JPH0637517B2 - N- (S-3-alkyl-heptanoyl) -D-γ-glutamyl-glycyl-D-alanine derivative and process for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention has the following formula: [In the formula, R is methyl or ethyl, R ⁴ and R ⁵ are each hydrogen, or one of R ⁴ and R ⁵ is hydrogen and the other is (C ₁ -C ₆ ) alkyl or (C
₆ -C ₈₎ advantageous method for producing an immunomodulatory agent represented by cycloalkyl methyl]; and the following absolute stereochemistry formula: [Wherein one of the dashed lines (but not both) represents a double bond, R is as defined above, and R ² and R ³
Are both hydrogen (IIIa, or a pharmaceutically acceptable salt thereof), or R ² and R ³ are each independently (C ₁ -C ₆ ) alkyl, (C ₆ -C ₈ ) cycloalkylmethyl , Or benzyl (IIIb), provided that when the double bond is in the terminal 6,7-position, R is methyl].

Optically pure S-3-methylheptenoic acid (II, R = CH
₃ ) is usually produced in indeterminate yields from the corresponding racemate by several crystallizations of the quinine salt at -15 ° C [Levene et al., J. Biol Chem. (J.Biol.Che
m. ) 95, pp . 1-24 (1932), p. 2-
It is named n-butyl-butyric acid-4]. Optically active 3-methylheptanoic acid is subsequently produced by many other methods [Soai et al., J.
Chem Suk. , Chem Commun. ( J.Chem.Soc., Chem.Com
mun. ) 1985, 469-470; Oppolzer (Oppo
lzer) etc., Helv.Chim.Acta.
68, 212-215 (1985); Ohn
U.S. Pat. No. 4,564,620 (1986);
ri) et al., Synthesis (Synthesis ) 1982, 752-753
Page; Helb . Chim. Actor. Volume 64, 28
08-2811 (1981); Mukaiyam
a), etc. Chem. Let. (Chem. Lett. ) 1981, 913
~ 916; Posner et al . , Jay. Am. Ke
Mu. Suk (J. Am . Chem . Soc. ) 103 volumes, 2886-28
88 (1981); Mukaiyama et al., The Chemical Society of Japan Bulletin
(Bull.Chem.Soc.Japan 51, pp. 3368 to 3372 (1978);. Myers (Meyers) and the like, J. A
Mu. Chem. Soku. Volume 98, pages 2290-2294 (19
76)] However, these preparation methods generally have one or more drawbacks (the maximum possible yield is 50%,
At least 50% of the by-products are required to be discarded, at least 50% of the by-products are selected until the final stage of the manufacturing process, the acid of the product is not optically pure and difficult to handle. Large-scale use of reagents is required, total yields are low, and / or required reagents are not readily available. The asymmetric epoxidation currently used (so-called Sharpless decomposition) has been previously reported. (Kats
uki) and Sharpless, Jay. Am. Ke
Mu. Soku. 102, 5974-5976 (198
0); pure pull, such as sharpless. Chem. (Pure A
ppl.Chem. ) 55, 589-604, 1983];
Similarly, stereospecific Claisen condensation is also known [Chan et al., US Pat. No. 4,045,475].

The relatively new field of immunopharmacology and, in particular, the area of immunomodulation, continues to develop rapidly. Various naturally occurring compounds have been investigated. Such compounds ^{N 2} - ^[1-N 2 -L- threonyl -L- lysyl) -L- prolyl] was chemically known as -L- arginine, tetrapeptide tufts thin. Synthetic peptidoglycan derivatives, especially those known as muramyl dipeptides, have received considerable attention.

Immunomodulators of formula (1) as amorphous lyophilizates, generally with R ⁴ = R ⁵ = H, and methods for their use are described in co-pending PCT application No. PCT / U, filed November 25, 1985.
It is disclosed in S85 / 02351. Since this application is not yet publicly available, methods of making such compounds and their methods of use are included herein to support the utility of the present invention.

Other immunostimulatory peptides have also been mentioned in numerous patent specifications: L-alanyl-α-glutaric acid N-acyl dipeptides are described in West German Patent 3,024,35 issued Jan. 15, 1981.
No. 5; D-alanyl-L-glutamyl group or L-alanyl-
Tetra-peptides containing a D-glutamyl group and penter peptides are described in British Patent No. 2,053, issued Feb. 4, 1981.
No. 231 and West German Patent No. 3,02 issued January 8, 1981
4,281 respectively; N-acyl-alanine-γ-D-glutamyl tripeptide derivatives whose C-terminal amino acids are lysine or diaminopimelic acid are described in West German Patent No. 3,024,369 issued January 15, 1981. A lactoyl tetrapeptide consisting of N-lactylalanyl, glutamyl, diaminopimeryl and carboxymethylamino components is described in EP 11283 issued May 23, 1980; Formula (A) : [Wherein R ^a is hydrogen or acyl, R ^b is especially hydrogen, lower alkyl, hydroxymethyl or benzyl, R ^c and R ^d are each hydrogen, carboxy, -CONR ^g
R ^h (R ^g is hydrogen, lower alkyl optionally substituted with hydroxy, R ^h is mono- or dicarboxy lower alkyl) and R ^e is hydrogen or carboxy. However, when one of R ^d and R ^e is hydrogen, the other is carboxy or —CONR ^g R ^h ; R ^f is hydrogen, m is 1 to 3, and n is 0 to 2. ] And a derivative thereof in which the carboxy and amino groups are protected are represented by U.S. Patent Nos. 4,311,640 and 4,322,341, European Patent Application Nos. 25,482, 5
0,856, 51,812, 53,388, 55,846 and 57,
No. 419 is disclosed. Further, a peptide similar to the peptide of the above formula (A) (however, R ⁴ forms a basic amino acid group) is described in Ives et al., US Pat. No. 4,565,653 and European Patent Application No. 157,572, R ⁴
Peptides forming a heterocyclic amino acid are described in Yves European Patent Application No. 178,845.

Kitaura and other J.MeD Chem (J.Me
d. Chem. 25, 335-337 (1982), N.
² (γ-D-glutamyl) -meso-2 (L), 2 (D) -diamino-pimelic acid was added to the compound of formula (A) (where n is 1 and R
^a is CH ₃ CH (OH) -CO-, R ^b is CH ₃ , R ^c and R
each of ^e is -COOH, R ⁴ is -CONHCH ₂ COOH,
R ^f is H) as the minimum structure capable of exhibiting a characteristic biological reaction.

The compound of formula (A) is known as FK-156.

We now use the above formula (1) (R = CH ₃ , R ⁴ = R ⁵ = hydrogen)
Have discovered a more stable crystalline form of the immunomodulatory compound N- (S-3-methylheptanoyl) -D-γ-glutamyl-glycyl-D-alanine.

Also, we are currently using organometallic reagents and R in the latter half of the synthesis.
An optically pure intermediate, R-3-alkyl-4-heptenoic acid (VIb) and S-3-alkyl-heptanoic acid (above II), which avoids uneconomical by-products of the enantiomers. ) Has been found to be an effective manufacturing method.

In one manufacturing route, the first step is a Sharpless decomposition of trans-4-alkene-3-ol represented by the following formula: wherein R is as defined above. This step is particularly performed in the presence of titanium tetraisopropoxide and L-(+)-tartaric acid-diisopropyl in a solvent inert to the reaction to oxidize the racemate of formula (IV) and the S-enantiomer to obtain the absolute stereochemical formula. Reacting with (V) sufficient t-butyl hydroperoxide to maintain the desired unreacted R-enantiomer.

In the second step, the compound of formula (V) is tri [(C ₁ -C ₃ )]
Alkyl] orthoacetate is condensed stereospecifically and the intermediate allyl-enol ether can be rearranged without isolation in the presence of an acid in a reaction-inert solvent to give the absolute stereochemical formula (VIa). Represented by (C _{1 to} C ₃ ).
This produces an alkyl R-3-alkyl-4-heptenoate.

(VIa) X = OR ¹ ; R ¹ = (C ₁ -C ₃ ) alkyl (VIb) X = OH (VIc) X = Cl (VId) X = H In one method, the ester is then hydrolyzed. Trans-R-3-alkyl-4-heptenoic acid (VIb) is formed and, if desired, hydrogenated to form S-3-alkylheptanoic acid. In another method, the ester is first hydrogenated to produce a (C ₁ -C ₃ ) alkyl S-3-alkylheptanoate and then hydrolyzed to give the acid of formula (II).

In yet another method, the decomposed trans-4- of formula (V) is
Alken-3-ols are converted to allyl vinyl ethers (IX) by the action of ethyl vinyl ether in the presence of mercuric acetate [Hg (OAc) ₂ ] catalyst. The allyl vinyl ether is then heated to form R-3-alkyl- of formula (VIa)
Rearrangement to 4-heptenal, which is then oxidized, for example by Jones reagent (chromic anhydride in dilute inorganic acid), to form the R-3-alkyl-4-heptenoic acid described above.

We also use organometallic reagents and the following absolute stereochemical formulas from readily available S-citronellol: And an uneconomical by-product of the R-enantiomer represented by
- heptenoic acid (below, Xd) and found S-3- heptanoate also an effective way of producing in (above, II, R = CH ₃₎ good overall yield.

The first step in this synthesis is a conventional method, which involves conventional silyl protecting groups (eg t-butyldimethylsilyl group).
With optional protection of the alcohol group by the following and ozonolysis by treatment with methyl sulphide, including the absolute stereochemical formula: [Wherein R ⁸ is hydrogen (XIIa) or a silylhydroxy protecting group (XIIb)]. The method to be used is described in detail in the production examples below, but the compound of the formula (V) (R
⁸ is the same as the method previously applied to R -citronellol in the preparation of the R -enantiomer of (t-butyldimethylsilyl). R-enantiomers not used here are used in the synthesis of proxiphomin [Tapolczay et al., J. Chem.
Soku. , Chem Commun 1985, 143-145
page〕.

The intermediate compound of formula (V) and the following formula: (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 2 and the next step: (a) reacting the compound of formula (XIII) with methylenetriphenylphosphorane CH ₂ ═P (C ₆ H ₅ ) ₃ to give formula (Xa) Or forming a compound of (Xb); (b) forming a compound of formula (Xb) by hydrolysis with a dilute inorganic acid when the compound is a compound of formula (Xa), this step being independent And (c) the compound of formula (Xb) is oxidized with chromic anhydride in dilute inorganic acid to give S-3 of formula (Xd). -Methyl-6-heptenoic acid is formed; and, if desired, (d) a compound of formula (Xd) is catalytically hydrogenated to form formula (II) (where R is methyl Of by forming a S-3- methyl heptanoic acid, a compound formula (Xd) and of formula (XII) (II,
It can be converted to an optically active acid with R = CH ₃ ).

When R ⁸ is hydrogen, there is the advantage of fewer manufacturing steps, but when R ⁸ is a silyl protecting group,
There is the advantage of easy purification when the intermediate product is isolated from step (a). Preferred hydroxy protecting groups are trimethylsilyl, p-t-butylphenethyldimethyl-silyl and t-butyldimethylsilyl.
The preferred dilute inorganic acid is H ₂ SO ₄ . By the ozonolysis of S-citronellol with methyl sulfide treatment, the formula
By synthesizing the compound of Ia) and further reacting the following steps: (e) S-citronellol is reacted with t-butyldimethylsilyl chloride and (f) ozonolysis of the hydroxy-protected citronellol produced by treatment with methyl sulfide to give the formula ( It is preferable to synthesize the compound of XIIb) (R ⁸ is t-butyldimethyl-silyl).

The present invention comprises the following steps: (a) R-3-alkyl-4-in a solvent inert to the reaction
An activated form of heptenoic acid or S-3-methyl-6-heptenoic acid (eg acid chloride VIc or Xe) is represented by the formula: [Wherein R ⁶ and R ⁷ are both 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] and an intermediate compound of the formula (IIIb) (R ² and R ³ are the same as R ⁶ and R ⁷⁾ . And (b) hydrogenate the intermediate compound in a solvent inert to the reaction in the presence of a hydrogenation catalyst to simultaneously reduce the double bond and form a benzyl group (s) It also relates to an improved process for the preparation of an immunomodulator of formula (I) which comprises hydrolyzing

The invention also relates to an immunomodulator and / or a precursor of compound (I) of formula (III) above. The compound of formula (IIIa) (R ² = R ³ = hydrogen) is obtained by ester hydrolysis of the diester compound of formula (IIIb).

The expression "reaction-inert solvent" as used herein means a solvent that does not interact with the starting materials, intermediates or products to the extent that it adversely affects the yield of the desired product.

A preferred group for R ¹ is ethyl. The preferred acid catalyst is a Lewis acid (eg dry HCl, AlCl ₃ ). Without limiting the invention, racemic trans-4-hexen-3-ol or trans-4-heptene-3-
Alls are preferably obtained by reaction of the ethyl Grignard reagent with crotonaldehyde or 2-pentenal, respectively.

The present invention is easily implemented. R-trans-4-alkene-3-ol (V) is obtained by the method of asymmetric epoxidation, the so-called Sharpless decomposition (reference above). According to this method, racemic undesired S-trans-2-hexen-4-ol (IV) was added to 1 molar excess of L-(+)-diisopropyl tartrate and substantially 1 molar equivalent of titanium tetraisopropoxide. In the presence of and t-butyl hydroxyperoxide (racemic hexenol 1
At least 0.5 mol per mol, but clearly less than 1 mol). This reaction is generally carried out in a reaction-inert solvent such as methylene chloride, generally. −
It is carried out under anhydrous conditions at low temperatures in the range of 20 to -80 ° C. The desired non-reactive R-trans-2-alkene-4-ol (V) is isolated by standard methods such as distillation or chromatography. Low boiling solvents such as pentene and ether are preferred for such chromatography due to the relatively high volatility of the desired product.

The next step is R-trans-4-alkene-3-ol (V) and (C ₁ -C ₃ ) alkyl-R-3-alkyl-4-heptenoate (VIa) to produce (C ₁ -C ₃ ) alkyl-R-3-alkyl-4-heptenoate (VIa).
C ₃ ) Condensation / Claisen rearrangement with trialkyl orthoacetate. Although this reaction is carried out in a solvent inert to the reaction, this reaction is generally performed at an elevated temperature (for example, 120 to
It is desirable to simply carry out in excess of orthoacetate ester at 160 ° C). When triethyl orthoacetate (boiling point 142 ° C.) is the reagent, it is convenient to use the reflux temperature. This reaction is carried out in the presence of an acid catalyst. Acids such as propionic acid, pivalic acid, 2,4-dinitrophenol, dry HCl and AlCl ₃ are effective. The preferred catalyst is a Lewis acid. The most preferred catalyst is AlCl ₃ .

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

The method of the present invention is also easy to implement. Commercially available S-citronellol (XI) is first converted to one of the optically active starting materials of formula (XII) by conventional methods, as described above and as illustrated in the preparation examples below. . Compound (XII) is described in detail in the following paragraphs, with S-3-methyl-6 of formulas (Xd) and (II) above, respectively.
-Stepwise conversion to heptenoic acid or S-3-methylheptanoic acid.

In the first stage, for example, Generally, (methyl) triphenylphosphonium halides (bromide favored) and butyl against hydroxy or hydroxy protected aldehydes (XIIa or XIIb) in a reaction-inert aprotic solvent such as a mixture of tetrahydrofuran and hexane. The Wittig reaction with methylenetriphenyl-phosphorane, which is newly formed in situ from lithium, is performed. Temperature is not a critical factor in phosphorane formation, but the preferred temperature is ±
Within the range of 25 ° C, most preferably within the range of ± 10 ° C. Reacting an aldehyde (XIIa) or a hydroxy protected aldehyde (XIIb) with a phosphorane in a similar solvent and in a similar temperature range to give the formula (Xb) or (Xa), respectively.
To form a hydroxy or hydroxy protected olefin.

In the second step, which is necessary only if a hydroxy protecting group is present, is by hydrolysis, most conveniently by the aqueous acidic conditions used in the third step, Jones oxidation of the primary alcohol (Xb) to the acid (Xa). , The protecting groups are removed. Jones oxidation uses a so-called Jones reagent, an aqueous solution of H ₂ CrO ₄ formed from CrO ₃ and a strong acid. Jones reagent is typically 1: 1 with excess concentrated H ₂ SO ₄ and CrO _3.
Prepared from water in a weight ratio of 1 and then diluted with water to the desired concentration, for example about 3M. The alcohol (XIIa), the protected alcohol (XIIb), as a solution in a reaction-inert organic solvent, which is generally miscible with water, for example acetone, is reacted with at least 2 molar equivalents of the Jones reagent. Under these conditions, the silyl ether groups are rapidly hydrolyzed to form an alcohol, which is then oxidized to the acid (Xd). The temperature is not critical, e.g. 0-50 <0> C is usually a satisfactory temperature, ambient temperature (e.g. 17-27 <0> C) being most convenient.

Jones oxidation occurs via the primary oxidation of an alcohol to an aldehyde of formula (Xc), which is usually not isolated by Jones oxidation. If isolation of the intermediate aldehyde is desired, oxidation of the alcohol with a more selective oxidant such as pyridinium chlorochromate clearly produces the intermediate aldehyde (Xc), which is then (Jones Reagent or other suitable reagent)
Oxidizes to form acid (Xd).

If desired, the unsaturated acid (XIb) or (Xd) may be used in the activated form (eg, as the acid chloride of formula (VIc) or (Xe), as a conventional mixed anhydride, or in a conventional manner such as dicyclohexylcarbodiimide). In the form activated by the dehydration binder of the formula: [In the formula, R ⁹ and R ¹⁰ are each independently (C ₁ to
C ₆ ) alkyl, (C ₆ -C ₈ ) cycloalkylmethyl or benzyl] in the conventional manner to form a compound of formula (III
Form the immunomodulatory diester compound of b). This is hydrolyzed in the usual way to form the immunomodulatory diacid compound of formula (IIIa) or a pharmaceutically acceptable salt thereof.

Alternatively, the compound of formula (IIIb) (R ² and / or R ³ is benzyl) can be hydrogenated to form an immunomodulatory compound of formula (1). In this hydrogenation, the double bond is saturated with hydrogen and at the same time the benzyl group is hydrolyzed. Hydrogenation is supported (eg Raney nickel, Pd /
C) or with a non-supported (eg RhCl ₃ ) reaction in a solvent inert to the reaction over a hydrogenation catalyst such as nickel or a noble metal. The solvent, temperature and pressure are not critical. Suitable solvents include, but are not limited to, lower alcohols; ethers such as tetrahydrofuran or dimethoxyethane; and esters such as ethyl acetate. It is preferred to avoid the expense of heating or cooling and use ambient temperature without cooling, even if the reaction is somewhat exothermic. The pressure is not critical, but to avoid the use of expensive high pressure equipment, 7
It is preferably below atmospheric pressure. Hydrogenation over Pd / C at a pressure of 3 to 5 times atmospheric pressure is particularly well suited for the reaction according to the invention.

Alternatively, an unsaturated acid of formula (VIb) or (Xd) is hydrogenated under the same conditions detailed in the preceding paragraph to form S-3-alkylheptanoic acid (II); By reversing the hydrocracking / hydrocracking step,
S-3-alkylheptanoic acid (II) is (C ₁ -C ₃ ) alkyl-trans-R-3-alkyl-4-heptenoate (VIa) to (C ₁ -C ₃ ) alkyl S-3-alkyl-heptanoate Obtained through. Finally, S-3
-The alkylheptanoic acid is activated in the manner detailed above, coupled with the diester of formula (VII) and hydrogenated in the manner described above to form the immunomodulatory compound of formula (1).

In yet another method of the present invention, R-trans-4-alkene-3-ol (V) is reacted by the action of ethyl vinyl ether in the presence of Hg (OAc) ₂ as a catalyst in a solvent inert to the reaction ( It is preferably in excess) and is converted to the corresponding vinyl ether of formula (IX) at a temperature in the range of 25-40 ° C., preferably at the reflux temperature of ethyl vinyl ether (boiling point 36 ° C.).

The resulting vinyl ether (IX) is generally stereospecific by heating in a high-boiling, reaction-inert, lipophilic solvent such as xylene or decalin at 140-200 ° C., if necessary under pressure. Rearranged to the formula (IV
The unsaturated aldehyde of d) is formed. To obtain the unsaturated acid of formula (IVd), this aldehyde (IVd) is conveniently the so-called Jones reagent (H ₂ formed from CrO ₃ and a strong acid).
It is easily oxidized by CrO ₄ aqueous solution). The Jones reagent is typically made from excess concentrated sulfuric acid and CrO ₃ with water in a 1: 1 weight ratio to achieve the desired concentration (eg, about 3M).
Diluted with water. To form the unsaturated acid (VIb), the unsaturated aldehyde (IVd) is generally used as a solvent in a water-miscible, reaction-inert solvent, such as acetone, at least 1 molar equivalent of Jones. React with reagents. The temperature is not critical, for example 0-50 ° C is usually sufficient, but ambient temperature (eg 17-27 ° C) is most convenient.

The crystalline form of the compound of formula (1) (R is methyl, R ⁴ and R ⁵ are hydrogen) is obtained by crystallization from an organic solvent or a combination of organic solvents. Suitable solvents are acetone, acetonitrile / ethanol and tetrahydrofuran / ether. A preferred solvent is acetonitrile / ethanol from the viewpoint of product recovery, but acetone is preferred from the viewpoint of product yield. This novel form has distinct, stability advantages over the prior art amorphous lyophilizates. This new form is easier to handle, thicker, less electrostatic, and allows the production of more complex dosage forms.

The pharmaceutically acceptable monobasic salts or monobasic salts of the compounds of formula (I) or (IIIa) are generally prepared as a solution, preferably an aqueous solution of an acid, in suitable stoichiometric proportions of NaOH, KOH, Na ₂ CO
Obtained by treatment with a base such as ₃ or an amine. The salt is isolated by evaporation or precipitation.

The products of the invention of formula (I) or (III) are useful as agents for the clinical and therapeutic treatment of diseases caused by various pathogenic bacteria in mammals, including humans, in particular Gram-negative bacteria. These products also have an increased risk of infection due to pre-existing or clinically induced immunosuppression.
It is useful as an immunostimulant in mammals including humans.

The test uses normal or immunocompromised C ₃ H / HeN male mice from the Charles River Breeding Laboratory. Mice were acclimated for 5 days before use and then subcutaneously (SC) with 0.2 ml of various dilutions (100, 10, 1 and 0.1 mg / kg) of test compound or placebo (pyrogen-free saline). ) Treated or orally (PO) treated. The treatment plan depended on the infectious organism used. 24 hours and 0 hours before testing for Klebsiella pneumonia in normal mice; and 3 days, 2 days and 1 day for testing for E. coli or Staphylococcus aureus in immunocompromised mice.
The treatment was performed day before. The test was carried out by intramuscular administration (1M) in the lumbar region for Pneumococcus, and intraperitoneal administration (1P) for Escherichia coli and Staphylococcus aureus. A 0.2 ml volume was used for the test. In the case of S. pneumoniae, after 7 days,
Mortality was recorded after 3 days in the case of the other two tests.

Culture Preparation: S. pneumoniae, E. coli or Staphylococcus aureus: Cultures were streaked on brain heart infusion (BHI) agar for purification from frozen blood stock. Three colonies were picked from the 18 hour plate culture and placed in 9 ml of BHI broth. Place the broth culture on a rotary shaker 37
Grow at room temperature for 2 hours, then add 0.2 ml of some B
Streaks occurred on the surface of the HI agar slant medium. After 18 hours of incubation at 37 ° C, the gradient medium was incubated with BH.
Wash with I broth and spectronic 2
0 was used to control the culture density and LD90 in normal mice
Appropriate dilution to reach reagent level.

When the compound (I) or (III) of the present invention is used as an anti-infection agent or immunostimulant in humans, it is generally used as a standard composition in the formulation method, and is orally, subcutaneously, intramuscularly, intravenously or intraperitoneally. It is convenient to administer by route. For example, these compounds can be administered as tablets, pills, powders or granules containing excipients such as starch, lactose, certain clays and the like. These can also be mixed with the same or similar excipients and administered as capsules. These compounds may also be administered as oral suspensions, solutions, emulsions, syrups and elixirs containing flavoring and coloring agents. For oral administration of the therapeutic agent of the present invention, the active ingredient is about 50 to about 500.
Tablets or capsules containing mg are suitable for most uses.

The doctor will determine the most suitable dosage for each patient, but the dosage will vary with the particular age, weight and response and route of administration. A preferred oral dose range is from about 1.0 to about 300 mg / kg / day in single or divided doses.
A preferred parenteral dose is about 1.0 to about 100 mg / kg / day, and a more preferred range is about 1.0 to about 20 mg / kg / day.

The following examples are set forth to illustrate the invention, but they should not be construed as limiting the invention and many variations of the invention are possible within the scope and spirit of the invention.

Reference Example 1 R-trans-4-hexen-3-ol In a 500 ml-three-necked round bottom flask equipped with a magnetic stirrer, a septum, a thermometer and an N ₂ supply port, 270 ml of CH ₂ Cl ₂ was added.
Then, 7.2 g of 4A type molecular sieves was charged. Ti 〔O
CH (CH _{3) 2] 4} (10.7 ml, 0.036 mol) was added from a syringe, the mixture was cooled to -66 ° C., L-diluted in order to reduce the viscosity at this point in CH ₂ Cl ₂ 10ml ( +)-Diisopropyl tartrate (10.1 g, 0.043 mol) was added via cannula, and 5 ml of CH ₂ Cl ₂ was added while washing the cannula. The temperature was raised to −62 ° C. at which point racemic trans-2-hexen-2-ol (3.60 g, 0.036 mol) was added and 5 ml CH ₂ Cl ₂ was added while washing the cannula. After stirring for 8 minutes in an acetone / dry ice bath, the temperature dropped to -68 ° C, at which point t-butyl hydroperoxide (7.18 ml0.22 as a 3M solution in toluene) was added.
Mol) was added by syringe. The reaction mixture is -35 ° C.
And kept at this temperature in the refrigerator for 18 hours. Then pass the cold mixture through a grooved paper and add acetone 5
It was placed in a stirred mixture of 40 ml and 11 ml of water cooled to -20 ° C. The mixture was gradually warmed to room temperature and stirred for 20 hours. The mixture was passed over diatomaceous earth and washed with CH ₂ Cl ₂ . The combined liquor and washings were stripped without heating to give an oil (17.62g). Avoid overstripping as the desired product is actually volatile. Oil on 179 g of silica gel with hexane: isopropyl ether (4: 1) as eluent, tl
Chromatographic analysis was performed while monitoring by c (thin layer chromatography). The solvent was evaporated using a short glass helix packed column with a variable take-off head. This removes the solvent having a boiling point up to a distillation outlet temperature of 69.5 ° C. (from which the distillation outlet temperature begins to drop), leaving a substantially pure title product as a bite residue;
tlc Rf 0.25 (hexane: ether 3: 1). The repeat of this experiment used the highly volatile pentane: ether (4: 1) as the eluent to facilitate solvent removal and minimize product loss during stripping.

Reference Example 2 Ethyl R-3-methyl-4-hexenoate The product of the above Reference Example (0.4 g), thienyl orthoacetate (3 ml) and pivalic acid were heated under reflux for 2.5 hours, cooled and the whole reaction mixture on silica gel. Chromatographic analysis showed that hexane: ether (6:
0.45 g of product was obtained using 1) and then eluted with hexane: ether (7: 1) to give 0.35 g of the purified title product.

IR (film): 2960, 1736, 1456, 1367, 1334,
1280, 1232, 1172, 1028, 962, 8
40 cm ^-1 . Instead of this, pure S-trans-2-hexene-4-
The pentane / ether column fraction containing oar was introduced directly to this stage and pentane and ether were first distilled off from the reaction mixture.

Substantially the same results were obtained using propionic acid instead of pivalic acid. Although dried HCl and 2,4-dinitrophenol have been successfully used as acid catalysts, AlCl ₃ used in the following examples is the preferred catalyst.

Reference Example 3 R-3-methyl-4-heptenoic acid Method A A 35 ml-flask equipped with a magnetic stirrer and a Soxhlet extractor containing 4A type molecular sieves in a single product was added to the product of Reference Example 2 (1.5 g, 0.015 mol). Add thienyl orthoacetate (9 ml, 0.049 mol), then AlCl ₃ (0.12 g, 0.009 mol),
The mixture was refluxed on a Soxhlet extractor for 2 hours. By this time tlc showed the conversion to the intermediate ethyl R-3-methyl-4-hexanoate was complete; tlc Rf
0.75 (4: 1 hexane: ethyl acetate), Rf 0.85 (1
5: 5: 2 hexane: ether: acetic acid).

The reaction mixture was cooled, diluted with 15 ml of 2N NaOH and 12 ml of CH ₃ OH and stirred at ambient temperature for 27 hours. By this time, tlc showed that the hydrolysis was complete.
Strip the methanol from the reaction mixture to remove H ₂ O 1
It was diluted with 2 ml and extracted with CH ₂ Cl ₂ (25 ml × 3). The combined CH ₂ Cl ₂ was backwashed with 2N NaOH (25 ml × 1). The aqueous layer and the backwash solution were combined, the pH was adjusted to 1 with concentrated hydrochloric acid, and the mixture was extracted 3 times with CH ₂ Cl ₂ . Combine the CH ₂ Cl ₂ layers and add MgSO 4.
Dried over ₄ , stripped to give the title product as an oil (1.28 g, 60%); tlc Rf 0.65 (15: 5: 2 hexane: ether: acetic acid); ¹ H-nmr (CDCl ₃ ) Delta (ppm) 9.4 (s, 1H), -CO ₂
H), 5.5 (m, 2H, -CH = CH-), 3.0-1.6 (m, 5
H), 1.3-0.8 (m, 6H); IR (film) 340
0-2400, 2960, 2925, 2860, 170
8, 1458, 1410, 1380, 1295, 122
4,1190,1152,1100,930 cm ^-1 . Method B 25 ml of CH ₃ OH and 1N NaOH 1 were added to the ester product of Reference Example 2.
1.5 ml and was added. The mixture was stirred at ambient temperature for 3.5 hours. At this point tlc showed the hydrolysis was complete. The mixture was extracted with ether (35 ml x 2),
The pH was adjusted to 2 with concentrated HCl and extracted with ether (35 ml x 3). The acid extracts were combined, dried and stripped to give the title product (0.20 g) which was identical to the product of Method A.

Reference Example 4 Ethyl S-3-methylheptanoate The product of Reference Example 2 (0.20 g) was added to 5% Pd / C in 40 ml of ethyl acetate under 4 atm of hydrogen in a Paar hydrogenation apparatus.
Hydrogenate over 0.2 g (moistened with 50% water) for 3 hours. The catalyst is recovered by filtration over diatomaceous earth. The title product is recovered by stripping the solvent solution.

Reference Example 5 S-3-Methylheptanoic acid Method A Ethyl acetate 40 under hydrogen at 4 atm in a Parr hydrogenation apparatus
The product of Reference Example 3 (0.20 g) in 5 ml of 5% Pd / C 0.2 g
Hydrogenated over (moistened with 50% water) for 3 hours. The catalyst was recovered by filtration over diatomaceous earth and the title product was recovered as an oil (0.2g) by stripping the liquor. If desired, the title product was obtained purified by distillation under high vacuum; bp 77-79 ° C. /0.2 mm. ¹ H-nmr (CDCl ₃ ) delta (ppm): 12.0 (s, -COOH), 1.0 (d, -C)
H ₃ ), 0.6-2.8 (m, residual 13H); IR (film)
3400-2400, 2960, 2925, 2860,
1708, 1458, 1410, 1380, 1295,
1228, 1190, 1152, 1100, 930c
m ^-1 ; ▲ [α] ²⁵ _D ▼ = -6.41 ° (= 1% in CH ₃ OH); ▲ n ^22.5 _D ▼ = 1.427. Method B The product of Reference Example 4 is hydrolyzed by Reference Example 3, Method B to give the title product.

Reference Example 6 S-3-methylheptanoyl chloride Acid product from Reference Example 5 or Reference Example 18 (8.5 g, 0.0
62 mol) was dissolved in 18 ml CH ₂ Cl ₂ . Oxalyl chloride (5.36 ml, 7.80 g, 0.0614 mol) was mixed into this solution,
The mixture was left for 4 hours, the reaction was complete as evidenced by no more gas evolution during this time. This solution of acid chloride was immediately used directly in Example 1, Method C. Alternatively, the acid chloride was isolated by stripping the solvent and further purified by distillation used in Method A of Example 8, if desired (boiling point 45 ° /
1.5mm). Reference Example 7 R-3-methyl-4-heptanoyl chloride The acid product of Reference Example 3 (0.747 g, 5 mmol) was converted to a CH ₂ Cl ₂ solution of the title product by the method of Reference Example 6,
This is used directly in Example 9 below. Alternatively, the reaction mixture is stripped to give the title product, which is vacuum distilled if desired.

Example 1 N- (S-3-methylheptanoyl) -D-γ-glutamyl (α-benzyl ester) -glycyl-D-alanine benzyl ester Method A D-γ-glutamyl (α-benzyl ester in 50 ml of methylene chloride. ) -Glycyl-D-alanine benzyl ester hydrochloric acid (Preparation Example A) (1.0 g, 2.03 mmol) and triethylamine (616 mg, 6.09 mmol) were added to a solution of S-
660 mg (4.06 mmol) 3-methylheptanoyl chloride were added and the reaction mixture was stirred at room temperature for 80 hours. 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 phase was separated, dried over magnesium sulfate and concentrated in vacuo. The residue was triturated with diethyl ether and passed under a nitrogen atmosphere to give the title product, all of which was used directly in Example 3, Method A.

Method B Product of Preparation 4 (0.75 g, 1.53 mmol), CH ₂ Cl ₂ 5
ml and triethylamine (0.212 ml, 1.53 mmol) were combined and stirred under N ₂ atmosphere. S- in 4 ml of CH ₂ Cl ₂
3-Methylheptanoic acid (Reference Example 5; 0.20 g, 1.39 mmol) was added, followed by dicyclohexylcarbodiimide (0.28
6 g, 1.37 mmol) was added and the mixture was stirred for 16 hours. Pass the reaction mixture, strip the solution,
The residue is taken up in 10 ml of ethyl acetate and this solution is treated with 2.5% HCl.
Wash successively with 5 ml, 5 ml of H ₂ O, 5 ml of 10% K ₂ CO _{3 and} 5 ml of brine and dry over MgSO ₄ to give 71 m of the title product.
g (88%) was obtained.

METHOD C stirrer, 500Ml- in a four-neck round bottom flask equipped with a thermometer insertion funnel and a N ₂ inlet, was dissolved the product of preparation 4 (32.8 g, 0.059 mol) in CH ₂ Cl ₂ 175 ml,
Cooled to 0-5 ° C. While maintaining this temperature range, triethylamine (24.7 ml, 17.9 g, 0.177 mol, 3 eq) was slowly fed in over 15 minutes. Maintaining an ice-water bath, the entire batch of S-3-methylheptanoyl chloride in CH ₂ Cl ₂ from Example 6 was added over 15 minutes while increasing the temperature to 21 ° C. Stirring in the ice-water bath was continued for 45 minutes, during which time the gelatinous mixture became thick and could not be stirred. Destroy the gelatinous mass for 10
% HCl 125 ml and CH ₂ Cl ₂ 50 ml were added and mixed. The organic layer was separated, and H ₂ O (125 ml × 2), 10% K ₂ CO ₃ (1
It was washed successively with 25 ml x 2) and H ₂ O (125 ml x 1), dried over MgSO _{4 and} stripped to give a wet white solid 82.3 g. Add these solids to hot ethyl acetate 5
Placed in 00 ml. Upon gradual cooling to ambient temperature, a large amount of the title product precipitated. The mixture was further diluted with 40 ml of ethyl acetate in order to maintain easy stirring. The purified title product was collected by filtration and dried under vacuum at 40 ° C (31.1%, 90.5%). ¹ H-nmr (CDCl ₃ ) delta (ppm): 8.4-8.1 (m, 3
H), 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 2 N- (R-3-methyl-4-heptenoyl) -D-γ-
Glutamyl (α-benzyl ester) -glycyl-D-alanine benzyl ester By the method C of Example 1 the product of Preparation 4 (2.77
g, 5 mmol) is combined with the entire batch of acid chloride in CH ₂ Cl ₂ from Reference Example 7. The product is recovered by stripping the washed and dried organic layer, redissolving the residue in ethyl acetate and re-stripping.

Example 3 N- (S-3-methylheptanoyl) -D- [gamma] -glutamyl-glycyl-D-alanine Method A All the products of Example 1, Method A were dissolved in 65 ml of methanol. Palladium hydroxide was added to this solution, and the mixture was shaken in a hydrogen atmosphere at 4 atm for 3 hours. The catalyst was passed and the catalyst was removed under vacuum. The residue was dissolved in water and freeze-dried to obtain the desired product.

NMR spectrum (DMSO-d ₆ showed the following absorption.

8.27-8.03 (m, 3H), 4.32-4.1 (m, 2H), (3.72
(D, J = 6Hz, 2H), 2.22 (t, J = 8Hz, 2
H), 2.27-1.68 (m, 6H), 1.42-1.0 (m, 10
H) and 0.94-0.8 (m, 6H). The title product of Example 1 (0.50 g) was weighed into CH ₃ OH 2
When this method was carried out with 90 mg of 20% Pd (OH) ₂ / C in 5 ml (wet with 30% water), 0.24 g of the same fluffy, electrostatic title product was obtained. IR [nujol mull] 3300, 2940,
1740, 1650, 1540, 1468 and 138
0 cm ^-1 ; all peaks except the last two are broad,
The decomposition was insufficient.

Method B 2-The product of Example 1, Method C (30.8 g) was slurried in 300 ml of absolute ethanol in an autoclave. 1.54 g of 5% Pd / C (wet with 50% water) was added and the mixture was hydrogenated at 4 atm for 1 hour. During this time, absorption of hydrogen was completed. The catalyst was passed first on paper and then on 0.45 micron nylon Millipore, using 100-150 ml of ethanol for migration and washing. The liquor and wash were combined and stripped to give a damp white solid. Add this to absolute ethanol and acetonitrile (1: 1
0) dissolved in 150 ml of hot mixture, clarified by heating, boiled to 35 ml, gradually cooled to room temperature, granulated, and passed over to give a crystalline, thick, non-electrostatic title. 20.1 g (94%) of product were obtained, which was characterized by an IR (nujol mul) spectrum containing the following fully resolved major peaks: 3340, 3300, 2900, 2836, 1725,
1650, 1628, 1580, 1532, 1455,
1410, 1370, 1280, 1240, 1216an
d1175 cm ^-1 . Method C The crystalline product (9.4 g) prepared by Method B above was heated to reflux for 1 hour to give 1000 ml of acetone.
Dissolved in. The solution was cooled to room temperature and inoculated with trace amounts of the Method B product to induce crystallization. After stirring for 6 hours, the purified title product was collected by filtration, washed with a minimal amount of acetone, and dried under vacuum at 35 ° C (7.
25 g). It showed the same IR peak as the crystals from Method B acetonitrile / ethanol.

Method D The product of Example 2 (0.50 g) was added to 0.026 g of 5% Pd / C (50% water) in 125 ml of absolute ethanol in a Parr hydrogenator.
% Wet) was added. The mixture under 4 atmospheres of hydrogen
Hydrogenated for 2.5 hours. The catalyst was collected by filtration and the liquor stripped to give the title product as a sticky solid which was crystallized by the method of Example 2.

Example 4 N- (R-3-methyl-4-heptenoyl) -D-γ-
Glutamyl-glycyl-D-alanine The product of Example 2 (1 g) is dissolved in ₅ ml CH3OH.
1N NaOH (2.50 ml) is added and the mixture is stirred at ambient temperature for 3 hours. CH ₃ OH is stripped, the aqueous residue is diluted with 7.5 ml H ₂ O, extracted with ethyl acetate (75 ml × 2) and acidified to pH 3.0 with 1N HCl. The acidified aqueous phase is successively extracted with fresh ethyl acetate and the extract is stripped to give the title product, which is lyophilized by Example 3, Method A.

Reference Example 8 R-trans-4-hepten-3-ol The title product of Preparation 5 (10 g, 0.088 mol) was prepared by the method of Reference Example 1, but using a reaction temperature of −20 ° C. at all stages during reagent mixing. ) Was converted to the title product of Reference Example 8. After adding all reagents, the mixture is allowed to stand at -20 ° C.
Stir for 1.5 h, then warm to room temperature and cool by dropwise addition of 25 ml H ₂ O and then 6 ml 30% NaOH (saturated with NaCl). The cooled mixture is stirred for 20 minutes and CH ₂ C
Dilute with 30 ml of 1 ₂ and separate the aqueous layer and use fresh CH ₂ Cl ₂
It was washed with (50 ml × 2). The organic layers were combined and MgSO ₄
It was dried above and distilled to a residual volume of 50 ml, and the residue was chromatographed on silica gel using pentane first, then pentane: ether (4: 1) as eluent. The initial fraction containing low polar impurities was discarded. Pure product fraction with Rf 0.3 (4: 1
(Pentane: ether) are combined and stripped to give the title product (5.7g) as a clear colorless liquid.

Reference Example 9 R-3-Ethyl-4-heptenoic Acid By Reference Example 3, Method A, but using ether instead of CH ₂ Cl ₂ for isolation, the product of Reference Example 8 (5.7 g, 0.05 m
l) is condensed with ethyl orthoacetate and rearranged,
Saponification gave the title product (1.4 g) as an oil; tlc Rf 0.4 (2: 1 hexane: ether), the intermediate ethyl ester showed Rf 0.25 in the same tlc system.

Reference Example 10 S-3-Ethylheptanoic acid The product of Reference Example 9 (1.4 g) was added to 5% Pd (O) in 20 ml of CH ₃ OH.
Hydrogenated over 0.1 g of H) ₂ / C at 4 atm for 1 hour. The catalyst was recovered by filtration. The solvent was stripped from the liquor and the residue was distilled to give 1.0 g of the title product; boiling point 7
6 to 77 ° C / 0.4 torr; [α] ▲ ²⁵ _D ▼ -1.6 ° (C =
2% in CH ₃ OH).

Under _{N 2} was Example 11 S-3- ethyl-heptanoyl chloride drying, the product of Example 10 (1.0
g, 0.00633 mol) was dissolved in 10 ml of CH ₂ Cl ₂ . Oxalyl chloride (0.547 ml, 0.00627 mol) was added and the mixture was stirred for 1 hour. During this time, the gas generation ended. The resulting title product solution was evaporated under a stream of N ₂ to 7 ml and used directly in the next step.

By the same method, the unsaturated acid of Reference Example 9 is converted to a solution of R-3-ethyl-4-heptenoyl chloride.

Example 5 N- (S-3-Ethylheptanoyl) -D-γ-glutamyl (αbenzyl ester) -glycyl-D-alanine benzyl ester The title product of Preparative Example 4 (1.1 g, 0.00222 mol) was added to CH ₂ Cl ₂
Dissolved in 30 ml and triethylamine (0.935 ml, 0.00666 mol). A CH ₂ Cl ₂ solution (2.3 ml, 0.00211 mol) of S-3-ethylheptanoyl chloride of Reference Example 11 was added,
The mixture was stirred under N ₂ for 0.75 h, 20 ml each of 10% HC
It was washed successively with 1, H ₂ O, 10% K ₂ CO ₃ and saturated brine, dried over MgSO _{4 and} stripped to give a solid residue which was triturated with ether and triturated. Recovered (0.8 g). The product was taken up in ethyl acetate and rewashed and re-striped as above to give a second solid (0.7 g) which was eluted on CH 2 Cl 2 as CHC eluent.
Chromatographic purification using l ₃ : CH ₃ OH (97: 3) gave the purified title product (467 mg).

3-Ethyl-4-heptanoyl chloride was coupled with the title product of Preparation 4 by the same method to give N- (R-3
-Ethyl-4-heptanoyl) -D-γ-glutamyl (α-benzyl ester) -glycyl-D-alanine benzyl ester is obtained.

Example 6 N- (S-3-methylheptanoyl) -D-γ-glutamyl-glycyl-D-alanine The title product of Example 5 (467 mg) was hydrogenated by the method of Reference Example 10. After recovering the catalyst, the liquor was stripped to give a foam which was taken up in H ₂ O, filtered and lyophilized to give 238 mg of the title product. ¹ H-nmr (DMSO-d ₆ ) delta (ppm): 8.20-8.00 (m, 3
H), 4.24-4.16 (m, 2H), 3.74-3.60 (m, 2)
H), 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, 6
H). By the same method, the unsaturated product of Example 5 is converted to the same product.

Example 7 N- (R-3-ethyl-4-heptenoyl) -D-γ-
Glutamyl-glycyl-D-alanine The unsaturated product of Example 5 is hydrolyzed by the method of Example 4 to give the above title product.

Example 8 N- (S-3-ethylheptanoyl) -D-γ-glutamyl (αbenzyl ester) -glycyl-D-alanine butyl ester By the method of Example 6, S-3-ethylhepta of Example 5 Noyl chloride was combined with D-γ-glutamyl (αbenzyl ester) -glycyl-D-alanine butyl ester (1.0 g, 0.00222 mol) to produce the above title product, isolated and similarly purified. The yield was 0.7 g. ¹ N-nmr (DMSO-d ₆ ) delta (ppm): 8.22 (d, J = 7,
1H), 8.12-8.00 (m, 2H), 4.40-4.16 (m, 2
H), 4.08-3.95 (m, 2H), 3.75-3.62 (m, 2
H), 2.18 (t, J = 6, 2H), 2.02 (d, J = 6,
2H), 2.04-1.62 (m, 3H), 1.60-1.46 (m, 2
H), 1.38-1.10 (m, 15H), 0.90-0.75 (m, 9
H). By the same method, R-3-ethyl-4-heptenoyl chloride was converted to N- (R-3-ethyl-4-heptenoyl).
Convert to -D-γ-glutamyl (α-benzyl ester) -glycyl-D-alanine butyl ester.

Example 9 N- (S-3-Ethylheptanoyl) -D- [gamma] -glutamyl-glusyl-D-alanine butyl ester The title product of Example 8 was hydrogenated by the method of Reference Example 10 to yield the above titled product. Manufactured. After recovering the catalyst,
The liquor was stripped to give a solid which was triturated with ether and the product recovered by passing (256 mg, mp 130-131 ° C).

By the same method, the unsaturated product of Example 8 is converted to the same product as above.

Reference Example 12 R-3-Ethyl-4-heptenal A solution of R-trans-4-hepten-3-ol (3.0 g) of Reference Example 8 in 300 ml of dried ethyl vinyl ether was heated under reflux while Hg (OAc ) Add ₂ times 5 times (2 g each, every 2 hours). After refluxing for a further 18 hours, the clear solution is cooled to room temperature, treated with 0.5 ml glacial acetic acid and stirred for 3 hours. The solution formed is diluted with ether, poured into 200 ml of 5% KOH solution and washed with ether 3 times.
Extract twice. The ether extracts were combined, dried over K ₂ CO _3, stripped to allyl vinyl ethers, R-
Trans-O-vinyl-4-hepten-3-ol is obtained.

Allyl vinyl ether (2.3 g) in 20 ml decalin
The solution of is refluxed for 10.5 hours. The resulting solution is then cooled to room temperature and fed directly onto a silica gel column. After removing the decalin by hexane flush, the title product is recovered by elution from the column with ether and stripping.

Reference Example 13 S-3-Ethyl-4-heptenoic acid The product of Reference Example 12 (100 mg) is dissolved in 4 ml of acetone and cooled to 0-5 ° C. In another flask CrO ₃ (72.1
g, 0.72 mol) in a mixture of H ₂ O50ml, stirred at 0 to 5 ° C., was slowly added concentrated H ₂ SO ₄ 62.1ml, the mixture was diluted with H ₂ 0, in the 250 ml, H ₂ C Obtain a 2.88 M solution of _r O ₄ (Jones reagent). This solution (1 ml) is added dropwise to the above acetone solution over 1 hour. The temperature rises with the addition of reagents and is maintained at 17-25 ° C. The mixture is recooled to 6 ° C. and 70 ml of 2-propanol are added over 10 minutes (while the temperature rises to 20 ° C.), then concentrated in vacuo to give an oil which is stirred with 2.5 N NaO 2.
Add 8 ml H over 50 minutes, maintaining a temperature of 22 ± 5 ° C. Heat the mixture, pass over diatomaceous earth, heat
Wash with 2.5N NaOH. The solution and the washing solution are combined and extracted with isopropyl ether (100 ml × 3). The combined organic layers are back-extracted with 200 ml 2N NaOH. To the combined aqueous layers was slowly added 0.5 ml of concentrated HCl to acidify the aqueous layer to pH 1.0 and the product was added to 300 ml of fresh isopropyl ether.
Extract into × 3. The organic extracts are combined and stripped to give the title product as an oil.

Reference Example 14 S-7 (t-butyldimethylsilyloxy) -5-methyl-1-heptene In a 500 ml four-neck round bottom flask equipped with a stirrer, thermometer, N ₂ inlet and addition funnel, (methyl) Triphenylphosphonium bromide (25.7g, 0.072mol,
1.25 eq) was slurried in 77 ml THF (tetrahydrofuran) and cooled in an acetone / ice water bath. Charge n-butyllithium (43.2 ml of a 1.6N solution in hexane, 0.069 mol, 1.20 eq) into the addition funnel. Initially -8 ℃
Butyllithium was added to the above slurry over 1 hour, the temperature rose and the temperature was maintained at ± 1 ° C. The mixture was stirred at 0-2 ° C for an additional 0.5 hours to give an intermediate in a thin suspension of LiBr. Ensured complete formation of the body methylenetriphenylphosphorane. The aldehyde product of Preparative Example 2 (14.1 g, 0.0576 mol) in 14 ml of THF was added at a temperature of 3-.
While maintaining at 7 ° C, added in small portions over 40 minutes. After stirring for a further 15 minutes, no starting aldehyde was detected by tlc using hexane: ether (3: 1) as eluent (starting aldehyde Rf 0.
6; product Rf 0.95). Warm the reaction mixture to ambient temperature,
It was diluted with 150 ml of ethyl acetate and 90 ml of H ₂ O.
The organic layer was separated and washed with H ₂ O (100 ml × 2). Three
The aqueous layers were combined and backwashed with 40 ml of ethyl acetate. The organic layers were combined, dried over MgSO _{4 and} stripped to give 25 g of an oil, which was triturated with 10 ml of hexane, passed over a sinter glass funnel and in situ solid hexane (10 ml). × 4) was added to re-pulp, and the combined hexane solution and washings were stripped to give the title product (13.5 g, 96.6%) as an oil. ¹ H-nmr (CDCl ₃ ) delta (ppm): 5.4-6.2 (m, = CH),
_{4.8-5.3 (m, = CH 2)} , 3,7 (t, J = 6.5Hz, -OCH
_2- ), 0.08 (s, C (CH ₃ ) ₃ ) and 0.0 (s, Si (C
H ₃ ) ₂ ), 8 mol% (C ₆ H ₅ ) ₃ PO contaminated (7.6, s, 1.25
H). Reference Example 15 S-3-Methylhept-6-en-1-ol Method A By the method of Reference Example 14, but using 2,2 equivalents of methyl (triphenylphosphonium bromide and n-butyllithium, respectively) Aldehyde product of 6 (2
6.3 g, 0.20 mol; amended for purity) was reacted with methylenetriphenylphosphorane. The formation of a gummy solid was observed during the addition of the aldehyde solution, but once heated to ambient temperature for processing, the reaction became a thin slurry. The reaction mixture was added with 500 ml of H ₂ O and ethyl acetate 3 times.
Diluted with 00 ml. The layers were separated and H ₂ O (250 ml x 3)
Washed with. The combined aqueous layers were combined with ethyl acetate (300 ml x
Backwashed in 2). The 3 organic layers were combined, dried over MgSO ₄ , stripped to give 25.6 g (100%) of the title product and about 40 g of triphenylphosphine oxide, suitable for further processing in Method B below. Oil containing 6
5.7 g was obtained.

The pure title product is more easily obtained by conventional hydrolysis of the product of Reference Example 14, eg in dilute sulfuric acid of Reference Example 17 below. Extracting the title product into ethyl acetate, isolated by drying and stripping over MgSO _4.

Reference Example 16 S-3-Methyl-6-heptenal The title product of Reference Example 15 (1.14 g, 0.01 mol) was added to CH ₂ Cl ₂
It was dissolved in 20 ml and cooled to 0 ° C. Pyridine chlorochromate (4.30 g, 0.02 mol) is added in small portions and the temperature is maintained at 0-5 ° C. The mixture is warmed to ambient temperature, stirred for 2 hours, passed through a pad of silica gel and the solution is stripped to give the title product as an oil, which is further purified by distillation if desired.

Reference Example 17 S-3-Methyl-6-heptenoic acid Method A 2000 equipped with stirrer, thermometer and addition funnel.
The title product of Reference Example 14 (81 g, corrected for purity, 0.33 mol) was added to 4 ml of acetone in a ml 3-neck round bottom flask.
It was dissolved in 00 ml and cooled to 0-5 ° C. In a separate flask, mix CrO ₃ (72.1 g, 0.72 mol) with 50 ml H ₂ O,
Stir at 0-5 ° C., slowly add 62.1 ml of concentrated H ₂ SO _{4 and} dilute the mixture with H ₂ O to 250 ml, add 2.8 of H ₂ CrO ₄ .
Obtain an 8M solution (Jones reagent). This solution (240 ml, 0.67 mol) was added in small portions to the above acetone solution over 1.2 hours. When the reagent is added, the temperature rapidly rises to 1
Raised to 7 ° C and maintained at 17-25 ° C. No exotherm was noted upon completion of the addition. The mixture was recooled to 6 ° C and 70 ml of 2-propanol was added over 10 minutes (while the temperature rose to 20 ° C) then concentrated in vacuo to give an oil which was stirred and the temperature was raised. 22
While maintaining at ± 5 ° C, 400 ml of 5N NaOH was added. The thick reaction mixture was diluted with 400 ml H ₂ O and passed over diatomaceous earth. Damp cake with 400 ml H ₂ O and 5N NaOH 5
Pulped in 0 ml, warmed on a steam bath and passed again. Combine the liquids and use isopropyl ether (300 ml).
It was washed with × 3). The combined organic layers are 2N NaOH200
Back extracted with ml. To the combined aqueous layers was added 50 ml of concentrated HCl slowly to acidify it to pH 1.0 and the product was extracted into fresh isopropyl ether (300 ml x 3).
The organic extracts were combined and stripped to give the title product as an oil (29.1 g, 61%). Secondary basic extraction of the diatomaceous earth filter cake and subsequent similar isolation yielded an additional 7.7 g (16%). The ¹ H-nmr (CDCl ₃ ) delta (ppm) of the combined product 1 was 11.9.
(S, -COOH), 5.8 (m, = CH), 5.0 (m, = CH ₂ ) and 1.0 (d, -CH ₃ ), the isopropyl ether peaks of 3.7 and 1.1 were 8.6 mol% (6.3 wt% ) With isopropyl ether. This contamination does not prevent the use of this product for other treatments.

Method B Product of Reference Example 15, Method A (65.7 g, S-3-methylhepto-6-en-1-ol 26.3 g, containing 0.20 mol)
Was oxidized by this Example, Method A. After cooling with isopropanol and stirring for 1 hour at 0-5 ° C. (during which the reaction mixture became completely green), the organic solvent was stripped off, the aqueous residue was diluted with 250 ml H ₂ O and isopropyl ether (250 ml) was added. It was extracted in × 3). The organic extracts were combined and treated with 160 ml of 2N NaOH, which caused a large amount of (C ₆ H ₅ ) ₃ PO (contaminant in the starting material) to precipitate, which was separated and washed with complete 1N NaOH. The liquids were combined, the layers were separated and the organic layer was washed with 80 ml 1N NaOH. All the aqueous layers were combined, washed with isopropyl ether (250 ml x 3), acidified to pH 1.0 with 50 ml concentrated HCl, and the desired product was purified with fresh isopropyl ether (25 ml).
Extracted into 0 ml x 3). The combined acidic organic extracts
Dry over MgSO _{4 and} strip to give the title product 14.0
g was obtained. If desired, it is further purified by distillation.

Reference Example 18 S-3-methylheptanoic acid 10% Pd / C (wet with 50% water, 1.64 g) in a Parr hydrogenation apparatus, 150 ml of ethyl acetate and the unsaturated acid of Reference Example 17 (3.28 g, purity) Modified) and the slurry was hydrogenated at 4 atm for 1.5 hours. During this period, hydrogen absorption was completed. The catalyst was recovered by filtration over diatomaceous earth and the liquor stripped to give the title product as an oil (3.20 g, 96%).

Instead of this, 5% Pd / C (water 5
Charge 0% moistened, 2 g), then charge the title product of Reference Example 17 (33.3 g, corrected for purity) in 150 ml of ethyl acetate at 4 atm, 30-. Hydrogenation was carried out at 31 ° C. for 2 hours, during which hydrogen absorption had ceased. The catalyst was recovered by filtration over diatomaceous earth and the liquor was stripped to give 35.4 g of an oil which was distilled under high pressure to give the purified title product (29.6 g, 87.6%).

Boiling ^{point: 77-79 ℃ / 0.2mm; 1 H} -nmr (CDCl 3) delta
(ppm): 12.0 (s, -COOH), 1.0 (d, -CH 3), 0.6-2.8
(M, residual 13H); IR film) 3400-240
0,2960,2925,2860,1708,145
8, 1410, 1380, 1295, 1228, 119
0,1152,1100,930 cm ^-1 ; [α] ▲ ²⁵ _D
▼ = -6.41 ° (C = 1% in CH ₃ OH); n ▲ ^22.5 _D
▼ = 1.427. Reference Example 19 S-3-Methyl-6-heptenoyl chloride By the method of Reference Example 6, the acid product of Reference Example 4 was converted to a CH ₂ Cl ₂ solution of the title product to give Example 10 below. Use directly. Instead, strip the reaction mixture,
The title product is obtained and, if desired, distilled under reduced pressure.

Example 10 N- (S-3-methyl-6-heptenoyl) -D-γ-
Glutamyl (α-benzyl ester) -glycyl-D-alanine benzyl ester By the method C of Example 1 the product of Preparation 4 (2.77
g, 5 mmol) was combined with the entire batch of CH ₂ Cl ₂ acid chloride from Reference Example 19. The first product, 2.77, recovered by stripping the washed and dried organic layer.
g in 20 ml of hot ethyl acetate, this solution was diluted with 20 ml of hexane and the purified product was recovered by filtration (2.24 g, 77%), mp 137.5-139.5 ° C.

Example 1 N- (S-3-methyl-6-heptanoyl) -D-γ-
Glutamyl-glycyl-D-alanine The product of Example 10 (1 g) is eluted in ₅ ml CH3OH. 1N NaOH (2.50 ml) is added and the mixture is stirred at ambient temperature for 3 hours. CH ₃ OH is stripped, the aqueous residue is diluted with 7.5 ml H ₂ O, extracted with ethyl acetate (7.5 ml × 2) and acidified to pH 3.0 with 1N HCl. The acidified aqueous layer is extracted sequentially with fresh ethyl acetate and the extract is stripped to give the title product, which is lyophilized by Example 10, Method A.

Example 12 N- (S-3-methyl-6-heptenoyl) -D-γ-
Glutamyl (α-benzyl ester) -glycyl-D-
Alanine butyl ester By Example 11, Method A, D-γ-glutamyl (α
Benzyl ester) -glycyl-D-alanine butyl ester (Preparation Example 4) is coupled with S-3-methyl-6-naphthenoyl chloride (Reference Example 19) to give the above title product.

Example 13 N- (S-3-Methylheptenoyl) -D-γ-glutamyl-glycyl-D-alanine butyl ester By the hydrogenation method of Example 3, the product of Example 12 is converted to the above title product. Invert.

Production Example 1 400 ml of THF was charged into a 3-four-necked flask equipped with a racemic trans-4-hexen-3-ol stirrer, an addition funnel, a thermometer and a nitrogen inlet. THF was cooled to −70 ° C. and ethylmagnesium bromide (2M in THF, 600 ml, 1.2 mol) was added. Crotonaldehyde (78 ml, 0.94 mol) was added over 7 minutes while maintaining the temperature between -70 ° C and -60 ° C. After stirring for 20 minutes at −70 ° C. and then for 1 hour while gradually warming the mixture to −20 ° C., the mixture was cooled again to −70 ° C. and carefully quenched with 200 ml saturated NH ₄ Cl (first bubbling), Warm to ambient temperature,
Diluted with 200 ml CH ₃ COOH and 100 ml water. The two-layer system is saturated with NaCl. The aqueous layer was separated and extracted with ether (500ml x 2). The original organic layer and ether extract were combined, washed with saturated NaHCO ₃ (250 ml × 4), dried over MgSO ₄ , stripped and the residue was distilled at atmospheric pressure (69 g, bp 133-). 137 ° C., ¹ H-nmr (CDCl ₃ ) delta (ppm): 5.53 (composite m, 2
H), 3.93 (m, 1H), 2.26 (s, 1H), 1.71
(D, 3H), 1.43 (m, 2H), 0.84 (t, 3H). Production Example 2 Glycyl-D-alanine benzyl ester Nt-butyloxycarbonylglycine (10 g, 5
7 mmol), D-alanine benzyl ester p-toluenesulfonate (20 g, 57 mmol) and triethylamine (5.77 g, 57 mmol) in a cold (0 ° C.) solution of 100 ml methylene chloride in dicyclohexylcarbodiimide (12.3 g). , 60 mmol),
The resulting reaction mixture was allowed to warm to room temperature. After 18 hours, the mixture was passed and the liquid was concentrated in vacuo. The residue was dissolved in 200 ml of ethyl acetate and the organic layer was added with 2.5% hydrochloric acid,
It was washed with water, saturated sodium hydrogen carbonate solution and brine solution. The organic layer was separated, dried over magnesium sulfate and evaporated under reduced pressure. 200 ml of hydrogen chloride-saturated dioxane was added to the resulting oil. After 30 minutes, 400 ml of diethyl ether was added and the product was passed under a nitrogen atmosphere (10.9 g, 70% yield).

Production Example 3 Nt-butoxycarbonyl-D-γ-glutamyl (α
Benzyl ester) hydrosuccinamide ester Nt-butoxycarbonyl-D-γ-glutamic acid α
-Benzyl ester (50 g, 143 mmol) and N-
Dicyclohexylcarbodiimide (30.9 g, 15 mmol) was added to 1500 ml of methylene chloride containing hydroxy-succinamide (17.3 g, 150 mmol), and the resulting reaction mixture was stirred at room temperature for 18 hours. The solid was passed and the solution was concentrated in vacuo. The residue was triturated with diethyl ether and the solid separated under nitrogen (43.7
g, yield 68%).

Production Example 4 D-γ-glutamyl (α-benzyl ester) -glycyl-D-alanine benzyl ester hydrochloride Nt-butoxycarbonyl-D-γ-glutamyl (α-benzyl ester) hydroxysuccinamide ester (in 100 ml of methylene chloride) 4.3 g, 9.45 mmol), glycyl-D-alanine benzyl ester hydrochloric acid (2.71 g, 9.92 mmol) and triethylamine (1.
A solution containing 0 g, 9.92 mmol) was stirred at room temperature for 18 hours and then concentrated in vacuo. The residue was dissolved in 200 ml of ethyl acetate and this solution was washed with 2.5% hydrochloric acid, water, 10% potassium carbonate and brine solution. The organic layer was separated, dried over magnesium sulfate and evaporated under reduced pressure. The residue was treated with 200 ml of hydrogen chloride saturated dioxane and stirred for 2 hours. The solution was concentrated under vacuum to dryness and the residue was triturated with diethyl ether. The solid was separated under nitrogen (3.41 g, 73% yield).

By the same method, the product of Preparation Example 3 and glycyl-D-
Alanine butyl ester is D-γ-glutamyl (α
Benzyl ester) -glycyl-D-alanine butyl ester.

Preparative Example 5 Racemic trans-4-hepten-3-ol 2-pentenal (25 g, 0.30 mol) was converted to the title product and distilled by the method of Preparative Example 1, but using normal ether as the solvent. (25.6g; boiling point 145
~ 150 ° C).

Production Example 6 O- (t-butyldimethylsilyl) -S-citronellol In a 500 ml four-neck round bottom flask equipped with a stirrer, thermometer, N ₂ inlet and addition funnel, S-citronellol (547 g, 3.5 mol). Was dissolved in 547 ml of DMF (dimethylformamide) at ambient temperature (21 ° C). Imidazole (262.1 g, 3.85 mol, 1.1 eq) was added. The temperature dropped to 13 ° C, but was further lowered to -6.5 ° C with an ice / acetone bath. T-Butyldimethylchlorosilane (580.3 g, 3.85 mol, 1.1 eq) pre-dissolved in 1160 ml DMF by vigorous stirring was added over 1.25 hours and the temperature was gradually raised to 11 ° C over the same time. After an additional 0.25 h, tlc (3: 1 hexane: ether) showed complete conversion to the desired product (starting material Rf 0.2; product Rf 0.9).

For isolation, the mixture is treated with 500 ml of hexane and 10 ml of ice / water.
It was added to 00 ml. The layers were separated and the organic layer was washed with 2000 ml ice-cold 0.25N HCl. The two aqueous layers were combined and backwashed with 500 ml of hexane, the hexane washes were combined with the organic layer, washed with 500 ml of saturated NaHCO ₃ , dried over MgSO ₄ , stripped and the title product chemistry. A stoichiometric yield (986.7 g) was obtained. This yield is 104% of theory due to a small retention of solvent. ¹ H-nmr (CDCl ₃ ) delta (ppm): 5.2 (t, J = 7 Hz, =
CH), 3.65 (t, J = 6.5 Hz, -O-CH _2- )), 1.7 and 1.
65 (2S, 2 = C ( CH 3)), 0.8 (s, -SiC (CH 3) 3), and _{0.0 (s, -Si (CH 3} ) 2). Production Example 7 S-6- (t-Butyldimethylsilyloxy) -4-methylhexanal Mechanical stirrer, straight glass feed tube with O ₃ / O ₂ flow, thermometer and 500 ml with outlet connected to saturated KI trap. The product of Preparation 6 (81.2 g, corrected for solvent content, 0.30 mol) was CH 2 in a 4-neck round bottom flask.
Dissolved in 120 ml ₂ Cl ₂ and 81 ml CH ₃ OH. NaHCO
₃ (6.3 g, 0.075 mol, 0.25 equiv) was added and the mixture was cooled to -10 ° C in an acetone / dry ice bath. O ₃ / O while further lowering the temperature and maintaining it at -72 ° C to 75 ° C
₂ was bubbled into the reaction mixture for 6 hours. After less than 1 hour, no O ₃ was absorbed by the reaction mixture as evidenced by the yellow formation in the KI trap.
Tlc with hexane as eluent showed that the reaction was complete (starting material Rf 0.3, intermediate material Rf 0.0) The reaction mixture was purged of excess O ₃ with N ₂ and dimethyl sulfide (26.4 ml, 22.4 g, 0.36 mol, 1.2 eq) was added, the bath was removed, the mixture was allowed to warm to ambient temperature and stirred under N ₂ for 16 h, by which time tlc
(6: 1 hexane: ether) no longer showed residual intermediate (intermediate Rf 0.8; product Rf 0.05). The mixture is then stripped and the residue is washed with ethyl acetate 15
Partitioned between 0 ml and 300 ml H ₂ O. The organic layer is fresh H ₂ O 30
Wash with 0 ml. The combined H ₂ O layers were backwashed with 150 ml of fresh ethyl acetate. The organic layers are combined, dried over MgSO ₄ , stripped to an oil and finally stripped under high vacuum for 16 hours to give a stoichiometric yield of 74.7 g of the title product. This yield is 101.9% of theory due to mild solvent contamination. ¹ H-nmr (CDCl ₃ ) delta (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). Production Example 8 S-6-Hydroxy-4-methylhexanal Method A A 250 ml three-neck round bottom flask was equipped with a magnetic stirrer, thermometer, gas supply tube and gas outlet tube leading to a gas wash bottle containing saturated KI. CH ₂ Cl ₂ 81
ml and CH ₃ OH54ml in S- citronellol (31.25 g, 0.
(20 mol) and charged to -8 ° C. Temperature from -2 °
While maintaining the 10 ° C., it was 4.5 hours bubbling O ₂ / O ₃ in the reaction mixture. By this time, an excess of O 2 due to the KI solution
Capture of ₃ was seen, demonstrating the completion of the reaction by a positive starch / KI paper test on the reaction mixture. The mixture was maintained at -5 ° C., and purged with N _2, was added methyl sulfide (17.7ml, 15.0g, 0.24 mol). The reaction mixture was allowed to warm to ambient temperature and then stirred for 16 hours. During this time tlc using isopropyl ether as eluent showed that the reaction was complete (S-citronellol Rf 0.7, product Rf 0.4) and finally stripped off the solvent to give 50.8 g of an oil. It was The oil was diluted with 30 ml of ethyl acetate and 25 ml of H ₂ O to give a single phase. An additional 150 ml of ether formed two phases. The layers were separated and the organic layer was washed with H ₂ O (25 ml x 2), dried over MgSO _{4 and} stripped to a colorless oil (23.
9g) was obtained. The combined aqueous layers were combined with ethyl acetate (50 ml x
Extracted in 2), combined the extracts, dried and stripped to give an additional 7.8 g of colorless oil. The colorless oils were combined and stripped to give the title product (28.3 g,
108.7% of theory) was obtained. This is tlc (as described above)
It is pure except for solvent contamination and is suitable for use in other reactions as described above.

Method B The product of Preparative Example 2 is hydrolyzed by conventional methods, such as the dilute sulfuric acid method of Reference Example 4 above. The title product is isolated by extraction into ethyl acetate and stripping as in Method A above.

Claims (5)

[Claims] 1. The following absolute stereochemical formula: [In the formula, one of the broken lines represents a double bond, R is methyl or ethyl, R ² and R ³ are each hydrogen, or R ² and R ³ are each independently (C _1- C
₆ ) alkyl, (C ₆ -C ₈ ) cycloalkylmethyl or benzyl; provided that when the double bond is at the terminal 6,7-position, R is methyl] or R ² A pharmaceutically acceptable cation salt thereof when R ³ is hydrogen. 2. The compound according to claim 1, wherein R ² and R ³ are each hydrogen. 3. The following absolute stereochemical formula: The compound according to claim 1, which is a crystalline compound represented by: 4. The following absolute stereochemical formula: [Wherein R is methyl or ethyl, R ⁴ and R ⁵ are each hydrogen, or one of R ⁴ and R ⁵ is hydrogen and the other is (C ₁ -C ₆ ) alkyl or (C ₆ ~ C
₈ ) Cycloalkylmethyl], wherein (a) R-trans-3-methyl-4-heptenoic acid, R
-Trans-3-ethyl-4-heptenoic acid or S-3
The activated form of -methyl-6-heptenoic acid has the following absolute stereochemical formula: [Wherein R ⁶ and R ⁷ are each 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], and reacts with a compound of the following absolute stereochemical formula: Wherein one of the dashed lines (but not both) represents a double bond, provided that the double bond is at the terminal 6,7-position, then R is methyl. Forming and (b) simultaneously hydrogenating the compound of formula (IIIc) to reduce the double bond and hydrolyze the benzyl ester group to form a compound of formula (1). . 5. A process according to claim 4, wherein the activated form of the acid is the acid chloride, R ⁶ and R ⁷ are both benzyl and R ⁴ and R ⁵ are both hydrogen.