Glutamic acid derivatives

All isomeric forms and mixtures of isomers of glutamic acid compounds of the formula ##STR1## wherein the glutamic aid is of D- or L- configuration, R.sub.1 is selected from the group consisting of hydrogen, alkyl of 1 to 5 carbon atoms, an amino acid, a peptide of 2 to 4 amino acids and an amino acid or a peptide of 2 to 4 amino acids in which the amine is esterified with an optionally unsaturated aliphatic carboxylic acid of 6 to 24 carbon atoms or R.sub.1 is selected from the group consisting of a residue of a C.sub.6 -C.sub.24 optionally unsaturated aliphatic acid. R.sub.5 is selected from the group consisting of hydrogen or an alkyl radical of 1 to 5 carbon atoms, R.sub.3 is selected from the group consisting of hydroxy, alkoxy of 1 to 5 carbon atoms, an amino acid with the amine optionally substituted with alkyl of 1 to 5 carbon atoms, Z is ##STR2## R.sub.2 is selected from the group consisting of hydrogen, an amino acid and a peptide of 2 to 4 amino acids, R.sub.4 is selected from the group consisting of hydroxy, alkoxy 1 to 5 carbon atoms and an amino acid optionally substituted on the amine with alkyl of 1 to 5 carbon atoms, U is selected from the group consisting of ##STR3## --CH.dbd.CH--CH.sub.2 -- (E or Z isomer), --CH.sub.2 --CH.dbd.CH-- (E or Z isomer) and ##STR4## or U and Y together are .dbd.CH--CH.sub.2 --CH.sub.2 -- (E or Z isomer) and X is hydrogen and their salts with non-toxic, pharmaceutically acceptable acid or bases having immunomodulatory properties.

STATE OF THE ART 
French Patent No. 2,566,410 describes diaminopimelic acid compounds having 
antibacterial properties and U.S. Pat. No. 4,311,640 describes related 
compounds. 
OBJECTS OF THE INVENTION 
It is an object of the invention to provide the novel compounds of formula 
I and their non-toxic, pharmaceutically acceptable addition salts and a 
process for their preparations. 
It is another object of the invention to provide novel immunomodulating 
compositions and a novel method of inducing immunomodulating activity in 
warm-blooded animals. 
These and other objects and advantages of the invention will become obvious 
from the following detailed description. 
THE INVENTION 
The novel compounds of the invention are selected from the group consisting 
of all isomeric forms and mixtures of isomers of glutamic acid compounds 
of the formula 
##STR5## 
wherein the glutamic acid is of D- or L- configuration, R.sub.1 is 
selected from the group consisting of hydrogen, alkyl of 1 to 5 carbon 
atoms, an amino acid, a peptide of 2 to 4 amino acids and an amino acid or 
a peptide of 2 to 4 amino acids in which the amine is esterified with an 
optionally unsaturated aliphatic carboxylic acid of 6 to 24 carbon atoms 
or R.sub.1 is selected from the group consisting of a residue of a C.sub.6 
-C.sub.24 optionally unsaturated aliphatic acid. R5 is selected from the 
group consisting of hydrogen and an alkyl of 1 to 5 carbon atoms, R.sub.3 
is selected from the group consisting of hydroxy, alkoxy of 1 to 5 carbon 
atoms and an amino acid with the amine optionally substituted with alkyl 
of 1 to 5 carbon atoms, Z is 
##STR6## 
R.sub.2 is selected from the group consisting of hydrogen, an amino acid 
and a peptide of 2 to 4 amino acids, R.sub.4 is selected from the group 
consisting of hydroxyl, alkoxy of 1 to 5 carbon atoms and an amino acid 
optionally substituted on the amine with alkyl of 1 to 5 carbon atoms, U 
is selected from the group consisting of 
##STR7## 
--CH.dbd.CH--CH.sub.2 -- (E or Z isomer), --CH.sub.2 --CH.dbd.CH-- (E or Z 
isomer) and 
##STR8## 
or U and Y together are .dbd.CH--CH.sub.2 --CH.sub.2 -- (E or Z isomer) 
and X is hydrogen or U and X together are .dbd.CH--CH.sub.2 --CH.sub.2 -- 
(E or Z isomer) and Y is hydrogen and their salts with non-toxic, 
pharmaceutically acceptable acid or bases. 
The amino acid in the compounds of formula I is preferably an .alpha.-amino 
acid and examples of such acids are Ala, Val, Ival, Leu, lle, Asp, Asn, 
Glu, Gln, Ser, Thr, Cys, Met, Lys, Arg, Phe, Tyr, Trp, His and Pro, Nva, 
Nle, Hyp, Orn, in the D or L form, as well as Sar and Gly , it being 
possible for all the said acids to be N-esterified or N-alkylated. In the 
case of a peptide of 2,3 or 4 amino acids, the latter is chosen from the 
group consisting of the above amino acids. It will be understood by 
convention that the symbols for the -amino carboxylic acids denote these 
acids in their D or L configuration (for example, the term Ala denotes 
alanine in D form or in L form). 
The saturated or unsaturated aliphatic acid of 6 to 24 carbon atoms, 
preferably from 12 to 22 carbon atoms, may be stearic acid, palmitic acid, 
lauric acid, caprylic acid, myristic acid, .alpha.- or .gamma.-linolenic 
acid, linoleic acid, arachidonic acid or docosapentaenoic acid. 
Examples of alkyl of 1 to 5 carbon atoms are pentyl, isobutyl, butyl and 
isopropyl, and preferably propyl, ethyl or methyl. Examples of alkoxy of 1 
to 5 carbon atoms are pentoxy or butoxy, but preferably propoxy, ethoxy or 
methoxy. 
The products of formula I contain one or more asymmetric carbon atoms and, 
as stated above, the subject of the invention is the said compounds of 
formula I in all their possible isomeric forms and in the form of 
mixtures. 
The non-toxic, pharmaceutically acceptable salts of the derivatives of the 
invention can be formed with organic and inorganic bases such as alkali 
metal and alkaline earth metal hydroxides such as, for example, sodium, 
potassium, lithium and calcium hydroxides, and magnesium or ammonium 
hydroxide. Among organic bases, there may be mentioned substituted or 
unsubstituted alkylamines such as trimethylamine, methylamine, 
propylamine, N,N-dimethylethanolamine or tris(hydroxymethyl)methylamine. 
Basic amino acids such as lysine or arginine may also be mentioned as well 
as glucosamine or procaine may be mentioned. 
The addition salts with inorganic or organic acids can be, for example, the 
salts formed with hydrochloric acid, hydrobromic acid, nitric acid, 
sulfuric acid, phosphoric acid, acetic acid, formic acid, propionic acid, 
maleic acid, fumaric acid, succinic acid, tartaric acid, citric acid, 
oxalic acid, glyoxylic acid and aspartic acid, alkanesulfonic acids such 
as methane- and ethanesulfonic acid, arylsulfonic acids such as benzene- 
or p-toluenesulfonic acid, and arylcarboxylic acids such as benzoic acid. 
Among the preferred compounds of formula I are those wherein the glutamic 
acid has the D-configuration, those wherein R.sub.3 and R.sub.4 are 
hydroxy, those wherein R.sub.5 is hydrogen and their non-toxic, 
pharmaceutically acceptable acid addition salts. Specific preferred 
compounds are 2-amino-6-.gamma.-D-glutamylamino)-4-methyleneheptanedioic 
acid and 
2-(L-alanylamino)-6-(.gamma.-D-glutamylamino)-4-methyleneheptanedioic 
acid, and their non-toxic, pharmaceutically acceptable acid addition 
salts. 
The novel process of the invention for the preparation of the compounds of 
formula I comprises reacting a glutamic acid compound of the formula 
##STR9## 
wherein Alk is alkyl of 1 to 3 carbon atoms and R'.sub.1 has the 
definition of R.sub.1 other than hydrogen or an amino protective group 
with a compound of the formula 
##STR10## 
as an isomer or mixture of isomers wherein U has the above definition, Alk 
and Alk.sub.2 are alkyl of 1 to 3 carbon atoms, X' and Y' are hydrogen or 
--COOAlk.sub.3, Alk.sub.3 is alkyl of 1 to 3 carbon atoms or together with 
U form an additional bond and R'.sub.2 is amine protecting group, an amino 
acid or a peptide of 2 to 4 amino acids with the amine protected to obtain 
a compound of the formula 
##STR11## 
in all isomeric forms or isomeric mixtures which is optionally isolated 
and salified or subjected to the following reactions in any order: 
a) if X' or Y' is COO--Alk.sub.3 : decarboxylation, 
b) deprotection of the amino groups, 
c) dealkylation of the hydroxyl groups, 
d) amidation of the free amino groups with an amino acid or a peptide of 
2,3 or 4 amino acids in which the amino group is protected, followed by 
deprotection of this amino group, 
e) esterification or salification of the carboxy groups with a base, and 
f) salification of the amino groups with an acid. 
In a preferred mode of the process of the invention, the glutamic acid 
compound of formula II is activated by formation of a mixed anhydride such 
as with isobutyl chloroformate or by the presence of a condensation agent 
such as dicyclohexylcarbodiimide, N,N'-carbonyldiimidazole or 
bis(alkylamides) of sulfurous acid such as 
##STR12## 
The decarboxylation is preferably effected with a concentrated inorganic 
acid such as 12N hydrochloric acid. A direct decarboxylation may be 
effected directly by the procedure of Krapcho or Keinan et al [J. Org. 
Chem., Vol. 51 (1986), p.3615-3619]. 
The deprotection of the amino groups (formyl or BOC, for example) is 
preferably carried out with a dilute mineral acid such as hydrochloric 
acid. The dealkylation of the carboxyl groups is preferably carried out by 
saponification with an inorganic base such as potassium hydroxide and, 
preferably sodium hydroxide. It can, if necessary, be performed in two 
steps in the event of being incomplete. In the latter case, the second 
dealkylation is preferably carried out after the deprotection of the amino 
groups and/or the decarboxylation. The amidation of the free amino groups 
is carried out, for example, using a functional derivative such as a 
halide of the amino acid or peptide, or in the presence of a condensation 
agent such as those mentioned above. The esterification of the carboxyl 
groups is carried out, for example, under the same conditions as above 
(condensing agents). 
The compounds of formula I may have a basic or acidic nature and if so, the 
addition salts thereof may be prepared by reaction with the appropriate 
base or acid in substantially stoichiometric form. 
In a preferred embodiment of the process, the compound of formula I.sub.A 
is subjected to the following reactions: dealkylation of the carboxyl 
groups, deprotection of the amino groups and decarboxylation, amidation or 
salification of the amino groups with an acid and/or esterification or 
salification of the carboxyl groups with a base. 
Some of the compounds of formula III are known and if they are not known, 
they may be prepared by reacting a compound of the formula 
##STR13## 
wherein Alk.sub.1, Alk.sub.2, U, X', Y' and R'.sub.2 have the above 
definitions with an alkanesulfonyl halide of 1 to 3 alkyl carbon atoms, 
preferably methane sulfonyl chloride, in the presence of a condensation 
agent such as pyridine and then with an alkali metal azide such as sodium 
azide or diphenylphosphoryl azide to form a compound of the formula 
##STR14## 
reducing the latter with triphenyl phosphine, for example, followed by 
aqueous hydrolysis or by catalytic hydrogenation in the presence of a 
Lindlar catalyst (poisoned palladium or charcoal) to form a compound of 
the formula 
##STR15## 
wherein R.sub.2, X', Y', U, Alk.sub.1 and Alk.sub.2 have the above 
definitions and when X' and/or Y' are other than hydrogen, reacting the 
latter an agent to protect the amino group to form a compound of the 
formula 
##STR16## 
wherein R.sub.2, Alk.sub.1, Alk.sub.2, U, X' and Y' have the above 
definitions and Rp is an amino protective group, subjecting the latter to 
a decarboxylating agent to obtain the corresponding compound with 1 or 2 
asymetrical carbon atoms, separating the latter into the isomers or 
diastereoisomers such as by crystallization or chromatography and then 
removing the amino protective group to form the compound of formula III in 
the form of separate isomers or isomeric mixtures. 
The compounds of formulae II and IV are known. 
The novel immunomodulatory compositions of the invention are comprised of 
an immunomodulatorily effective amount of at least one compound of formula 
I and its non-toxic, pharmaceutically acceptable salts with acids and 
bases and an inert pharmaceutical carrier or excipient. The compositions 
may be in the form of tablets, dragees, gelatin capsules, granules, 
suppositories and injectable solution or suspensions. 
Examples of suitable excipients are talc, gum arabic, lactose, starch, 
magnesium stearate, cocoa butter, aqueous or non-aqueous vehicles, fats of 
animal or vegetable origin, paraffinic derivatives, glycols, the various 
wetting, dispersing or emulsifying agents and preservatives. 
The compositions of the invention have exceptional immunomodulatory 
properties, particularly by activation of human monocytes and production 
of monokines such as TNF (tumor necrosis factor) and IL-1, interleukin-1. 
They are useful in the treatment of autoimmune diseases, whether conditions 
affecting organs nonspecifically (rheumatoid polyarthritis, lupus 
erythematosus, haemolytic anaemia, autoimmune leukopenia, and the like) or 
specific diseases of organs (thyroiditis, Basedow's disease, Addison's 
disease, multiple sclerosis, pemphigus, haemorrhagic rectocolitis, some 
types of nephropathy, and the like). Compositions are also be useful in 
the treatment of haemopathies, cancer, AIDS, viral and microbial 
conditions, especially those which are chronic and recurrent (bronchitis, 
influenza, and the like), diseases of the oral cavity, and the like. They 
can constitute adjuvants of viral therapy, antibiotic therapy or 
anticancer chemotherapy. 
The compositions also find use in the treatment of many secondary or 
acquired immune deficiency states observed during a wide variety of 
conditions: deficiencies associated with metabolic disorders, deficiencies 
of iatrogenc origin (corticoids, ionizing radiation, etc.), deficiencies 
observed in major burns patients, and the like. 
The novel method of the invention for inducing immunodulatory activity in 
warm-blooded animals, including humans, comprises administering to 
warm-blooded animals an immunodulatorily effective amount of at least one 
compound of formula I and its non-toxic, pharmaceutically acceptable salts 
with acids and bases. The compounds may be administered orally, rectally 
or parenterally and the usual daily dose is 0.007 to 0.7 mg/kg depending 
on the compound, condition treated and method of administration. For 
example, the compound of Example may be orally administered at a dose 
0.007 to 0.7 mg/kg for the treatment of rheumatoid polyarthritis.

In the following examples there are described several preferred embodiments 
to illustrate the invention. However, it is to be understood that the 
invention is not intended to be limited to the specific embodiments. 
EXAMPLE 1 
2-amino-6-(.gamma.-D-glutamylamino)-4-methylene-heptanedioic acid 
STEP A: Triethyl 1-formylamine-3-methylen-5-[(N-trifluoroacetyl 
-O-methyl-.gamma.-D-glutamyl)-amino-1,1,5-pentanetricarboxylate 
A solution of 4 g of 1-methyl N-trifluoracetyl-D-glutamate in 20 ml of 
dimethoxyethane was added to a solution of 5.1 g of triethyl 
5-amino-1-(formylamino)-3-methylene-1,1,5-pentanetricarboxylate in 280 ml 
of dimethylethylamine and the mixture was cooled to 0.degree. C. 3.2 g of 
dicyclohexylcarbodiimide were added in small portions and the mixture was 
stirred for 16 hours at 0.degree. C. and filtered. The filtrate was 
evaporated to dryness and the residue was taken up with cold 
dimethoxyethane. The mixture was filtered and the filtrate was taken to 
dryness. The residue was taken up in methylene chloride and the mixture 
was washed with N hydrochloric acid, with saturated sodium bicarbonate 
solution and then with water saturated with sodium chloride, dried and 
evaporated to dryness. The residue was purified by chromatography on 
silica (eluant:ethyl acetate/cyclohexane 5:5) to obtain 5 g of the 
expected product with a specific rotation of [.alpha.].sub.D =+8 (c=1% in 
methylene chloride). 
STEP B: 2-amino-6-(.gamma.-D-glutamylamino)-4-methylene-heptanedioic acid 
Saponification: 
10.9 ml of N sodium hydroxide were added dropwise over 5 minutes at 
0.degree. C. to a solution of 1.28 g of the product Step A in 20 ml of 
ethanol and the mixture was allowed to return to room temperature and 
stirred for 24 hours. It was neutralized with 0.9 ml of 12N hydrochloric 
acid and taken to dryness under reduced pressure. 
Decarboxylation - deformylation: 
The residue was taken up in 10 ml of ethanol and 1 ml of 12N hydrochloric 
acid was added and the mixture was stirred for 40 minutes at 80.degree. C. 
Saponification: 
The above product was chilled and 15.2 ml of 2N sodium hydroxide were added 
dropwise over 5 minutes. The mixture was stirred for 24 hours at room 
temperature, brought to pH 6 using 12N hydrochloric acid and taken to 
dryness. 
Purification: 
The above product was taken up in a few ml of water and the pH was adjusted 
to 6 with N sodium hydroxide. The mixture was dealkalinized on Dowex 50 
W.times.8 H.sup.+ resin (50-100 mesh) and elution was performed first 
with water and then with N ammonia solution. The product was purified by 
chromatography on silica (eluant: ethanol/ammonia solution 95:5, then 
ethanol/ammonia solution 8:2) and the eluate was evaporated to dryness. 
The residue was taken up in 200 ml of water and the mixture was filtered. 
The filtrate was lyophilized for 16 hours to obtain 400 mg of the expected 
product with a specific rotation [.alpha.].sub.D 
=-9.5.degree..+-.1.degree. (c=1% in 4N hydrochloric acid). 
A) Preparation of 1-methyl-N-trifluoroacetyl-D-glutamate 
STEP 1: 5-(1,1-dimethylethyl)-N-trifluoroacetyl-D-glutamate 
4.1 ml of triethylamine and then, while cooling 4.1 ml of trifluoroacetic 
anhydride were added dropwise to a solution of 5.5 g of 
5-(1,1-dimethylethyl)-1-methyl-D-glutamate in 180 ml of methylene 
chloride. The mixture was then stirred for 2 hours at room temperature and 
poured into 70 ml of ice-cold water. The resulting mixture was extracted 
with methylene chloride and the extract was washed with saline solution, 
dried and taken to dryness to obtain 7.1 g of the expected product with a 
specific rotation of [.alpha.].sub.D =+5.degree..+-.1.degree. (c=1.3% in 
dioxane). 
STEP 2: 1-methyl-N-trifluoroacetyl-D-glutamate 
6.8 g of the product of Step 1 were dissolved in 50 ml of methylene 
chloride and 50 ml of trifluoroacetic acid were added. The mixture was 
stirred for 1 hour and evaporated to dryness. The residue was taken up in 
ethyl acetate and the mixture was poured into water. The resulting mixture 
was extracted with ethyl acetate and the extract was washed with saline 
solution and taken to dryness to obtain 5.5 g of the expected product with 
specific rotation of [.alpha.].sub.D =+9.degree..+-.2.degree. (c=0.6% in 
dioxane) 
B) Preparation of the starting triethyl 
5-amino-1-(formylamino)-3-methylene-1,1,5-pentanetricarboxylate 
STEP 1: Diethyl 2-(formylamino)-(2-methyl-2-propenyl)-propanedioate 
40 g of potassium carbonate, 0.380 g of 18-crown-6-crown ether catalyst and 
39.9 g of chloromethylpropene were added to a solution of 30 g of ethyl 
formamidomalonate in 300 ml of cyanomethyl and the mixture was stirred at 
reflux for 3 hours and filtered. The filtrate was evaporated to dryness 
and the residue was cooled to 0.degree. C. to 5.degree. C. and taken up in 
10 ml of isopropyl ether. The mixture was filtered, washed with isopropyl 
ether and dried under reduced pressure to obtain 25.2 g of the expected 
product with a melting point of 72.degree. C. 
STEP 2: Triethyl 
1-(formylamino)-5-hydroxy-3-methylene-1,1,5-pentanetricarboxylate (mixture 
of isomers) 
A solution of 26.5 g of ethyl glyoxylate in 180 ml of methylene chloride 
was added dropwise over 10 minutes to a solution of 84 g of ferric 
chloride in 180 ml of methylene chloride and the mixture was stirred for 1 
hour and cooled to -20.degree. C. A solution of 34 g of the product of 
Step A in 180 ml of methylene chloride was added dropwise over 20 minutes, 
and the mixture was stirred for 1 hour at -20.degree. C. and was then 
poured into 300 ml of ice-cold water. The resulting mixture was extracted 
with methylene chloride and the extract was washed with 2N HCl and then 
with saline solution, dried and evaporated to dryness under reduced 
pressure to obtain 56.5 g of the expected product (mixture of isomers) 
with a melting point of 68.degree. C. after purification by chromatography 
on silica (eluant:ethyl acetate/cyclohexane 6:4). 
STEP 3: Triethyl 
5-azido-1-(formylamino)-3-methylen-1,1,5-pentanetricarboxylate 
46 g of the product (mixture of isomers) of Step 2 were dissolved in 500 ml 
of pyridine and the mixture was cooled to 0.degree. C. 12 ml of 
methanesulfonyl chloride were added dropwise at 0.degree. C. and the 
mixture was stirred for 3 hours at room temperature and poured into 400 ml 
of ice-cold 4N hydrochloric acid and 200 ml of methylene chloride. The 
resulting mixture was extracted with methylene chloride and the extract 
was washed with 4N hydrochloric acid, with saturated sodium bicarbonate 
solution and with saline solution, dried and evaporated to dryness under 
reduced pressure. The 56 g of residue obtained were dissolved in 250 ml of 
dimethylformamide and 9.98 g of sodium azide were added. The mixture was 
stirred for 16 hours at room temperature and the solvent was removed. The 
residue was taken up in methylene chloride and the mixture was washed with 
saturated sodium bicarbonate solution and then with saline solution, dried 
and taken to dryness to obtain 59 g of expected product (mixture of 
isomers) which was purified by chromatography on silica 
(eluant:cyclohexane/ethyl acetate 8:2) to obtain 26 g of the expected 
product. 
STEP 4: Triethyl 
5-amino-1-(formylamino)-3-methylene-1,1,5-pentanetricarboxylate 
10.9 g of triphenylphosphine were added to a solution of 13 g of the 
product of Step 3 in 250 ml of tetrahydrofuran at approximately -5.degree. 
C. and the mixture was stirred for 5 hours at room temperature. 8.5 ml of 
water were added and the mixture was stirred for 24 hours at room 
temperature. The tetrahydrofuran was evaporated off and the residue was 
taken up in methylene chloride. The mixture was poured into ice-cold 2N 
hydrochloric acid, followed by extraction with 2N hydrochloric acid, 
neutralization with sodium bicarbonate and extraction with methylene 
chloride. The extract was washed with saline solution, dried and 
evaporated to dryness to obtain 10 g of the expected product melting at 
50.degree. C. after crystallization from isopropyl ether. 
EXAMPLE 2 
2-(L-Alanylamino)-6-(.gamma.-D-glutamylamino)-4-methyleneheptanedioic acid 
STEP A: Triethyl 
1-amino-3-methylene-5-(N-trifluoroacetyl-0-methyl-.gamma.-D-glutamyl)-amin 
o]-1,1,5-pentanetricarboxylate 
1.5 ml of 12N hydrochloric acid were added to a solution of 1.22 g of 
product of Step A of Example 1 in 15 ml of ethanol and the mixture was 
stirred for 40 minutes at 80.degree. C. The solvent was removed under 
reduced pressure and the residue was taken up in 10 ml of water. The 
mixture was neutralized with sodium bicarbonate and extracted with 
methylene chloride. The extract was washed with saline solution, dried and 
taken to dryness. The residue was purified by chromatography on silica 
(eluant:ethyl acetate/cyclohexane 8:2) to obtain 0.850 g of the expected 
product. 
STEP B: Triethyl 1- N-[(1,1-dimethylethoxy)-carbonyl]-L-alanylamino 
-3-methylene-5-[(N-trifluoroacetyl-0-methyl-.gamma.-D-glutamyl)amino]-1,1, 
5-pentanetricarboxylate 
0.310 g of BOC-L-alanine were dissolved in 50 ml of tetrahydrofuran and 
0.43 ml of triethylamine were added. The mixture was cooled to 
approximately 5.degree. C. and 0.23 ml of isobutyl chloroformate were 
added. The mixture was stirred for 30 minutes at approximately +5.degree. 
C. and a solution of 0.800 g of the product of Step A in 10 ml of 
tetrahydrofuran was added dropwise over 5 minutes. The mixture was allowed 
to return to room temperature and was stirred for 16 hours. The 
tetrahydrofuran was removed and the residue was taken up in a 
water/methylene chloride mixture. The mixture was extracted with methylene 
chloride and the extract was washed with saline solution, dried and taken 
to dryness. The residue was purified by chromatography on silica (eluant: 
ethyl acetate/cyclohexane 5:5) to obtain the expected product with a 
specific rotation of [.alpha.].sub.D =12.degree..+-.1.degree. (c=1% in 
methylene chloride). 
STEP C: 
2-L-alanylamino)-6-(.gamma.-D-glutamylamino)-4-methyleneheptanedioic acid 
Using the procedure of Step B of Example 1, 700 mg of the product of Step B 
were reacted. Saponification, deprotection of the BOC-protected amine, 
saponification and purification were performed to obtain 170 mg of the 
expected product with a specific rotation of [.alpha.].sub.D 
=-13.degree..+-.1.degree. (c=1% in 4N HCl). 
EXAMPLE 3 
2-amino-4-methylene-6-[N-1-oxooctadecyl)-L-alanyl-.gamma.-D-glutamylamino]- 
heptanedioic acid 
Using the procedure of Example 1, 1-methyl 
N-[N-(1-oxooctadecyl)-1-alanyl]-D-glutamate was used in place of the 
corresponding N-trifluoroacetyl derivative obtained in the preparation A 
of Example 1, to obtain the expected product with a specific rotation of 
[.alpha.].sub.D =-25.degree. (c=0.6% in water). 
Preparation of 1-methyl N-[N-(1-oxooctadecyl)-L-alanyl]-D-glutamate 
STEP 1: Methyl N-(1-oxooctadecyl)-L-alaninate 
24.9 ml of triethylamine were added to a solution of 10 g of 1-alanine 
methyl ester hydrochlorde in 250 ml of methylene chloride, followed, by 
dropwise addition over 30 minutes of a solution of 21.7 g of stearyl 
chloride in 50 ml of methylene chloride. The mixture was stirred for 150 
minutes and filtered and the filtrate was washed with 2N hydrochloric 
acid, then with saline solution, dried and taken to dryness under reduced 
pressure. The residue was made into a paste with isopropyl ether, vacuum 
filtered and crystallized in methanol to obtain 22 g of the expected 
product with melting point of 84.degree. C. and with a specific rotation 
of [.alpha.].sub.D =-15.degree. (c=1%, pyridine) 
STEP 2: N-(1-oxooctadecyl)-L-alanine 
A suspension of 21 g of above product in 500 ml of methanol was cooled to 
0.degree. C. and 62 ml of N potassium hydroxide were added dropwise. The 
mixture was stirred for 16 hours at room temperature and 500 ml of 
methanol were added. The mixture was stirred for 4 hours, cooled to 
0.degree. C., brought to a pH of 3 using 2N hydrochloric acid and 
evaporated. The residue was taken up in methylene chloride and the mixture 
was washed with saline solution, dried and taken to dryness under reduced 
pressure. The residue was made into a paste with isopropyl ether vacuum 
filtered and crystallized from methanol to obtain 11.8 g of the expected 
product melting at 102.degree. C. and having a specific rotation of 
[.alpha.].sub.D =-18.degree. (c=0.9% in pyridine). 
STEP 3: 5-(1,1-dimethylethyl) 1-methyl N-[N-(1-oxooctadecyl) 
-L-alanyl]-D-glutamate 
A suspension of 1.77 g of N-(1-oxooctadecyl)-L-alanine and 1.08 g of 
5-(1,1-dimethylethyl)-1-methyl D-glutamate in 170 ml of dimethoxyethane 
was cooled to 0.degree. C. and a solution of 1.23 g of 
N,N-dicyclohexylcarbodiimide in 5 ml of dimethoxyethane was added dropwise 
over 10 minutes. The mixture was stirred for 3 hours at room temperature, 
cooled and filtered. The filtrate was evaporated to dryness and the 
residue was taken up in 250 ml of ether in the heated state. The mixture 
was filtered and the filtrate was evaporated to dryness under reduced 
pressure. The residue was made into a paste in isopropyl ether and vacuum 
filtered to obtain 2.4 g of the expected product melting at 
.perspectiveto.100.degree. C. after crystallization in methanol and having 
a specific rotation of [.alpha.].sub.D =-32.degree..+-.1.degree. (c=1% in 
CH.sub.2 Cl.sub.2). 
STEP 4: 1-Methyl N-[N-(1-oxooctadecyl)-L-alanyl]-D-glutamate 
31 ml of trifluoroacetic acid were added dropwise to a solution of 11.4 g 
of the product of Step 3 in 150 ml of methylene chloride and the mixture 
was left for 16 hours with stirring and then evaporated to dryness under 
reduced pressure. The residue was taken up in methylene chloride and the 
mixture was washed with saline solution, dried and filtered. The filtrate 
was evaporated to dryness under reduced pressure and the residue was then 
crystallized from ethanol to obtain 6.8 g of the expected product melting 
at .perspectiveto.100.degree. C. and having a specific rotation of 
[.alpha.].sub.D =-16.degree..+-.1.degree. (c=0.9% in pyridine). 
EXAMPLE 4 
2-Amino-6-(.gamma.-D-glutamylamino)-3-heptenedioic acid 
Using the procedure of Example 1, triethyl 
5-amino-1-formylamino-2-pentene-1,1,5-tricarboxylate was reacted to obtain 
the expected product. 
Preparation of trietyl 5-amino-1-formylamino-2-pentene-1,1,5-tricarboxylate 
Using the procedure for the preparation of triethyl 
5-amino-1-(formylamino)-3-methylene-1,1,5-pentanetricarboxylate, 
chloro-methylpropene replaced the allyl bromide to obtain the expected 
product in the form of an oil with an Rf=0.4 in ethyl acetate/ethanol 4:1. 
EXAMPLE 5 
2-Glycylamino-6- 
[N-(1-oxododecyl)-L-alanyl-.gamma.-D-glutamyl]-amino-2-heptenedioic acid 
Using the procedure of Example 1, 1-methyl 
N-[N-(1-oxododecyl)-L-alanyl]-D-glutamate [prepared like 1-methyl 
N-[N(1-oxooctadecyl)-L-alanyl]-D-glutamate in Example 3 by replacing 
stearyl by lauryl (dodecyl)] was reacted with 7-ethyl 1-methyl 
6-amino-2-[(N-formylglycyl)-amino]-3-heptenedioate. In this case, 
deformylation with hydrochloric acid was not necessary and the 
neutralization and the desalification were carried out simultaneously 
using a H.sup.+ resin (Amberlyst 15). The expected product, which became 
resinous at 150 C, was obtained and had a specific rotation of 
[.alpha.].sub.D =-14 (c=0.8% in water). 
EXAMPLE 6 
Mixture of (2D, 6L)- and (2L, 
6D)-2-amino-6-(.gamma.-D-glutamylamino)-4-methyleneheptanedioic acid 
STEP A: Mixture of(2D, 6L)-and (2L, 6D)-2-formylamino-4-methylene 
-6-[N-(trifluoroacetyl)- -D-glutamylamino]-heptanedioic acids 
1.3 g of dicyclohexylcarbodiimide were added at 0 C to a solution 
containing 1.5 g of the mixture of diethyl (2D,6L)- and (2L, 
6D)-2-formamido-4-methylene-6-amino-1,7-heptanedioate and 1.48 g of 
D-glutamic acid in 150 ml of dimethoxyethane, and the resulting mixture 
was stirred for 16 hours while the temperature is maintained at 0.degree. 
C. The precipitate was filtered off and the filtrate was concentrated to 
dryness under reduced pressure. The 10.5 g of crude product were purified 
by chromatography on silica (eluant:cyclohexane/ethyl acetate 2:8) to 
obtain the expected product with a specific rotation of [.alpha.].sub.D 
=-14.degree. (c=0.8% in water). 
STEP B: Mixture of (2D,6L)- and 
(2L,6D)-2-amino-6-(.gamma.-D-glutamylamino)-4-methyleneheptanedioic acids 
5 ml of 12N hydrochloric acid were added to 1.9 g of the product of Step A 
dissolved in 100 ml of ethanol, and the mixture was stirred for 30 
minutes. The solvent was removed under reduced pressure and the residue 
was taken up in 50 ml of ethanol. The pH was adjusted to 7 with 0.1N 
sodium hydroxide solution and the mixture was cooled to 0.degree. C. and 
16 ml of N sodium hydroxide were added dropwise. The reaction medium was 
allowed to return to room temperature, stirred for 16 hours and 
neutralized with N hydrochloric acid. The solvents were removed under 
reduced pressure and the residue was chromatographed on silica 
(eluant:ethanol/ammonia solution 95:5, 90:1 and then 80:20). The resin 
obtained was taken up in water and the mixture was filtered and the 
filtrate was lyophilized to obtain 0.92 g of expected product with a 
specific rotation of [.alpha.].sub.D =-13.degree..+-.1.degree. (c=1% in 3N 
HCl). 
EXAMPLE 7 
Mixture of (2D, 6D) , 
6L)-2-amino-6-(.gamma.-D-glutamylamino)-4-methyleneheptanedioic acids 
STEP A: Mixture of (2D, 6D)- and (2L,6L)-2-formylamino-4-methylene 
-6-[N-(trifluoroacetyl)-.gamma.-D-glutamylamino]-heptanedioic acids 
Using the procedure of Step A of Example 6, a mixture of diethyl (2D,6D)- 
and (2L,6L)-2-formamido-4-methylene-6-amino-1,7-heptanedioates was reacted 
to obtain the expected product. 
STEP B: Mixture of (2D,6D)- and (2L,6L)-2-amino-6-(.gamma.-D-glut 
amylamino)-4-methyleneheptanedioic acids 
Using the procedure of Step B of Example 6, the product of Step A was 
reacted to obtain the expected product having a specific rotation of 
[.alpha.].sub.D =-12.5.degree..+-.1.degree. (c=[lacuna], 3N HCl) 
The mixture of diethyl (2D,6D)- and 
(2L,6L)-2-formamido-4-methylene-6-amino-1,7-heptanedioates used at the 
beginning of Example 7 was prepared as described in the preparation of the 
Example (Step C), starting with the mixture of diethyl (2D,6D)- and 
(2L,6L)-2-formamido-4-methylene-6-tert-butoxycarbonylamino-1,7-heptanedioa 
tes (isomer I) obtained in Step B of this preparation. 
Preparation of 7-ethyl 1-methyl 
6-amino-2-[(N-formylglycyl)amino]-3-heptenedioate 
Using the procedure of preparation B of Example 1, diethyl 
(formylamino)-(2-methyl-2-propenyl)-propanedioate in Step 2 was replaced 
by methyl 2-[(N-formylglycyl)-amino]-2-(2-propenyl)ethanoate to obtain the 
expected product with an Rf=0.2 in a methylene chloride/methanol (9:1) 
mixture. 
Comment: 
The reduction of the azide to amine (Step 4 of preparation B of Example 1) 
was carried out by hydrogenation in the presence of palladium on calcium 
carbonate and of quinoline, instead of triphenyl phosphine. 
Preparation of the mixture of diethyl (2D,6L)- and (2L, 6D)-2-formamido 
-4-methylene-6-amino-1,7-heptanedioates 
STEP A: Triemethyl 
1-formamido-3-methylene-5-(tertbutoxycarbonylamino)-1,1,5-pentanetricarbox 
ylate 
13.6 g of di-tert-butyl pyrocarbonate dissolved in 25 ml of methylene 
chloride were added at room temperature to 19.5 g of triethyl 
1-formamido-3-methylene-5-amino-1,1,5-pentanetricarboxylate dissolved in 
75 ml of methylene chloride, and the mixture was maintained with stirring 
at room temperature for 36 hours. The reaction medium was diluted with 150 
ml of methylene chloride and the organic phase was washed with water and 
dried. The solvents were removed under reduced pressure and the mixture 
was left to crystallize at +4.degree. C. to obtain 25.8 g of expected 
product melting at 68.degree. C. 
STEP B: Mixture of diethyl (2D,6D)- and (2L,6L-2-formamido-4-methylene 
-6-(tert-butoxycarbonylamino)-1,7-heptanedioates and mixture of (2D,6L) 
and (2L,6D) isomers 
2.21 g of diethylcaesium and 9.42 g of p-aminophenol were added to 17.25 g 
of product of Step A dissolved in 250 ml of dimethylformamide, and the 
mixture was heated for 3 hours to 85.degree. C. The mixture was allowed to 
return to room temperature and was filtered. The solvents were removed 
under reduced pressure and the residue was taken up in 300 ml of methylene 
chloride. The organic phase was washed with 0.5N hydrochloric acid and 
then with aqueous sodium bicarbonate solution and dried. The solvent was 
removed under reduced pressure and the residue was chromatographed on a 
silica column (eluant:cyclohexane/ethyl acetate 6:4) to obtain 4.70 g of 
isomer I (DD,LL isomer), 3.84 g of isomer 11 (DL,LD isomer) and 2.80 g of 
a mixture of the 2 isomers. 
STEP C: Mixture of diethyl (2D,6L)- and 
(2L,6D)-2-formamido-4-methylene-6-amino -1,7-heptanedioates 
25 ml of 2N hydrochloric acid in ether were added to 3 g of product II of 
Step B (DL,LD isomer) dissolved in 50 ml of methylene chloride. The 
mixture was stirred at room temperature for 30 minutes and concentrated 
under an inert atmosphere. The amine hydrochloride was taken up in 
methylene chloride to which triethylamine had been added, and the mixture 
was then evaporated to dryness under reduced pressure. After 
chromatography on silica (eluant:ethyl acetate/cyclohexane 7:3), 1.86 g of 
expected product were recovered. 
EXAMPLE 8 
Tablets were prepared containing 50 mg of 
2-amino-6-(.gamma.-D-glutamylamino)-4-methyleneheptanedioic acid and 
sufficient excipient of lactose, starch, talc, magnesium stearate for a 
final weight of 100 mg. 
EXAMPLE 9 
Tablets were prepared containing 50 mg of 
2-(L-alanylamino)-6-(.gamma.-D-glutamylamino)-4-methylene-heptanedioic 
acid and sufficient excipient of lactose, starch, talc, magnesium stearate 
for a final weight of 100 mg. 
PHARMACOLOGICAL STUDY 
Stimulation of monocytic cells with an immunostimulant 
The mononuclear cells of the circulating blood of normal donors were 
separated by the classical technique described by Boyum using a Ficoll 
gradient. After being washed, the mononuclear cells were incubated at 
37.degree. C. for 1 hour in the proportion of 5.times.10.sup.6 monocytes 
(NSE.sup.+ cells) per ml of culture medium, 5 ml per culture bottle. The 
culture medium used in this experiment was composed of RPMI 1640 to which 
antibiotics and HEPES buffer had been added. After one hour, the 
non-adherent cells were removed by washing the bottles with medium 
previously brought to 37 C, and the adherent cells, composed essentially 
of monocytes (&gt;90%), were cultured again in the presence of different 
amounts of test products in a PBS medium (Dulbecco) without Ca.sup.2+ or 
Mg.sup.2+. Culturing was continued for 24 or 48 hours and the cell 
supernatants were then withdrawn, centrifuged, aliquoted and stored either 
at -80.degree. C. or at -20.degree. C. The culture supernatants were 
replaced in the bottles by the same amount of pyrogen-free distilled water 
to Lyse the cells. The Lysate was recovered, aliquoted and also stored at 
-20.degree. C. The following experiments were carried out in the presence 
or absence of interferon-.gamma. (10.sup.3 U/ml) at a dose at which 
IFN-.gamma. alone was inactive. 
Tests for the presence in the supernatants of monokines (interleukin-1 and 
tumor necrosis factor) on the basis of their biological activity. 
Interleukin-1 (IL-1) Test: 
This test was first described by Gery et al in 1972 [Gery et al, 1972 
Potentiation of the T Lymphocyte response to mitogens. II. The Cellular 
Source of Potentiating Mediator(s). J. Exp. Med., 136-143]. It is based on 
the co-mitogenic action of IL-1 in the presence of an antigen (mimicked by 
phytohaemagglutinin in the test) on mouse thymocytes. 1.5.times.10.sup.6 
thymocytes of C.sub.3 H/HeJ mice (supplied by C.S.E.A.L. of Orleans) were 
cultured for 3 days in the presence of different dilutions of cell 
supernatants and lysates likely to contain IL-1 activity, and Wellcome 
PHA-P (1 .mu.g/ml), in culture plates having 96 flat-bottomed wells, in a 
final volume of 200 .mu.l of medium composed of RPMI 1640 containing, in 
addition to antibiotics (penicillin 1 .gamma./ml streptomycin 1000 U/ml , 
1 mM HEPES buffer, 2 mM glutamine, 5% calf serum and 5.times.10.sup.-5 M 
2-mercaptoethanol. After 68 hours at culturing, 1 .mu.Ci of tritiated 
thymidine was added to each well ([methyl.sup.3 H]thymidine, CEA Saclay, 
TMM79A, specific activity 1 .mu.Ci/mM), and the radioactivity incorporated 
by the cells was measured after the cultures had been filtered on a Skaton 
type semi-automatic collecting apparatus and the filters counted in a 
scintillation counter (LKB). The results were expressed as the difference 
between the pulses per minute incorporated by the cultures in the presence 
of supernatants and the pulses per minute incorporated by the control 
cultures. 
Tumor Necrosis Factor (TNF) Test: 
TNF activity was demonstrated by the toxicity of this factor on L-929 
target cells (sub-clone .alpha.). The technique was sensitized by adding 
actinomycin D to the test. The L cells were distributed in the proportion 
of 2.times.10.sup.4 cells per well of a flat-bottomed microplate in 100 ul 
of RPMI 1640 medium enriched with 5% calf serum, glutamine, HEPES buffer 
and antibiotics. After 24 hours, different dilutions of the test 
supernatants were added in a volume of 100 .mu.l, as well as a dose of 
actinomycin D of 1 .mu.g/ml. After 24 hours of culturing, the number of 
unlysed viable cells was measured by staining the plates with crystal 
violet and measuring the optical density of different wells on a multiscan 
reader. 
RESULTS 
The products of Examples 1 and 2 stimulated monocytes and their IL-1 and 
TNF production. In addition, there was synergy between the products of 
Examples 1 and 2 and interferon-.gamma.. 
Various modifications of the products and processes of the invention may be 
made without departing from the spirit or scope thereof and it should be 
understood that the invention is intended to be limited only as defined in 
the appended claims.