CN1021436C - Process for the preparation of crystalline compounds of absolute stereochemical formula - Google Patents

Abstract

Immunoregulatory N-(S-3-alkylheptanoyl)-D-gamma-glutamyl-glycyl-D-alanine and esters thereof; and an improved process and intermediates of a novel immunoregulatory N-(R-3-4-heptanoyl and S-3-methyl-heptanoyl)-D-gamma-glutamyl-glycyl-D-alanine,and esters.

Description

The present invention relates to an advantageous preparation method of the immunomodulator represented by formula (I),

In formula (I), R is methyl or ethyl; and each of ^R4 and ^R5 is hydrogen, or one of ^R4 and ^R5 is hydrogen, and the other is ( _C1 - _C6 ) alkyl or (C ₆ -C ₈ ) cycloalkylmethyl; The present invention also relates to trans-R-3-alkyl-4-heptenoic acid (VIb, see below), S-3-methyl-6-heptenoic acid ( Xb, see later) and the preparation method and intermediate of S-3-alkyl-heptanoic acid, the absolute stereochemical formula of S-3-alkyl-heptanoic acid is

In the formula (II), R is defined as above, and they can be used as an intermediate for the compound of the synthetic formula (I); the present invention relates to an immunomodulator (or precursor) whose absolute stereochemical formula is shown in (III) or it can be used as a medicine of salt,

Either one of the two dotted lines in the formula (Ⅲ) represents a double bond rather than both of them representing a double bond at the same time, R is defined as above, ^R2 and ^R3 both are hydrogen (Ⅲa) or they can be used for medicine Salt or R ² and R ³ are each independently (C ₁ -C ₆ ) alkyl, (C ₆ -C ₈ ) cycloalkylmethyl or benzyl (Ⅲb), with the proviso that when the terminal 6,7-position When it is a double bond, R is a methyl group.

Optically pure S-3-methylheptanoic acid (II, R=CH ₃ ) was previously obtained from the corresponding racemate by multiple recrystallizations of its quinine salt at -15°C in unspecified yields [Levene et al. J. Biol. Chem., 1932, Vol. 95, pp. 1-24, on p. 18, referred to in the literature as 2-n-butylbutyric acid-4]. Optically active 3-methylheptanoic acid has been successively reported to be prepared by some other methods [Soai et al., J.Chem.Soc., Chem.Commun, 1985 pp. 469-470; Oppolzer et al., Helv.Chim.Acta .1985 Vol. 68 pp. 212-215; Ohno et al., U.S. Patent 1986 4,564,620; Mori et al., Synthesis 1982 pp. 752-753; Oppolzer et al., Helv.Chim.Acta. pp. 2808-2811; Mukaiyama et al., Chem. Lett. 1981 pp. 913-916; Posner et al., J.Am.Chem.Soc. Vol. .Japan, 1978 51 volumes 3368-3372 pages; Meyers et al., J.Am.Chem.Soc. 1976 98 volumes 2290-2294 pages], but these preparation methods have one or more shortcomings (maximum possible yield is 50%, at least 50% of the undesired by-products need to be disposed of; at least 50% of the undesired by-products carry over to the final steps of the process; the resulting acid is not optically pure; it needs to be used in large-scale preparations Difficult to handle organometallic reagents; low overall yield; and/or poor availability of required reagents).

Now used asymmetric epoxidation (so-called Sharpless splitting) have been previously reported (Katsuhi and Sharpless, J.Am.Chem.Soc., 1980, Vol. 102, pp. 5974-5976; Sharpless et al., Pure Appl. Chem., 1983, Vol. 55, pp. 589-604); Stereospecific Claisen condensations (Chan et al., U.S. Patent 4,045,475).

The relatively new field of immunopharmacology, especially as it relates to immunomodulation, is developing rapidly. Various naturally occurring compounds have been studied, including the tetrapeptide tuffsin, chemically named N-[1-(N-L-threonyl-L-lysyl)-L-prolyl]-L- arginine. Much attention has focused on the synthesis of glycan-peptide derivatives, particularly those compounds known as muramyl dipeptides.

Immunomodulators of formula (I) (generally amorphous freeze-dried substances when R ⁴ =R ⁵ =H) and their methods of use were filed as early as November 25, 1985 in the relevant international patent (PCT ) described in Application Serial No. PCT/US85/02351. Since this application is not publicly available, the preparation of these compounds and their methods of use are incorporated into this disclosure in support of this application.

Other immunostimulatory peptides have been described in some patent specifications:

L-alanyl-α-glutaric acid N-acyl dipeptides, (see West German Patent 3,024,355, issued January 15, 1981);

Tetra- and penta-peptides containing a D-alanyl-L-glutamyl moiety or an L-alanyl-D-glutamyl moiety (see British Patent 2,053, issued February 4, 1981, respectively , 231 and West German Patent 3,024, 281);

N-acyl-alanyl-r-o-glutamyl tripeptide derivatives, wherein the C-terminal amino acid is lysine or diaminopimelic acid (see West German Patent 3,024 published on January 15, 1981, 369);

Lactoyl tetrapeptides including N-lactoylalanyl, glutamyl, diaminopimeloyl and carboxymethylamino moieties (see European Patent EP-11283 published on May 23, 1980);

Polypeptides of formula (A) and derivatives thereof are disclosed in U.S. Patents 4,311,640 and 4,322,341; European Patent Applications 25,482,50,856, 51,812, 53,388, 55,846 and 57,419 middle,

In formula (A), R ^a is hydrogen or acyl; R ^b is first hydrogen, and can also be lower alkyl, hydroxymethyl, benzyl; R ^c and R ^d are each hydrogen, carboxyl, CONR ^g R ^h (wherein R ^g is hydrogen, lower alkyl optionally substituted with hydroxy, R ^h is monocarboxy, dicarboxy lower alkyl); R ^e is hydrogen or carboxyl, with the proviso that when one of R ^d and R ^e is hydrogen, the other One is carboxyl or -CONR ^g R ^h ; R ^f is hydrogen; m is 1 to 3 and n is 0 to 2, and the carboxyl and amino groups in the polypeptide and its derivatives are protected; and

Peptides similar to formula (A) above, but wherein ^Rd forms a basic amino acid moiety (IVes et al., U.S. Patent 4,565,653; European Patent Application 157,572), or a heterocyclic amino acid ( IVes, European Patent Application 178,845).

Kitaura et al. reported in J.Med.Chem.1982, 25 volumes 335-337 pages, that N ² (rD-glutamyl)-meso-2(L), 2(D)-diaminopimelic acid is (A) The compound of the formula can lead to the minimum structure of biological response characteristics, (A) in the formula, n is 1; m is 2; R ^a is CH ₃ CH (OH)-CO-; R ^b is CH ₃ ; R ^c and R ^e is each -COOH; R ^d is -CONHCH ₂ COOH; and R ^f is H. The compound of formula (A) is called FK-156.

We have now found a more stable _crystalline form of the immunomodulator, namely ^the compound N-(S- ^{methylheptanoyl} )-Dr- Glutamyl-Glycyl-D-Alanine.

We have now found an efficient method for preparing intermediates R-3-alkyl-4-heptenoic acid (VIb, see below) and S-3-alkylheptanoic acid (II, see above) with high optical purity. high overall yield and avoid Application of organometallic reagents and unwanted by-products of the R-enantiomer in later synthesis.

In the first route, the initial step involves a Sharpless type resolution of trans-4-alken-3-ols of the formula,

The definition of R in the formula is as above. Specifically, this step comprises making the racemate of formula (IV) react with tert-butyl hydroperoxide in the reaction inert solvent that titanium tetraisopropoxide and L-(+)-diisopropyl tartrate exist, and then The amount of either should be sufficient to oxidize the S-enantiomer while retaining the desired unreacted R-enantiomer of absolute stereochemical formula (V).

In the second step, the compound of formula V is stereospecifically condensed with tri[(C ₁ -C ₃ ) alkyl] orthoacetate without isolation, and the intermediate allyl- Enol ether rearrangement to give R-3-alkyl-4-heptenoic acid (C ₁ -C ₃ ) alkyl esters of absolute stereochemical formula (VIa),

(VIa) X = OR ¹ ; R ¹ = (C ₁ -C ₃ )alkyl

(VIb)X=OH

(VIc)X=Cl

(VId)X=H

In another route, the ester is then hydrolyzed to trans-R-3-alkyl-4-heptenoic acid (VIb) and, if desired, hydrogenated to S-3-alkylheptanoic acid (II). In an alternative route, the ester is first hydrogenated to give (C ₁ -C ₃ )alkyl S-3-alkylheptanoate, which is then hydrolyzed to the acid of formula (II).

In another route, resolved trans-4-alken-3-ols (Formula V) were converted to alkenes with ethyl vinyl ether in the presence of catalyst mercuric acetate [Hg(OAc) ₂ ]. Propyl vinyl ether (IX). Heating then rearranges allyl vinyl ether to R-3-alkyl-4-heptenal shown in formula (VId), and oxidation, for example, with Jones reagent (chromic anhydride in dilute mineral acid), The above R-3-alkyl-4-heptenoic acid is formed.

We have also found the intermediates S-3-methyl-6-heptenoic acid (Xd, see below) and S-3-methylheptanoic acid (II, R= _CH3 , see above) of high optical purity. An effective method with high overall yield, avoiding the use of organometallic reagents and useless R-enantiomer by-products, and using the readily available S-citronellol (Ⅺ) with an absolute stereochemical formula as a raw material.

The initial steps of the synthesis are conventional and include selective protection of alcohol groups with conventional silyl protecting groups such as tert-butyldimethylsilyl, followed by ozonolysis and treatment with dimethylsulfide to give absolute A new compound with the stereochemical formula shown in (XII),

^R8 in formula (XII) is hydrogen (XIIa) or a silyl hydroxy protecting group (XIIb). The method used is specifically explained in the following preparations, and is similar to the method previously used for R-citronellol in the preparation of the R-enantiomer of the compound of formula (V) above, wherein R ⁸ is tert-butyldimethyl silyl group. The latter (R-citronellol is not useful here) can be used for the synthesis of proxipomin (Tapolczay et al., J. Chem. Soc., Chem. Commun. 1985 pp. 143-145).

The present invention particularly relates to formula (V) intermediate compound and formula (X) compound,

(Xa) R ⁹ = CH ₂ OR ¹⁰ , R ¹⁰ = silyl protecting group

(Xb) R ⁹ =CH ₂ OH

(Xc) R ⁹ =CHO

(Xd) R ⁹ =COOH

(Xe) R ⁹ =COCl

It also relates to a method for converting a compound of formula (XII) into an optically active acid [formula (Xd) and (II, R= _CH3 )], which method comprises the following steps:

(a) reacting a compound _of formula (XII) with methylenetriphenylphosphine _CH2 =P( _C6H5 ) ₃ to form a compound of formula (Xa) or (Xb);

(b) when dilute mineral acid is used to hydrolyze the compound of formula (Xa) to the compound of formula (Xb), it can be carried out as a separate step or simultaneously with the next step; and

(c) Oxidation of a compound of formula (Xb) to S-3-methyl-6-heptenoic acid (Formula Xd) with chromic anhydride in dilute mineral acid; a further step can be carried out if desired,

(d) Catalytic hydrogenation of a compound of formula (Xd) to S-3-methylheptanoic acid of formula (II) wherein R is methyl.

^R8 being hydrogen has the advantage of fewer steps and ^R8 being a silyl protecting group has the advantage of easier purification when isolating the intermediate product from step (a). Preferred hydroxy protecting groups are trimethylmethyl, p-tert-butylphenethyldimethylsilyl and tert-butyldimethylsilyl. _A preferred dilute mineral acid is _H2SO4 . Preferably select to use ^S -citronellol ozonolysis, the method synthetic formula (XIIa) compound of treating with dimethyl sulfide; A compound of formula (XIIb).

(e) reacting S-citronellol with tert-butyldimethylsilyl chloride; and

(f) The hydroxyl-protected citronellol obtained from the reaction was decomposed by ozonolysis and treated with dimethyl sulfide.

The present invention also relates to an improved method for preparing an immunomodulator of formula (I), the method comprising the steps of:

(a) make R-3-alkyl-4-heptenoic acid or the activated form of S-3-methyl-6-heptenoic acid (such as acid chloride VIc or Xe) react inertly with the compound shown in formula (VII) Coupling in a solvent to form the intermediate compound of the above formula (Ⅲb),

In formula (VII), both R ⁶ and R ⁷ are benzyl, or one of R ⁶ and R ⁷ is benzyl, and the other is (C ₁ -C ₆ ) alkyl or (C ₆ -C ₈ ) cycloalkylmethyl. ^R2 and ^R3 in formula (IIIb) correspond to ^R6 and ^R7 in formula (VII); and

(b) hydrogenation of the above-mentioned intermediate compound in a reaction-inert solvent in the presence of a hydrogenation catalyst so that reduction of the double bond and hydrogenolysis of one or more benzyl groups take place simultaneously.

Finally, the present invention relates to immunomodulators and/or precursor formula (III) of the above-mentioned compound (I). Compounds of formula (IIIa) (i.e. R ² =R ³ =hydrogen) are obtained from diester compounds of formula (IIIb) by conventional ester hydrolysis.

The term "reaction inert solvent" as used herein refers to a solvent which does not react with the starting materials, intermediates or products, but which adversely affects the yield of the desired product.

Preferred R is ethyl. Preferred acid catalysts are Lewis acids (ie dry HCl, _AlCl₃ ). The preferred racemate of trans-4-hexen-3-ol or trans-4-hepten-3-ol is obtained by reacting ethyl Grignard reagent with crotonaldehyde or 2-pentenal, respectively, but The present invention is not limited thereto.

The present invention is easy to realize. R-trans-4-alken-3-ol (V) is obtained by asymmetric epoxidation, the so-called Sharpless resolution (cited above). According to this method, the undesired S-trans-2-hexen-4-ol in the racemate (IV) has more than 1 mole of L-(+)-diisopropyl tartrate and substantially 1 Selectively react with t-butyl hydroperoxide (at least 0.5 mole but certainly less than 1 mole per mole of racemic hexenol) in the presence of a molar equivalent of titanium tetraisopropoxide. The reaction is carried out under anhydrous conditions in a reaction-inert solvent such as dichloromethane at low temperature, typically in the range of -20 to -80°C. The desired unreacted R-trans-2-alken-4-ol (V) is isolated by standard methods such as distillation or chromatography. Because of the high volatility of the desired product, low boiling point solvents such as pentane and diethyl ether are preferred for chromatographic separation.

The next step is to condense R-trans-4-alken-3-ol (Ⅴ) with (C ₁ -C ₃ ) trialkyl ester of orthoacetic acid (Claisen condensation rearrangement) to obtain R-3-alkyl -(C ₁ -C ₃ )alkyl 4-heptenoate (VIa). The reaction can be carried out in a reaction-inert solvent, but is preferably carried out with only an excess of orthoacetate, generally at elevated temperature (e.g. at 120-160°C). When triethyl orthoacetate (boiling point 142°C) is the reagent, the reflux temperature is usually applied. The reaction is carried out in the presence of an acid catalyst. Acids such as propionic acid, pivalic acid, 2,4-dinitrophenol, dry HCl and _AlCl3 are effective, the preferred catalysts are Lewis acids, most preferably _AlCl3 .

In another route, the resulting ester (formula VIa) is hydrolyzed by conventional methods to give the corresponding unsaturated acid (formula VIb).

Another alternative route of the present invention is also easy to realize. First, the commercially available S-citronellol (XI) is converted into the optically active starting material represented by formula (XII) by a conventional method, which has been pointed out above and will be described in detail in the preparation below. Then, compound (XII) is converted into S-3-methyl-6-heptenoic acid (Xd) or S-3-methylheptanoic acid (II), respectively, step by step as detailed below.

In the first step, a hydroxy-unprotected or protected aldehyde (XIIa or XIIb) undergoes a Wittig reaction with methylenetriphenylphosphine. Typically, methylenetriphenylphosphine is freshly prepared, usually from methyltriphenyl halide (conveniently bromide) and n-butyllithium in a reaction-inert aprotic solvent such as a mixture of tetrahydrofuran and hexane form.

For example

Although temperature is not critical in the formation of the phosphine, the preferred temperature is within ±25°C, most preferably within ±10°C. The hydroxy-unprotected aldehyde (XIIa) or hydroxy-protected aldehyde (XIIb) is then reacted with the phosphine in the same solvent and within the same temperature range to form the hydroxy-unprotected or protected alkene (Xb) or (Xa), respectively.

In the second step, the protecting group is deprotected by hydrolysis, which is only required when a hydroxyl protecting group is present, preferably using the aqueous acid conditions used in the third step, which involves the Jones oxidation of the primary alcohol (Xb ) is oxidized to acid (Xd). The Jones oxidation method uses the so-called Jones reagent, that is, an aqueous solution of HCrO ₄ formed from CrO ₃ and a strong acid. A typical Jones reagent is prepared from excess concentrated _H2SO4 , about 1:1 (by weight) _CrO3 and water, then diluted _with water to the desired concentration, eg, about 3M. The alcohol (Xb), protected alcohol (Xa), is generally dissolved in a water-miscible, reaction-inert organic solvent such as acetone, and they are reacted with at least two molar equivalents of Jones' reagent. Under these conditions, any silyl ether groups are rapidly hydrolyzed to the alcohol (Xb) and then oxidized to the acid (Xd). The temperature is not critical, eg 0-50°C is generally satisfactory, most suitably room temperature eg 17-27°C.

The Jones oxidation begins with the oxidation of alcohols to aldehydes (Xc), which are usually not isolated. If the intermediate aldehyde needs to be isolated, a more selective oxidizing agent, such as pyridinium chlorochromate, should be used to oxidize the alcohol. The resulting pure intermediate aldehyde (Xc) is reoxidized (using Jones reagent or another suitable reagent) to the acid (Xd).

If desired, the unsaturated acid (VIb) or (Xd) can be prepared in an activated form [e.g., acid chloride (VIc) or (Xe), commonly used mixed anhydrides, or with commonly used dehydrating coupling agents such as dicyclohexylcarbodiazine Activation], coupled with the compound represented by formula (Ⅷ) according to conventional methods to form the desired diester compound (formula IIIb) with immunomodulatory effect. The latter is hydrolyzed by a conventional method to obtain a dibasic acid compound (formula IIIa) having an immunomodulatory effect or a salt thereof which can be used as medicine.

In formula (VIII), R ⁹ and R ¹⁰ are each independently (C ₁ -C ₆ )alkyl, (C ₆ -C ₈ )cycloalkylmethyl or benzyl.

Alternatively, when ^R2 and/or ^R3 is benzyl, the compound of formula (IIIb) can be hydrogenated to form an immunomodulator of compound of formula (I). In this hydrogenation reaction, the double bonds are saturated with hydrogen and the benzyl groups (1 or 2) are hydrogenolyzed. The hydrogenation reaction is carried out in a reaction inert solvent and a hydrogenation catalyst, such as nickel or noble metal; the hydrogenation catalyst can be supported (such as Raney nickel, Pd/C) or unsupported (such as RhCl ₃ ). Solvent, temperature and pressure are not critical. Suitable solvents are not limited to lower alcohols, but may include ethers such as dioxane, tetrahydrofuran or dimethoxyethane; esters such as ethyl acetate. Preferably room temperature is used and cooling is not required even if the reaction is somewhat exothermic, thus avoiding the expense of heating or cooling. The pressure is not critical, but preferably below 7 atmospheres in order to avoid the use of expensive high pressure equipment. Use Pd/C to carry out hydrogenation reaction, the pressure 3-6 times of atmospheric pressure is especially suitable for this reaction.

Alternatively, the unsaturated acid (VIb) or (Xd) can be hydrogenated under the same conditions as detailed in the previous paragraph to give S-3-alkylheptanoic acid (II). The reverse hydrogenation/hydrolysis procedure can also be used to generate trans-R-3-alkyl-4-heptenoic acid (C ₁ -C ₃ ) alkyl ester (VIa) via S-3-alkylheptanoic acid (C ₁ - C ₃ ) Alkyl esters are obtained. Finally, the S-3-alkylheptanoic acid is activated by the method detailed above, coupled with the diester of the above formula (VII), and hydrogenated by the above method to obtain the immunomodulator of the compound of the above formula (I).

In another alternative route of the present invention, R-trans-4-alken-3-ol (V) is converted into the corresponding vinyl ether (IX), which is reacted in an inert solvent (preferably Excess ethyl vinyl ether), obtained by Hg(OAc) ₂ as a catalyst and ethyl vinyl ether, the temperature range is 25-40°C, and it is convenient to use ethyl vinyl ether (boiling point 36°C) at the reflux temperature. The resulting vinyl ether (IX) is heated to 140-200°C, typically in a high-boiling, lipophilic, reaction-inert solvent such as xylene or decalin, and stereospecifically rearranges under pressure, if necessary, to the unsaturated aldehyde ( VId). To obtain the unsaturated acid (VIb), the aldehyde is conveniently oxidized with the so-called Jones reagent (i.e. an aqueous solution of _H2CrO4 formed from _CrO3 and _a strong acid). A typical Jones reagent is prepared from an excess of concentrated sulfuric acid, about 1:1 (by weight) of _CrO3 and water, then diluted with water to the desired concentration, eg, about 3M. To form the unsaturated acid (VIb), the unsaturated aldehyde (VId) is usually dissolved in a water-miscible, reaction-inert organic solvent such as acetone and reacted with at least 1 molar equivalent of Jones' reagent. The temperature is not critical, eg generally 0-50°C is satisfactory, most suitably room temperature eg 17-27°C.

The crystalline form of the compound of formula (I) wherein R is methyl and R ⁴ =R ⁵ =hydrogen is obtained by recrystallization from an organic solvent or a mixture of organic solvents. Suitable solvents are acetone, acetonitrile/ethanol and tetrahydrofuran/ether. In terms of product recovery, the preferred solvent is acetonitrile/ethanol, but in terms of product purity, acetone is preferred. This novel form is certainly more stable than previous amorphous lyophilizates, it is easier to handle, denser, has far less electrostatic force, and can be prepared in more precise dosage forms.

The monobasic and dibasic salts of the compounds of formula ₍ I) or (IIIa) which can be used as medicine, usually in acid solution, preferably in aqueous solution and base (such as NaOH, KOH, _Na2CO3 or amine) Prepared by reacting in appropriate stoichiometric ratios, salts can be separated by evaporation or precipitation.

The compounds of formula (I) and (III) of the present invention are used as medicines for treating diseases in mammals (including humans) caused by various pathogenic microorganisms, especially Gram-negative bacteria. The compounds of the present invention are also useful as immunostimulants in mammals, including humans, since preexisting and clinically induced immunosuppression increases the risk of infection.

The test method was performed with normal or _CH3H /HeN immunized male mice from Charles River Breeding Laboratories. Mice were acclimatized for five days before use, and then administered subcutaneously (SC) or orally (PO) different dilutions (100, 10, 1 and 0.1 mg/kg) of the test compound or placebo (without a heat source of salt), the volume used was for 0.2 ml. The administration method is related to the microorganisms used for infection: Klebsiella pneumoniae were administered 24 and 0 hours before the challenge to normal mice; Escherichia coli or Staphylococcus aureus were administered 3, 2 and 1 day before the challenge to immunized mice. Intramuscular injection (IM) into the buttocks to attack Klebsiella pneumoniae, intraperitoneal injection (IP) to attack Escherichia coli or Staphylococcus aureus. A volume of 0.2 ml was used for challenge. Mortality was recorded after seven days with K. pneumoniae and after three days with the other two microorganisms.

Culture specimens:

Klebsiella pneumoniae, Escherichia coli, or Staphylococcus aureus: For purification, cultures were streaked on brain heart infusion (BHI) agar with frozen blood stocks. Three colonies were picked from 18-year-old plate cultures and placed in 9 ml of BHI broth. broth cultures in rotation After 2 hours of growth at 37°C on a shaker, 0.2 ml was streaked on several brain heart infusion agar slants. Then cultured at 37°C for 18 hours, the slant was washed out with brain heart infusion medium, the turbidity of the culture was checked with Spectronic 20 and appropriate dilution was made to achieve the LD90 challenge level in normal mice.

When the compounds (I) and (III) of the present invention are used in humans as anti-infective agents or immunostimulants, they are usually administered orally, subcutaneously, intramuscularly, intravenously, or intraperitoneally, usually in the form of Administered in the form of a composition conforming to specifications in pharmaceutical practice. For example, tablets, pills, powders, or granules that contain excipients (such as starch, lactose, some binders, etc.) may be taken. Capsules can be taken, which are obtained by mixing with the same or equivalent excipients mentioned above, and then putting the mixture into capsules. Oral suspensions, solutions, emulsions, syrups and elixirs can also be used, and they may contain flavoring and coloring agents. For oral administration of the therapeutic formulations of the present invention, tablets or capsules containing from about 50 to 500 mg of active ingredient are suitable for most uses.

Physicians will determine the most appropriate dose based on each patient's individual age, weight, individual patient response, and route of administration. The preferred oral dosage range is from about 1.0 to about 300 mg/kg/day in single or divided doses. Dosages for parenteral administration are preferably from about 1.0 to about 100 mg/kg/day; more preferably in the range from about 1.0 to about 20 mg/kg/day.

The following examples are given to illustrate the present invention, but should not be regarded as a limitation of the present invention. Within the scope and spirit of the present invention, many changes can be made to the following examples.

Example 1

R-trans-4-hexen-3-ol

In a 500 ml three-necked round bottom flask equipped with electromagnetic stirring, a septum, a thermometer and a _N2 tube, put 270 ml of _CH2Cl2 and _7.2 g of type 4A molecular sieves. Ti[OCH(CH ₃ ) ₂ ] (10.7 ml _, 0.036 mol) was added via syringe, the mixture was _cooled to -66°C, and the viscosity-reduced L-(+ ) diisopropyl tartrate (10.1 g, 0.043 mol) and rinsed with an additional 5 ml _{of CH2Cl2} . When the temperature had risen to -62°C, rac trans-2-hexen-4-ol (3.60 g, 0.036 mol) was added and _rinsed with 5 ml _CH₂Cl₂ . After stirring in an acetone/dry ice bath for 8 minutes, when the temperature had dropped to -68°C, tert-butyl hydroperoxide (7.18 mL of a 3M solution in toluene, 0.022 mol) was added by syringe. The reaction mixture was warmed to -35°C and maintained at this temperature for 18 hours in a freezer. The cooled mixture was filtered through fluted filter paper into a stirred mixture of 540 mL acetone and 11 mL _H2O which had been cooled to -20°C. The mixture was slowly warmed to room temperature and stirred for 20 hours. The mixture was filtered through celite and _washed with _CH2Cl2 . The combined filtrate and washings were evaporated without heating to give an oil (17.62g). Avoid excessive evaporation as the desired product is volatile. The oil was chromatographed on 176 g of silica gel using 4:1 hexane:isopropyl ether as eluent and monitored by thin layer chromatography. The solvent was removed by distillation using a short column equipped with a variable discharge head and packed with a glass single helical ring. The solvent was boiled to a head temperature of 69.5°C (at which point the head temperature began to drop) and the residue was pure title product; TLC Rf 0.25 (3:1 hexane:ether). When this experiment was repeated, the more volatile 4:1 pentane:ether was used as the eluent, which allowed for faster solvent removal to minimize product loss on evaporation.

Example 2

R-3-methyl-4-hexenoic acid ethyl ester

The product of the previous example (0.4 g), triethyl orthoacetate (3 ml) and trimethylacetic acid were heated at reflux for 2.5 hours, cooled, and the whole reaction mixture was chromatographed twice on silica gel, starting with 6:1 hexane:ether Elution gave 0.45 g of product, then elution with 7:1 hexane:ether gave 0.35 g of pure title product; , 840 cm ^-1 .

Alternatively, the pentane/ether column fractions containing pure S-trans-2-hexen-4-ol were all carried directly to this step, initially distilling pentane and ether from the reaction mixture.

Propionic acid instead of trimethylacetic acid gave virtually the same results. Dry HCl and 2,4-dinitrophenol can also be used successfully as acid catalysts, but in the next example, _AlCl3 is the best catalyst.

Example 3

R-3-methyl-4-heptenoic acid

Method A

Into a 35 ml flask equipped with a magnetic stirrer and a Soxhlet extractor fitted with a type 4A molecular sieve in the cannula was charged the product of the previous example (1.5 g, 0.015 mol). Triethyl orthoacetate (9 ml, 0.049 mol) was added, followed by AlCl ₃ (0.12 g, 0.009 mol), and the mixture was refluxed on a Soxhlet extractor for 2 hours, during which time TLC showed that Complete conversion to intermediate R-3-methyl-4-hexenoic acid ethyl ester; TLC Rf0.75 (4:1 hexane:ethyl acetate), Rf0.85 (15:5:2 hexane : ether: acetic acid).

The reaction mixture was cooled, diluted with 15 mL of 2N NaOH and 12 mL of methanol, and stirred at room temperature for 27 hours, during which time TLC showed that the hydrolysis was complete. Methanol was removed from the reaction mixture, diluted with 12 mL _H2O , extracted with 3 x 25 mL _{CH2Cl2 ,} the combined _CH2Cl2 was backwashed with 1 x 25 mL 2N NaoH, the combined aqueous layer and backwash _were washed with Concentrated HCl was adjusted to pH 1 and _extracted with 3 x _CH2Cl2 . The combined organic layers were dried over _MgSO4 and evaporated to give 1.28 g (60%) of the title product as an oil; TLC Rf 0.65 (15:5:2 hexane:ether:acetic acid);

¹ H-NMR (CDCl ₃ ) δ (ppm) 9.4 (S, 1H, -CO ₂ H), 5.5 (m, 2H, -CH=CH-), 3.0-1.6 (m, 5H), 1.3-0.8 ( m, 6H); IR (film) 3400-2400, 2960, 2925, 2860, 1708, 1458, 1410, 1380, 1295, 1228, 1190, 1152, 1100, 930 cm ^-1 .

Method B

The preceding ester product (0.24 g) was combined with 25 ml CH ₃ OH and 11.5 ml 1N NaOH and the mixture was stirred at room temperature for 3.5 hours, during which time tlc showed complete hydrolysis. The mixture was extracted with 2 x 35 ml ether, adjusted to pH 2 with conc. HCl, and extracted 3 x 35 ml ether. The acid extracts were combined, dried and evaporated to give 0.20 g of the title product corresponding to the Method A product.

Example 4

S-3-methylheptanoic acid ethyl ester

The product of Example 2 (0.20 g) was hydrogenated in 40 ml of ethyl acetate with 0.20 g of 5% Pd/C (50% water wet) in a Paar hydrogenation apparatus under 4 atmospheres of hydrogen for 3 hours. The catalyst was recovered by filtration through celite. Evaporation of the filtrate gave the title product.

Example 5

S-3-methylheptanoic acid

Method A

The product of Example 3 (0.20 g) was hydrogenated in 40 ml of ethyl acetate with 0.2 g of 5% Pd/C (50% water wet) in a Paar hydrogenation apparatus under 4 atmospheres of hydrogen for 3 hours . The catalyst was recovered by filtration through celite and the filtrate was evaporated to give 0.2 g of the title product as an oil. If necessary, distill under high vacuum to give the pure title product, bp 77-79 °C/0.2 mm; ¹ H-NMR) CDCl ₃ ) δ) ppm): 12.0 (S, COOH), 1.0 (d, - CH ₃ ), 0.6-2.8 (m, rest 13H); IR (film) 3400-2400, 2960, 2925, 2860, 1708, 1458, 1410, 1380, 1295, 1228, 1190, 1152, 1100 ^{, 930} cm- ¹ ; [α] = -6.41° (c = 1% in methanol); n ^22.5 _D = 1.427.

Method B

The preceding product was hydrolyzed according to method B of Example 3 to obtain the title product.

Example 6

S-3-Methylheptanoyl chloride

The acid product from _the previous Example or Example 27 (8.5 g, 0.062 mol) was dissolved in 18 ml _CH2Cl2 . Oxalyl chloride (5.36 ml, 7.80 g, 0.0614 mol) was added to the solution and the mixture was allowed to stand for 4 hours, during which time the reaction was complete as evidenced by no further gas evolution. The acid chloride solution was immediately used directly in Example 8, Method C. Alternatively the acyl was isolated after evaporation of the solvent and used in Example 8 Method A and, if desired, distilled for further purification, bp 45°/1.5 mm.

Example 7

R-3-Methyl-4-heptenoyl chloride

The acid product of Example 3 (0.747 g, 5 mmol) was converted into a solution of the title product in _CH2Cl2 by the procedure of the previous example and used directly in _Example 9 below. Alternatively, the reaction mixture is evaporated to give the title product, which can be distilled off under reduced pressure if desired.

Example 8

N-(S-3-methylheptanoyl)-D-gamma-glutamyl (alpha benzyl ester)-glycyl-D-alanine benzyl ester.

Method A

Add 660 mg (4.06 mmol) of S-3-methylheptanoyl chloride to 1.0 g (2.03 mmol) of D-γ-glutamyl (α-benzyl ester-glycyl-D-alanine benzyl ester hydrochloride salt (Preparation 4) and 616 mg (6.09 mmol) of triethylamine in 50 mL of dichloromethane, and the reaction mixture was stirred at room temperature for 80 hours. The dichloromethane was distilled off in vacuo, and the residue was dissolved in in ethyl acetate. The resulting solution was washed successively with 2.5% hydrochloric acid, water, 10% potassium carbonate solution, water and sodium chloride solution. The organic phase was separated, dried over magnesium sulfate and concentrated in vacuo. The residue was triturated with diethyl ether Filtration under nitrogen afforded the title product, which was used in its entirety directly in Example 10, Method A.

Method B

The product of Preparation 4 (0.75 g, 1.53 mmol), 5 mL of dichloromethane and triethylamine (0.212 mL, 1.53 mmol) were combined, stirred under nitrogen, and S-3-methylheptanoic acid (Example 5 , 0.20 g, 1.39 mmol) in 4 ml of CH ₂ Cl ₂ , then dicyclohexylcarbodiimide (0.286 g, 1.37 mmol) was added, and the reaction mixture was stirred for 16 hours. The reaction mixture was filtered, the filtrate was evaporated, the residue was dissolved in 10 mL of ethyl acetate, the solution was washed successively with 5 mL of 2.5% HCl, 5 mL of water, ₅ mL of 10% _K2CO3 solution and 5 mL of brine, passed over _MgSO4 Drying yielded 71 mg (88%) of the title product.

Method C

In a 500-ml four-necked round-bottom flask equipped with a stirrer, thermometer, dropping funnel, and N _inlet tube, the product from Preparation 4 (32.8 g, 0.059 mol) was dissolved in 175 ml _{of CHCl} and _chilled . to 0-5°C. Maintaining this temperature range, triethylamine (24.7 ml, 17.9 g, 0.177 mol, 3 equiv) was slowly added dropwise over 15 minutes. Under cooling in an ice-water bath, the entire _CH2Cl2 solution of S-3-methylheptanoyl chloride obtained in Example ₆ was added within 15 minutes, and its temperature rose to 21°C. Stirring was continued in the ice-water bath for 45 minutes, during which time the gelled mixture became too thick for stirring to occur. The gelatinous mass was broken up and mixed with 125 ml 10% HCl and ₅₀ ml _CH2Cl2 . The organic layer was separated, washed sequentially with 2 x 125 mL HO, 2 x 125 mL 10% _K2CO3 and 1 x 125 mL _H2O , dried over _MgSO4 and evaporated to yield 82.3 _g of a damp white solid. This solid was dissolved in 500 mL of hot ethyl acetate. Slowly cooling to room temperature, the title product slowly crystallized, and the mixture was diluted with an additional 40 ml of ethyl acetate to maintain good stirring. Filtration and vacuum drying at 40°C gave 31.1 g (90.5%) of pure title product.

¹ H-NMR (CDCl ₃ ) δ (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), 0.92-0.8 (m, 6H).

Example 9

N-(R-3-Methyl-4-heptenoyl)-D-γ-glutamyl-(α-benzyl ester)-glycyl-D-alanine benzyl ester

Following Procedure C of the previous example, the product of Preparation 4 (2.77 g, 5 mmol) was reacted with the entire acid chloride from Example ₇ in _CH2Cl2 . The washed and dried organic layer was evaporated, and the residue was dissolved in ethyl acetate, evaporated, and repeated several times to obtain the product.

Example 10

N-(S-3-Methylheptanoyl)-D-γ-glutamyl-glycyl-D-alanine.

Method A

The entire product of Example 8, Method A, was dissolved in 65 mL of methanol. To the solution was added palladium hydroxide (250 mg), and the mixture was shaken under hydrogen at 4 atm for 3 hours. The catalyst was filtered off and the solvent was removed in vacuo. The residue was dissolved in water and lyophilized to obtain the desired product.

NMR (DMSO-d ₆ ) at 8.27-8.03 (m, 3H), 4.32-4.1 (m, 2H) 3.72 (d, J=6Hz, 2H), 2.22 (t, J=8Hz, 2H), 2.27 Absorption at -1.68 (m, 6H), 1.42-1.0 (m, 10H) and 0.94-0.8 (m, 6H).

When carried out with the product of Example 8 (0.50 g), 90 mg of 20% Pd/(OH) ₂ /C (31% water wet) in 25 ml of methanol, the procedure yielded 0.24 g of the same soft, static title product ; Ir (paraffin paste) 3300, 2940, 1740, 1650, 1540, 1468 and 1380 cm ^-1 ; all but the last two peaks are broad and poorly resolved.

Method B

Example 8 Process C product (30.8 g) was slurried in 300 ml of absolute ethanol in a 2 liter autoclave and 5% Pd/C (1.54 g, 50% water wet) was added. The mixture was hydrogenated at 4 atmospheres for 1 hour, during which time hydrogen uptake was complete. The catalyst can be recovered by passing through filter paper first, then through 0.45 micron pore nylon filter, and washed with 100-150 ml of ethanol. The filtrate and washings were combined and evaporated to give a wet white solid, which was dissolved in 150 ml of hot 1:10 mixture of absolute ethanol and acetonitrile, hot filtered to make it clear, evaporated to reduce the volume to 35 ml, slow Cool slowly to room temperature, pulverize and filter to obtain the title product in a dense crystalline form without static electricity, weighing 20.1 g (94%), identified by IR (paraffin paste), and its main sharp peaks with good resolution are 3340, 3300, 2900 , 2836, 1725, 1650, 1628, 1580, 1532, 1455, 1410, 1370, 1280, 1240, 1216 and 1175 cm ^-1 .

Method C

The crystalline product (9.4 g) prepared by the previous method B was directly placed in 1000 ml of acetone, Heat to reflux for 1 hour to dissolve. The solution was cooled to room temperature, and crystallization was induced by using a trace amount of the product obtained in method B as a seed crystal. After stirring for 6 hours, the further purified title product was collected by filtration, washed with a small amount of acetone, and dried in vacuo at 35°C to yield 7.25 g, which had IR peaks consistent with Method B crystallization from acetonitrile/ethanol.

Method D

The product of the previous example (0.50 g) was placed in a Paar hydrogenation bottle with 0.026 g of 5% Pd/C (50% water wet) and 125 ml of absolute ethanol. The mixture was hydrogenated under 4 atmospheres of hydrogen for 2.5 hours. The catalyst was recovered by filtration, and the filtrate was evaporated to obtain the title product as a viscous solid, which was directly crystallized according to the previous method.

Example 11

N-(R-3-Methyl-4-heptenoyl)-D-γ-glutamyl-glycyl-D-alanine

The product of Example 9 (1 g) was dissolved in 5 ml CH ₃ OH. 1N NaOH (2.50 mL) was added and the mixture was stirred at room temperature for 3 hours. The methanol was evaporated and the aqueous residue was diluted with 7.5 mL of water, extracted with 2 x 7.5 mL of ethyl acetate and acidified to pH 3.0 with 1N HCl. Continuous extraction with fresh ethyl acetate through acidified water and evaporation of the extract gave the title product, which was lyophilized as in Example 10, Method A.

Example 12

R-trans-4-hepten-3-ol

The title compound of Preparation 5 (10 g, 0.088 mol) was converted to the title product by the procedure of Example 1, except that the reaction temperature was -20°C during all reagent addition stages. After all the reagents had been added, the mixture was stirred at -20°C for 1.5 hours, then warmed to room temperature and quenched by the dropwise addition of 25 ml HO followed by 6 ml 30% NaOH (saturated with NaCl). The cooled mixture was stirred _for 20 minutes, diluted with 90 mL of _CH2Cl2 , and the aqueous layer was separated and _washed with 2 x 50 mL of fresh _CH2Cl2 . The combined organic layers were dried over _MgSO4 and evaporated to a residual volume of 50 mL and chromatographed on silica gel using pentane and then 4:1 pentane:ether as eluents. The initial fraction containing a small amount of polar impurities was discarded. Pure product fractions with Rf 0.3 (4:1 pentane:ether) were combined and evaporated to give 5.6 g of the title product as a clear colorless liquid.

Example 13

R-3-Ethyl-4-heptenoic acid

Using method A of Example 3, except that diethyl ether was used instead of _CH2Cl2 during isolation, the _previous product (5.7 g, 0.05 mol) was condensed with ethyl orthoacetate, rearranged and saponified to give the title product as an oil, TLC Rf0 .4 (2:1 hexane:ether). In the same thin-layer system, the intermediate ethyl ester has an Rf of 0.25.

Example 14

S-3-ethylheptanoic acid

The product of the previous example (1.4 g) was hydrogenated in 20 ml CH ₃ OH with 0.1 g 5% Pd(OH)/C at 4 atm for 1 hour, and the catalyst was recovered by filtration. The solvent was removed from the filtrate and the residue was distilled to give 1.0 g of the title product; bp 76-77°C/0.4 Torr; [α] ²⁵ _D -1.6° (c=2% in methanol).

Example 15

S-3-Ethyl-heptanoyl chloride

Under dry _N2 , the product of the _previous example (1.0 g, 0.00633 mol) was dissolved in 10 mL of _CH2Cl2 , oxalyl chloride (0.547 mL, 0.00627 mol) was added, and the mixture was stirred for 1 hour, during which time the evolution ceased. gas. The resulting solution of the title product was evaporated to 7 mL under a stream of _N2 . can be used directly in the next step.

In the same way, the unsaturated acid of Example 13 can be converted into a solution of R-3-ethyl-4-heptenoyl chloride.

Example 16

N-(S-3-Ethylheptanoyl)-D-γ-glutamyl(2-benzyl ester)-glycyl-D-alanine benzyl ester

The title product of Preparation 4 (1.1 g, 0.00222 mol) was dissolved in 30 ml of _CH2Cl2 and _{triethylamine} (0.935 ml, 0.00666 mol). The CH ₂ Cl ₂ solution of S-3-ethylheptanoyl chloride (2.3 ml, 0.00211 mol) from the previous example was added, and the mixture was stirred under N ₂ for 0.75 hours, followed by 20 ml of 10% HCl, H ₂ O, 10% K Washing with ₂ CO ₃ and saturated brine, drying over MgSO ₄ and evaporation gave a solid residue which was triturated with ether and recovered by filtration to yield 0.8 g. This was dissolved in ethyl acetate, washed again as above and evaporated again to give another 0.7 g of solid which was chromatographed on silica gel using 97:3 CHCl:CHOH as eluent to give 467 mg of pure title product .

In the same manner, 3-ethyl-4-heptenoyl chloride was coupled with the title product of Preparation 4 to give N-(R-3-ethyl-4-heptenoyl)-D-γ-glutamyl ( α-Benzyl Ester)-Glycyl-D-Alanine Benzyl Ester.

Example 17

N-(S-3-Ethylheptanoyl)-D-γ-glutamyl-glycyl-D-alanine

The title product of the previous example (467 mg) was hydrogenated as in Example 14. After recovery of the catalyst, the filtrate was evaporated to give a foam which was dissolved in water, filtered and lyophilized to give 238 mg of the title product.

¹ H-NMR (DHSO-d ₆ ) δ (ppm): 8.20-8.00 (m, 3H), 4.24-4.16 (m, 2H), 3.74-3.60 (m, 2H), 2.18 (t, J=7, 2H), 2.02 (d, J=7, 2H), 2.02-1.60 (m, 3H), 1.26 (d, J=6, 3H), 1.26-1.08 (m, 8H), 0.92-0.74 (m, 6H ).

In the same way, the unsaturated product of the previous example can be converted into the same product.

Example 18

N-(R-3-Ethyl-4-heptenoyl)-D-γ-glutamyl-glycyl-D-alanine

The unsaturated product of Example 16 can be hydrolyzed to the title product by the method of Example 11.

Example 19

N-(S-3-Ethylheptanoyl)-D-γ-glutamyl(α-benzyl ester)-glycyl-D-alanine butyl ester

Using the method of Example 17, S-3-ethylheptanoyl chloride and D-γ-glutamyl (α benzyl ester)-glycyl-D-alanine butyl ester (1.0 g, 0.00222 mol) of Example 16 were mixed. Coupling gave the title product, which was isolated and purified in the same way to obtain 0.7 g.

¹ H-NMR (DMSO-d ₆ ) δ (ppm): 8.22 (d, J=7, 1H), 8.12-8.00 (m, 2H), 4.40-4.16 (m, 2H), 4.08-3.95 (m, 2H), 3.75-3.62 (m, 2H), 2.18 (t, J=6, 2H), 2.02 (d, J=6, 2H), 2.04-1.62 (m, 3H), 1.60-1.46 (m, 2H ), 1.38-1.10 (m, 15H) 0.90-0.75 (m, 9H).

In the same way, R-3-ethyl-4-heptenoyl chloride can be converted into N-(R-3-ethyl-4-pentenoyl)-D-γ-glutamyl (α-benzyl ester) - Glycyl-D-alanine butyl ester.

Example 20

N-(S-3-Ethylheptanoyl)-D-γ-glutamyl-glycyl-D-alanine butyl ester

The title product of the previous example was hydrogenated by the method of Example 14 to obtain the title product. After recovery of the catalyst, the filtrate was evaporated to give a solid which was triturated with ether and filtered to give 256 mg, mp 130-131°C.

In the same way, the unsaturated product of the previous example can be converted to the same product.

Example 21

R-3-Ethyl-4-heptenal

A solution of R-trans-4-hepten-3-ol (3.0 g) of Example 12 in 300 ml of dry ethyl vinyl ether was heated to reflux during which time five portions of Hg(OAc) ₂ (each 2 g at 2 hour intervals), and after an additional 18 hours of reflux, the clear solution was cooled to room temperature, treated with 0.5 ml of glacial acetic acid and stirred for 3 hours. The resulting solution was diluted with ether, poured into 200 ml of 5% KOH solution, and extracted three times with ether. The combined ether extracts were dried with K ₂ CO ₃ and evaporated to give allyl vinyl ether, R-trans-O-vinyl-4-hepten-3-ol.

A solution of the above allyl vinyl ether (2.3 g) in 20 ml of decalin was refluxed for 10.5 hours. The resulting solution was then cooled to room temperature; directly placed on a silica gel column and chromatographed. After rinsing with hexane to remove the decalin, the column was eluted with ether and evaporated to give the title product.

Example 22

S-3-Ethyl-4-heptenoic acid

The product of the previous example (100mg) was dissolved in 4ml of acetone and cooled to 0-5°C. In another flask, CrO ₃ (72.1 g, 0.72 mol) and 50 ml H ₂ O were mixed, stirred at 0-5°C, 62.1 ml concentrated H ₂ SO ₄ was slowly added, and the mixture was diluted to 250 ml with H ₂ O , to obtain 2.88 moles of H ₂ CrO ₄ solution (Jones reagent). A solution (1 ml) was added to the above acetone solution in portions over 1 hour. The temperature was raised as the reagents were added and was maintained at 17-25°C. The mixture was recooled to 6°C and 70 ml of 2-propanol was added over 10 minutes (during which time the temperature was allowed to rise to 20°C), then concentrated under vacuum to give an oil which was added to the oil within 50 minutes with stirring. Add 8 ml of 2.5N NaoH and keep the temperature at 22±5°C. The mixture was heated and filtered through celite, washing with 2.5N hot NaoH. The combined filtrates were extracted with 3 x 300 ml isopropyl ether. The combined organic layers were back extracted with 200 mL of 2N NaoH. The aqueous layers were combined, acidified to pH 1 by the slow addition of 0.5 mL of concentrated HCl, and the product was extracted into 3 x 300 mL of fresh diisopropyl ether. The organic layers were combined and evaporated to give the title product as an oil.

Example 23

S-7-(tert-butyldimethylsiloxaneoxy)-5-methyl-1-heptene

In a 500-mL four-neck round-bottom flask equipped with a stirrer, thermometer, N _inlet tube, and addition funnel, methyltriphenylphosphonium bromide (25.7 g, 0.072 mol, 1.25 equiv) was mixed with 77 mL of THF (tetrahydrofuran) Slurry together and cool in an acetone/wet ice bath. Put n-butyllithium (43.2ml 1.6N hexane solution, 0.069mol, 1.20eq) into the addition funnel, add butyllithium to the slurry with an initial temperature of -8°C within 1 hour, the temperature rises and maintains within ±1°C. The mixture was stirred for an additional 0.5 h at 0-2°C to ensure complete formation of the intermediate methylenetriphenylphosphine in a thin LiBr suspension. A solution of the aldehyde product of Preparation 2 (14.1 g, 0.0576 mol) in 14 ml of THF was added portionwise over 40 minutes, controlling the temperature at 3-7°C. After stirring for an additional 15 minutes, it was checked by thin layer chromatography using 3:1 hexane:ether as the eluent, which showed the absence of starting aldehyde (starting aldehyde Rf 0.6; product Rf 0.95). The reaction mixture was warmed to room temperature and diluted with 150 mL ethyl acetate and 90 mL _H2O . The organic layer was separated and washed with 2 x 100 mL _H2O . The combined three aqueous layers were back extracted with 40 mL of ethyl acetate. The combined organic layers were dried over _MgSO4 and evaporated to give 25 g of an oil which was triturated with 10 mL of hexane and filtered on a fritted glass funnel, washing the solid well with 4 x 10 mL of hexane, combining the hexane filtrate and washing The liquid was evaporated; the title product was obtained as an oil, 13.5 g (96.6%).

¹ H-NMR (CDCl ₃ ) δ (ppm): 5.4-6.2 (m, =CH), 4.8-5.3 (m, =CH ₂ ), 3.7 (t, J = 6.5H, -OCH ₂ O-), 0.08 (s, C(CH ₃ ) ₃ ) and 0.0 (s, Si(CH ₃ ) ₂ ), mixed with 8 mol % (C ₆ H ₅ ) ₃ PO (7.6, s, 1.25H).

Example 24

S-3-Methyl-hept-6-en-1-ol

Method A

Using the procedure of the previous example, but using 2.2 equivalents each of methyltriphenylphosphonium bromide and n-butyllithium, the aldehyde product of Preparation 6 (26.3 g, 0.20 moles; purity acceptable) was reacted with methylenetriphenylphosphine. Although a gummy solid formed upon addition of the aldehyde solution, the reaction became a thin slurry as soon as the reaction was allowed to warm to room temperature. The reaction mixture was diluted with 500 mL _H2O and 300 mL ethyl acetate. The layers were separated and washed with 3 x 250 mL _H2O . The combined aqueous layers were backwashed with 2 x 300 mL ethyl acetate. The three organic layers were combined, dried over _MgSO4 and evaporated to give 65.7 g of an oil containing 25.6 g (100%) of the title product and about 40 g of triphenylphosphine oxide, which was further processed as in Example 26 below.

Method B

The pure title product can be obtained relatively easily by hydrolyzing the product of Example 23 in a conventional manner, for example in dilute sulfuric acid as described in Example 26 below. The title product was isolated by extraction into ethyl acetate, drying over _MgSO4 and evaporation.

Example 25

S-3-Methyl-6-heptenal

The title product of the previous example (1.14 g, 0.01 mol) was dissolved in 20 ml _{of CH2Cl2} and cooled to 0°C. Pyridinium chlorochromate (4.30 g, 0.02 mol) was added in portions while maintaining the temperature at 0-5°C. The mixture was warmed to room temperature, stirred for 2 hours, filtered through a pad of silica gel and the filtrate evaporated to give the title product as an oil which was further purified by distillation if necessary.

Example 26

S-3-methyl-6-heptenoic acid

Method A

In a 2000 ml three-necked flask equipped with agitator, thermometer and addition funnel, the title product of Example 23 (81 g, acceptable purity, 0.33 moles) was dissolved in 400 ml of acetone and cooled to 0-5°C. In another flask, mix CrO ₃ (72.1 g, 0.72 mol) with 50 ml of water, and slowly add 62.1 ml of concentrated sulfuric acid dropwise under stirring at 0-5°C, and dilute the mixture with 250 ml of water to obtain 2.88 molar H ₂ CrO ₄ solution (Jones reagent). The latter solution (240 ml, 0.67 mol) was added portionwise to the above acetone solution over 1.2 hours. The temperature quickly rose to 17°C, and the temperature was maintained at 17-25°C when the reagent was added. At the end of the addition, there was no temperature rise, and the mixture was cooled to 6°C again. Within 10 minutes, 70 ml of 2-propanol was added. (At this point the temperature rises to 20°C), and then concentrate in vacuo to form an oil. With stirring while maintaining the temperature at 22±5°C, add 400ml of 5N NaoH within 50 minutes, and dilute the viscous oil with 400ml of _H2O . The reaction mixture was filtered on celite, and the wet cake was slurried with 400 ml of H ₂ O and 50 ml of 5N NaoH, heated on a steam bath, re-filtered, washed with 3×300 ml of isopropyl ether and combined of the filtrate. The combined organic layer solution was back extracted with 200 mL of 2N NaOH, the combined aqueous layer solution was acidified to pH 1.0 by the slow addition of 50 mL of concentrated hydrochloric acid, and the product was extracted into 3 x 300 mL of fresh isopropyl ether. The combined organic extracts were evaporated to give 29.1 g (61%) of the title product as an oil. The diatomaceous earth filter cake extract was basified again, separated by the same method, and another 7.7 g (16%) of the product was obtained. ¹ H-NMR of the combined product (CDCl ₃ ), δ (ppm): 11.9 (S, -COOH), 5.8 (m, =CH), 5.0 (m, =CH ₂ ), 1.0 (d, -CH ₃ ) , isopropyl ether peaks at 3.7 and 1.1, indicating the presence of 8.6 mole % (6.3 weight %) of isopropyl ether, but this does not affect the use of this product in further operations.

Method B

The product of Example 24, Method A (65.7 g, containing 26.3 g, 0.20 moles of S-3-methylhept-6-en-1-ol) was oxidized according to Method A of this Example. After addition of isopropanol and stirring at 0-5 °C for 1 hour, the reaction mixture was completely green, the organic solvent was evaporated, the aqueous residue was diluted with 250 mL of water, extracted with 3 x 250 mL of isopropyl ether, and the combined organic The extract was treated with 160 ml of 2N NaoH to generate a large amount of precipitate (C ₆ H ₅ ) ₃ PO (mixed in the raw material), which was recovered by filtration, washed with 1N NaoH, the filtrate and washings were combined, the solution was separated, and the organic The layers were washed with another 80 mL of 1N NaOH, all aqueous layers were combined, washed with 3 x 250 mL of isopropyl ether, acidified to pH 1.0 with 50 mL of concentrated HCl, and the desired product was extracted into 3 x 250 mL of freshly prepared The combined acidic organic extracts were dried over _MgSO4 in isopropyl ether and evaporated to give the title product, 14.0 g, which was further purified by distillation if necessary.

Example 27

S-3-methylheptanoic acid

10%Pd/C (1.64 grams, containing 50% water), 150 milliliters of ethyl acetate and the unsaturated acid (3.28 grams, qualified in purity) in the previous example are packed in a Parr hydrogenation bottle, and the slurry is carried out at 4 atmospheres Hydrogenation takes about 1.5 hours for hydrogen absorption to complete. The catalyst is recovered by filtration through celite, and the filtrate is evaporated to obtain 3.20 g (96%) of the title product as an oil.

In addition, 5% Pd/C (2 grams, containing 50% water), the title product of the previous example (33.3 grams, qualified in purity) and 150 milliliters of ethyl acetate were charged together in a 1-liter autoclave, at 30-31 ° C, 4 Hydrogenation under atmospheric pressure for 2 hours, at this time _H2 has been completely absorbed. The catalyst was recovered by filtration on celite, and the filtrate was evaporated to give 35.4 g of an oil, which was distilled under high vacuum to give 29.6 g (87.6%) of the pure title product; bp 77-79°C/0.2 mm; ¹ H-NMR (CDCl ₃ ) δ (ppm): 12.0 (s, -COOH), 1.0 (d, -CH ₃ ), 0.6-2.8 (m, rest 13H); IR (thin film) 3400-2400, 2960, 2925, 2860 , 1708, 1458, 1410, 1380, 1295, 1228, 1190, 1152, 1100, 930 ^cm -1, [α] ²⁵ _D = -6.41° (C = 1% in methanol); n ^22.5 _D = 1.427.

Example 28

S-3-Methyl-6-heptenoyl chloride

The product acid _of Example 4 (0.747 g, 5 mmol) was converted by the method of Example 6 into a solution of the title product in _CH2Cl2 , which was used directly in Example 9 below. Alternatively, the reaction mixture is evaporated to give the title product, which can be distilled under reduced pressure if desired.

Example 29

N-(S-3-Methyl-6-heptenoyl)-D-γ-glutamyl-(α-benzyl ester)-glycyl)-D-alanine benzyl ester

Using Example 8, Method C, the product of Preparation 4 (2.77 g, 5 mmol) was coupled with the entire acid chloride from Example ₂₈ in _CH2Cl2 . The initial product 2.77 g was obtained by evaporating the washings and the dried organic layer. This product was dissolved in 20 ml of ethyl acetate, diluted with 20 ml of hexane, cooled and filtered to give 2.24 g of pure product ( 77%), melting point 137.5-139.5°C.

Example 30

N-(S-3-Methyl-6-heptenoyl)-D-γ-glutamyl-glycyl-D-alanine

The product of Example 29 (1 g) was dissolved in 5 mL of methanol, 1N NaoH (2.50 mL) was added, the mixture was stirred at room temperature for 3 hours, the methanol was evaporated, the water residue was diluted with 7.5 mL of HO and washed with 2 x 7.5 mL of acetic acid Extracted with ethyl ester, acidified to pH 3.0 with 1N HCl. Continuous extraction of the acidic aqueous solution with fresh ethyl acetate and evaporation of the extract afforded the title product, which was converted to a lyophilizate by following Method A of Example 10.

Example 31

N-(S-3-Methyl-6-heptenoyl)-D-γ-glutamyl (α-benzyl ester)-glycyl-D-alanine butyl ester

Coupling of D-γ-glutamyl(α-benzyl ester)-glycyl-D-alanine butyl ester (Preparation 4) with S-3-methyl-6-heptenoyl chloride using Example 8, Method A , get the product of this title.

Example 32

Butyl N-(S-methylheptanoyl)-D-γ-glutamyl-glycyl-D-alanine

The preceding product can be converted to the title product by hydrogenation as in Example 10.

preparation 1

rac-trans-4-hexen-3-ol

Put 400 milliliters of THF into a 3-liter four-necked flask equipped with stirring, an addition funnel, a thermometer and a nitrogen inlet tube. THF was cooled to -70 ° C, and ethylmagnesium bromide (600 milliliters of 2M THF solution, 1.2 molar ). Keeping the temperature at -70°C to -60°C, crotonaldehyde (78 ml, 0.94 mol) was added within 7 minutes. After stirring at -70°C for 20 minutes, the mixture was slowly warmed to -20°C and stirred for 1 hour. The mixture was cooled again to -70°C, quenched carefully by the addition of 200 ml sat. NH ₄ Cl (foaming started), warmed to room temperature, diluted with 200 ml acetic acid and 100 ml water, and the two layers were saturated with NaCl. The aqueous layer was separated and extracted with 2 x 500 ml ether. The initial organic layer and ether extract were combined, washed with 4 x 250 ml saturated NaHCO, dried over MgSO ₄ , evaporated, and the residue was distilled at atmospheric pressure to give 69 g; boiling point 133-137°C., ¹ H-NMR (CDCl ₃ ) δ (ppm): 5.53 (complex m, 2H), 3.39 (m, 1H), 2.26 (s, 1H), 1.71 (d, 3H), 1.43 (m, 2H), 0.84 (t, 3H) .

preparation 2

Glycyl-D-alanine benzyl ester hydrochloride

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

preparation 3

N-tert-butoxycarbonyl-D-γ-glutamyl (α-benzyl ester) hydroxysuccinamide ester

Add 30.9 g (15 mmol) of dicyclohexylcarbodiimide containing 50 g (143 mmol) of N-tert-butoxycarbonyl-D-γ-glutamic acid α-benzyl ester and 17.3 g (150 mmol) ) N-hydroxysuccinamide in 1500 ml of dichloromethane, and the resulting reaction mixture was stirred at room temperature Stir for 18 hours. The solid was filtered and the filtrate was concentrated in vacuo. The residue was triturated with ether and the solid was collected by filtration under a stream of nitrogen, yielding 43.7 g (68% yield).

preparation 4

D-γ-glutamyl (α-benzyl ester)-glycyl-D-alanine benzyl ester hydrochloride

At room temperature, 4.3 g (9.45 mmol) of N-tert-butoxycarbonyl-D-γ-glutamyl (α-benzyl ester) hydroxysuccinamide ester, 2.71 g (9.92 mmol) of glycyl- A solution of D-alanine benzyl ester hydrochloride and 1.0 g (9.92 mmol) of triethylamine in 100 ml of dichloromethane was stirred for 18 hours, then concentrated in vacuo, and the residue was dissolved in 200 ml of ethyl acetate, 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 evaporated under reduced pressure. The residue was treated with dioxane saturated with hydrogen chloride, stirred for 2 hours, and in vacuo The solution was concentrated to dryness and the residue was triturated with ether and filtered under a stream of nitrogen to give a solid 3.41 g (73% yield).

In the same manner, the product of the previous preparation and butyl glycyl-D-alanine were converted to butyl D-γ-glutamyl(α-benzyl ester)-glycyl-D-alanine.

Preparation 5

rac-trans-4-hepten-3-ol

2-Pentene (25 g, 0.30 mol) was converted to the title product by the method of Preparation 1, but using ordinary diethyl ether as solvent, yielding 25.6 g; bp 145-150°C.

Preparation 6

0-(tert-butyldimethylsilyl)-S-citronellol

At room temperature (21°C), 5-citronellol (547 g, 3.5 moles) was dissolved in 547 ml of DMF (dimethylformamide), and the solution was placed in an N tube equipped with a stirrer, a thermometer, and Add imidazole (262.1 g, 3.85 mol, 1.1 equiv) to a 500 ml four-neck round bottom flask with a return funnel. The temperature was lowered to 13°C and further to -6.5°C with an ice/acetone bath. tert-Butyldimethylsilyl chloride (580.3 g, 3.85 mol, 1.1 eq) previously dissolved in 1160 mL of DMF was added with vigorous stirring while slowly raising the temperature to 11°C over 1.25 hours. in another After an additional 0.5 hour, thin layer chromatography (3:1 hexane:ether) indicated complete conversion to the desired product (Rf 0.2 starting material; Rf 0.9 product).

The mixture was added into 500 milliliters of hexane and 1000 milliliters of ice and water to be divided into two layers, and the organic layer was washed with 2000 milliliters of ice-cold 0.25N hydrochloric acid. The two aqueous layers were combined and washed with 500 milliliters of hexane. Combined, washed with 500 ml saturated _NaHCO3 , dried over _MgSO4 and evaporated to give 986.7 g of the title product, 104% of theory due to the presence of a small amount of solvent, ¹ H-NMR ( _CDCl3 ) δ (ppm), 5.2 (t, J = 7Hz, = CH), 3.65 (t, J = 6.5Hz, -O-CH ₂ -), 1.7 and 1.65 (2S, 2×C (CH ₃ )), 0.8 (S, -SiC (CH ₃ ) ₃ ), and 0.0 (S,-SiC(CH ₃ ) ₂ ),

Preparation 7

S-6-tert-butyl-dimethylsilyloxy)-4-methyl-hexanal

The previously prepared product (81.2 g, corrected for solvent content, 0.3 mol) was dissolved in 120 ml of _CH2Cl2 and 81 ml of _CH3OH , and the solution was placed under a flow of _O3 / _O2 with a _mechanical stirrer. A 500 ml four-neck round bottom flask connected with a straight glass tube, a thermometer and an outlet tube connected to a saturated KI trap. NaHCO ₃ (6.3 g, 0.075 mol, 0.25 equiv) was added and the mixture was cooled to -10°C with an acetone/dry ice bath. When O ₃ /O ₂ was blown into the reaction solution, the temperature was lowered and maintained at -72°C to -75°C for 6 hours. After less than 1 hour, the reaction mixture no longer absorbed _O3 , as evidenced by the formation of a yellow color in the KI trap. Thin layer chromatography using hexane as developing solvent indicated the completion of the reaction, (Rf 0.3 starting material; Rf 0.0 intermediate).

The reaction mixture containing _excess O was washed with N, dimethylsulfide (26.4 mL, 22.4 g, 0.36 mol, 1.2 equiv) was added, the cooling bath was removed, the mixture was warmed to room temperature, and stirred under _N flow for 16 hours, at which time Thin layer chromatography (6:1 hexane:ether) indicated that no intermediate material was present (intermediate material Rf 0.8; product Rf 0.05). The mixture was evaporated and the residue was partitioned between 150 mL ethyl acetate and 300 mL _H2O , the organic layer was washed with 300 mL water and the combined aqueous layers were backwashed with 150 mL fresh ethyl acetate. The combined organic layers were dried over _MgSO4 and evaporated to an oil which was finally evaporated under high vacuum for 16 hours to give quantitative title product 74.7 g, 101.9% of theory (due to a small amount of solvent impurity) ¹ H-NMR (CDCl ₃ ) δ (ppm); 9.75 (t, -CHO), 3.6 (t, J=6Hz, -O-CH ₂ -), 0.9 (s, -SiC(CH ₃ ) ₃ ), 0.0 (s, -Si(CH ₃ ) ₂ ).

Preparation 8

S-6-Hydroxy-4-methylhexanal

Method A

To a 250 mL three-neck round bottom flask equipped with an electromagnetic stirrer, a thermometer, gas inlet and outlet pipes connected to a wash bottle containing saturated KI, was added 81 mL of S-citronellol (31.25 g, 0.20 mol) CH ₂ Cl ₂ and 54 ml CH ₃ OH solution, and cooled to -8°C. Keep the temperature between -2°C and -10°C, blow O ₂ /O ₃ bubbles into the reactant within 4.5 hours, at this time the excess O ₃ is captured by the KI solution, and the reaction is indicated by the positive starch/KI paper test When complete, the mixture was kept at -5°C, purged with _N2 gas, and dimethylsulfide (17.7 mL, 15.0 g, 0.24 mol) was added. The reaction mixture was warmed to room temperature, then stirred for 16 hours, at which time thin layer chromatography (using isopropyl ether as a developing solvent) indicated that the reaction was complete (S-citronellol Rf was 0.7; product Rf was 0.4), and finally the solvent was evaporated , 50.8 g of oil were obtained. The oil was diluted with 30 mL of ethyl acetate and 25 mL of _H2O to give a single phase, followed by 150 mL of ether to obtain two phases. The solution layers were separated and the organic phase was washed with 2 x 25 mL of water, dried over _MgSO4 and evaporated to give 23.9 g of a colorless oil. The combined aqueous layers were extracted with 2 x 50 ml ethyl acetate and the combined extracts were dried and evaporated to give an additional 7.8 g as a colorless oil. The combined colorless oils were evaporated to give 28.3 g (108.7% of theory) of the title product. Thin layer chromatography (mentioned above) showed that the product was pure, except for the mixed solvent, and was suitable for the next reaction as described above.

Method B

The product of Preparation 2 is hydrolyzed by conventional methods, such as the dilute sulfuric acid method used in Example 4 above. The title product was isolated by extraction into ethyl acetate, evaporation, and work-up as in Method A above.

Claims (1)

1, preparation has the method for following absolute stereo chemical formula crystalline compounds,

This method comprises makes this compound from a kind of organic solvent that is selected from ethanol, acetonitrile or acetone, or crystallization from ethanol and acetonitrile mixture.