FI86858B - FOERFARANDE FOER FRAMSTAELLNING AV TERAPEUTISKT ANVAENDBARA PEPTIDER, VILKA INNEHAOLLER ACYLGLUTAMINSYRA. - Google Patents

Description

1 86858

This invention relates to a process for the preparation of novel acylglutamic acid-containing peptides which can be used as immunostimulants and anti-infectives and which have the formula 1 0

II

10 R-CNH .. D ^ C0R, 1 2 4 ^ CH ^ LO (1)

B

(CH2) r 3-2conhchc

K

Wherein Rx is alkyl of 2 to 10 carbon atoms, cycloalkyl of 4 to 7 carbon atoms or cycloalkylmethyl of 5 to 8 carbon atoms; R 2 is hydrogen or alkyl of 1 to 3 carbon atoms; R 3 is a hydroxy or amino acid residue of formula 20

D

-NHCH (CH 2) CO 0 R c I z n / b

X

Wherein X is hydrogen, alkyl of 1 or 2 carbon atoms or hydroxymethyl and n is an integer from 0 to 4; and R 4 and R 5 are each independently hydrogen, alkyl of 1 to 6 carbon atoms, cycloalkylmethyl or benzyl of 6 to 8 carbon atoms, and salts thereof.

30 The relatively new field of immunopharmacology, and in particular its part dealing with immunomodulation, is constantly evolving rapidly. Numerous naturally occurring compounds have been studied, including the tetra-peptide tuftsin, which has the chemical structure N2- [1- (35-N2-L-threonyl-L-lysyl) -L-prolyl] -L-arginine. Attention has been paid to synthetic peptidoglycan derivatives, especially muramyl dipeptides. Summaries of the numerous compounds studied as immunomodulators, and in particular as immunostimulants, can be found in 5 articles by Duker et al., Ann. Rep. Med. Chem. 14 (1979) 146-167, Lederer, J. Med. Chem. 23 (1980 819-825 and Kralovec, J., Drugs of the Future 8 (1983) 615-638.

Immunostimulant peptides have been described in several patents: 10 L-alanyl-alpha-glutaric acid N-acyl dipeptides in DE Patent 3,024,355, published January 15, 1981; tetra- and pentapeptides containing D-alananyl-L-glutamyl groups or L-alanyl-D-glutamyl groups, in GB Patent 2,053,231, published February 4, 1981, and DE Patent 3,024,281 , published January 8, 1981, respectively; and N-acylalanyl-gamma-D-glutamyl tripeptide derivatives in which the C-terminal amino acid is lysine or diaminopimelic acid, in DE Patent Publication 3,024,369, published January 15, 1981; and lactoyl tetrapeptides consisting of the N-lactylalanyl, glutamyl, diaminopimelyl and carboxymethylamino components in EP Patent Publication No. 11,283, issued May 23, 1980.

Immunostimulant polypeptides of formula (A) R 1 - (HNCHCO) -HN-CH-R 3 I 2 n '30 R (CHO) m I 2 m.

CO-NH-CH-R (A) (<R 2 -hn-ch-r 5 35 3 868 58 wherein R 1 is hydrogen or acyl; R 2 is, inter alia, hydrogen, former alkyl, hydroxymethyl, benzyl; R 3 and R 4 are each hydrogen, carboxy or -CONR 5 R 0, wherein R 7 is hydrogen, lower alkyl optionally substituted by hydroxy and R 8 is mono- or dicarboxy-lower alkyl, R 5 is hydrogen or carboxy, provided that when either R 4 or R 5 is hydrogen, the other is carboxy or -CONR7R8, R6 is hydrogen, m is 1-3 and n is 0-2, and derivatives thereof in which the carboxy and amino groups are protected are disclosed in U.S. Patent Nos. 4,311,640. and 4,322,341, and in EP patent applications 25 4-22, 50,856, 51,812, 53,388, 55,846 and 57,419.

None of the polypeptides disclosed in the art has a heterocyclyl group at the position where the variable R4 is in the above formula, with the exception of U.S. Patent No. 4,619,915. U.S. Patent No. 4,565,653 describes polypeptides in which the variable R4 is a basic amino acid group.

Kitaura et al., J. Med. Chem. 25 (1982) 335-337, reports N2- (gamma-D-glutamyl) -meso-2 (L), 2 (D) -diamino-20 as the smallest structure of pimelic acid capable of eliciting a biological response characteristic of formula (A ) wherein n is 1; R1 is CH3CH (OH) -CO-; R2 is CH3; both R3 and R5 are -C00H; R4 is -CONHCH2C00H; and R 6 is H. Said compound of formula (A) is known as FK-156.

A preferred group of compounds of formula 1 are those wherein R 1 is alkyl of 5 to 8 carbon atoms, R 2 is hydrogen, R 3 is said amino acid residue, wherein X, n and R 5 are as defined in formula 1 and R 4 is hydrogen. Particularly preferred are those in question. compounds of the group wherein n is 0 and R 5 is hydrogen, said alkyl or cyclohexylmethyl. Particularly preferred are compounds of formula 1 wherein Rx is (R, S) -2-ethyl-1-butyl, R5 is hydrogen and X is methyl; R 3 is (R, S) -3-heptyl, R 5 is hydrogen and X is methyl; R 3 is (R, S) -2-methyl-1-pentyl-4,885,81, R 5 is hydrogen and X is methyl; Rx is (R, S) -2-heptyl, R5 is hydrogen and X is methyl; R 1 is (R, S) -2-ethyl-1-pentyl, R 5 is hydrogen and X is methyl; Rx is (R, S) -1-hexyl, R5 is hydrogen and X is methyl; R 1 is (R, S) -2-ethyl-1-hexyl, R 5 is hydrogen and X is methyl; Rx is (S) - or (R, S) -2-methyl-1-hexyl, R5 is hydrogen and X is methyl; R 3 is (S) - or (R, S) -2-ethyl-1-hexyl, X is methyl and R 5 is hydrogen; and Rt is 1-hexyl, X is methyl and R5 is hydrogen. Also particularly preferred is a compound wherein R 1 is 1-hexyl, X is hydrogen and n is 3. Particularly preferred esters are esters wherein R x is (R, S) -2-ethyl-1-pentyl, X is methyl and R 5 is n-butyl, i-butyl or cyclohexylmethyl;

R 1 is (S) - or (R, S) -2-methyl-1-hexyl, X is methyl and R 5 is n-butyl, i-butyl or cyclohexylmethyl; And R 1 is (S) - or (R, S) -2-ethyl-1-hexyl, X is methyl and R 5 is n-butyl, i-butyl or cyclohexylmethyl.

Another preferred group of compounds of formula 1 are those wherein R 1 is cycloalkyl of 4 to 7 carbon atoms, r 2 is hydrogen and R 3 is said amino acid residue wherein n is 0, X is alkyl of 1 to 2 carbon atoms and R 4 and R 5 are both hydrogen. Particularly preferred in this group is a compound wherein Rt is cyclohexyl and X is methyl.

A third preferred group of compounds of formula 1 are those wherein Rx is alkyl of 5-8 carbon atoms, R2 is hydrogen, R3 is said amino acid residue wherein X is hydrogen or alkyl of 1-2 carbon atoms, n is 0-4 and R 5 is hydrogen, and R 4 is alkyl of 1 to 6 carbon atoms, cycloalkylmethyl of 6 to 8 carbon atoms, or benzyl.

By pharmaceutically acceptable base salts of said compounds of formula 1 are meant salts formed with inorganic or organic bases such as alkali metal and alkaline earth metal hydroxides, ammonia, tri-ethylamine, ethanolamine and dicyclohexylamine.

, 86858 b

The configuration of the amino acid groups constituting the compounds of formula 1 is significant for the pharmacological activity of said compounds. The most active are the compounds of formula 1, the stereochemistry of which is of said formula. The preferred stereochemistry of the compounds of formula 1 in which R 2 and X are other than hydrogen on said carbon is indicated by the prefixes L and D, respectively.

The compounds of formula 1 can be prepared by any of a number of methods known to those skilled in the art. The methods involve the formation of peptide bonds between amino acids, which due to their amino and carboxy groups as well as often the presence of other reactive groups make the protection of said groups and / or the activation of these groups, in particular the carboxy group, necessary to effect or can be optimized.

The process according to the invention for the preparation of the novel compounds of the formula I20 and their salts is characterized in that a) the compound of the formula 25 is dehydratingly coupled.

I II

(CH 2) 2CONHCHC-OH

* 2 30 wherein R 1 and R 2 are as defined above, with a compound of formula

D

H0NCH (CHn) COOrc 2, 2 n 2 5

35 X

6 86858 wherein the non-reactive functional groups can be blocked and wherein X, n and R 5 are as defined above, followed by an optional step of selectively removing the inhibitor groups and, if desired, preparing a pharmaceutically acceptable base salt of the product; or b) acylating a compound of formula H 2 N 1 D 2 CO 2 R 5 10 L 0

I II

(ch2) 2conhch-c-r3 k wherein R2, R3 and R5 are as defined above, by a compound of formula R1CO-Hal wherein the non-reactive functional groups can be blocked and wherein R: is as defined above and Hal is halogen, followed by an optional step of selectively removing the inhibitor groups and, if desired, preparing a pharmaceutically acceptable base salt of the product.

The examples presented herein specifically describe certain protecting groups and activating groups. However, one skilled in the art will recognize that other protecting groups or activating groups could have been used. The choice of a particular protecting group will depend greatly on the availability of the required reagent, its effect on the solubility of the "protected" compound, its ease of removal, and the presence of other groups that may be affected by its use; this means its selectivity or elimination.

For example, in many reactions it is necessary or at least desirable to protect amino groups and / or carboxy groups. The synthetic route chosen for peptide synthesis may require the removal of one or both of said protecting groups in order for an additional reaction to occur in the reconstituted amino or carboxy group; this means that the protecting groups used are reversible and in most cases can be removed independently. In addition, the choice of a protecting group for a particular amino group depends on the proportion of said amino acid in the overall reaction scheme. The protecting groups of the amino group 10 are used, the variability of which, i.e. the ease of removal, varies. The same is true for the protecting groups of the carboxy group. Such groups are known in the art and reference is made to the overviews in Bodansky et al., "Peptide Synthesis", 2nd Edition, John Wiley & Sons, 15 N.Y. (1976); Greene, "Protective Groups in Organic Synthesis," John Wiley & Sons, N.Y. (1981); McOmie, Protective Groups in Organic Chemistry, Plenum Press, N.Y. (1973); and Sheppard, in "Comprehensive Organic Chemistry, The Synthesis and Reactions of Organic Com-20 Pounds," Pergamon Press, N.Y. (1979), E. Haslam, ed., Part 23.6, pp. 321-339.

Conventional protecting groups for amino and carboxy groups are known to those skilled in the art. Representative amino protecting groups include, but are not limited to, benzyloxycarbonyl, substituted or unsubstituted aralkyl such as benzyl, trityl, benzhydryl, and 4-nitrobenzyl; benzylidene; arylthio such as phenylthio, nitrophenylthio and trichlorophenylthio; phosphoryl derivatives such as dimethylphosphoryl and 0,0-di-benzylphosphoryl; trialkylsilyl derivatives such as trimethylsilyl; and others, as described in U.S. Patent 4,322,341, which is incorporated herein by reference. The preferred amino protecting group is benzyloxycarbonyl. Methods for substituting said group 35 for a particular amino group are well known. In general, they involve the acylation of a suitable amino compound with benzyloxy-8 86858 carbonyl chloride (benzyl chloroformate) in a reaction-inert solvent, e.g. water, methylene chloride or tetrahydrofuran, in the presence of a base (acid acceptor), e.g. water, sodium hydroxide or sodium hydroxide. and using an organic solvent in the presence of a tertiary amine such as C 1-4 trialkylamines and pyridine. When an aqueous solvent system is used, the pH of the reaction is maintained at about pH 8-10 and preferably at pH 9. Alternatively, when the reactant (i.e., the compound whose amino group is to be protected) contains basic groups, it can act as an acid. recipient.

The acyl group RjCO is introduced into the peptide using standard acylation methods, such as reacting said peptide with a suitable acid chloride or bromide in a reaction-inert solvent. Preferred conditions are anhydrous conditions, including the addition of a suitable base such as an organic base, preferably a tertiary amine such as triethylamine, N-methylmorpholine or pyridine. The preferred solvent is methylene chloride.

Representative carboxy protecting groups include numerous esters such as silyl esters, including trialkylsilyl esters, trihalosilyl esters, and haloalkylsilyl esters; certain hydrocarbyl esters such as C 1-4 alkyl, especially t-butyl groups, benzyl and substituted benzyl esters, benzhydryl and trityl; phenacyl and phthalimidomethyl esters; certain substituted hydrocarbyl esters such as chloromethyl, 2,2,2-30 trichloroethyl, cyanomethyl; tetrahydropyranyl; methoxymethyl; methylthiomethyl; protected carbazoyl such as -CONH-NHR0, wherein R 0 is the amino protecting group disclosed above, especially benzyloxycarbonyl; and others, as described in U.S. Patent 4,322,341, which is incorporated herein by reference. A very preferred carboxy group protecting group is a t-butoxycarbonyl group.

9 86858

Protected amino and carboxy groups are converted to unprotected amino and carboxy groups using methods known to those skilled in the art. The benzyl group, the preferred carboxy group protecting groups (as part of the protected carbazoyl group) are removed by catalytic hydrogenation using a palladium catalyst, especially a palladium-carbon catalyst. Alternatively, said protecting groups are removed with trifluoromethanesulfonic acid dissolved in trifluoroacetic acid and in the presence of anisole to prevent alkylation, the t-butoxycarbonyl group is easily removed by treating the mixture with dioxane saturated with hydrogen chloride.

Activation of carboxyl groups as a means of performing a particular reaction is a method known to those skilled in the art. It is particularly useful in the reaction sequence described herein to use anhydrides, especially cyclic anhydrides, as well as activated esters such as N-hydroxyphthalimide and N-hydroxysuccinimide derivatives, both of which are used in peptide syntheses.

20 Activated N-hydroxysuccinimide esters promote (expedite) subsequent reactions in said activated ester groups. As will be appreciated by one skilled in the art, other activating groups may be used. A particularly interesting group is the N-hydroxyphthalimido group, which is used in the same manner as the N-hydroxysuccinimido group. In both cases, a dehydrating coupling agent is used to form the activated ester. Such coupling agents include 1-cyclohexyl-3- (2-morpholinoethyl) -carbodiimide metho-p-toluenesulfonate, dicyclohexyl-30-carbodiimide, N, N'-carbonyldiimidazole, N- (3-dimethyl- ethylaminopropyl) -N'-ethylcarbodiimide hydrochloride, ethoxyacetylene, diphenyl ketene and N-ethyl-5-phenylisoxazolene-3'-sulfonate. The reaction conditions using these coupling agents are well described in the literature. They generally use a reaction-inert solvent and temperatures ranging from the ambient ambient temperature to 100 ° C. The above-mentioned carbodiimide reagents are preferred because they can be used at ambient temperature and provide the desired esters in satisfactory yields.

Once the coupling reactions leading to the final products have been carried out, the various protecting groups can be removed using the suitable methods mentioned above and the compounds of formula 1 can be isolated.

Pharmaceutically acceptable base salts of compounds of formula 1 wherein R 3 is hydroxy or R 4 or R 5 is hydrogen are obtained by treating a solution thereof, preferably an aqueous solution thereof, with a base such as those listed above in generally stoichiometric proportions.

The salts are isolated by evaporation or mixing.

The products of this invention can be used in mammals, including humans, as agents in the clinical and therapeutic treatment of diseases caused by various pathogenic microorganisms, especially gram-negative bacteria. They can also be used as immunostimulants in mammals, including humans, who are at increased risk of infection due to pre-existing inflammation or due to increased risk of infection due to existing or clinically induced immunosuppression.

The experimental method using C3H / HeN male mice from the Charles River Breeding Laboratory animal is shown below. Mice were acclimated for five days prior to use and then treated with either subcutaneous (SC) or oral (PO) dilutions (100, 10, 1, and 0.1 mg / ml) of test compound or placebo (pyrogen-free saline). kg) using a volume of 0.2 ml. Treatment depended on the infectious organism used: -24 and 0 hours before Klebsiella pneumoniae 35 exposure in normal mice and -3, -2 and -1 days before Escherichia coli or Staph, aureus exposure in il 8 6 8 58 mice with compromised immune systems . Exposure was given to pelvic muscle (IM) in the case of K. pneumoniae or intraperitoneally (IP) in the case of E. coli and Staph, aureus. A volume of 0.2 ml was used for exposure. Mortality was recorded after seven days for K. pneumoniae and after three days for exposure to two other microorganisms.

Preparation of cultures: 10 K. pneumoniae, E. coli or Staph, aureus: the culture was cultured to check the purity of the frozen blood stock on brain-heart infusion (BHI) agar. Three colonies were picked from an 18 hour culture plate and placed in 9 ml of BHI broth. The broth culture was grown for 15 hours at 37 ° C on a rotary shaker, after which 0.2 ml was applied to several BHI agar surfaces.

After 18 hours of incubation at 37 ° C, the slant surfaces were washed with BHI broth, the culture density was adjusted using a Spectronic 20, and appropriate dilutions were made to obtain the LD90 exposure level in normal mice.

Test results

The following experimental results (Tables I and II) show that the new compounds of formula 1 protect the carbon from Klebsiella pneumoniae infection. Experimental results have been obtained by administering to mice certain amounts of test compounds; survivors means the percentage of experimental animals that survived at a given dose in each case, and placebo means the percentage of experimental animals that survived even though they had not received a protective agent of formula 1. Table III shows the corresponding results for the known standard compound FK-156 described in J. Med. Chem. 25 (1982) 337-9.

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-y <tr Λί tr e (Λ ΓΟ rH r-4 o 3 o 3 <86858 23 When the compounds of this invention are used as anti-infectives or immunostimulants, they are administered orally, subcutaneously, intramuscularly, intravenously or intraperitoneally as a composition. For example, they may be administered in the form of tablets, pills, powders, or granules containing excipients such as starch, milk sugar, certain types of clays, etc. They may be administered in capsules in admixture with the same or equivalent excipients. also in the form of oral suspensions, solutions, emulsions, syrups and elixirs, which may contain flavoring and coloring agents For oral administration of the therapeutic agents of this invention, tablets or capsules containing about 50-500 mg are suitable for most applications.

The physician will determine the dosage which will be most suitable for an individual patient and it will vary with the age, weight and response of the particular patient and the mode of administration. The preferred oral dose range, as a single or divided dose, is about 1.0 to 300 mg / kg / day. The preferred parenteral dose is about 1.0 to 100 mg / kg / day; the preferred range is from about 1.0 to 20 mg / kg / day.

The following examples are provided solely to further illustrate the invention. For brevity, the following abbreviations are used for the peak forms of the NMR spectrum: s, singlet, d, doublet, t, triplet, q, quartet, m, multiplet. The terms mole and millimole are abbreviated to m and 30 mm, respectively.

86858 24

Example 1 N-Heptanoyl-D-gamma-glutamyl-glycyl-D-alanine

D

Rx = CH3 (CH2) 5; R2 = H; and R 3 = NHCH (CH 3) CO 2 H) IA. N-heptanoyl-D-gamma-glutamyl (alpha-benzyl ester) glycine_

To a solution of 879 mg (13.0 mm) of glycine and 1.3 g (13.0 mm) of triethylamine in 10 mL of water was added 5.0 g (11.2 mm) of N-heptanoyl-D-gamma. -glutamyl- (alpha-benzyl ester) hydroxysuccinimide ester in 100 ml of dioxane and the resulting reaction mixture was allowed to stir at room temperature for 80 hours. The solution was poured into 300 ml of ethyl acetate and the separated organic phase was washed with 10% hydrochloric acid, water and brine. The organic phase was separated, dried over magnesium sulfate and concentrated to dryness in vacuo. The residue was triturated with diethyl ether and filtered under nitrogen, 3.43 g (74% yield-20 to).

IB. N-heptanoyl-D-gamma-glutamyl-glycyl-D-alanine_

To a solution of 2.0 g (4.78 mm) of N-heptanoyl-D-gamma-glutamyl (alpha-benzyl ester) glycine was 1.75 g (5 mm) of D-alanine benzyl ester p-toluenesulfonic acid salt. , 206 mg (5 mm) of triethylamine and 675 mg (5 mm) of 1-hydroxybenzotriazole in 100 ml of tetrahydrofuran were added, 3.03 g (7.17 mm) of 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide -meto-p-toluenesulfonate and the reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was poured into 300 ml of ethyl acetate, and the organic phase was separated and washed with 10% hydrochloric acid, water, saturated sodium bicarbonate solution and brine. The organic phase was separated, dried over magnesium sulfate and concentrated in vacuo. The residue was triturated with diethyl ether and filtered under nitrogen, 2.7 g. 2 g of solid in 75 ml of methanol containing 400 mg of 10% palladium hydroxide-carbon catalyst were shaken for 4 hours under an atmosphere of hydrogen at an initial pressure of 345 kPa. The catalyst was filtered off, the filtrate was evaporated under reduced pressure and the residue was dissolved in water and lyophilized to give 1.23 g (90% yield) of the desired product as a white solid.

The NMR spectrum (DMSO-d 6) showed absorption at 4.35-4.2 (m, 2H), 3.83 (s, 2H), 2.35 (t, J = 7 Hz, 2H), 2 , 17 (t, J = 7 Hz, 2H), 2.1 - 1.8 (m, 2H), 1.55 - 1.45 (m, 2H), 1.3 (d, J = 6 Hz, 3H), 1.17 (bs, 6H) and 0.75 (b, 3H) ppm.

Example 2 N-heptanoyl-D-gammaglutamyl-glycine (R 1 = CH 3 (CH 2) 5; R 2 = H; and R 3 = OH)

A solution of 1.0 g of N-heptanoyl-D-gamma-glutamyl (alpha-benzyl ester) glycine in 50 ml of methanol was treated with 100 mg of 10% palladium hydroxide-carbon catalyst and the mixture was shaken. in a hydrogen atmosphere at 345 kPa for three hours. The catalyst was filtered off and the filtrate was concentrated in vacuo. The residue was dissolved in hot water and evaporated to dryness. The residue was redissolved in water and lyophilized to give 630 mg (83% yield) of the desired product as a white solid.

The NMR spectrum (DMSO-d 6) showed absorption at 4.37 - 4.25 (m, 1H), 3.9 (s, 2H), 2.35 (t, J = 73 Hz, 2H), 2, 18 (t, J = 6 Hz, 2H), 2.4-1.8 (m, 2H), 1.6-1.4 (m, 2H), 1.8 (bs, 6H) and 0.7 (bt, 3H) ppm.

26 86858

Example 3 N-heptanoyl-D-gamma-glutamyl-glycyl-glycine (Rt = CH3 (CH2) 5; R2 = H; and R3 = NHCH2CO2H) 3A. N-Heptanoyl-D-gamma-glutamyl (alpha-benzyl-5 ester) glycine hydroxysuccinamide ester

To a cold solution (0 ° C) containing 13.0 g (31 mm) of N-heptanoyl-D-gamma-glutamyl (alpha-benzyl ester) glycine and 3.91 g (34 mm) of N-hydroxysuccinimide in 400 ml: in tetrahydrofuran, 7.0 g (34 mm) of dicyclohexylcarbodiimide was added and the mixture was allowed to stir at 0 ° C for 1 hour and at room temperature for 18 hours. The solids were filtered and the filtrate was concentrated under reduced pressure. The residue was triturated with diethyl ether and filtered under nitrogen to give 15.4 g (98%) of the desired intermediate.

3B. To 2.0 g (3.97 mm) of N-heptanoyl-D-gamma-glutamyl-glycylglycine-N-heptanoyl-D-gamma-glutamyl- (alpha-benzyl ester) glycine hydroxysuccinimide ester-20 in 100 ml of dioxane was added. 446 mg (5.95 mm) of glycine and 0.55 ml (3.9 mm) of triethylamine in 10 ml of water and the resulting reaction mixture were allowed to stir at room temperature for 18 hours. The solution was poured into 100 ml of ethyl acetate and the organic layer was washed with 2.5% hydrochloric acid, water and brine.

The organic layer was separated, dried over magnesium sulfate and concentrated to dryness. The residue was triturated with diethyl ether and filtered under nitrogen to give 1.7 g of a white solid.

One and a half grams of solid in 75 ml of methanol containing 200 mg of 10% palladium hydroxide-carbon catalyst was shaken under a hydrogen atmosphere at 345 kP for 3 hours. The catalyst was separated and the filtrate was concentrated in vacuo. The residue was dissolved in water and lyophilized to give 1.12 g (90% yield) of the desired product.

86858 27 NMR spectrum (DMSO-d 6) showed absorption at 8.2 to 8.0 (m, 3H), 4.19 (m, 1H), 4.8 to 4.6 (m, 4H), 2, 25 (t, J = 7 Hz, 2H), 2.1 (t, J = 6 Hz, 2H), 2.05-1.7 (m, 2H), 1.5 (m, 2H), 1, 25 (bs, 6H) and 0.85 δ (t, J = 6 Hz, 3H) ppm.

Example 4 N-Heptanoyl-D-gamma-glutamyl-glycyl-D-serine

D

10 (R 1 = CH 3 (CH 2) 5; R 2 * H; and R 3 = -NHCH (CH 2 OH) CO 2 H) Using 2.0 g (3.98 mm) of N-heptanoyl-1-D-gammaglutamyl (alpha-benzyl ester) as starting material glycine hydroxysuccinimide ester, 780 mg (4.02 mm) of O-benzyl-D-serine and 0.556 ml (4.02 mm) of triethylamine and following the procedure of Example 3B, 902 mg (76% yield) of the desired product were isolated, mp. 130-132 ° C.

Nuclear Magnetic Resonance Spectrum (DMSO-d 6) showed absorption at 8.36 to 7.94 (m, 3H), 4.46 to 4.28 (m, 1H), 4.28 to 4.08 m, 1H), δ , 94 - 3.50 (m, 4H), 2.25 (t, J = 9 Hz, 2H), 2.17 t, J = 0 Hz, 1H), 2.10 - 1.04 (m, 14H) and 0.9 (t, J = 6 Hz, H) ppm.

Example 5 N-Heptanoyl-D-gamma-glutamyl-glycyl-D-alpha-aminobutyric acid

25 D

(Rx = CH3 (CH2) 5-; R2 = H; and R3 = -NHCH (CH2CH3) CO2H)

The procedure of Example 3B was repeated using 2.0 g (3.98 mm) of N-heptanoyl-D-gamma-glutamyl-alpha-benzyl ester) glycylhydroxysuccinimide ester, 30,400 mg (4.02 mm) of D-alpha-aminobutyric acid and 0.556 ml ( 4.02 mm) of triethylamine to give 632 mg (57% yield) of the desired product, m.p. 140-141 ° C.

Nuclear Magnetic Resonance Spectrum (DMSO-d 6) showed absorption at 8.16 - 8.04 (m, 3H), 4.22 - 4.08 (m, 2H), 3.84 - 35 3.58 (m, 2H) , 2.2 (t, J = 9 Hz, 2H), 2.12 (t, J = 9 Hz, 2H), 2.04 - 1.0 (m, 15H) and 0.85 (t, J = 6 Hz, 6H) ppm.

28 86858

Example 6 N-Heptanoyl-D-gamma-glutamyl-glycyl-3-aminopropionic acid (Rx = CH3 (CH2) 5-; R2 = H; and R3 is -NH (CH2) 2CO2H) Following the procedure of Example 3B and using starting materials 1.5 g (3.0 mm) of N-heptanoyl-D-gamma-glutamyl (alpha-benzyl ester) glycine hydroxysuccinimide ester, 350 mg (3.9 mm) of 3-aminopropionic acid and 0.55 ml (3 , 9 mm) of triethylamine gave 500 mg (43% yield) of the desired product, m.p. 135-138 ° C.

The NMR spectrum (DMSO-d 6) showed absorption at 8.19 to 8.02 (m, 2H), 7.98 to 7.87 (t, J = 5 Hz, 1H), 4.25 to 4.1 ( m, 2H), 3.8-3.49 (m, 2H), 3.44-3.1 (m, 2H), 2.4 (t, J = 6 Hz, 2H), 2.22 (t , J = 7 Hz, 2H), 2.14 (t, J = 7 Hz, 2H), 2.1-1.77 (m, 2H), 1.6-1.17 (m, 8H) and 0.88 (t, J = 6Hz, 3H) ppm.

Example 7 N-Heptanoyl-D-gamma-glutamyl-glycyl-4-aminobutyric acid 20 (R 1 = CH 3 (CH 2) 5; R 2 = H; and R 3 = -NH (CH 2) 3 CO 2 H)

The procedure of Example 6 was repeated replacing 3-aminopropionic acid with 410 mg (4.0 mm) of 4-aminobutyric acid to give 600 mg (50% yield) of the desired product, m.p. 140-142 ° C.

The NMR spectrum (DMSO-d 6) showed absorption at 8.18 to 8.03 (m, 2H), 7.88 (bt, J = 4 Hz, 1H), 4.17 to 4.09 (m, 2H), 3.81 - 3.48 (m, 2H), 2.32 - 2.08 (m, 6H), 2.08 - 1.08 (m, 12H) and 0.88 (t, J = 6 Hz, 3H) ppm. Example 8 N-Heptanoyl-D-gamma-glutamyl-glycyl-5-aminopentanoic acid (Rx = CH3 (CH2) 5-; R2-H; and R3 = -NH (CH2) 4CO2H) Substituting 3-aminobutyric acid 470 mg 11a (4.0 mm) of 5-aminopentanoic acid and following the procedure of Example 6 gave 520 mg (42% yield) of the desired product, m.p. 122-124 ° C.

Δ 6 8 58 29 NMR spectrum (DMSO-d 6) showed absorption at 8.25 - 7.94 (m, 2H), 7.85 (t, J = 5 Hz, 1H), 4.25 - 4.1 (m, 2H), 3.82 - 3.46 (tn, 2H), 3.24 - 2.9 (m, 2H), 2.21 - 2.08 (tn, 6H), 2.08 - 1 , 2 (m, 14H) and 0.88 (t, 5 J = 6 Hz, 3H) ppm.

Example 9 N-Heptanoyl-D-gamma-glutamyl-glycyl-6-aminohexanoic acid (R 1 = CH 3 (CH 2) 5 -; R 2 = H; and R 3 = -NH (CH 2) 5 CO 2 H) The procedure of Example 6 was repeated, substituting 3-Aminobutyric acid 530 mg (4.0 mm) of 6-aminohexanoic acid to give 520 mg (40% yield) of the desired product as a white foam.

The NMR spectrum showed absorption (DMSO-d 6) at δ 8.28-7.9 (m, 2H), 7.82 (bt, J = 4 Hz, 1H), 4.27-4.1 (m , 2H), 3.81 - 3.47 (m, 2H), 3.15 - 2.90 (m, 2H), 2.3 - 2.08 (m, 6H), 2.08 - 1.18 (m, 16H) and 0.88 (t, J = 6 Hz, 3H) ppm.

Example 10 N-Isovaleryl-D-gamma-glutamyl-glycyl-D-alanine

D

(R 1 = (CH 3) 2 CHCH 2 -; R 2 = H; and R 3 = -NHCH (CH 3) CO 2 H) 10A. Glycyl-D-alanine benzyl ester hydrochloride in 100 ml of a cold solution of methylene chloride containing 10 g (57 mm) of Nt-butyloxycarbonylglycine, 20 g (57 mm) of D-alanine benzyl ester p-toluenesulfonic acid salt and 5.77 g (57.7 g) of ) triethylamine, 12.3 g (60 mm) of dicyclohexylcarbodiimide were added and the resulting reaction mixture was allowed to warm to room temperature. After 18 h, the mixture was filtered and the filtrate was concentrated in vacuo. The residue was dissolved in 200 ml of ethyl acetate, and the organic layer was washed with 2.5% hydrochloric acid, water, saturated sodium bicarbonate solution and brine. The organic layer was separated, dried over magnesium sulfate and evaporated under reduced pressure. To the resulting oil was added 200 ml of dioxane saturated with hydrogen chloride.

After 30 minutes, 400 ml of diethyl ether was added to the mixture and the product was filtered under nitrogen, 10.9 g (70% yield).

IOB. Nt-butoxycarbonyl-D-gamma-glutamyl (alpha-benzyl ester) hydroxysuccinimide ester to 1,500 ml of methylene chloride containing 50 g of 10 (143 mm) Nt-butoxycarbonyl-D-gamma-glutamic acid alpha-benzyl ester and 17.3 g (150 mm) of N-hydroxysuccinimide, 30.9 g (15 mm) of dicyclohexylcarbodiimide were added and the resulting reaction mixture was allowed to stir at room temperature for 18 hours. The solids were filtered and the filtrate was concentrated in vacuo. The residue was triturated with diethyl ether and the solids were filtered under nitrogen, 43.7 g (68% yield).

IOC. D-gamma-glutamyl (alpha-benzyl ester) Glycyl-D-alanine benzyl ester hydrochloride To a solution of 4.3 g (9.45 mm) of Nt-butoxycarbonyl-D-gamma-glutamyl (alpha-benzyl ester) roxisuccinimide ester, 2.71 g (9.92 mm) of glycyl-D-alanine benzyl ester hydrochloride and 1.0 g (9.92 mm) of triethylamine in 100 ml of methylene chloride were allowed to stir at room temperature for 18 hours, after which it was concentrated in vacuo. The residue was dissolved in 200 ml of ethyl acetate and the solution was washed with 2.5% hydrochloric acid, water, 10% potassium carbonate and brine. The organic phase was separated, dried over magnesium sulphate and evaporated under reduced pressure. The residue was treated with 200 ml of dioxane saturated with hydrogen chloride and the mixture was allowed to stir for 2 hours. The solution was concentrated to dryness in vacuo and the residue was triturated with di-ethyl ether. The solids were filtered under nitrogen, 3.41 g (73% yield).

i 86858 31 10D. N-isovaleryl-D-gamma-glutamyl-glycyl-D-alanlini__

To a solution of 1.0 g (2.03 mm) of D-gamma-glutamyl (alpha-benzyl ester) glycyl-D-alanine benz-5 silyl ester hydrochloride and 616 mg (6.09 mm) of triethylamine in 50 ml of methylene chloride was added 490 mg (4.06 mm) of isovaleryl chloride and the reaction mixture was stirred at room temperature for 80 hours. The methylene chloride was evaporated in vacuo and the residue was dissolved in ethyl acetate. The resulting solution was washed 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, filtered under nitrogen (910 mg) and dissolved in 700 mg of methanol. 200 mg of palladium hydroxide was added to the solution and the mixture was shaken under a hydrogen atmosphere at 345 kPa for three hours. The catalyst was filtered off and the solvent was removed in vacuo. Residue 20 was dissolved in water and lyophilized to give 364 mg (65% yield) of the desired product.

Nuclear Magnetic Resonance Spectrum (DMSO-d 6) showed absorption at 8.25-8.05 (m, 3H), 4.33-4.12 (m, 2H), 3.72 (d, J = 6 Hz, 2H) , 2.21 (t, J = 8 Hz, 2H), 1.88 - 1.68 (m,. 1H), 2.08 - 1.9 (m, 4H), 1.28 (d, J = 9 Hz, 3H) and 0.9 (d, J = 7 Hz, 6H) ppm.

Example 11 Using the appropriate acid chloride and D-gamma-glutamyl (alpha-benzyl ester) glycyl-D-alanine-30-nibenzyl ester hydrochloride as starting materials and using the procedure of Example 10D, the following compounds were prepared: 32 86858 o

II

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Us) V> 110 (dec.) (DMSO-d 6) 8.3-7.9 '-' (m, 3H), 4.33-4.17 (m, 2H), 3.72 (d, 35 J = 5Hz, 2H), 2.3-2.1 (τη, 2H) and 2.10-1.0 (m, 16H) 33 86 8 5 8 ftl SP · »cC» HMR, ppm CH3 (CH2 ) 2-> 110 (dec.) (DMSO-d 6) 8.25-8.05 δ (m, 3H), 4.35-4.22 (m, 2H), 3.72 (d, J = 6Hz) , 2H), 2.22 (t, J = 8Hz, 2H), 2.12 (t, J = 8Hz, 2H), 2.08-10.77 (m, 2H), 1.55 tq, J = 8Hz, 2H), 1.28 (d, J = 8Hz, 3H) and 0.88 (t, J = 7Hz, 3H) δ CH 3 (CH 2) 4 "> 190 <ha 3 ·) (DMSO-d 6) δ , 33-8.0 (m, 3H), 4.35-4.1 (m, 2B), 3.7 (d, J = 6Bz, 2H), 2.17 (t, J = 8Hz, 2H) , 2.1 (t, 20 J = 8Hz, 2H), 2.05-1.63 (m, 2H), 1.48 (tf J = 7Hz, 2H), 1.2-1.1 (m, 7H) and 0.85 (t, J = 7Hz, 3H) CH 3 (CH 2) 6-> 180 (dec.) (D 2 O) 4.42-4.28 (m, 2H), 3.91 (s, 1H), 2.4 (t, J = 7Hz, 2H), 2.27 (t, J = 7Hz, 2H), 2.22-1.94 (m, 2H), 1.65-1.55 (m, 2H), 1.39 (d, J = 8 Hz, 3H), 1.34-1.17 (m, 8H) and 0.73 (m, 3H) 35 34 86858 mp, * C. NME , ppm CE-, I 3 5 CH3 (CH2) 3CHCH2- - (DKSO-dg) 8r27-8r03 (6) (·, 3H), 4.32-4.1 (, 2H), 3.72 (d, J = 6Hz, 2H) t 2.22 (t, J-10H2, 2H), 2.27-1.68 (m, 6H) , 1.42-1.0 (m, 10H) and 0.94-0.8 (n, 6H) CH-CH- 2 3 15 CH 3 (CH 2) 3 CH- - (DMSO-d 6) 8.22-8.0 (R, S) (e, 3H), 4.32-4.1 (K, 2H), 3.8- 3.6 (.2H), 2.28-1.68 (e, 6H), 1.6-1.0 (m, 12H) and 0.94-0.7 (m, 6H).

(CH 3 CH 2) 2 CH- (DMSO-d 6) 8.29-7.97 (m, 3H), 4.33-4.1 (b, 2H), 3.81-3.59 (, 2H), 2.32-1.65 (b, 6H), 1.65-1.17 (a, 8H) and 1.02-30.68 (tn, 6H) 35 80858 Rf mp, ° C. NMR, ppm

Us) VcH 2 - (DMSO-d 6) 8.3-8.0 (B, 3H), 4, 32-4, 15 (b, 2H), 3.85-3, 62 (1.2H), 2.21 (t, J = 8Bz, 2H), 2.02 (d, J = 8Hz, 2H), 2.01-1.9 (m, 1H), 1.85-1.5 (m, 8H), lr28 (d, J = 8Hz, 3H) and 1.28-0.8 (m, 5H) (CH3CH2CH2) 2CHCH2- (DMSO-d6) 8.18-8.0 (*, 3B), 4 , 31-4.1 (c, 2H), 3.84-3.6 (m, 2H), 2.22 (t, J = 6Bz, 2H), 2.07 2Q (df J = 8Bz, 2H) , 2.03-1.7 (m, 3H), 1.4-1.15 (m, 11H) and O- 87 (t, J = 6Hz, 6H) δ (CH 3 CH 2) 2 CHCH 2 - (DMSO-d 6) 8.27 -7.95 (n, 3B), 4.3-4.1 (m, 2H), 3.78-3.6 (m, 2B), 2.3-1.57 (m, 8B), 1.46-1.13 (m, 8H) and 0.84 (t, J = 8Ez, 6B) 36 86858 R 1 SP ·, ° C. NMR, ppm CH-I 3 CH 3 t CH 2 * 3CHCH 2 "- (DHSO-d 6) 8.18-8.0 δ (RrS) (m, 3H), 4.24-4.06 (m, 2H), 3.74 -3.56 (m, 2H), 2.17 (t, J = 9 Hz, 2H), 2.12-2.0 (m, 1H), 2.0-1.64 10 7 (m, 4H) , 1.24 (d, J = 6Hz, 7H), 1.14-0.98 (m, 2H) and 0.81 (d, J = 6Hz, 6H) CH3, CH3 (CH2) 2CHCH2- - (DMSO-d 6) 8.2-8.04 (R, S) (m, 3H), 4.26-4.08 (m, 2H), 3.76-3.6 δ (m, 2H) , 2.28-1.64 (m, 7H), 1.4-0.96 (m, 7H) and 0.96-0.74 (m, 6H).

(CH 3) 2 CH (CH 2) 3 - (DMSO-d 6) 8.24-7.95 (m, 3H), 4.3-4.08 (m, 2H), 3.81-3.59 ( m, 2H), 2.21 (t, J = 6Hz, 2H), 2.11 (t, J = 8Hzf 2H), 2.05-1.38 (m, 7H), 1.27 (d, J = 8Hz, 3H), 1.17-1.05 (m, 2H) and 0.86 (d, J = 10Hz, 6H) 37 86858

Rl SP · i * C. NKR, ppm CH 3 '- (DMSO-d 6) 8.24-8.0 δ CH 3 (CH 2) 4 CH- (m, 3H), 4.33-4.11 (R, S) (m, 2H), 3 , 79-3.6 (m, 2H), 2.41-2.29 (m, 1H), 2.22 (t, J = 8Hz, 2H), 2.11-1.66 (m, 2H); ), 1.59-1.43 (m, 1H), 1.38-1.11 (m, 11H), 1.06-0.95 (m, 3H) and 0.87 CH PH, J = 6Hz 13H 2 CH 3 (CH 2) 3 CHCH 2 - (DMSO-d 6) 8.24-8.02 (R # S) (m, 3H), 4.32-4.11 (m, 2H), 3 .84-3.6 (m, 2H), 2.24 (t, J = 8Hz, 2H), 2.08 (d, J = 8Hz, 2H), 2.03-1.63 (m, 4H ), 1.44-1.11 (m, 12H) and 0.97-0.71 (in, 6H) 2 O (CH 3) 2 CH <CH 2) 4- - (DMSO-d 6) 8.24-8.04 (m, 3H), 4.28-4.1 (m, 2H), 3.76-3.6 (tn, 2H), 2.18 (t, J = 30 Hz, 2H), 2.1 ( t, J = 6 Hz, 2H), 2.04-1.86 (m, 1H), 1.84-1.66 (m, 1H), 1.56-1.38 (m, 3H), 1, 23 35 (d, J = 6Hz, 3H), 1.2-1.06 (τη, 3H) and 0.82 (d, J = 6Hz, 6H) 38 86858

Rj sp. , * C. NMR, ppm CH, I 3 CH3 (CH2) 4CHCH2- - (DMSO-d6) 8.23-7.98 δ (R, S) <*, 3H), 4 r 3-4.13 (, 2H), 3.81-3.61 (e, 2H), 2.22 (t, J = 8 Hz, 2H), 2.18-1.68 (m, 6H), 1.45-10.07 (m, 12H) and 0.98-0.8 (m, 6H) ch 3 (CH 3) 2 CH (CH 2) 2 CHCH 2 - (DMSO-d 6) 8.37-8.03 (R, S) (r, 3H), 4.31-4.1 (e, 2H), 3.78-3.6 (*, 2H), 2.26 (t, J = 8Hz, 2H), 2.2-1.36 (m, 7H ), 1.3 (d, J = 20 8 Hz, 5H), 1.26-1.05 (*, 2H) and 1.05-0.73 (m, 9H) CH 2 - (DMSO-d 6) 8.23-7.98 I 3 D '25 (CH3) 2CHCH2CHCH2- (m, 3H), 4.3-4.13 (m, 2H), 3.81-3.61 (m, 2H), 2 , 21 (t, J = 8 Hz, 2H), 2.15-2.0 (m, 2H), 1.9 (t, J = 8Hz, 2H), 1.85-1.52 (m, 3H ), 1.4-1.22 (m, 3H), 1.22-0.94 (m, 3H) and 0.94-0.80 (m, 6H) 35 L! 39 86 8 58 R1 sd. In ° C. NMR, ppm CH-CH-31 2 CH 3 (CH 2) 2 CHCH 2 - - (DMSO-d 6) 8.25-7.98 δ (R, S) (m, 3H), 4.3-4.08 (in, 2H), 3.81-3.62 (m, 2H), 2.22 (t, J = 8Hz, 2H), 2.06 (d, J = 8Hz, 2H), 2.02-1.89 (m, 1H), 1.87-1.65 (m, 2H), 1.41-1.06 (m, 11H) and 0.98-0.7 (m, 6H) δ DMSO-d 6) 8.33-7.95 (m, 3H) f 4.3-4.06 (m, 2H), 3.83-3.59 (m, 2H), 2.21 (t, J = 8Hz, 2H), 2.11 (s, 3H), 2.08-1.87 (m, 1H), 1.87-1.35 (m, 8H), 1.25 (d, J = 8Hz, 3H) and 1.22-0.98 (m, 3H) δ (CH3CH2) 2CB (CH2) 2- - (DMSO-d6) 8.21-8.0 (m, 3H), 4.32- 4.1 (ra, 2H) f 3.83-3.6 (m, 2H), 2.21 (t, 30 J = 8Hz, 2H), 2.11 (t, J = 8Hz, 2H), 2 .05-1.89 (m, 1H), 1.87-1.67 (m, 1H), 1.57-1.38 (m, 3H), 35 1.38-1.08 (m, 9H) and 0.83 (t, J = 6 Hz, 6B) 408 868 58 Rf. In ° C. NMR, ppm CH, 13 CH 3 (CH 2) 2 CH (CH 2> 2- - (DMSO-d 6) 8.24-7.97 (R, S) <B, 3H), 4.33-4.1 (b , 2H), 3.86-3.59 (n, 2H), 2.35-2.08 (a, 4H), 2.08-1.9 δ. 1H), 1.89-1.67 (Bf 1H), 1.63-1.46 (b, 1H), 1.46-1.02 (b, 11H) and 0.98-0.73 (m , 6H) CH-CH-31 3 CH3 (CH2) 4CHCH2- - (DMSO-d6) 8.2-7.94 (R, S) (m, 3H), 4.26-4.06 (b, 2H), 3.76-3.56 (b, 2H), 2.16 (t, J = 6Hz, 2H), 2.1-1.84 (b, 3H), 1.84-1.6 (m, 2H), 1.24 (d, J = 6Hz, 9H), 1.12-0.92 (b, 3H) and 0.92-0.64 (in, 9B) CH-.

I 3 CH 3 (CH 2) 5 CHCH 2 - (DMSO-d 6) 8.23-8.0 (R, S) (m, 3H), 4.32-4.06 (m, 2H), 3.72 ( d, J = 8Hz, 2H), 2.22 (t, J = 10Hz, 2H), 2.16-1.7 (m, 6H), 1.42-1.08 (m, 14H) 35 and 0 .92-7.0 (m, 6H) 4i 86858 1 HP-r NMR, ppm δ 2 H 5 CH 3 (CH 2) 3 CHCH 2 - (DMSO-d 6) 8.2-8.0 δ (m, 3H) .4.24-4.16 (m, 2H); 3.74-3.60 (m, 2H); 2.18 (t, J1, 2H); 2.02 (d, J * 7H); , 2.02-1.6 (m, 3H), 1.26 (d, J = 6.3H), 1.26-1.08 (m, 8H) and, 92-, 74 (π, 6H) 15

Example 12 N- (3- (S) -methylheptanoyl) -D-gamma-glutamyl-L-alanyl-D-alanine (R 1 = (S) CH 3 (CH 2) 3 CH (CH 3) CH 2 -;

D

R2 = CH3 'R3 = -NHCH (CH3) CO2H) 12A. N-t-butoxycarbonyl-L-alanyl-D-alanine benzyl ester _______

To a solution of 23.0 g (0.121 m) of Nt-butoxycarbonyl-L-alanine, 42.6 g (0.121 m) of D-alanine benz-25-ethyl ester p-toluenesulfonic acid salt and 17 ml (0.121 m) of triethylamine in 400 ml in cold (0 ° C) methylene chloride, 25.0 g (0.121 m) of dicyclohexylcarbodiimide in 100 ml of methylene chloride were added dropwise. After stirring overnight at room temperature, the solids were filtered and the filtrate was concentrated to an oil. The residue was dissolved in 400 ml of ethyl acetate, which was washed with 1% hydrochloric acid solution, 10% potassium carbonate solution, water and brine. The organic phase was separated, dried over magnesium sulfate and concentrated to an oil. The residue was triturated with diethyl ether and the resulting solids were filtered under 42 86858 nitrogen, 16.0 g. An additional 12.7 g of the desired product crystallized from the filtrate.

12B. L-Alanyl-D-alanine benzyl ester hydrochloride To a slurry of 28.7 g of Nt-butoxycarbon-5-yl-L-alanyl-D-alanine benzyl ester in 150 ml of dioxane was added 150 ml of dioxane saturated with hydrogen chloride, and the mixture was stirred for 4 hours. time at room temperature. The solvent was removed in vacuo and the residue was triturated with diethyl ether. The resulting solids were filtered, redissolved in methylene chloride, and the solution was concentrated to a volume of about 150 mL. Ether was added and the solids were filtered under nitrogen, 22.0 g.

12C. N-t-butoxycarbonyl-D-gamma-glutamyl (alpha-15 benzyl ester) -L-alanyl-D-alanine benzyl ester_

To a slurry containing 5.0 g (9.64 mm) of Nt-butoxycarbonyl-D-gamma-glutamine (alpha-benzyl ester) dicyclohexylamine and 2.76 g (9.64 mm) of L-alanyl-D- ala-20-benzyl ester hydrochloride in 100 ml of methylene chloride cooled to 0 ° C, 2.0 g (9.64 mm) of dicyclohexylcarbodiimide in 20 ml of the same solvent were added. After stirring overnight at room temperature, the solids were filtered and the filtrate was concentrated in vacuo. The residue was treated with 150 mL of ethyl acetate, the solids were filtered, and the filtrate was washed with 1% hydrochloric acid, 10% potassium carbonate solution, water, and brine. The organic phase was dried over sodium sulfate and concentrated to give a white solid which, when triturated with ether and filtered, gave 4.1 g of the desired product.

12D. D-gamma-glutarnyl (alpha-benzyl ester) -L-alanine-D-alanine benzyl ester hydrochloride To a slurry containing 4.1 g (7.21 mm) of Nt-butox-35-carbonyl-D-gamma-glutamyl (alpha-benzyl ester) ) -L-alanyl-D-alanine benzyl ester in 50 ml of dioxane, 86858 43 was added 100 ml of dioxane saturated with hydrogen chloride and the reaction mixture was stirred for 3 hours at room temperature. The solvent was removed in vacuo and the residue was triturated with diethyl ether, 3.5 g.

5 12E. N- (3- (S) -methylheptanoyl) -D-gamma-glutamyl (alpha-benzyl ester) -L-alanyl-D-alanine benzyl ester ____

To a mixture of 1.0 g (1.98 mm) of D-gamma-Glu-tamyl) alpha-benzyl ester) -L-alanyl-D-alanine benzyl-10 ester and 0.833 ml (5.93 mm) of triethylamine in 50 ml: methylene chloride, 390 mg (2.37 mm) of 3- (S) -methylheptanoyl chloride was added and the reaction mixture was stirred under nitrogen for 45 minutes. The reaction mixture was poured into 150 ml of ethyl acetate and the organic phase was washed with 10% hydrochloric acid, 10% potassium carbonate solution, water and brine. The organic phase was dried over sodium sulfate and concentrated to dryness. The residue was triturated with ether and filtered under nitrogen, 900 mg.

20 12F. N- (3- (S) -methylheptanoyl-D-gamma-glutamyl-L-alanyl-D-alanine_

A mixture of 200 mg of palladium hydroxide-carbon catalyst and 900 mg of N- (3- (S) -methylheptanoyl) -D-gammaglutamyl (alpha-benzyl ester) -L-alanyl-D-alanine-25-nibenzyl ester in 50 ml of methanol , was shaken under an atmosphere of hydrogen at a pressure of 345 kPa for one hour. The catalyst was filtered off and the solvent was removed in vacuo. To the residue was added water, which was removed under reduced pressure to give 492 mg of product as a white solid, 30 m.p. 165-168 ° C.

Nuclear Magnetic Resonance Spectrum (DMSO-d 6) showed absorption at 8.21 to 7.98 (m, 3H), 4.41 to 4.1 (m, 3H), 2.3 to 2.06 (m, 4H). ), 2.06-1.56 (m, 6H), 1.43-1.02 (m, 11H) and 1.02-0.73 (m, 6H) ppm.

44 86 8 58

Example 13 N- (3- (S, R) -ethylhexanoyl) -D-gamma-glutamyl- (alpha-n-butyl ester) glycyl-D-alanine (R1 = CH3 (CH2) 2CH {C2H5) CH2-, R2 = B; R3 =

5 D

NH CH (CH3) C02H? R4 = n-C4H9) 13A. Nt-butoxycarbonyl-D-gamma-glutamine (alpha-n-butyl ester) dicyclohexylamine salt A solution of 39.5 g (0.172 m) of Nt-butoxy-10-carbonyl-D-glutamic anhydride in 75 ml of dry tetrahydrofuran was added dropwise. over two hours to a solution of 47 ml (0.516 m) of n-butanol and 34.3 ml (0.172 m) of dicyclohexylamine in 300 ml of ether at 0 ° C. The reaction mixture was allowed to stir at 0 ° C for 3 hours and kept in the refrigerator overnight. The solids were filtered, slurried in ethanol and filtered, 43.3 g.

13B. D-gamma-glutamyl (alpha-n-butyl ester) Glycyl-D-alanine benzyl ester hydrochloride The product of Example 13A (10 g, 0.021 m) and 6.7 g (0.024 m) of glycyl-D-alanine benzyl ester hydrochloride were slurried in 200 ml of methylene chloride. under nitrogen and the mixture was cooled to 0 ° C. Dicyclohexylcarbodiimide (4.25 g, 0.021 m) was added and the mixture was allowed to warm to 25 room temperature overnight. The urea by-product was filtered and the solvent removed in vacuo. The residue was treated with ethyl acetate and filtered. The filtrate was washed successively with water, 2.5% hydrochloric acid, water, 10% potassium carbonate and brine. The organic phase was dried over magnesium sulfate, the solvent was removed in vacuo and the residue was dissolved in 300 ml of dioxane saturated with hydrogen chloride. After stirring at room temperature for 4 hours, the solvent was removed and the residue was triturated with ethyl acetate-hexane (1: 1) and filtered, 7.4 g.

45 86 8 58 13C. N- (3- (S, R) -etyyliheksanoyyli-D-glutamyl (alpha-n-butyyljesteri) glycyl-D-alanine

To a mixture of the product of Example 13B (1.0 g, 2.35 mm) and 0.99 mL (7.05 mm) of triethylamine in 50 mL of ethylene chloride was added 460 mg (2.83 mm) of 3- (S, R ) -ethylhexanoyl chloride and the reaction mixture was stirred overnight under nitrogen. The solvent was removed in vacuo and the residue was dissolved in ethyl acetate. The organic phase was washed successively with 10% hydrochloric acid, water, 10% potassium carbonate and brine. The organic phase was separated, dried over magnesium sulphate and the solvent removed in vacuo. The residue was dissolved in 10 ml of methanol and the mixture was shaken with 170 mg of 10% palladium hydroxide for 1.5 hours under a hydrogen atmosphere at an initial pressure of 345 kP.

The spent catalyst was filtered off and the solvent was removed in vacuo, 100 mg.

NMR (DMSO-d 6): 8.18 (d, J = 6, 1H), 8.10 (d, J = 6, 2H 1H), 8.02 (t, J = 5, 1H), 4.28 -4.10 (m, 2H), 4.00 (t, J = 6.2H), 3.78-3.56 (m, 2H), 2.18 (t, J = 6.2H), 2 .02 (d, J = 6f 2H), 2.00-1.60 (m, 3H), 1.58-1.42 (m, 2H), 1.28-1.08 (m, 8H), 1.24 (a, J = 61 3H), 0.92-0.76 (m, 9H).

Example 14 Using the general procedure of Example 13 and using the reagents required as starting materials, the following compounds were prepared: 46 86858

O

II

R.CNH D .C0oR.

1 \ / 2 4

CH D

5 I

(CH 2) 2CONHCH2CONHCH (CH3) C02H

Ri R.

1_ 4 NKR

3-methylheptanoyl methyl NMR (DMSO-d 6): 8.23 (d, 10 J = 6, 1H), 8.15 (d, 3 = 6, 1H), 8.09 (t, 3 = 6, 1H) , 4.28-4. 14 (m, 2H); 3.72 (d, J = 6.2H), 3.61 (s, 3H), 2.23 (t, 3 = 1.2H), 2.16-1.70 (m, 6H), 1 .34 - 1.04 (m, 8H), 1.25 (d, 3 = 1.3H), 0.92-0.76 (m, 6H).

3-ethylheptanoyl methyl NMR (DMSO-d 6): 8.19 (d, 3 = 6, 1H), 8.11 (d, J = 6, 1H), 8.03 (t, J = 6, 1H) , 4.24-4.10 (m, 2H), 3.74-3.62 (m, 2H), 3.57 (s, 3H), 2.18 (t, J = 9, 2H), 2.01 (d, J = 6.2H), 1.97-1.60 (m, 3H), 1.32-1.10 (m, 8H), 1.23 (d, 3 = 1.3H); ), 0.90-0.72 (m, 6H) δ 3-methylheptanoyl ethyl NMR (DMSO-d 6): 8.24-8.02 (m, 3H), 4.26-3.96 (m, 2H ), 4.04 (q, J = 9, 2H), 3.76-3.56 (m, 2H), 2.17 (t, 3 = 1.2H), 2.12-1.63 ( m, 6H); 1.74-0.94 (m, 6H), 1.23 (d, J = 5.3H), 1.13 (t, J = 9R), 0.88-0.72 (m, 6H); ) 47 868 58

R1 R4 gHR

3-ethylheptanoyl ethyl NMR (DMSO-d 6): 8.16 (d, δ J = 6, 1H), 8.09 (d, J = 6, 1H), 8.02 (t, J = 6, 1H) , 4.22-4.08 (in, 2H), 4.02 (q, J = 7.2H), 3.74-3.54 (m, 2H), 2.16 (t, J = 7, 2H), 2.00 (d, J = 6.2H), 1.96-1.58 (m, 3H), 1.30-1.08 (m, 8H), 1.21 (d, J) -7.3H), 1.13 (t, J = 7.3H), 0.88-0.70 (m, 6H) δ-methylheptanoyl 1-iso-NMR (DMSO-d 6): δ .14 (d, butyl J = 6, 1H), 8.08-7.98 (m, 2H), 4.20-4.04 (m, 2H), 3.75 (d, J = 6.2H); ), 3.68-20.54 (ro, 2H), 2.14 (tf J = 61 2H), 2.08-1.64 (xnr 6H), 1.28-0.96 (m, 6H ) f 1.19 (d, J = 7.3H), 0.88-0.70 (ra, 12H) 3-ethylheptanoyl iso-NMR (DMSO-d 6): 8.21 (d, butyl) , 1H), 8.14-8.04 (m, 2H), 4.24-4.08 (m, 2H) r 3.79 (d, J = 6, 2H), 3.72-30 58 (η, 2H), 2.19 (t, J = 7.2H), 2.03 <d, J = 6.2H), 1.99-1.60 (η, 4H), 1.32- 1.10 (m, 8H), 1.23 (d, J = 6.3H), 0.92-0.72 35 (m, 12H) 48 86858

* 1 R4 NMR

3-ethylhexanoyl iso-NMR (DMSO-d 6): 8.18 (d, 5-butyl J = 6, 1H), 8.10-8.00 (m, 2H), 4.26-4.08 (m , 2H), 3.79 (d, J = * 6.2H), 3.72-3.58 (m, 2H), 2.18 (t, J = 6.2H), 2.02 (d, J = 6.2H), 1.98-1.62 (m, 4H), 1.34-1.08 (m, 6H), 1.23 (d, J = 7.3H), 0.96- 0.72 (m, 12H) 3-ethylhexanoyl methyl NMR (DMSO-dfc): 8.21 (d, J * 7, 1H), 8.10 (d, J = 7, 1H), 8.05 (t , Js6, 1H), 4.26-4.10 (m, 2H), 3.76-3.30 (m, 2H), 3.59 (s, 3H), 2.18 (t, J = 6, 2H), 2.02 (d, J = 6.2H), 2.00-1.60 (0.3H), 1.32-1.08 (m, 7H), 0.90-0.72 (in, 6H).

3-ethylhexanoyl ethyl NMR (DMSO-d 6): 8.22 (d, J = 7, 1H), 8.18-8.06 (m, 2H), 4.26-4.10 (in, 2H) , 4.06 (q, J = 5.2H), 3.78-3.58 (m, 2H), 2.20 (t, J = 6.2H), 2.04 (d, J = 6H), , 2H), 2.02-1.60 (m, 3H), 1.26-1.20 (m, 7H), 1.18 (t, J = 5.3H), 0.90-0.78 35 (m, 6H) 86858 49 R, R.

1 <_KMR

3-methylheptanoyl butyl NMR (DMSO-d 6): 8.20 (d, J = 7, 1H), 8.16-8.04 (.5H), 4.24-4.06 (m, 2H), 4.00 (t, J = 6.2H), 3.74-3.56 (m, 2H), 2.17 (t, J = 6.2H), 2.12-1.60 <E, 5H ), 1.58-1.40 (m, 2H), 1.36-1.00 (m, 8H), 1.21 (d, J = 6.3H), 0.90-0.74 ( m, 9H) 3-Ethyl-5-octanoyl-butyl NMR (DMSO-d 6): 8.16 (d. 15.76 J = 7, 1H), 8.11 (d, J = 7, 1H), 8.03 ( t, J = 5, 1H), 4.26-4.09 (m, 2H) f 3.99 (t, J = 7.2H), 3.79-3.58 2Q and (2H), 2, 17 (t, J = 6.2H), 2.01 (d, J = 6.2H), 2.00-1.60 (mv 3H), 1.58-1.42 (m, 2H), 1 , 36-1.08 (m, 10H), 1.24 (d, J = 5.23H), 0.92-0.72 (m, 9H), 86858 50

Example 15 N- (3- (R, S) -ethylhexanoyl) -D-gamma-glutamylglycyl-D-alanine ethyl ester (R 1 = CH 3 (CH 2) 2 CH (C 2 H 5) CH 2 -; R 2 = H; R 2 =

5 D

-HNCH (CH3) C02C2H5; R 5 = H) 15A. D-gamma-glutamyl (alpha-benzyl ester) glycyl-D-alanine ethyl ester hydrochloride_

To a slurry containing 14.8 g (0.0285 m) of Nt-bu-10 toxicocarbonyl-D-gamma-glutamic acid (alpha-benzyl ester) dicyclohexylamine hydrochloric acid and 6 g (0.0285 m) of Glycyl-D-alanine ethyl ester hydrochloride In 200 ml of methylene chloride, 5.6 g (0.0270 m) of dicyclohexylcarbodiimide were added and the mixture was stirred under a nitrogen atmosphere overnight. The urea was filtered off and the solvent was removed in vacuo. The residue was treated with 300 mL of ethyl acetate, filtered and the filtrate was washed successively with 2.5% hydrochloric acid, water, 10% potassium carbonate solution and brine. The organic phase was separated, dried over magnesium sulfate and concentrated in vacuo. The residual oil was dissolved in 450 ml of dioxane saturated with hydrogen chloride. The solution was stirred for two hours and the solvent was removed in vacuo. The residue was triturated with ether and filtered, 11.2 g.

15B. N- (3- (R, S) -ethylhexanoyl) -D-gamma-glutamylglycyl-D-alanine ethyl ester

To a mixture of the product of Example 15A (1.0 g, 2.33 mm) and 0.98 mL (6.98 mm) of triethylamine in 30 mL of methylene chloride under nitrogen was added 378 mg (2.33 mm) of 3- ( R, S) -etyyliheksanoyylikloridia. After stirring at room temperature for 1.5 hours, the mixture was poured into 100 ml of ethyl acetate and the organic phase was washed successively with 10% potassium carbonate solution and brine. The organic phase was separated, dried over magnesium sulfate and concentrated in vacuo. The white solid residue was dissolved in 30 ml of methanol and hydrogenated with 0.1 g of palladium hydroxide under an atmosphere of hydrogen at an initial pressure of 345 kPa. After 2 hours, the catalyst was filtered off, the filtrate was concentrated to dryness and the residue was triturated with ether and filtered, 275 mg.

NMR (DMSO-d 6) 8.26 (d, J = 9, 1H), 8.14-8.02 (m, 2H), 4.31-4.00 (m, 2H), 4.06 δ q , J = 10, 2H) f 3.78-3.60 (m, 2H), 2.17 (t, J = 8, 2H), 2.08-1.65 (m, 1H), 2.03 (df J = 8, 10H), 1.82-1.53 (m, 3H), 1.40-0.96 (ro, 5H) f 1.23 td, J »6, 3H), 1, 14 (t, J = 10, 3H), 0.90-0.64 (m, 6H).

Example 16 Using the appropriate reagents as starting materials and the procedure of Example 15A-15B, the following compounds were prepared:

O

R t CNH D C ° 2h

20 Q.H '' D

(CH 2) 2 CONHCH 2 CONHCH (CH 3) CO 2 R 5 R 5 NMR_ 3-methylheptanoyl iso-NMR (DMSO-d 6): 8.24 butyl (d, J = 6, 1H), 8.10-8.00 (m, 2H), 4.30-4.18 (m, IB), 4.18-4.08 (m, 1H), 3.86-3.72 (m, 2H), 3.72-3.58 (m, 2H), 2.16 (t, J * 6, 2H), 2.12-1.64 (m, 6H), 1.52-1.00 (m, 6H), 1.27 <d, J = 7, 3H), 35 0.90-0.76 (m, 12H) 52 86858

--- HUB

Β-ethylhexanoyl iso-NMR (DMSO-d 6): 8.23 (d, butyl J = 6, 1H), 8.08-7.99 (m, 2H), 4.29-4.17 (m, 1H), 4.17-4.07 (m, 18), 3.83-3.71 (m, 2H) r δ 3.71-3.58 (m, 2H), 2.15 (t, J = 7.2H), 2.04-1.60 (m, 4H), 2.00 (d, J = 6.2H), 1.31-1.09 (m, 6H), 1.25 (d); , J = 6.3H), δ 0.90-0.72 (m, 12H) 3-ethylheptanoyl isobutyl NMR (DMSO-d 6): 8.23 (d, J = 6, 1H) t 8.08- 7.98 (m, 2H), 4.29-4.18 (m, 1H), 4.18-4.07 (m, 1H), 3.86-3.72 (n, 2H), 3 .70-3.57 (in, 2H), 2.15 (t, J = 7.2H), 2.04-1.59 (m, 4H), 2.00 (d, J = 6.25H) ), 1.30-1.11 (m, 8H), 1.25 (d, J = 6.3H), 0.89-0.70 (m, 12H) 3-methylheptanoyl methyl NMR (DMSO-d 6 ): 8.25 (d, 30 cyclo-J = 6, 1H), 8.13-8.00 (m, hexyl 2H), 4.32-4.20 (m, 1H), 4.20-4 .08 (m, 1H), 3.91-3.76 (m, 2H), 3.76-3.59 (m, 2H), 2.18 35 (t, J = 6, 2H), 2, 13-1.48 (m, 8H), 1.36-1.01 (m, 12H), 1.27 (d, J = 6.38), 1.02-0.76 (m, 8H) 53 868 58

R1 r5 HMR

3-ethylhexanoyl methyl NMR (DMSO-d 6): 8.25 (d, cyclo-J = 6, 1H), 8.13-8.00 (m, hexyl 2H), 4.32-4.20 (m , 1H), 4.20-4.08 (©, 1H), 3.92-3.74 (m, 2H), 3.74-3.59 (m, 2H), 2.18 (t, J = 6, 2H), 2.09-1.86 (m, 1H), 2.03 (d, J = 6.2H), 1.82-1.43 (m, 8H), 1.36- 1.01 (m, 9H), 1.27 (d, J = 6.3H), 1.01-0.70 (ro-NMR) 3-ethylheptanoyl methyl NMR (DMSO-d6): 8.26 ( d, cyclo- J = 6, 1H), 8.12-8.02 (m, hexyl 2H), 4.31-4.19 (m, IB), 4.19-4.08 (m, 1H) , 3.93-3.72 (m, 2H), 3.72-3.58 (m, 2H), 2.18 (t, J = 6.2H), 2.08-1.86 (m , 1H), 2.03 (d, J = 6, 2H), 1.82-1.48 (m, 8H), 1.34-1.02 (m, 11H), 1.27 (d, J = 6, 3H), 1.00-0.74 (m, 8H) 3-methylheptanoyl ethyl HMR (DMSO-d 6): 8.25 (d, J-6, 1H), 8.12-8, 00 (m, 2H), 4.28-3.96 (n, 2H), 4.03 (q, J-7, 2H), 3.74-3.56 (m, 2H), 2.16 t, J-9, 2H), 2.11-1.62 35 (m, 6B), 1.32-0.98 (m, 6H), 1.24 (d, J-7, 3H), 1 .14 (t, J-7, 3H), 0.88-0.76 (m, 6H) 54 86858

R1 R5 NMR

3-ethylheptanoyl ethyl NMR (DMSO-d 6): 8.28 (d, J-6, 1H), 8.16-8.04 (in, δ 2B), 4.32-4.04 (m, 2H) , 4.10 (q, J = 6.2H), 3.78-3.64 (m, 2H), 2.22 (t, J = 6.2H), 2.11-1.92 (m, 1H), 2.07 (d, J = 6, 2H), 1.86-1.64 (m, 2H), 1.40-1.14 (m, 8H), 1.30 (d, J = 6.3H), 1.21 (t, <J = 6.3H), 0.94-0.76 (m, 6B).

3-ethylheptanoyl butyl NMR (DMSO-d 6): 8.27 (d, J = 8, IB), 8.14-8.02 (m, 2H), 4.32-4.10 (m, 2H) , 4.10-3.94 (m, 2H), 3.78-3.60 (m, 2H), 2.18 (t, J = 6.2H), 2.04 (d, J = 6); , 2B), 2.04-1.62 (m, 3H), 1.60-1.46 (m, 2B), 1.38-1.10 (m, 12H), δ 1.27 (d, J = 6.3H, 0.90-0.75 (id, 9H) 3-S-methylheptabutyl NMR (DMSO-d 6): 8.30 (d, noyl J = 8, IB), 8.15 -8.04 (m, 2H), 4.45-4.12 (m, 2H), 4.12-3.98 (ra, 2H), 3.78-3.65 (m, 2H), 2.22 (tf J = 7.2H), 2.18-1.69 (m, 7H), 1.61-1.48 <m, 35H, 1.40-1.11 (m (11H)); ), 0.97-0.80 (m, 9H) 86858 55

Ri Rc

- 1 5 NMR

3-S-ethylheptanobutyl NMR (DMSO-d 6): 8.22 (d, 5-yl J = 7, IE), 8.12-8.0 (m, 2H), 4.4-4.16 ( m, 2H), 4.08-3.95 (m, 2H), 3.75-3.62 (m, 2H), 2.18 (t, J = 6.2H), 2.02 (d, 10J); -6, 2H), 2.04-1.62 (o, 3H), 1.60-1.46 (m, 2H), 1.38-1.1 (m, 15H), and, 9-, 75 (m, 9H) δ 3-ethylhexanoyl butyl NMR (DMSO-d 6): 8.28 (d, J = 8, 1H), 8.14-8.04 (m, 2H), 4.34-4, Δ (m, 2H), 4.10-3.95 (m, 2H), 3.75-3.62 (m, 2H), 2.19 δ (t, J = 6, 2H), 2.04 (d , J = 6, 2H), 2.04-1.60 (m, 3H), 1.60-1.45 (m, 2H), 1.40-1.10 (m, 13H), 0.90 -0.76 (m, 9 H)

Example 17

The procedure of Example 15 was repeated again using the appropriate reagents as starting materials, except that no hydrogenation was performed to give the following compounds:

3C

0

II

RCNH D CO, Ra

1 \ / 24 n CH D

35 (CH2) 2CONHCH2CONHCH (CH3) CO2R5 56 868 58

R, R. Rc NMR

14 D-3-ethylbutyl butyl NMR (DMSO-d 6): 8.27 (d, J = 7.5 h, 1H), 8.20 (d, J = 7, 1H), 8.07 sanoyl (t, J = 7, 1H), 4.37-4.13 Jm, 2H), 4.02 (t, J = 6, 4R), 3.80-3.62 (m, 2H), 2.20 ( t, J = 6, 2H), 2.05 (d, J = 6, 2H), 2.02-1.64 (m, 3H), 1.60-1.77 (m, 4H), 1 , 40-1.13 (m, 13H), 0.95-0.77 (m, 12H) 3-ethylbutyl butyl NMR (DMSO-d 6): 8.27 (d, J = 7, 15 h. 1H), 8.20 (d, J = 7, 1H), 8.06 tanoyl (t, J = 6, 1H), 4.36-4.13 (m, 2H), 4.02 (t, J = 6.4H), 3.80-3.60 (m, 2H), 2.20 (t, J = 6.2H), 2.04 (d, J = 6.22H), 2.00-1, 60 (m, 3H), 1.60-1.49 (tn, 4H), 1.40-1.10 (m, 15H), 0.95-0.72 (m, 12H) 3-methylbutyl butyl nmr (DMSO-d 6): 8.26 (d, J = 7.25 h, 1H), 8.19 (d, J = 7, 1H), 8.07 noyl (t, J = 6, 1H) , 4.32-4.11 (m, 2H), 4.02 (t, J = 5.4H), 3.79-3.59 (m, 2H), 2.20 (t, J = 6, 2H), 2.14-1.68 (m, 5H), 1.61-1.46 (m, 4H), 1.40-1.06 (in, 13H), 0.95-0.81 (in, 12H) 57 86858 r2 r4 r5 nmr 5 3 - S-benzyl-benzyl-NMR (DMSO-d6): 8.33 (d, J = 7, style 1H), 24 (d, J = 7.1), Δr08 he? (T, J = 5, 1H), 7.33 (s, 10H), noyl 5.08 (s, 4H), 4.40-4.22 (m, 2H), 3.80 -3.60 (m, 2H), 2.21 (t, J = 5.2H), 2.14-1.64 (m, 5H), 1.26 (d, J = 7.3H), 1.22-0.98 (m, 6H), 0.88-0.73 (m, 6H) Example 1 8

Crystalline N- (3- (S) -methylheptanoyl) -D-gamma-Glu-tamyl-glycyl-D-alanine N- (3- (S) -methylheptanoyl) -D-gamma-glutamyl- (alpha-benzyl ester) glycyl -D-alanine benzyl ester 20 (30.8 g) was slurried in 300 ml of absolute ethanol in a 2 liter autoclave. 5% Pd / C, 1.54 g, 50% water content, was added and the mixture was hydrogenated four times at atmospheric pressure for 1 hour during which time hydrogen consumption had ceased. The catalyst was recovered by filtration using first paper and then a 0.45 micron Nylon Millipore filter and using 100-150 mL ethanol for transfer and washing. The combined filtrate and washings were removed to give a moist white solid which was dissolved in 150 ml of a hot absolute mixture of ethanol and acetonitrile, 1:10, clarified by hot filtration, boiled to a volume of 35 ml, slowly cooled to room temperature, granulated and filtered to give the crystalline, dense non-electrostatic title product, 20.1 g (94%), which was characterized by its IR (nujol Mull) containing well-separated, sharp main peaks at 3340, 3300, 2900, 2836, 86858 58 1725, 1650, 1628, 1580, 1532, 1455, 1410, 1370, 1280, 1240, 1216 and 1175 cm-1.

This crystalline product (9.4 g) was further purified by dissolving it in 1000 ml of acetone under reflux for one 5 hours. The solution was cooled to room temperature and seeded using a small amount of the above crystals. After stirring for 6 hours, the title product was collected by filtration and washed with as little acetone as possible and dried in vacuo at 35 ° C, 7.25 g, having identical IR properties.

Example 19 N- (3- (R) -methyl-4-heptenoyl) -D-gamma-glutamyl- (alpha-benzyl ester) glycyl-D-alanine benzyl ester Following the procedure of Example 10D, 2.77 g (5 mm ) D-gamma-glutamyl (alpha-benzyl ester) glycyl-D-alanine benzyl ester hydrochloride and acid chloride prepared from 747 mg (5 mm) of 3- (R) -methyl-4-heptenoic acid gave the title compound.

Example 20 N- (3- (S) -methyl-4-heptanoyl) -D-gamma-glutamyl-glycyl-D-alanine

A mixture of 500 mg of the product of Example 19 and 26 mg of 5% palladium-on-carbon catalyst (50% water content) in 125 ml of ethanol was shaken for 2.5 hours under an atmosphere of hydrogen at an initial pressure of 4 x atmospheric pressure. The catalyst was filtered off and the solvent was removed in vacuo. The product was purified using the method of Example 18 and was identical in all respects to the product of that Example 30.

Preparation A

Cyclohexylacetyl chloride A1. Ethyl cyclohexyl acetate To 4.9 g of 60% sodium hydride in oil was added enough hexane to dissolve the oil. to oil-free sodium hydride under nitrogen was added 100 ml of dry 59,885,88 tetrahydrofuran followed by a solution of 22.2 ml of triethylphosphonoacetate in 80 ml of dry tetrahydrofuran. After the mixture was stirred at room temperature for 1 hour, 10.5 ml of cyclohexanone in 40 ml of tetrahydrofuran was added thereto, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was poured into water and extracted with diethyl ether. The organic phase was washed with 1N sodium hydroxide solution, water and brine. The organic phase was separated, dried over magnesium sulfate and concentrated under reduced pressure.

The residue was dissolved in 250 ml of methanol, treated with 1.5 g of 10% palladium hydroxide-carbon catalyst and the mixture was stirred under a hydrogen atmosphere at 345 kPrn for 4 hours. The catalyst was filtered off and the filtrate was concentrated in vacuo. The residue was distilled at 45-50 ° C under 0.4 torr to give 15.4 g (90% yield) of the desired intermediate.

A2. Cyclohexylacetyl chloride To 100 ml of methanol containing 15.4 g of ethyl-20-cyclohexyl acetate was added 15.2 g of potassium hydroxide, and the solution was refluxed for three hours. The methanol was removed in vacuo and the residue was treated with water. The solution was extracted with diethyl ether, then acidified with 10% hydrochloric acid. The acidic solution was extracted with fresh ether and the organic phase was separated and washed with water and brine. Removal of the solvent after drying afforded a liquid residue.

The residue was dissolved in 60 ml of methylene chloride and treated with 18 ml of oxalyl chloride. After stirring at room temperature for 4 hours, the reaction mixture was concentrated in vacuo and the residue was distilled, 45/50 ° C / 0.4 torr, 12.5 g (86% yield).

Preparation B

Following the general procedure of Preparation A 35 and starting from triethylphosphonoacetal and the appropriate aldehyde or ketone, the following acid chlorides were prepared: 86858 I COCO R 1 K 2 - ° C. / torr 5 (CH 3 CH 2 CH 2) 2 CHCH 2 - 50-55 / 0 , 4 (CH3CH2) 2CHCH2- 22-25 / 0.5 CH., I 3 10 CH. (CH2) -CHCH9-23-30 / 0.5 3 J (R, sr CH-.

CH- (CH -) - CHCH-- 22-25 / 0.5 15 3 Z ^ {R, Sr (CH3) 2CH (CH2) 3-24-31 / 0, 7 CB, CH0 I 3 2 20 CH3 ( CH 2) 3CHCH? 34-37 / 0.5 (R »S] CH0 I 3 25 CH, (CH0) -CHCH- 45-47 / 0.6 3 1 4 (R, sf [^ \ cB2- 25-30 / 0.5 30 (CH3CH2) 2CH (CH2) 2-32-36 / 0.4 CH, I 3 CH7 (CH) CH (CH} - 30-38 /, 06 3 MR, sr 35 ,, 8 6 8 S 8 o 1 Rp. ° C. / torr CH-CH- 5 I 3 2 CH3 (CH2) 4CHCH? 63-65 /; 95 (R »Sf f3 10 CH, (CH _) ^ CHCHO- 89-92 / 5 <R , Sf CH, I 3 (CH3) 2CH (CH2), CHCH2 46-50 / 0j5 15 tR / S) Γ3 (CH ~) _CHCH ^ CHCH_- 30-34 / 0.5 (R, Sf 20 CH, CH ~ 31 2 CH, (CH2) 5CHCH5- 31-35 / 0; 7 mr, sr

Preparation C

25 6-Methylheptanoyl chloride C1. 3-Hydroxy-4-methyl-1-pentene To 90 ml of 1.0 M vinyl magnesium bromide in tetrahydrofuran cooled to 5 ° C was added dropwise 6.3 ml of isobutyraldehyde in 30 ml of tetrahydrofuran, followed by warm to room temperature. After 2 hours, the reaction mixture was added to saturated ammonium chloride solution and extracted with ether. The ether extracts were combined, washed with saturated arammonium chloride solution, saturated sodium bicarbonate solution and brine, and dried over magnesium sulfate 62 86 8 58. The solvent was removed in vacuo to give 6.0 g of the desired product.

C2. 6-Methyl-4-heptenoic acid ethyl ester A mixture of 18.2 g of 3-hydroxy-4-methyl-1-5 pentene, 200 ml of triethyl orthoacetate and 500 ml of p-toluenesulfonic acid was treated with 400 ml of toluene and heated to reflux 4A. on a molecular sieve for 24 hours. The solvent was removed in vacuo and the residue was distilled. The fraction distilled at 45-64 ° C at 10 and 0.5 torr gave 7.5 g of the desired product. C3. 6-Methylheptenoic acid ethyl ester To a mixture of 7.5 g of 6-methyl-4-heptanoic acid ethyl ester in 75 ml of methanol was added 700 mg of 10% palladium hydroxide-carbon catalyst, and the mixture was shaken under a hydrogen atmosphere at 345 kP under a pressure of 1.5. for an hour. The catalyst was filtered off and the solvent was removed in vacuo to give 5.7 g of the desired product.

C4. 6-metyyliheptanoyylikloridi

Following the procedure of Preparation A2, 5.7 g of 20 6-methylheptanoic acid ethyl ester gave 2.0 g of the desired product, b.p. 30-34 ° C / 0.5 torr.

Preparation D

2-methylheptanoyl chloride D1. 2-Methylheptanoic acid To a cold (0 ° C) solution of 100 ml of dry tetrahydrofuran in 11.8 ml of dry diisopropylamine and 55 ml of 1.6 mol / l n-butyllithium were added 5.4 ml of n-heptanoic acid and the mixture was allowed to stir at room temperature for one hour. The resulting solution was cooled to 0 ° C and 7.2 ml of methyl iodide was added. The reaction mixture was stirred at room temperature under nitrogen for 1.5 hours, then poured into 10% hydrochloric acid and extracted with diethyl ether (3 x 100 mL). The extracts were combined, washed with 10% hydrochloric acid, water, 20% sodium bisulfite and brine, and dried over magnesium sulfate 63 86858. The solvent was removed in vacuo and the residue, 5.61 g, was dissolved in methanol containing 5.1 g of potassium hydroxide. After stirring overnight, the methanol was removed and the residue was dissolved in 150 mL of water. The aqueous layer was washed with ether (2 x 100 mL) and acidified with 10% hydrochloric acid. The product was extracted with ether, washed with 20% sodium bisulfite solution and brine, and dried over magnesium sulfate. Removal of the ether afforded 5.0 g of product as a yellow liquid.

D2. 2-Methylheptanoyl chloride Using 5 g of 2-methylheptanoic acid and 7.6 ml of oxalyl chloride and the method of Preparation A2, 3.3 g of the desired product were obtained, b.p. 32-34 ° C / 0.6 torr.

15 Preparation E

3- (S) -methylheptanoyl chloride E1. 3- (R) -Methylglutaric acid monomethyl ester In a 5-liter four-necked flask fitted with a stirrer and a pH electrode, 2.5 L of 0.01 mol / L potassium acid phosphate buffer, pH 7.0, and then 150 mg of porcine liver esterase and 150 g of dimethyl-3-metyyligluta-stearate. The pH of the mixture was maintained at about pH 6.85 by the occasional addition of 10% potassium carbonate solution. After 2.5 hours, the reaction mixture was acidified with 10% chloro-hydrochloric acid to pH 2.0 and the product was extracted with diethyl ether. The extracts were combined, dried over magnesium sulfate and concentrated in vacuo to give 114 g of the desired product / alpha_7D = -1.48 (CH 2 OH C = 0.086 g / ml).

E2. Methyl 3- (R) -methyl-5-hydroxypentanoate To a mixture of 114 g of 3- (R) -methylglutaric acid monomethyl ester in 715 ml of dry tetrahydrofuran cooled to 0 ° C was slowly added 391 ml of 2 mol / l a solution of borane dimethyl sulfide in tetrahydrofuran. After the addition was complete, the reaction mixture was stirred overnight at room temperature. The reaction mixture was cooled and 50 ml of water was added slowly. The reaction mixture was extracted (3 x 100 mL) with 64,885,88 ether and the extracts were combined, washed with water, saturated sodium bicarbonate solution and brine, and dried over magnesium sulfate. Removal of the solvent gave 37 g of the desired product.

5 E3. Methyl 3- (R) -methyl 5- (t-butyldimethylsilyl) pentanoate_

To a solution of 37 g (0.253 m) of methyl 3- (R) -methyl-5-hydroxypentanoate and 37 g (0.543 m) of imidazole in 500 ml of dimethylformamide was added 37 g of 10 (0.249 m) of t-butyl] dimethylsilyl chloride. and the reaction mixture was stirred at room temperature for two hours. The reaction mixture was poured into water and extracted (4 x 100 mL) with ether. The combined extracts were washed with 10% hydrochloric acid, saturated sodium bicarbonate solution, water and brine and dried over magnesium sulfate. Removal of the solvent gave 121.88 g of crude product, which was distilled to give 107.12 g of pure product, b.p. 80-81 ° C / 0.4 torr.

E4. 3- (S) -methyl-5- (t-butyldimethylsilyloxy) -2 0 1 -pentanol ___________

To a mixture of 8.5 g (0.224 m) of lithium aluminum hydride in 250 ml of diethyl ether under nitrogen was added 53.5 g (0.206 m) of methyl 3- (R) -methyl 5- (t-butyldimethylsilyloxy) pentanoate. In 125 ml of eet-25 blades. The reaction mixture was stirred for 1 hour at 0 ° C, then treated dropwise with 8.4 g of water, 8.4 ml of 15% sodium hydroxide solution and 25.2 ml of water. The solids were filtered and the organic phase was separated and washed with water, 2.5% hydrochloric acid and brine. The organic phase was dried over magnesium sulfate and concentrated in vacuo to give 46 g of product.

E5. 3- (R) -Methyl-5- (t-butyldimethylsilyloxy) -1-pentanal_35 To a mixture of 56.3% oxalyl chloride in 300 mL of dry methylene chloride cooled to -60 ° C under a nitrogen atmosphere was added dropwise 74.81 g. g of dimethyl sulfoxide in 100 ml of dry methylene chloride. After 15 minutes, 92.0 g of 3- (S) -methyl-5- (t-butyldimethylsilyloxy) -1-pentanol in 250 ml of the same solvent were added dropwise. After 30 minutes, 206.1 g of triethylamine was added to the -60 ° C reaction mixture, after which the cooling bath was removed. The reaction mixture was stirred at room temperature for 1.5 hours, after which it was poured into water and extracted with methylene chloride.

The extracts were washed with 2.5% hydrochloric acid, saturated sodium bicarbonate solution, water and brine, then dried over magnesium sulfate. The solvent was removed and the residue was dissolved in ether and washed again and dried as before for 15 min. Removal of ether afforded 90.9 g of the desired product.

E6. 5- (S) -methyl-7- (t-butoxydimethylsilyloxy) -2-heptene_

To a slurry containing 80 g (0.2155 m) of triphenylethylphosphonium bromide in 800 ml of dry tetrahydrofuran cooled to 0 ° C was added 165.7 ml of a 1.3 mol / L n-butyllithium solution (0.2155 m) in the same manner. solvent. After 2 hours, 45 g (0.196 m) of 3- (R) -methyl-5- (t-butyldimethylsilyloxy) -1-pentanal in 200 ml of dry tetrahydro-furan were added dropwise to the reaction mixture. The reaction was allowed to stir for 2 hours at room temperature, after which it was poured into water and extracted with ether. The combined extracts were washed with water and brine and dried over magnesium sulfate. Removal of the solvent in vacuo gave a yellow oil-30 which distilled to give 37.4 g of product, b.p. 74-79 ° C / 0.2 - 0.1 torr.

E7. 3- (S) -methyl-1-heptanol

To a solution of 74.8 g of 5- (S) -methyl-7- (t-butyldimethylsilyloxy) -2-heptene in 500 ml of methanol-35 was added 7.5 g of 10% palladium hydroxide-carbon catalyst and the mixture was shaken under an atmosphere of hydrogen 86858 66 for 1.5 hours at a pressure of 345 kP. The catalyst was filtered off and the solvent was removed in vacuo, 30 g.

E8. 3- (S) -methylheptanoic acid To 10 g of 3- (S) -methyl-1-heptanol in 175 ml of acetone was added dropwise over 45 minutes and at 15-20 ° C 90 ml of Jones reagent. After 15 minutes, 15 ml of isopropanol was added to the mixture and stirring was continued for 30 minutes. The reaction mixture was poured into water and the product was extracted with ether. The extracts were combined, washed with water, sodium bisulfite solution and brine, and dried over magnesium sulfate. The solvent was removed to give 10 g of product in liquid form, b.p. 84-88 ° C / 0.4 torr, wall value = -4.46 (CH 3 OH C = 0.105 g / ml).

15 E9. 3- (S) -metyyliheptanoyylikloridi

Following the procedure of Preparation A 2, 5.0 g of 3- (S) -methylheptanoic acid and 7.5 ml of oxalyl chloride gave 2.9 g of the desired acid chloride, b.p. 29-32 ° C / 0.25 torr.

Claims (4)

Wherein Rx is alkyl of 2-10 carbon atoms, cycloalkyl of 4-7 carbon atoms or cycloalkylmethyl of 6-8 carbon atoms; R 2 is hydrogen or alkyl of 1-3 carbon atoms; R 3 is hydroxy or an amino acid residue of the formula D -NHCH (CHO) COORc I 2 n 2 X where x is hydrogen, alkyl of 1 or 2 carbon atoms or hydroxymethyl and n is a fully t 0-4; and R 4 and R 5 are each independently hydrogen, alkyl of 1-6 carbon atoms, cycloalkyl methyl of 6-8 carbon atoms or benzyl, and salts thereof, characterized in that a) a dehydratively couples a compound of formula R C02CH2-phenyl 30. CH 2 O (ch 2) 2conhchc-oh * 2 where R 1 and R 2 are the same as above, with a compound of formula t. 71 86858 D HONCH (CH is blocked as not participating in the reaction and wherein X, n and R5 are the same as above, following an optional step wherein selectively the blocking groups are removed and, if desired, a pharmaceutically acceptable base salt is prepared from the product; or b) acylates a compound of formula h 2 N v D CO 2 R 5 CH L 0 I II (CH 2) 0 COHHCH-C-Ro Δ ZI 3 R 2 wherein R 2, R 3 and R -Hal wherein the functional groups can be blocked which do not participate in the reaction and wherein Rx represents the same as above and Hal is halogen, then follows an optional step wherein selectively the blocking groups are removed and, if desired, a pharmaceutically acceptable base salt is prepared of the product. A process for the preparation of novel therapeutically useful peptides containing acylglutamic acid and having the formula D 1 2 4 LO (1) 2. A process according to claim 1, characterized in that the coupling is carried out in a manner using N-hydroxysuccinimide or 1-hydroxybenzotriazole and a carbodiimide. 3. A process according to claim 1, characterized in that the acylation is carried out with an acid chloride in the presence of a tertiary amine. 86858 72 4. A process according to claim 2 or 3, characterized in that the O-benzyl groups are removed by catalytic hydrogenolysis.