HU205062B - Process for producing n-(s-3-methylheptanoyl or -heptenoyl)-d-gamma-glutamyl-glycine-d-alanine - Google Patents

Abstract

S-3-methyl-6-heptenoic acid and analogues of formula (I) are new. R is CH2OR1, CH2OH, CHO, COOH or COCl or another activated form of the acid COOH; R1 is H or silyl hydroxy protecting gp. Alanine derivs. of formula (II) and their cationic salts when R2 and R3 are H are new. R2=R3=H; pr R2,R3=1-6C alkyl, 6-8C cycloalkylmethyl or benzyl. Prepn. of an acid or ester of formula (III) comprises coupling an activated form of (I) with an amino acid ester of formula (IV) to form a cpd. (II), followed by hydrogenation to reduce the double bond and remove the benzyl gp.(s). R4=R5=H; or one=H and the other=1-6C alkyl or 6-8C cycloalkylmethyl; R6=R7=PhCH2; or one of R6 and R7=PhCH2 and the other=1-6C alkyl or 6-8C cycloalkylmethyl.

Description

Scope of the description: 12 pages (including 3 sheets)

EN 205 062

The present invention relates to a novel process for the production of known immunoregulatory agents of formula (I), wherein: - represents a single or double bond.

Immunopharmology is a relatively new area, and especially the branch that deals with immunomodulation is developing rapidly. Numerous naturally occurring substances have been tested including tuftsin tetrapeptide chemically N ² - [1- (N ² -L-threonyl-L-lysyl) L-prolill-L-arginine. Great attention was paid to synthetic peptidoglycan derivatives, especially those known as murami dipeptides.

The immunoregulatory agents of formula (I) and the procedure for their use have been previously described in PCT / US85 / 02351, filed November 25, 1985. Since this patent application has not yet been disclosed at the time of filing this application, the preparation and use of these compounds are disclosed in the present application to support applicability.

Other immunostimulatory peptides are described in several patents.

L-Alanyl alpha-glutaric acid N-acyl dipeptides are described in German Patent No. 3,024,355 (published January 15, 1981):

Tetra- and pentapeptides containing D-alanyl-L-glutamyl moieties or L-alanyl-D-glutamyl moieties are disclosed in British Patent No. 2,053,231 (issued February 4, 1981) and in German Patent No. 3,024,281. specifications;

N-acylalanyl gamma-D-glutamyl tripeptide derivatives wherein the C-terminal amino acid is lysine or diaminopimelic acid, German Patent No. 3,024,369 (issued January 15, 1981);

Lactyl tetrapeptides consisting of N-lactylalanyl, glutamyl, diaminepimelyl and carboxymethylamino components, EP-11283 (published May 23, 1980);

Polypeptides of formula (A) wherein R ^a is hydrogen or acyl: R ^b is hydrogen, lower alkyl, hydroxymethyl, benzyl; R ^c and R ^d are hydrogen, carboxy, -CONR ^g R ^h wherein R ^g is hydrogen, lower alkyl optionally substituted by hydroxy; and R ^h is mono or dicarboxy-lower alkyl; R ^e is hydrogen or carboxy with the proviso that when one of R ^d and R ^e is hydrogen and the other is carboxy or -CR ^{g is} R ^h ; m is 1,

2,3 and n are 0,1,2 and derivatives thereof, in which the carboxy and amino groups are protected, U.S. Patent Nos. 4,311,640 and 4,322,341; and 25,482; 50,856; 51,812; 53,388; European Patent Applications Nos. 55 846 and 57 419; and peptides similar to those of formula (A) above, wherein R ^{4 is} a basic amino acid moiety (U.S. Patent No. 4,565,653 to European Patent Application No. 157,572) or a heterocyclic amino acid (Arch, 178) European Patent Application 845).

Kitaura et al. [J. Med, Chem., 25, 335-337 (1982)] report that N ² - (gamma-D-glutamyl) meso-2 (L), 2 (D) -diaminimimic acid is the minimum structure capable of eliciting a biological response to compounds of formula A wherein n is 1; R ^a is CH 3 CH (OH) -CO; ^Rb is methyl; R ^c and R ^e are -COOH; R ⁴ is CONHCH 2 COOOH; and R ^f is hydrogen. This compound of formula A is known as Fk-156.

The N- (S-3-methyl-heptanoyl) -D-gamma-glutamylglycyl-D-alanine immunoregulatory compound, i.e., the compound of formula (I), wherein - a single bond, has been described as a more preferred process.

The present invention relates to an improved process for the preparation of immunoregulatory agents of formula (I). According to the procedure a) the following steps are taken:

a) S-3-methyl-6-heptenic acid in activated form with a compound of the formula (VII) in absolute stereochemical form wherein R ⁶ and R ⁷ are benzyl and then a compound of formula (I) wherein single bond is present; yielded (IIIc) absolute stereochemistry of the compound in which R ⁶ and R ⁷ are simultaneously each as defined above, by hydrogenation reduction of the double bond and hydrogenolysis of benzyl ester group (cat), or formula

b) for the preparation of the compound of formula (IIIa) absolute stereochemical from the obtained compound of formula (IHc) absolute stereochemistry, wherein R ⁶ and R ⁷ are as defined in a), the benzyl ester groups are removed by hydrolysis.

The term "reaction-inert solvent" is used for a solvent that does not interact with the starting materials, intermediates or end products so as to adversely affect the yield of the desired product. By way of example, for example, in the solvent of step a), water is considered to be a solvent which is unsatisfactory for the reaction, even though it may be directly or indirectly involved in the desired hydrolysis of the silyl ether.

The process of the invention can be easily accomplished. The commercially available S-cytronellol is first converted to an optically active starting compound of formula (V) by conventional methods as mentioned above and described below for the compositions. The compound of formula (V) is converted to step (VId), S-3-methyl-6-heptenoic acid or S-3-methyl-heptanoic acid of formula (II) step by step as described below.

In the first step, the hydroxy compound or the hydroxyl protected aldehyde (Va or Vb compounds) is a Wittig reaction2.

EN 205 062 is subjected to methylene triphenylphosphorane, which is generally freshly formed from in situ (methyl) triphenylphosphonium halide (preferably bromide) and butyl lithium, in a reaction-aprotic solvent, e.g. in a mixture of tetrahydrofuran and hexane, e.g. the

Scheme 1. Although the temperature is not a critical feature of phosphorane formation, the preferred temperatures are in the range of ± 25 ° C, most preferably in the range of ± 10 ° C. The aldehyde (Va) or the hydroxy-protected aldehyde (Vb) is then reacted in a solvent similar to phosphorane and in a similar temperature range to give the hydroxy compound or the hydroxy-protected olefin of formula (VIb) or (V1). equivalent to.

In the second step, which is only required when a hydroxy protecting group is present, said protecting group is removed by hydrolysis, most preferably in an aqueous acid medium used in the third step, which is the Jones oxidation of the primary alcohol of formula (VIb). to the acid of formula II. In Jones oxidation, so-called A Jones reagent, which is an aqueous solution of H ₂ CrO ₄ , is prepared from CrO ₃ and a strong acid. The Jones reagent is typically prepared from excess concentrated sulfuric acid and CrO _{3 at} ca. 1: 1 water, then diluted to the desired concentration, e.g. 3M with water. The alcohol of formula (Va), the protected alcohol (Vb), which is generally dissolved in a water miscible solvent such as acetone, is reacted with at least two equivalents of Jones reagent. Under these conditions, rapid hydrolysis of any silyl ether group occurs, resulting in the formation of an alcohol of Formula VIb which is then oxidized to the acid of Formula VId, the temperature is not critical, and generally 0 to 50 ° C is suitable, e.g. 17-27 ° C is the most favorable.

Jones oxidation occurs through the initial oxidation of the alcohol to the aldehyde of formula VIc, this aldehyde is not isolated in Jones oxidation. If the intermediate aldehyde is to be isolated, the alcohol is oxidized with a more selective oxidizing agent, such as pyridinium chlorochromate, to form the intermediate aldehyde of formula VIc, which is then oxidized to the acid of formula VId (with Jones reagent or other suitable reagent). ).

If desired, the unsaturated acid of formula (VId) is in activated form [e.g. to (VIe) acid chloride of formula conventional mixed anhydride, or a conventional dehydrative coupling agent such as dicyclohexylcarbodiimide activated switch conventional manner with a compound of formula (VIII) wherein R ⁹ and R ¹⁰ is benzyl, to give a diester. If desired, the latter is hydrolyzed by conventional methods to the immunoregulatory compound of formula (IIIa) or a pharmaceutically acceptable salt thereof. Alternatively, the diesters, where R ² and R ³ are as defined above for R ⁶ and R ⁷ , are hydrogenated to give the immunoregulatory compounds of formula (I). In this hydrogenation process, both double bonds are saturated with hydrogen and subjected to hydrogenolysis to the benzyl group (s). The hydrogenation is carried out in a reaction-inert solvent in the presence of a hydrogenation catalyst, e.g. nickel or precious metal; supported (eg. Raney nickel, Pd / C) or unsupported (for example, RhCl ₃₎ catalyst. The solvent, temperature and pressure are not critical. Suitable solvents include, but are not limited to, lower alcohols, ethers such as dioxane, tetrahydrofuran or dimethoxyethane and esters such as ethyl acetate. Preferably, room temperature is used without cooling, even if the reaction is somewhat exothermic, thereby eliminating the costs of heating and cooling. The pressure is not critical, but preferably less than 0.7 MPa is used to avoid expensive high pressure equipment. Hydrogenation at 3-6 times the atmospheric pressure with the Pd / C catalyst is particularly suitable for these conversions.

Alternatively, the unsaturated acid of formula (VId) is hydrogenated in the same manner as described in the previous paragraph to give S-3-methyl heptanoic acid. Finally, the S-3-methyl heptanoic acid is activated as described above and coupled with a diester of formula (VII) and then hydrolyzed as described above to give the immunoregulatory compounds of formula (I).

The compound of formula (I), wherein the single bond of the crystalline form of the present invention is obtained by crystallization from an organic solvent or a combination of organic solvents. Suitable solvents include acetone, acetonitrile / ethanol or tetrahydrofuran / ether. The preferred solvent for the yield is acetonitrile / ethanol, but acetone is preferred for the purity of the product. This novel crystalline form is definitely advantageous for stability against the previous amorphous lyophilisate. It is much easier to handle because it is more compact and substantially less electrostatic, allowing you to produce more complicated dosage forms.

The pharmaceutically acceptable mono- or dibasic salts of compounds of formula (I) or (IIIa) are generally prepared by treating a solution of the free acid, preferably an aqueous solution thereof, with a base such as sodium hydroxide, potassium hydroxide, sodium carbonate or an amine. , usually according to the appropriate stoichiometric ratios. The salts are isolated by evaporation or precipitation.

The products of formula (I) or (IIIa) according to the invention are suitable agents for the clinical and therapeutic treatment of diseases in mammals, including man, caused by various pathogenic microorganisms, in particular gram-negative bacteria. They can also be used as immunostimulants in mammals, including humans, who are at higher risk for clinical induced immunosuppression.

Applied normal or immunocompromised C ₃ H / HeN male mice in the assay, which were obtained from Charles River Breeding Laboratöryból. Mice were acclimated for 5 days before use, either subcutaneously (SC) or

Orally (PO) treated with various dilutions (100.10, 1 and 0.1 mg / kg) of test compound or placebo (containing pyrogen-free saline) using a volume of 0.2 ml. The treatment regimen depends on the infectious organism used: 24 and 0 hours before infection with Klebsiella pneumoniae in normal mice; and 3.2 and 1 day on the Esherichia coli; or Staph. in mice exposed to immunosuppression before aureus infection. The infection is administered intramuscularly (IM) to the hip

K. pneumoniae or intraperitoneally (IP) E. coli and Staph. for aureus. A volume of 0.2 ml was used for the infection. Mortality was recorded after 7 days for K. pneumoniae and 3 days after infection with the other two microorganisms.

Creating a culture:

K. pneumoniae, E. coli or Staph. aureus: culture was plated for purity from frozen blood agar slopes on brain blood infusion (BHI) agar. Three colonies were removed from the 18-hour plate culture and placed in 9 ml BHI medium. The culture medium was grown for 2 hours at 37 ° C on a rotary shaker, then 0.2 ml was plated on the surface of several BHI agar sloping tubes. The tubes were incubated for 18 hours at 37 ° C, then washed with BHI medium and the culture density adjusted using spectronic 20 and diluted appropriately to achieve LD ₉₀ infection in normal mice.

For use as an anti-infective or immunostimulant for human use, the compounds of formula (I) or () of the present invention may be administered orally, subcutaneously, intramuscularly, intravenously or intraperitoneally, generally in the form of compositions customary in pharmaceutical practice. For example, the dosage forms may be in the form of tablets, pills, powders or granules containing binders such as starch, milk sugar, clarifier, etc. Dosage can also be in the form of capsules mixed with the former or equivalent binders. They can also be administered in the form of oral suspensions, solutions, emulsions, syrups and elixirs, which may contain flavoring and coloring agents. For the oral administration of the therapeutic agents of the present invention, the approx. Tablets and capsules containing 50,100 mg of active ingredient are suitable for most applications.

The doctor will determine the dose that is best suited to treat each patient, and this will vary with age, body weight, patient response, and dosage. The preferred oral dose is from about one to two. 1-300 mg / kg / day in single or divided doses. A preferred parenteral dose is about 30 minutes. 1-100 mg / kg / day; most preferred is approx. 1-20 mg / kg / day.

The following examples are given by way of illustration of the invention and are not intended to limit the scope of the invention.

l.példa

S-7- (t-butyldimethylsilyloxy) -5-methyl-1-heptene

A 500 ml 4-necked round bottomed flask equipped with a thermometer, N ₂ inlet and dosing funnel, 25.7 g (0.072 mol,

1.25 equivalents) (methyl) triphenylphosphonium bromide was slurried in 77 ml tetrahydrofuran (THF) and cooled in a bath containing acetone / aqueous ice. 43.2 ml (0.069 mol, 1.20 equivalents) of a 1.6 n solution of n-butyl lithium in hexane was added in the dosing funnel. To the slurry initially cooled to -8 ° C, butyl lithium is added for 1 hour before the temperature rises and maintained at +1 ° C. The mixture was stirred for an additional 0.5 h at 0-2 ° C to ensure complete formation of the methylene triphenylphosphorane intermediate in a dilute LiBr suspension. Preparation 2 The aldehyde product (14.1 g, 0.0576 mol) dissolved in 14 mL of THF was added portionwise over 40 min while maintaining the temperature at 3-7 ° C. After stirring for a further 15 minutes, no starting aldehyde was detected by thin layer chromatography (TLC) using 3: 1 hexane: ether as eluent (R _f initial aldehyde = 0.6; R _f product = 0.95). The reaction mixture was warmed to room temperature and diluted with 150 mL of ethyl acetate and 90 mL of water. The organic layer was separated and washed with water (2 x 100 mL). The three aqueous phases were combined and washed with ethyl acetate (40 mL). The organic phases are combined, dried over MgSO ₄ and stripped to an oil (25 g), triturated with 10 ml of hexane, filtered through a sintered glass funnel, the solids are pulverized locally with 4 x 10 ml hexane and the combined hexane filtrate and washings are concentrated to give The title compound was obtained as an oil (13.5 g, 96.6%). 1 H-NMR (CDCl ₃ ) delta (ppm) 5.4-6.2 (m, = CH), 4.8-5.3 (m, = CH ₂ ), 3.7 (t, J = 6) , 5 Hz, -OCH ₂ -), 0.08 [s, C (CH ₃ ) ₃ ] and 0.0 [s, Si (CH ₃ ) ₂ ], 8 mol% (C ₆ H ₅ ) ₃ PO ( 7.6, s, I, 25H).

Example 2

S-3-methyl-hept-6-eri-l-ol

The method

The above example was followed by the difference that 2.2 equivalents of both methyl (triphenylphosphonium bromide and n-butyl lithium, the aldehyde product of Preparation 6 (26.3 g, 0.20 mol) were used. Although a gummy solid is observed during the addition of the aldehyde solution, the reaction mixture becomes dilute slurry as soon as it is heated to room temperature for processing.The reaction mixture is diluted with 500 ml of water and 300 ml of ethyl acetate. The phases were separated and washed with water (3 x 250 mL) and the combined aqueous phases were washed with ethyl acetate (2 x 300 mL) .The three organic layers were combined, dried over MgSO ₄ and stripped to give 65.7 g of the title product, 25.6 g. (100%) and about 40 g of triphenylphosphine oxide suitable for further processing in Example 4 below.

Method B

The pure title product is more readily prepared by conventional hydrolysis of the product of Example 1, for example with the dilute sulfuric acid of Example 4 below.

EN 205 062 B. The title product was isolated by extraction into ethyl acetate, dried over MgSO ₄ and stripped.

Example 3

S-3-methyl-6-heplanol

1.14 g (0.01 mol) of the title compound of the previous example was dissolved in 20 ml of CH ₂ Cl ₂ and cooled to 0 ° C. 4.30 g (0.02 mole) of pyridinium chloroformate was added portionwise, maintaining the temperature between 0-5 ° C. The mixture was warmed to room temperature, stirred for 2 hours, filtered through a pad of silica gel and the filtrate was stripped to give the title product as an oil which was further purified by distillation if desired.

Example 4

S-3-methyl-6-heptenoic acid

The method

In a 2000 ml 3-neck round-bottomed flask equipped with a stirrer, thermometer and dosing funnel, the title product of Example 1 (corrected for 81 g purity, 0.33 mol) was dissolved in 400 ml of acetone and cooled to 0-5 ° C. . In another flask, 72.1 g (0.72 mole) of CrO ₃ were mixed with 50 mL of water and stirred at 0-5 ° C, slowly adding 62.1 mL of conc. H ₂ SO ₄ and the mixture were diluted with water to 250 mL to give a 2.88 M H ₂ CrO ₄ solution (Jones reagent). The latter solution (240 mL, 0.67 mol) was added portionwise to the above acetone solution for 1.2 h. The temperature rises rapidly to 17 ° C and is maintained at 17-25 ° C during the addition of the reagent. There is no exotherm at the end of the dosing. The mixture was cooled to 6 ° C, 2-propanol (70 ml) was added over 10 minutes (while the temperature rose to 20 ° C) and concentrated in vacuo to an oil, and 400 ml of 2N NaOH were added under stirring for 50 minutes. maintaining the temperature at 22 ± 5 ° C. The thick reaction mixture was diluted with water (400 mL) and filtered through diatomaceous earth. The wet cake is again converted into pulp in 400 ml of water and 50 ml of 5N NaOH, heated in a steam bath and resubmitted. The combined filtrates were washed with 3 x 300 mL of isopropyl ether. The combined organic phases were back-extracted with 200 mL of 2N NaOH. The combined aqueous phases were acidified to pH 1.0 with 50 mL conc. HCl was slowly added and the product was extracted into 3 x 300 mL fresh isopropyl ether. The organic extracts were combined and evaporated to give 29.1 g (61%) of the title compound as an oil. An additional 7.7 g (16%) of product was obtained by a second basic extraction of the diatomaceous earth cake followed by similar isolation. The combined product was ¹ H NMR (CDCl 3) delta (ppm) at 11.9 (s, -COOH), 5.8 (m, = CH), 5.0 (m, = CH ₂ ) 1.0 (d , -CH ₃ ) and isopropyl ether peaks of 3.7; and 1.1, which is an isopropyl ether (8.6 mol%, 6.3% by weight) impurity, which does not interfere with the use of this product in further processing.

Method B

EXAMPLE 2 The product of the method (65.7 g

26.3 g, 0.20 mole of S-3-methyl hept-6-en-1-ol) are oxidized according to method A of the present example. After removal of the isopropanol and stirring at 0-5 ° C for 1 hour, the reaction mixture is completely stripped, the organic solvents are stripped, the aqueous residue is diluted with 250 ml of water and extracted with 3 x 250 ml of isopropyl ether. The organic extracts were combined and treated with 2N NaOH (160 mL) to give a heavy precipitate (C ₆ H ₅ ) ₃ PO (impurity in the starting material), which was collected by filtration with thorough washing with 1 N NaOH. The filtrate and washings were combined, the phases were separated and the organic phase was washed with additional 1N NaOH (80 mL). All aqueous phases were combined, washed with 3 x 250 mL of isopropyl ether, 50 mL of conc. Acidify with HCl to pH 1 and extract the desired product into 3 x 250 mL fresh isopropyl ether. The combined acidic organic extracts were dried over MgSO ₄ and isolated to give the title product (14.0 g) which was further purified by distillation if desired.

Example 5

To a Parr hydrogenation bottle, a 10% Pd / C catalyst (1.64 g of 50% water moisture), 150 ml of ethyl acetate and the above unsaturated acid (3.28 g, corrected for purity), are placed in the slurry. hydrogenation of atmospheric pressure four times over 1.5 hours, during which time the H ₂ uptake is complete. The catalyst was recovered by filtration through diatomaceous earth, the filtrate was isolated to give the title compound as an oil (3.20 g, 96%).

Alternatively, a 5% Pd / C catalyst (2 g, 50% water moisture) and the title compound (33.3 g, corrected for purity) in 150 ml ethyl acetate were placed in a 1 liter autoclave and hydrogenated four times at atmospheric pressure 2 for 30 hours at 3031 ° C, during this time the H ₂ uptake is complete. The catalyst was recovered by filtration through diatomaceous earth and the filtrate was isolated as an oil (35.4 g), which was distilled under high vacuum to give the purified title product (29.6 g, 87.6%); fp. 77-79 ° C / 0.2 mm; ¹ H-NMR (CDCl 3) delta (ppm): 12.0 (s, -COOH), 1.0 (d, -CH 3), 0.6-2.8 (m, residual 13 H); IR (film) 3400-2400, 2960, 2925, 2860, 1708,1458,1410,1380,1295,1228,1190,1152,1100, 930 ^cm-1; [α] D = 6.41 ° (c = 1% in CH3OH); rfg- ⁵ = 1.427.

Example 6

S-3-methyl-heptanoyl chloride

The acid product of the preceding example (8.5 g, 0.062 mol) was dissolved in 18 ml CH ₂ Cl ₂ . The solution was mixed with oxalyl chloride (5.36 mL, 7.80 g, 0.0614 mole) and allowed to stand for 4 hours, during which time the reaction was complete, as indicated by the cessation of further gas evolution. This acid chloride solution was used immediately in Method C of Example 8. Alternatively, the acid chloride is isolated from the solvent by stripping for use in method A of Example 8 and further purified by distillation if desired, fp. 45 '/ l, 5 mm.

EN 205 062

Example 7

S-3-methyl-6-heptenoyl chloride

The acid product of Example 4 (0.747 g, 5 mmol) was converted to a solution of the title product in CH ₂ Cl ₂ by the method of the previous example and used directly in Example 9 below. Alternatively, the reaction mixture was evaporated to give the title product, which was distilled if necessary under reduced pressure.

Example 8

NS-3-methyl-heptenoyl-D-gamma-glutamyl (alpha-benzyl ester) -glid-D-alanine benzyl ester Method A

1.0 g (2.03 mmol) of D-gamma-glutamyl (alpha-benzyl ester) glycyl-D-alanine benzyl ester hydrochloride (Preparation 5) and 616 mg (6.09 mmol) of triethylamine in 50 ml of methylene To a solution of the chloride in S-3-methyl-heptanoyl chloride (660 mg, 4.06 mmol) was added and the reaction mixture was stirred at room temperature for 80 hours. The methylene chloride was evaporated in vacuo, and ^- the residue was dissolved in ethyl acetate. The resulting solution was washed successively with 2.5% hydrochloric acid, water, 10% potassium carbonate, water and brine. The organic layer was separated, dried over magnesium sulfate and concentrated in vacuo. The residue was triturated with diethyl ether and filtered under nitrogen to give the title product, all of which was used directly in the mixture.

Example 10 In its method.

Method B

The product of Preparation 5 (0.75 g, 1.53 mmol), 5 mL of CH ₂ Cl ₂ and triethylamine (0.212 mL, 1.53 mmol) were combined and stirred under N ₂ . 4 ml of S-3-methyl heptane acid (Example 5; 0.20 g, 1.39 mmol) dissolved in CH ₂ Cl ₂ followed by dicyclohexylcarbodiimide (0.286 g, 1.37 mmol) was added. and the mixture was stirred for 16 hours. The reaction mixture is filtered, the filtrate is evaporated, the residue is taken up in 10 ml of ethyl acetate and the solution is washed successively with 5 ml of 2.5% hydrochloric acid, 5 ml of water, 5 ml of 10% K ₂ CO ₃ and 5 ml of brine. dried over magnesium sulfate to give 71 mg (88%) of the title product.

Method C

In a 500 ml 4-neck round bottom flask equipped with a thermometer, drip funnel and N ₂ inlet, the product of Preparation 5 (32.8 g, 0.059 mol) is dissolved in 175 ml CH ₂ Cl ₂ and 0- Cool to 5 ° C. Holding this temperature range, triethylamine (24.7 mL, 17.9 g, 0.177 mol 3 eq.) Was added in slow flow over 15 min. The ice-water bath is maintained and the total amount of S-3-methyl-heptanoyl chloride methylene chloride product of Example 6 is added over 15 minutes before the temperature rises to 21 ° C. Stirring in the ice-water bath is continued for 45 minutes, during which time the gelatinous mixture becomes too thick for mixing. The gelatin mass was crushed and mixed with 125 mL of 10% hydrochloric acid and 50 mL of CH ₂ Cl ₂ . The organic layer was separated, washed successively with 2x125 ml of water, 2x125 ml of 10% K ₂ CO ₃ and 1 x 125 ml of water, dried over MgSO ₄ and concentrated to 82.3 g of a wet white solid. This solid is taken up in 500 ml of warm ethyl acetate. Slowly cooled to room temperature, the title compound is difficult to crystallize and the mixture is diluted with additional 40 mL of ethyl acetate to allow easy mixing. 31.1 g (90.5%) of purified title product are obtained by filtration and vacuum drying at 40 ° C.

1 H-NMR (CDCl ₃ ) delta (ppm): 8.4-8.1 (m, 3H), 7.15 (s, 10H), 5.1 (s, 4H), 4.4-4 , 2 (m, 2H), 3.7 (d, 2H), 2.2 (t, 2H), 2.1-1.7 (m, 6H), 1.4-1.1 (m, 10H ), 0.920.8 (m, 6H).

Example 9

N- (S-3-methyl-6-heptenoyl-D-gamma-glutamyl (alfabenzil ester) glycyl-D-alanine benzyl ester

By the same method as in the previous example, the product of Formulation 5 (2.77 g, 5 mmol) was reacted with an entire amount of the methylene chloride chloride obtained according to Example 7. The initially obtained 2.77 g product, obtained by evaporation of the washed and dried organic phase, was taken up in 20 ml of warm ethyl acetate, diluted with 20 ml of hexane, cooled and the purified product was collected by filtration: 2.24 g (77%). ), op. 137.5-139.5 ° C.

Example 70

N- (S-3-methyl-heptanoyl) -D-gamma-glutamyl-alanine gliciTD

The method

Example 8 The whole product of the method is dissolved in 65 ml of methanol. 250 mg of palladium hydroxide was added to the solution and the mixture was shaken for 3 hours under a hydrogen atmosphere at 4 atmospheres. The catalyst was filtered off and the solvent was removed in vacuo. The residue was dissolved in water and lyophilized to give the desired product.

The NMR spectrum _(DMSO-d6) showed the following absorptions: 8.27 to 8.03 (m, 3H), 4.32 to 4.1 (m, 2H), 3.72 (d, J = 6Hz _r 2H), 2.22 (t, J = 8 Hz, 2H), 2.271.68 (m, 6H), 1.42-1.0 (m, 10H) and 0.94-0.8 (m, 6H) ).

When working with a measured amount of the title compound of Example 8 (0.50 g) using 90 mg of a 20% Pd (OH) ₂ / C (31% aqueous moisture) catalyst in 25 mL of CH ₃ OH, this method is 0.24 fluffy, electrostatic product; IR (nujol mixture) 3300.2940, 1740, 1650, 1540, 1468 and 1380 cm ^-1 ; except for the last two peaks, each is wide and badly split.

Method B

Example 8 Method C (30.8 g) was slurried in 300 mL of abscissa in a 2 L autoclave. ethanol. 1.54 g of a 5% Pd / C (50% water) was added to the mixture and hydrogenated for 1 hour at 4 atmospheres under which hydrogen uptake was complete. The catalyst was recovered by filtration, first on paper and then on a 0.45 micron nylon filter using 100-150 ml of ethanol for filtration.

EN 205 062 Β for washing. The combined filtrate and washings were evaporated to a wet white solid, which was dissolved in 150 ml of warm 1:10 abs. ethanol and acetonitrile, filtered through warm filtration, evaporated to 35 ml, slowly cooled to room temperature, granulated and filtered to give crystalline solid non-electrostatic title compound (20.1 g, 94%) as IR (nujol mixture). characterized by larger, well-defined, sharp peaks at 3340,3300,2900,2836,1725,1650,1628,1580,1532,1455,1410,1370,1280,1240,1216 and 1175 cm-1.

Method C

9.4 g of crystalline product prepared according to the previous method B were dissolved in 1000 ml of acetone under reflux for 1 hour. The solution was cooled to room temperature and seeded with trace amount of product B to initiate crystallization. After stirring for 6 hours, additional purified title compound was obtained by filtration, washed with a minimum amount of acetone and dried in vacuo at 35 ° C, the product being 7.25 g; the IR peaks of the product correspond to the crystals of acetonitrile / ethanol obtained by Method B.

Method D

To the product of the previous example (0.50 g) was added 0.026 g of a 5% Pd / C catalyst (50% aqueousness) in 125 ml of abs. in ethanol in a Parr hydrogenation vessel. The mixture was hydrogenated four times with atmospheric pressure for 2.5 hours. The catalyst was recovered by filtration and the filtrate was evaporated to give the title product as a sticky solid which was crystallized according to the previous method.

Example 11

N- (S-3-methylimino-6-heptenoyl) -D-gamma-glycyl glulamil

D-HEREBY RECOMMEND

1 g of the product of Example 9 was dissolved in 5 ml of CH ₃ OH. 2.50 ml of 1N NaOH was added and the mixture was stirred at room temperature for 3 hours. The CH ₃ OH was evaporated and the aqueous residue was diluted

Extract with 7.5 ml of water, 2x7.5 ml of ethyl acetate and acidify to pH 3.0 with 1N HCl. The acidified aqueous mixture was continuously extracted with fresh ethyl acetate and the extract was evaporated to give the title product, which was converted to a lyophilisate according to Method A, Example 10.

Example 12

N- (S-3-methyl-6-heptenoyl) -D-gamma gÍutami.l- (alpha

benzyl ester) glycyl-D-HEREBY RECOMMEND-butyl ester

D-gamma-glutamyl (alpha-benzyl ester) glycyl] -D-alanine butyl ester (Preparation 5) is reacted with S-3-methyl-6-heptenoyl chloride (Example 7) in Example 8 According to its method, the title compound is obtained.

Example 13

N- (S-3-methyl-heptanoyl) -D-gamma glulamil-glycyl-alanine butyl ester

By the hydrogenation method of Example 10, the product of the preceding method is converted to the present product.

Preparation 1

0- (t-butyl-dimethyl-silyl) -S-citronellol

In a 500 ml 4-neck round-bottomed flask equipped with a thermometer, N ₂ inlet and dosing funnel, 547 g (3.5 mol) of S-citronellol was dissolved in 547 ml of dimethylformamide (DMF) at room temperature (21 ° C). ). 262.1 g (3.85 mol of 1.1 eq imidazole was added. The temperature dropped to 13 ° C and further reduced to -6.5 ° C with an ice-acetone bath. * 580.3 g (3, 85 moles, 1.1 eq.) Of t-butyldimethylchlorosilane, previously dissolved in vigorously stirred DMF (1160 ml) over 1.25 hours, allowing the temperature to slowly rise to 11 ° C. After an additional 0.25 hours, TLC (3: 1 hexane ether) showed complete conversion to the desired product ₍ starting material R _f 0.2; Rf product 0.9).

For separation, the mixture was added to 500 ml of hexane and 1000 ml of ice and water. The phases are separated and the organic phase is washed with ice-cold 0.25 N hydrochloric acid (2000 mL). The two aqueous phases were combined and viszszamossuk 500 ml of hexane which was combined with the original organic layer, washed with 500 mL saturated _NaHCO3 -oldattal, dried on _MgSO4 and stripped to recover the title product in quantitative yield, 986.7 got this theory 104% of this solvent is attributable to low residual retention; 1 H-NMR (CDCl ₃ ) delta (ppm): 5.1 (t, J = 7 Hz, = CH), 3.65 (t, J = 6.5 Hz, -O-CH ₂ -), 1, 7 and 1.65 [25.2 = C (CH _3)], 0.8 [s, SiC (CH3) _3] and 0.0 [s, Si _{(CH3) 2].}

Preparation 2

S-6- (t -butyl-dimethyl-silyl) -4-methyl-hexanol

In a 500 ml 4-neck round bottom flask equipped with mechanical stirrers, straight glass O ₃ / O ₂ current inlet, thermometer and outlet connected to a saturated KI trap - product of the previous formulation (81.2 g, adjusted to solvent content) was dissolved in 120 ml CH ₂ Cl ₂ and 81 ml CH ₃ OH. NaHCO ₃ (6.3 g, 0.075 mol, 0.25 eq.) Was added and the mixture was cooled to -10 ° C in an acetone / dry ice bath. The temperature was further reduced and maintained at -72-75 ° C while O ₃ / O ₂ was bubbled into the reaction mixture for 6 hours. After 1 hour, the reaction mixture does not adsorb all O ₃ , as indicated by the yellow color formed in the KI trap. TLC (hexane eluent) indicates the completion of the reaction ₍ starting material R _f : 0.3; R _f intemed material: 0.0).

Excess O ₃ was removed from the reaction mixture with N ₂ , dimethyl sulfate (26.4 mL, 22.4 g, 0.36 mol, 1.2 eq.) Was added, the bath was removed and the mixture allowed to warm to room temperature. After stirring for 16 hours under N ₂ , TLC (6: 1 hexane: ether) indicated that no intermediate was retained ₍ intermediate R _f : 0.8, Rf product: 0.05). The mixture is then stripped and the residue

HU 205 062 juk is divided between 150 ml of ethyl acetate and 300 water. The organic layer was washed with fresh water (300 mL). The combined aqueous phases were washed with 150 mL of fresh ethyl acetate. The organic phases were combined, dried over MgSO ₄ and stripped to an oil, and then concentrated under high vacuum for 16 hours to give the title compound as a quantitative yield: 74.7 g, 101.9% of theory, which was the minor solvent. due to contamination.

Ή NMR (CDC1 ₃₎ delta (ppm): 9.75 (t, CHO), 3.6 (t, J = 6Hz, _-O-CH2 -), 0.9 [s, -SiC ( CH ₃ ) ₃ ], 0.0 [s, (-Si (CH ₃ ) ₂ ]

Preparation 3

Glycyl-D-alanine benzyl ester hydrochloride

To 100 ml of cold (0 ° C) methylene chloride solution containing 10 g (57 mmol) of N- (t-butyloxycarbonyl) glycine, 20 g (57 mmol) of D-alanine benzyl ester p. Toluene sulfonic acid salt and 5.77 g (57 mmol) of triethylamine were added, dicyclohexylcarbodiimide (12.3 g, 60 mmol) was added and the resulting mixture was allowed to warm to room temperature. After 18 hours, the mixture was filtered and the filtrate concentrated in vacuo. The residue was dissolved in 200 ml of ethyl acetate and the organic layer was washed with 2.5% hydrochloric acid, saturated sodium bicarbonate solution and brine. The organic layer was separated, dried over magnesium sulfate and concentrated under reduced pressure. To the resulting oil was added 200 ml of dioxane saturated with hydrochloric acid. After 30 minutes, diethyl ether (400 ml) was added and the product was triturated under nitrogen. Yield 10.9 g, 70%.

Preparation 4.

N- (tert-butoxycarbonyl) -D-gamma glutamyl (alpha-benzyl ester) -htdroscinamide ester

1500 ml, -50 g (143 mol) of N- (tert-butoxycarbonyl) D-gamma-glutamic acid alpha-benzyl ester and 17.3 g (150 mmol) of methylene chloride containing N-hydroxysuccinamide 30.9 Dicyclohexylcarbodiimide (g, 15 mmol) was added and the resulting reaction mixture was stirred at room temperature for 18 hours. The solid was filtered off and the filtrate was concentrated in vacuo. The residue was triturated with diethyl ether and the solid filtered through 43.7 g (68% yield) of nitrogen.

Preparation 5

D-gamma-glutamyl (alpha-benzyl ester) glycyl-D-alanine benzyl ester hydrochloride

One 4.3 g (9.45 mmol) of N- (tert-butoxycarbonyl) -gamma-glutamyl (alpha-benzyl ester) hydroxy succin amide ester, 2.71 g (9.92 mol) 100 ml of methylene chloride solution containing glycyl D-alanine benzyl ester hydrochloride and 1.0 g of 9.92 mmol of triethylamine are stirred for 18 hours at room temperature and then concentrated in vacuo. The residue was dissolved in 200 ml of ethyl acetate and the solution was washed with 2.5% hydrochloric acid, water, 10% potassium carbonate and brine. The organic layer was separated, dried over magnesium sulfate and concentrated under reduced pressure. The residue was treated with 200 ml of dioxane saturated with hydrochloric acid and stirred for 2 hours. The solution was evaporated to dryness in vacuo and the residue was triturated with diethyl ether. The solids were filtered under nitrogen, 3.41 g (73% yield).

The product of the previous formulation is reacted with the same method with glycyl-D-alanine butyl ester to form D-gamma-glutamyl (alpha-benzyl ester) -glycyl-Dalanine butyl ester hydrochloride.

Preparation 6

S-6-hydroxy-4-methyl-hexanol

The method

A 250 ml 3-neck round-bottomed flask was fitted with a magnetic stirrer, thermometer, gas inlet tube and gas outlet tube, which led to a saturated Kl gas scrubbing flask. The flask was charged with CH ₂ Cl ₂ (81 mL) and CH ₃ OH (54 mL) containing 31.25 g (0.20 mol) of S-citronellol and cooled to -8 ° C. While maintaining the temperature between -2 ° C and -10 ° C, O ₂ O ₃ was bubbled through the reaction mixture

For 4.5 hours, at which time the excess of O ₃ indicates a KI solution and the reaction was complete positive starch / KI paper test detects the reaction mixture. The mixture was kept at -5 ° C by rinsing with N ₂ and methyl sulfide (17.7 mL, 15.0 g, 0.24 mol) was added. The reaction was allowed to warm to room temperature and stirred for 16 hours, after which time TLC eluting with isopropyl ether was used, indicating that the reaction was complete (R _f: S-citronellol 0.7; product _Rf: 0.4). Finally, the solvent was stripped to give 50.8 g of an oil. The oil was diluted with 30 mL of ethyl acetate and 25 mL of water to form a phase. Addition of an additional 150 mL of ether results in 2 phases. The phases were separated and the organic layer was washed with water (2 x 25 mL), dried over MgSO ₄ , stripped to give 23.9 g of a colorless oil. The combined aqueous phases were extracted with ethyl acetate (2 x 50 mL) and the extracts were combined, dried and evaporated to give an additional 7.8 g of a colorless oil. Colorless oils are combined and further stripped to obtain the title product:

28.3 g (108.7% of theory), TLC pure material (see above), except for solvent contamination, suitable for further reactions as described above.

Method B

The product of Formulation 2 was hydrolyzed in a conventional manner as in Example 4 above with dilute sulfuric acid. The title compound was isolated by extraction with ethyl acetate and evaporated as in Method A above.

Claims (1)

Patent Claim Point A process for preparing the absolute (S) stereochemical formula wherein - represents a single or double bond, characterized in that S-3-methyl-6-heptenoic acid in an activated form, is a (VH) absolute stereochemistry of the compound of formula - wherein R ⁶ and R ⁷ represents a benzyl group, then a) for the preparation of a compound of the formula I in which - by a single bond, the resulting absolute (HIc) In a compound of the formula wherein R ⁶ and R ⁷ are as defined above, hydrogenation is carried out by reduction of the double bond and hydrogenation of the benzyl ester group (s); or b) preparation of compound (IIIa) absolute stereochemistry of the resulting compound of formula (IIIc) absolute stereochemistry of the compound of the formula wherein ^R6 and ^R7 are as defined in a), the benzilésztercsoportokat removed by hydrolysis.