate (McGuire and Coward, J. Biol. Chem. 260 : 6747 (1985)), it did not terminate polyglutamate chain elongation. Instead, F₂Glu promotes chain elongation. Thus, synthesis of products from [³H]methotrexate containing 1 and 2 additional amino acid residues occurs at a substantially higher rate in the presence of F₂Glu when compared to identical reactions in the presence of Glu; this is more pronounced for the product containing 2 additional residues. The increased rate of addition is not solely a function of ligating F₂Glu to the internal Glu or to a previously incorporated F₂Glu, since ligation of Glu to 4-NH₂-10-CH₃PteGlu-γ-(3,3-difluoroglutamate) is also enhanced. These results are consistent with F₂Glu enhancing the synthesis of poly(γ-glutamate) metabolites at the level of either the incoming amino acid (glutamate analog) or the γ-glutamyl acceptor species. F₂Glu is thus the first glutamate analog which enhances chain elongation catalyzed by folylpolyglutamate synthetase. The compound 3,3-difluoroglutamate (F₂Glu) is used to prepare conjugates with certain folates and antifolates of formula 1 wherein R₁ is -NH₂, H, or -CH₃; R₂ is -NH₂ or -OH; R₃ is H, (C₁-C₄)alkyl, allyl, or propargyl, and X, Y, and Z are each independently a nitrogen atom or a CH group; and the pharmaceutically acceptable salts thereof. The structure 1 conjugates are useful in the treatment of tumors and psoriasis and in this context crossreference is made to copending EP-A 451 836. The compounds of the invention, F₂Glu and certain PABA-F₂Glu compounds are intermediates used in the preparation of the formula 1 compounds. F₂Glu and the difluoro glutamate portion of other compounds of this invention possess a chiral center and thus the compounds of this invention exist as pairs of stereochemical isomers. While the enantiomers, corresponding to the natural configuration of L-glutamic acid, are preferred, applicants contemplate that both the individual isomers and mixtures of the individual isomers including the racemic mixture are within the scope of this invention. Unless otherwise indicated the compounds of this application are mixtures of the two stereoisomers. Further, those compounds of formula 1 wherein Z is a CH group and wherein R₃ is other than a hydrogen have a second chiral site. The stereochemical configuration around this second chiral site is not critical and applicants again intend that the separate isomers and mixtures be included within the scope of this invention. A racemic mixture of isomers with regard to this second chiral site is preferred. The compounds of formula 1 contain two carboxylic acid moieties and can form mono or di basic salts. Illustratively, these salts include those of alkali metals, as for example, sodium and potassium; alkaline earth metals, such as calcium and magnesium; light metals of Group IIIA including aluminum; and organic primary, secondary and tertiary amines, as for example, trialkylamines, including triethylamine, procaine, dibenzylamine, 1-ethenamine, N,N'-dibenzylethylenediamine, dihydroabietylamine, N-(lower)alkylpiperidine, and any other suitable amine. Sodium salts are preferred. The compounds of formula 1 can also form pharmaceutically acceptable acid addition salts with any non-toxic, organic or inorganic acid. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulphuric and phosphoric acid and acid metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids which form suitable salts include the mono, di and tricarboxylic acids. Illustrative of such acids are, for example, acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, salicylic, 2-phenoxybenzoic and sulfonic acids such as methane sulfonic acid and 2-hydroxy-ethane sulfonic acid. Hydrochloride salts are preferred. The compounds of formula 1 can be prepared by protecting group removal from the product of the reaction of the formula 2 folate or antifolate derivative wherein R₁, R₂, R₃, X, Y, and Z are as defined above for the formula 1 compounds and a suitably protected derivative of F₂Glu such as the t-butyl ester derivative of formula 3, F₂Glu(OtBu)₂. The coupling of the formula 2 compound and the protected F₂Glu derivative of formula 3 can be accomplished in any appropriate manner for preparing an amide from an amine and a carboxylic acid. Applicants have coupled the formula 2 and 3 compounds using diethyl phosphorocyanidate ((EtO)₂P(=O)CN). This reaction is performed by adding (slow dropwise addition) a solution of the appropriate formula 2 compound to a solution of ((EtO)₂P(=O)CN) and an acid scavenger such as pyridine, or preferably triethylamine. The resulting mixture is allowed to react at from about 0°C to about 60°C, preferably at ambient temperature for from about 1 hour to about 10 hours, preferably from about 2 to about 5 hours, until the formula 2 compound is substantially reacted with the phosphorocyanidate. To this mixture is then added a solution of F₂Glu(OtBu)₂ and the resulting reaction is allowed to proceed for from about 24 hours to about 100 hours, generally for about 72 hours at from about 0°C to about 60°C, preferably at ambient temperature. The crude product is isolated, for example, by solvent evaporation, washed with water and dried. The crude product is then treated with trifluoroacetic acid (neat) to remove the t-butyl ester protecting groups. The desired formula 1 compound is isolated and purified, for example, by column chromatography. Suitable solvents for the above described coupling reaction include any solvent in which the various reactants are soluble and which do not interfere with the reaction such as preferably dimethylformamide (DMF). The compounds of formula 1 wherein Z is a nitrogen atom can be prepared by an alternative procedure in which a p-aminobenzoic acid (PABA) derivative of formula 4 wherein R₃ is as defined above for the formula 1 compounds is reacted with the formula 3 compound, F₂Glu(OtBu)₂, to give the t-butoxy protected intermediate compound of formula 5 wherein R₃ is as defined above for the formula 1 compounds. This coupling can be accomplished in any suitable manner such as by the procedures described in McGuire, et al., Cancer Research 1989, 49, 4517-4525; Galican et al., Proc. Natl. Acad. Sci. , USA, 1985, 82, 2598-2602; Piper and Montgomery, J. Org. Chem., 42(2) 208-211 (1977); Taylor, et al., J. Med. Chem. 28 , 913-921 (1985); Taylor, et al., J. Med. Chem. 28 , 1517-1522 (1985); Jones, et al., J. Med.Chem. 32 , 847-852 (1989). The formula 5 intermediate is then condensed with the bromomethyl derivative of formula 6 wherein R₁, R₂, X and Y are as defined for the formula 1 compounds above to produce the t-butoxy protected compounds which upon hydrolysis to remove the t-butoxy protecting groups such as with trifluoroacetic acid yield the desired compounds of formula 1. This reaction can be accomplished by allowing a mixture of the formulae 5 and 6 compounds and dimethylacetamide (Me₂NAc) to react for about 2 to about 12 hours, preferably for about 4 hours, at a temperature of from about 25°C to about 80°C, preferably at about 50-55°C, and then leaving the reaction mixture stand at ambient temperature for from about 6 to about 24 hours, preferably for about 12 hours. The Me₂NAc is then removed under reduced pressure and the crude material placed in an ice bath and treated with trifluoroacetic acid, allowing the mixture to warm to room temperature after addition is complete. After standing for from about 1 to about 6 hours, typically for about 4 hours, the TFA is removed under reduced pressure to give the crude desired product of formula 1. This product can be purified by, for example, chromatography (DEAE-cellulose with an NH₄HCO₃ gradient). This condensation reaction is more completely described in the references mentioned in the preceding paragraph. The selection and utilization of particular blocking groups are well known to one of ordinary skill in the art. In general, blocking groups should be selected which adequately protect the amino or hydroxy groups in question during subsequent synthetic steps and which are readily removable under conditions which will not cause degradation of the desired product. Examples of suitable hydroxy protecting groups are C₁-C₆ alkyl, tetrahydropyranyl, methoxymethyl, methoxyethoxy-methyl, t-butyl, benzoyl, and triphenylmethyl. The term C₁-C₆ alkyl refers to a saturated hydrocarbyl radical of one to six carbon atoms of straight, branched, or cyclic configuration. The benzoylated derivative can be formed by reacting the unblocked compound with benzoyl chloride in the presence of pyridine. Examples of suitable amino protecting groups are benzoyl, formyl, acetyl, trifluoroacetyl, phthalyl, tosyl, benzenesulfonyl, benzyloxycarbonyl, substituted-benzyloxycarbonyl (e.g., p-chloro,p-bromo, p-nitro, p-methoxy, o-chloro, 2,4-dichloro, and 2,6-dichloro derivatives), t-butyloxycarbonyl (Boc), t-amyloxycarbonyl, isopropyloxycarbonyl, 2-(p-biphenyl)-isopropyloxycarbonyl, allyloxycarbonyl, cyclopentyloxycarbonyl, cyclohexyloxycarbonyl, adamantyloxycarbonyl, phenylthiocarbonyl, and triphenylmethyl. Preferred amino protected compounds include the benzoyl derivative, made by reacting the unblocked compound with benzoyl chloride, and the acetyl derivative, made by reacting the unblocked compound with acetic anhydride. Compounds 1 prepared using the intermediates of the present invention provide a method of treating a patient suffering from a neoplastic disease comprising administering to the patient a therapeutically effective antineoplastic amount of a compound of formula 1. By administering a therapeutically effective antineoplastic amount of a compound of formula 1 to a patient suffering from a neoplastic disease, an antineoplastic effect is provided. The term "patient" refers to a warm-blooded animal, such as primates, including humans, sheep, horses, cattle, pigs, dogs, cats, rats and mice. With regard to the end compounds of formula 1, cross reference is made to the copending EP-A 451 836. EXAMPLE 1 N-[4-([(2,4-Diamino-6-pteridinyl)methyl]methylamino)benzoyl]-4,4-D-Fluoroglutamic acid A mixture of N-CH₃PABA-F₂Glu(OBu)₂ (0.201 mmol) and 2,4-diaminopteridine-6-bromomethyl hydrobromide (75 mg, 0.223 mmol) in Me₂NAc (2.5 ml) is stirred at 50-55°C for 4 hr and then left at ambient temperature overnight. Me₂NAc is thenremoved under reduced pressure. This crude material is placed in an ice bath, and 2 ml of trifluoroacetic acid is added with stirring. After 10 min, the reaction mixture is allowed to warm to ambient temperature and is stirred for 4 hr. The trifluoroacetic acid is removed under reduced pressure giving the crude product. The crude product is purified by chromatography on DEAE-cellulose with an NH₄HCO₃ gradient (15-600 mM). Lyophilization of the column effluent containing the desired product gives the desired pure product. EXAMPLE 2 4-Amino-4-deoxy-10-methylpteroyl[D,L-erythro,threo-4,4-(difluoro)glutamic acid] (1, R₁,R₂=NH₂; X,Y,Z=N;R₃=CH₃) In a 15 mL round bottom flask fitted with a drying tube is placed 2.5 mL DMF, 23 µL Et₃N (0.16 mmol) and 25 µL (0.16 mmol) diethyl phosphorocyanidate. The diaminopteroate (1,R₁,R₂=NH₂; X,Y,Z=N;R₃=CH₃)(0.16 mmol) is then added and the reaction solution is stirred at ambient temperature. After 3 h, an additional 5 µL (0.04 mmol) of diethyl phosphorocyanidate is added. F₂Glu (0.16 mmol) is dissolved in 1 mL DMF and added to the reaction flask and stirring continued for 72 h at ambient temperature. Solvent DMF is removed in vacuo , the residue is dissolved in 35 mL CHCl₃ and washed with 1% NH₄OH (2 x 20 mL) to remove unreacted starting material. The organic layer is washed with 20 mL H₂O, then dried over Na₂SO₄ and evaporated in vacuo to provide crude blocked product. This coupled material is dissolved in neat TFA (3 mL) and progress of the de-esterification reaction is monitored by TLC. After 24 h, the solvent TFA is removed by rotary evaporation and the residue dried in vacuo. The crude product is dissolved in 20 mL H₂O, the pH adjusted to 8 with dilute NH₄OH, and the sample volume brought to 160 mL in order to obtain sufficiently low conductance prior to loading on a DEAE-cellulose (Whatman DE-52) column (30 x 1 cm). The column is washed with 100 mL water and the desired formula 1 compound is eluted from the column with a linear gradient formed from 175 mL of 15 mM NH₄HCO₃ and 175 mL of 500 mM NH₄HCO₃. EXAMPLE 3 β,β-Difluoroglutamic acid bis-t-butyl ester 3a 2,2-Difluoropent-4-enamide To a stirred solution of 2,2-difluoropent-4-enoic acid (27.2 g, 0.2 mol) in hexane (200 mL) in a three-neck flask equipped with a reflux condenser and N₂ inlet is added DMF (20 drops, catalytic) and oxalyl chloride (20 mL, 0.23 mol). The mixture is stirred at room temperature for 2 h, when no further evolution of gas is seen. The solution is cooled in an ice/brine bath, the N₂ inlet is replaced by an empty CaCl₂ tube (to trap any solid material blown out of the flask), and a stream of NH₃ gas is passed into the flask. The gas flow is maintained for 30 min after the initial vigorous reaction has subsided. The mixture is then poured into H₂O (1 L) and Et₂O (0.5 L). The glassware is washed with H₂O and Et₂O, and the washings are added to this mixture. Celite is added and insoluble material is removed by filtration. The phases are separated. The Et₂O phase is washed once with brine, dried over Na₂SO₄, and concentrated. The aqueous phase is extracted twice with CH₂Cl₂. The combined extracts are washed once with brine, dried over Na₂SO₄, combined with the residue of the ethereal phase, and concentrated. The residue is distilled to give the title amide (20.6 g, 76%) bp 100-110°C 1333,22 Pa (10 Torr) as a slightly yellow oil which solidifies on cooling, mp 33-35°C. ¹H NMR (CDCl₃) δ 7.0-6.0 (2H, br.d), 5.75 (1 H, ddt, J = 18, 9, 6 Hz), 5.3 (2 H, m), 2.87 (2 H, dt, J = 6, 17 Hz). ¹⁹F NMR (δ C₆F₆ = O) δ - 56.0 (t, J = 17 Hz). Anal. Calcd for C₅H₇F₂NO: C, 44.45; H, 5.22; N, 10.37. Found: C, 42.03; H, 4.74; N, 9.33. 3b 2,2-Difluoropent-4-enonitrile To a solution of the above amide (3a) (20.6 g, 0.152 mol) in dry pyridine (25 mL, 0.31 mol) under N₂ cooled in an ice/brine bath is added dropwise over 1½ h, trifluoroacetic anhydride (TFAA, 23.5 mL, 0.166 mol). The reaction mixture becomes solid, and is allowed to stand at room temperature for 4 h. The flask is then equipped for distillation under N₂ and heated in an oil bath (finally at 130°C). The distillate is collected to give 17.7 g of colorless oil which fumes in air, bp 70-80°C 101247.2 Pa (760 Torr). Analysis by NMR indicates that the composition of the distillate is nitrile:TFAA:pyridine 80:12:8. Hence the yield of nitrile is 0.116 mol (76%). ¹H NMR (CDCl₃) 6.0-5.5 (1 H, m), 5.4 (2 H, m), 2.93 (2 H, dt, J = 6, 15 Hz). ¹⁹F NMR (δ C₆F₆ = O) δ - 71.7 (t, J = 15 Hz). 3c 4,4-Difluoroocta-1,6-dien-5-ylamine hydrochloride Propenylmagnesium bromide is prepared by the addition of a solution of 1-propenyl bromide (27.22 g, 0.225 mol) in THF (225 mL) to magnesium turnings (5.47 g, 0.225 mmol) at such a rate as to maintain the mixture warm but not boiling. When the addition is complete the mixture is stirred for 20 min, then diluted with THF (300 mL) and cooled to -15°C. To this mixture is added a solution of the nitrile (0.116 mmol) prepared in example 3b in THF (80 mL) at a rate such that the temperature of the reaction mixture remains within the range - 15 < T < -10°C. The mixture is stirred for a further 1 h in this temperature range, and then cooled to -40°C. A cold (-40°C) solution of NaBH₄ (6.4 g, 0.169 mol) in MeOH (650 mL) and water (30 mL) is added in one portion. The cooling bath is removed and the mixture is stirred for 2 h, then poured into 6 N HCl (800 mL). The organic solvents are removed by rotary evaporation, and the aqueous solution is washed twice with CH₂Cl₂. The solution is then basified to pH 10 with NaOH pellets (ice-cooling is necessary), and saturated with NaCl. Celite is added and the precipitated Mg(OH)₂ is removed by filtration. The solid residue is washed with CH₂Cl₂. The filtrate is extracted with CH₂Cl₂ three times, and the combined extracts and residue washings are dried over Na₂SO₄, filtered, acidified with Et₂O/HCl, and evaporated to give the title amine salt as a pale yellow solid (3 g). This material is not purified at this stage. 3d N-(t-Butyloxycarbonyl)-4,4-difluoroocta-1,6-dien-5-ylamine The above crude salt of example 3c (35 g) is dissolved in water (200 mL) and dioxan (200 mL). Solid KHCO₃ is added to give a mixture which is neutral to pH paper, to which is added further KHCO₃ (12 g, 0.12 mol) and di-t-butyldicarbonate (34 g, 0.156 mol). The mixture is stirred at room temperature overnight and then saturated with NaCl. The phases are separated. The aqueous phase is extracted with hexane (x 3). The extracts are combined with the organic phase and washed with water (x 3). The combined aqueous washings are extracted once with hexane. This extract is combined with the organic phases which are then washed with brine, dried over Na₂SO₄, and concentrated. The residue is purified by flash chromatography (eluant CH₂Cl₂/pentane 1/1) to give the title carbamate (19 g, 65%), R f 0.29, as an oil which soldifies on standing. ¹H NMR (CDCl₃) δ 6.0-5.5 (2 H, m); 5.5-4.5 (5 H, m); 2.67 (2 H, dt, J = 7, 16 Hz); 1.73 (3 H, dd*, J = 7,1 Hz); 1.47 (9 H, s). ¹⁹F NMR (δ C₆F₆=0) δ - 53.2 (m). 3e β,β-Difluoroglutamic acid hydrochloride To an ice-cold solution of KMnO₄ (52.56 g, 0.33 mol) in H₂O (1.2 L) is added a solution of the carbamate prepared in example 3d (10.95 g, 0.042 mol) in AcOH (160 mL). The mixture is stirred overnight at room temperature, during which time a brown solid precipitates. Solid sodium disulphite is added to decolorize the mixture, followed by conc. HCl to give a solution of pH 2. This solution is extracted with Et₂O (3 x 500 mL), then saturated with NaCl and further extracted twice with Et₂O. The combined organic fractions are washed once with brine and concentrated. The residue is taken up in 1 N HCl (500 mL) and concentrated. The residue is once again taken up in 1 N HCl, the mixture is warmed, and activated charcoal is added. The mixture is stirred for 10 min, filtered, and the filtrate is evaporated to dryness. Extraction of the residue with hot isopropanol gives the crude amino acid (2.22 g). Further extraction of the isopropanol insoluble material with EtOH does not give any useful material. ¹⁹F NMR (D₂O: δ CF₃CO₂H = 0) δ + 23 (ddt, J = 280, 5, 21 Hz), + 27 (ddt, J = 280, 22, 9 Hz); + 28 (t); relative integrals 5:5:3. 3f β,β-Difluoroglutamic acid bis-t-butyl ester The crude acid of example 3e (2.22 g) is suspended in t-butyl acetate (500 mL), and HClO₄ (2.16 mL of 70% aqueous solution, 25 mmol) is added to the mixture. The mixture is stirred at room temperature for 48 h, and then extracted with H₂O (2 x 200 mL). The combined extracts are washed with CH₂Cl₂ (2 x 100 mL) and then basified to pH 10 (4 N NaOH). The solution is extracted with CH₂Cl₂ (3 x 100 mL). The combined extracts are washed with brine, dried over Na₂SO₄, and concentrated to one third of their volume. This solution can be used for the next step without further treatment. A sample evaporated more completely shows ¹H NMR (CDCl₃) δ 4.10 (1 H, t, J = 14 Hz), 3.10 (1 H, t, J = 15 Hz), 3.00 (1 H, t, J = 15 Hz), 1.5 (18 H, 2 s); ¹⁹F NMR (δ C₆F₆ = O) δ - 58.7 (dt, J = 14, 15 Hz). EXAMPLE 4 β,β-Difluoroglutamic acid hydrochloride To a solution of the BOC-bis-ester of example 3(300 mg, 0.76 mmol) in dry Et₂O (10 mL) is added saturated Et₂O/HCl (3 mL). The mixture is stirred at room temperature (CaCl₂ tube protection) for 6 days. The precipitated white solid is collected and washed with Et₂O. Analysis by TLC and NMR indicates that cleavage of the esters is not complete. The material is taken up in 1 N HCl (5 mL) and stirred at room temperature overnight. The reaction is still not complete. A few drops of conc. HCl are added, and the mixture is stirred for 3 days. The water is removed in vacuo and the residue is dried by azeotropic evaporation with CCl₄. Trituration with diisopropyl ether (x 2) gives a slightly green powder, which is collected and dried over P₂O₅ to give the title amino acid (175 mg, 100%). ¹H NMR (D₂O). Signals obscured by solvent. ¹⁹F NMR (δ CF₃CO₂H=O) δ + 23 (ddt, J = 280, 5, 21 Hz), + 27 (ddt, J = 280, 22, 9 Hz). MS (CI, NH₃) m/e 184 (MH+, 100%), 140 (75%), 120 (40%), 102 (40%). Rf (HOAc/H₂O/BuOH 1/1/3) 0.19. Anal. Calcd. for C₅H₇F₂NO₄.HCl: C, 27.35; H, 3.67; N, 6.38. Found: C, 28.13; H, 3.58; N, 6.52.

Claims: A compound of the formula 3A or a salt or a protected ester thereof. A compound of the formula 3 or a salt thereof. A compound of the formula 5A wherein R₃ is H, (C₁-C₄)alkyl, allyl, or propargyl, or a salt or a protected ester thereof. A compound of the formula 5 wherein R₃ is as defined in claim 3, or a salt thereof. Use of the compounds of any of claims 1 to 4 for the preparation of folates and anti-folates of formula 1 wherein R₁ is -NH₂, H, or -CH₃; R₂ is -NH₂ or -OH; R₃ is H, (C₁-C₄)alkyl, allyl, or propargyl, and X, Y, and Z are each independently a nitrogen atom or a CH group; and of the pharmaceutically acceptable salts and protected esters thereof. A process for the preparation of a compound according to claim 1 which comprises (a) adding HCl to a diethyl ether solution of a compound of formula 3 and allowing the ensuing reaction to proceed; (b) isolating the precipitate formed in the reaction mixture of (a); (c) adding an aqueous solution of HCl to the product of (b) and allowing the reaction to proceed; and (d) isolating from the reaction mixture of (c) the product of formula 3A. A process for the preparation of a compound according to claim 2 which comprises (a) mixing β,β-difluoroglutamic acid hydrochloride with t-butyl acetate; (b) adding a solution of perchloric acid to the mixture of (a) and allowing the ensuing reaction to proceed; and (c) isolating from the reaction mixture of (b) the product of formula 3. The process of claim 7 wherein the β,β-difluoroglutamic acid hydrochloride is prepared by a process which comprises (a) adding a solution of N-(t-butyloxycarbonyl)-4,4-difluoroocta-1,6-dien-5-ylamine to a solution comprising potassium permanganate and permitting the reaction to proceed and (b) isolating from the reaction mixture of (a) a product comprising the β,β-difluoroglutamic acid hydrochloride. A process for the preparation of compounds according to claim 4 which comprises (a) reacting a p-aminobenzoic acid derivative of formula 4 with a compound of formula 3 and (b) isolating from the reaction mixture of (a) the compounds of claim 4.

Inventors: BEY PHILIPPE, KOLB MICHAEL H, BEY, PHILIPPE, KOLB, MICHAEL H.
Applicants: MERRELL DOW PHARMA, MERRELL DOW PHARMACEUTICALS INC.
Application year: 1991
Main classification: C07C229
IPC codes: C07C229, C07C233, C07C237, A61K31, A61P35, C07D471, C07D475