Glutamine derivatives

Glutamine derivatives and non-toxic salts thereof have been found to have immunomodulating activities.

BACKGROUND OF THE INVENTION 
This invention relates to novel glutamine derivatives having 
immunomodulating activities. 
We previously found out that certain glutamine derivatives had 
immunosuppressive activities (Japanese Laid-Open Specifications Nos. 
36428/1980, 36453/1980 and 36454/1980). Upon further earnest 
investigation, we have now found out that certain novel glutamine 
derivatives possess immunomodulating activities and accomplished this 
invention. 
SUMMARY OF THE INVENTION 
Thus, the present invention resides in glutamine derivatives of the 
formula: 
##STR1## 
wherein X is (i) an alkylene of the formula: --CH.sub.2 --.sub.n where n 
is an integer of 1 to 4, or vinylene, or (ii) a group of the formula: 
##STR2## 
where R.sup.1 and R.sup.2 may be the same or different and are hydrogen or 
a lower alkyl with the proviso that at least one of R.sup.1 and R.sup.2 is 
a lower alkyl; and Z is hydrogen or a lower alkyl and non-toxic salts 
thereof. 
The term "immunomodulating activity" used herein is intended to encompass 
both of immunosuppressive activity and immunostimulating activity.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
The compounds according to this invention are those of the foregoing 
formula (I) and non-toxic salts thereof. These compounds include, for 
example, glutamine derivatives of the formula: 
##STR3## 
where X is as defined in Formula (I) and non-toxic salts thereof, as well 
as glutamine derivatives of the formula: 
##STR4## 
where X is as defined in Formula (I) and Y is a lower alkyl, and non-toxic 
salts thereof. 
The lower alkyl for group Y in Formula (III) is an alkyl having 1 to 4 
carbon atoms which includes methyl, ethyl, propyl, butyl, isopropyl, 
sec-butyl and tert-butyl. 
In Formula (I), the glutamine moiety may be any of L-, DL- and D-isomers. 
The glutamine derivatives of Formula (I) include, for example: 
##STR5## 
In the above structural formulas, the glutamic acid residue 
##STR6## 
is abbreviated as "Glu" for brevity. 
Also for brevity, the individual compounds may hereinafter be expressed by 
the above number, for example, as "Compound (1)". 
Non-toxic salts of these L-, D- or DL-glutamine derivatives are 
pharmaceutically acceptable salts which include, for example, salts with 
an inorganic base such as an alkali or alkaline earth metal, e.g., sodium, 
potassium, calcium, etc.; salts with an organic base such as procaine, 
N,N'-dibenzylethylenediamine, etc.; acid addition salts such as 
hydrochloride, sulfate, fumarate, maleate, formate, etc.; and the like. 
The compounds according to this invention may be prepared in the following 
manner. For convenience, the preparation of the compounds of this 
invention will be described in two groups of the compounds of Formula (II) 
(hereinafter referred to as "carboxylic acids of this invention") and 
those of Formula (III) (hereinafter referred to as "esters of this 
invention"). 
The carboxylic acids of this invention may be prepared by various synthetic 
methods. For example, they may readily be prepared from an appropriate 
ester of this invention by a conventional ester hydrolysis procedure. 
The esters of this invention may be prepared in any conventional manner. 
For example, they may be obtained by the reaction of amino-protected 
glutamic acid anhydride with an aniline derivative of the formula: 
##STR7## 
where X and Y are as defined in Formula (III), followed by elimination of 
the amino-protecting group. 
Another process for the preparation of the esters of this invention 
comprises reacting glutamic acid in which both the .alpha.-carboxyl and 
.alpha.-amino groups are protected with an aniline derivative of Formula 
(IV) in the presence of an activating agent or reacting a reactive 
derivative at the .gamma.-carboxyl group of such .alpha.-carboxyl- and 
.alpha.-amino-protected glutamic acid with the foregoing aniline 
derivative, followed by elimination of the amino- and carboxyl-protecting 
groups. 
Any activating agent or reactive derivative which can be used in the 
conventional peptide synthesis may be employed. Illustrative thereof are 
such activating agents as dicyclohexylcarbodiimide, carboxyldiimidazole, 
etc., and such reactive derivatives as mixed acid anhydrides, activated 
esters, etc. 
The protective group for amino may be any group which can be eliminated 
later under mild conditions and which is used in the conventional peptide 
synthesis. Examples of such group include benzyloxycarbonyl group which is 
eliminable with hydrogen bromide or by catalytic reduction, phthalyl group 
eliminable with hydrazine, tert-butoxycarbonyl and formyl groups which are 
eliminable under weakly acidic conditions, and the like. 
The protective group for carboxyl includes esters with benzyl or its 
derivative which is eliminated by catalytic reduction, tert-butyl esters 
which are stable to alkalis and eliminated by hydrolysis with an acid. 
By way of example, N-(4-ethoxycarbonylmethylphenyl)-L-glutamine can be 
prepared by either of the two synthetic routes which are shown by the 
following reaction scheme. 
##STR8## 
Similarly, N-[4-(1-ethoxycarbonyl-n-propyl)phenyl]-L-glutamine can be 
prepared, for example, by the following synthetic route. 
##STR9## 
The aniline derivatives of the foregoing Formula (IV) which are used as 
starting materials can be prepared by various synthetic routes. For 
reference, examples of such synthetic routes are shown below in two groups 
with respect to X. 
(1) In the case where X is an alkylene of the formula: --CH.sub.2 --.sub.n 
or vinylene: 
##STR10## 
The reduction may be performed by catalytic hydrogenation using palladium, 
palladium black or palladium on charcoal. Alternatively, reduction with 
iron powder/NH.sub.4 Cl or the like may be applied. 
The esterification may be conducted by heating along with the starting 
alcohol in the presence of hydrochloric, sulfuric or p-toluenesulfonic 
acid, if necessary, followed by azeotropic dehydration in the presence of 
an azeotropic agent, thereby readily providing the desired ester. 
The compound 
##STR11## 
can readily be obtained by reduction of 
##STR12## 
with iron powder/NH.sub.4 Cl. 
(2) In the case where X is a group of the formula: 
##STR13## 
A diethyl 2-alkyl-2-(4-nitrophenyl)malonate which is the starting material 
in the above Process (i) can be obtained by reacting a 2-alkyl malonate 
with a strong base such as sodium hydride in N,N-dimethylformamide and 
then with p-halonitrobenzene. 
Hydrolysis of the resulting ester may be effected by reaction with sodium 
hydroxide, potassium hydroxide or the like in water or an alcohol 
(methanol, ethanol, etc.) or a mixture thereof. 
An .alpha.-alkyl-nitrophenylacetic acid can be obtained by heating a 
diethyl 2-alkyl-2-(4-nitriophenyl)malonate along with excess sodium 
hydroxide or potassium hydroxide in water or an alcohol (methanol, 
ethanol, etc.) or a mixture thereof, or by heating a 
2-alkyl-2-(4-nitrophenyl)malonic acid along with a suitable acid such as 
hydrochloric, sulfuric or p-toluenesulfonic acid in a suitable solvent 
such as an alcohol (methanol, ethanol, etc.), benzene, or toluene. 
The subsequent esterification may be performed by heating with ethanol in 
the presence of hydrochloric, sulfuric or p-toluenesulfonic acid. 
The reduction of nitro group in the next step may be effected either by 
catalytic hydrogenation using palladium or palladium black or reduction 
with iron powder/NH.sub.4 Cl. 
Ethyl 2-methyl-2-nitrophenylpropionate which is the starting material in 
the above Process (ii) can be obtained by reacting ethyl 
nitrophenylacetate with a strong base such as sodium hydride in 
N,N-dimethylformamide and then with excess methyl iodide. 
The subsequent reduction of nitro group may be effected either by catalytic 
hydrogenation using palladium or palladium black or by reduction with iron 
powder/NH.sub.4 Cl. 
The desired glutamine derivatives obtained by these processes may be 
purified by means of recrystallization, ion-exchange treatment, 
chromatography, activated charcoal treatment or the like, according to the 
conventional practice in organic chemistry. 
The compounds according to this invention are valuable as immunomodulating 
agent for use in therapy and prevention of various diseases caused by 
immunoreactions. 
Although pharmaceutical compositions for immunomodulation which contain at 
least one compound of this invention may consist essentially of one or 
more of such compounds, the compounds of this invention are generally 
used, according to the conventional manner, in admixture with one or more 
auxiliaries and/or pharmaceutically acceptable carriers, in the form of 
conventional pharmaceutical preparations, for example, tablets, fine 
granules, powders, granules, capsules, syrups for oral administration, and 
ointments, liniments, suppositories, injections for parenteral 
administration. 
The formulations of these preparations vary depending on the administration 
route, administration plan and the like. 
The dosage may vary depending on the age, condition, weight and degree of 
symptoms of the patient, the type of concomitant treatment, if any, the 
frequency of treatment, the nature of the desired effect and the like. 
The daily therapeutic dose is generally in the range of 0.1 to 100 mg/kg 
for parenteral administration and in the range of 1 to 1,000 mg/kg for 
oral administration. 
The pharmaceutical compositions for immunomodulation which comprises at 
least one compound of this invention may further contain, for example, one 
or more other immunosuppressive or immunostimulating agents or may be used 
along with such agents. 
The compounds of this invention have low toxicity and are useful as 
immunomodulation agents for use in therapy and prevention of various 
diseases caused by immunoreactions. 
The pharmaceutical compositions for immunomodulation which comprise one or 
more compounds of this invention may be used in therapy, for example, of 
the following diseases: autoimmune diseases such as rheumatoid arthritis, 
systemic lupus erythematodes (SLE), collagent disease, etc.; allergic 
diseases such as asthma, etc.; cancer; bacterial infectious diseases and 
the like. 
A mutagenicity test of Compound (2) (.about.100 .mu.g/plate) using six 
strains as stipulated in the Labor Safety and Hygiene Law in Japan as test 
strains showed that it was negative in mutagenicity, within the range of 
such experiment. 
The present invention will be further illustrated by the following 
preparation and examples. It should be understood, however, that the 
examples are given only for the purpose of illustration and not intended 
to limit the present invention in any way. 
PREATION 1 
In 15% hydrogen chloride-ethanol, p-aminophenol was heated under reflux. 
After removal of ethanol by distillation, the residue was extracted with 
ethyl acetate and the extract was washed successively with water, 
saturated sodium bicarbonate solution and water and dried. The ethyl 
acetate was then distilled off to give p-aminophenylacetic acid ethyl 
ester. 
In a similar manner, the methyl, n-propyl and n-butyl esters were obtained 
from hydrogen chloride in the appropriate alcohols. 
In 15% hydrogen chloride-ethanol, m- or o-nitrophenylacetic acid was 
ethyl-esterified and the resulting ethyl ester was hydrogenated with 
palladium catalyst in ethanol to give m- or o-aminophenylacetic acid ethyl 
ester. 
In a similar manner, ethyl 4-(p-aminophenyl)butyrate was also obtained. 
Likewise, p-nitrocinnamic acid was subjected to ethyl esterification and 
then to hydrogenation with palladium catalyst to give ethyl 
3-(p-aminophenyl)propionate. When the hydrogenation was effected by 
heating under reflux in iron powder-ammonium chloride-water-methanol, the 
resulting product was ethyl p-aminocinnamate. 
EXAMPLE 1 
N-(4-Ethoxycarbonylmethylphenyl)-L-glutamine: Compound (1) 
(a) To a mixture of 250 ml of tetrahydrofuran and 250 ml of 
N,N-dimethylformamide were added 74.28 g (0.2 mole) of 
N-carbobenzoxy-L-glutamic acid .alpha.-benzyl ester and 28 ml (0.2 mole) 
of triethylamine. Under stirring with ice cooling, 26.4 ml (0.2 mole) of 
isobutyl chlorocarbonate was then added dropwise and stirred for 15 
minutes. Thereafter, a solution of 35.84 g (0.2 mole) of ethyl 
p-aminophenylacetate in 50 ml of tetrahydrofuran and 50 ml of 
N,N-dimethylformamide was added and the mixture was stirred for 30 minutes 
under ice cooling and then for 8 hours at room temperature. 
The reaction solvent was distilled off in vacuo and to the residue were 
added 1,200 ml of ethyl acetate and 200 ml of water. The water layer was 
removed. The ethyl acetate layer was washed successively with 2 N 
hydrochloric acid, saturated sodium bicarbonate solution and saturated 
sodium chloride solution and dried over anhydrous magnesium sulfate. After 
the ethyl acetate was distilled off in vacuo, the residue was 
recrystallized from ethyl acetate-n-hexane to give an intermediate. Yield: 
96.16 g (90.7%). 
To 53.26 g (0.1 mole) of the intermediate obtained above were added 1,600 
ml of ethanol and 600 ml of water and heated until a solution was formed. 
Thereafter 0.5 g of palladium black was added and the mixture was 
subjected to hydrogenation at atmospheric pressure to eliminate the 
protective group. 
Palladium was filtered off while hot and the filtrate was treated with 
activated charcoal and concentrated. The precipitated crystals were then 
collected by filtration, washed with ice water and dried to give 28.57 g 
(93% yield) of N-(4-ethoxycarbonylmethylphenyl)-L-glutamine, m.p. 
179.8.degree.-180.5.degree. C. 
______________________________________ 
Elementary analysis (wt. %) 
C H N 
______________________________________ 
Calc. for C.sub.15 H.sub.20 N.sub.2 O.sub.5 : 
58.43 6.54 9.09 
Found: 58.59 6.60 9.23 
______________________________________ 
[.alpha.].sub.D.sup.25 =+29.5.degree. (c=1, 2 N HCl) 
The NMR and IR spectra of the product are shown in FIGS. 1 and 2, 
respectively. 
The NMR spectrum was measured at room temperature in trifluoroacetic acid 
using tetramethylsilane as a reference, while the IR spectrum was measured 
in potassium bromide. (The measurement of these spectra was made in the 
same manner also in the following examples.) 
(b) To 100 ml of tetrahydrofuran were added 7.43 g (0.02 mole) of 
N-carbobenzoxy-L-glutamic acid .alpha.-benzyl ester and 3.58 g (0.02 mole) 
of ethyl p-aminophenylacetate and under ice cooling and stirring 2.2 ml 
(0.024 mole) of phosphorus oxychloride was added and stirred for 15 
minutes. A solution of 6.4 ml (0.046 mole) of triethylamine in 30 ml of 
tetrahydrofuran was added dropwise over 25 minutes under ice cooling and 
the mixture was stirred for 1 hour under cooling and for 3 hours at room 
temperature. 
After the tetrahydrofuran was distilled off in vacuo, ethyl acetate was 
added to the residue. The ethyl acetate solution was then washed 
successively with water, 2 N hydrochloric acid, saturated sodium 
bicarbonate solution and saturated sodium chloride solution and dried over 
anhydrous magnesium sulfate. Then the ethyl acetate was distilled off in 
vacuo and the residue was recrystallized from ethyl acetate-n-hexane to 
give 6.5 g (63% yield) of an intermediate. 
The intermediate was subjected to elimination of the protective group as 
described in (a) to give 3.4 g (90% yield) of 
N-(4-ethoxycarbonylmethylphenyl)-L-glutamine. 
(c) To 200 ml of tetrahydrofuran was added 5.18 g (0.02 mole) of 
N-phthalyl-L-glutamic acid anhydride and the mixture was heated under 
reflux for 3 hours. The tetrahydrofuran solvent was then distilled off in 
vacuo. 
To the residue were added 200 ml of ethanol and 1.33 ml (0.022 mole) of 80 
wt. % hydrazine hydrate and the mixture was stirred for 1 hour at room 
temperature and then heated under reflux for 3 hours. After the ethanol 
was distilled off in vacuo, 300 ml of 2 N hydrochloric acid was added to 
the residue and stirred. The insoluble matters were then removed and the 
solution was neutralized with conc. aqueous ammonia. The precipitated 
crystals were collected by filtration, washed with ice water and dried to 
give 3.4 g (55% yield) of N-(4-ethoxycarbonylmethylphenyl)-L-glutamine, 
m.p. 179.4.degree.-180.0.degree. C. 
______________________________________ 
Elementary analysis (wt. %) 
C H N 
______________________________________ 
Calc. for C.sub.15 H.sub.20 N.sub.2 O.sub.5 : 
58.43 6.54 9.09 
Found: 58.20 6.67 9.01 
______________________________________ 
[.alpha.].sub.D.sup.26 =+28.5.degree. (c=1 2 N HCl) 
The IR spectrum of the product is shown in FIG. 3. 
EXAMPLE 2 
N-(4-Carboxymethylphenyl)-L-glutamine: Compound (2) 
A solution of 0.62 g (0.0094 mole) of 85% potassium hydroxide in 10 ml of 
water was added to a suspension of 1.45 g (0.0047 mole) of 
N-(4-ethoxycarbonylmethylphenyl)-L-glutamine in 20 ml of water and the 
mixture was stirred for 4 hours at room temperature. 
Under ice cooling, the reaction mixture was acidified to pH 3 with 2 N 
hydrochloric acid and the precipitated crystals were collected by 
filtration, washed with ice water and dried to give 1.21 g (92% yield) of 
N-(4-carboxymethylphenyl)-L-glutamine, m.p. 197.7.degree.-199.6.degree. C. 
______________________________________ 
Elementary analysis (wt. %) 
C H N 
______________________________________ 
Calc. for C.sub.13 H.sub.16 N.sub.2 O.sub.5 : 
55.71 5.75 10.00 
Found: 55.69 5.58 9.75 
______________________________________ 
[.alpha.].sub.D.sup.26 =+16.6 (c=1 0.5 N sodium carbonate) 
The NMR and IR spectra of the product are shown in FIGS. 4 and 5, 
respectively. 
EXAMPLE 3 
N-(4-Methoxycarbonylmethylphenyl)-L-glutamine: Compound (3) 
Following the procedure described in (a) of Example 1, 11.14 g (0.03 mole) 
of N-carbobenzoxy-L-glutamic acid .alpha.-benzyl ester was reacted with 
4.96 g (0.03 mole) of methyl p-aminophenylacetate to give 13.38 g (88% 
yield) of an intermediate. 
The intermediate was dissolved in 500 ml of tetrahydrofuran, 200 ml of 
methanol and 100 ml of water. To the solution was added 0.5 g of palladium 
black and the mixture was subjected to hydrogenation at atmospheric 
pressure to give 6.09 g (79% yield) of 
N-(4-methoxycarbonylmethylphenyl)-L-glutamine, m.p. 
183.3.degree.-184.2.degree. C. 
______________________________________ 
Elementary analysis (wt. %) 
C H N 
______________________________________ 
Calc. for C.sub.14 H.sub.18 N.sub.2 O.sub.5 : 
57.13 6.16 9.52 
Found: 57.10 6.05 9.74 
______________________________________ 
[.alpha.].sub.D.sup.25 =+29.2.degree. (c=1 2 N HCl) 
The IR spectrum of the product is shown in FIG. 6. 
EXAMPLE 4 
N-(4-n-Propyloxycarbonylmethylphenyl)-L-glutamine: Compound (4) 
Following the procedure described in (a) of Example 1, 11.43 g (0.03 mole) 
of N-carbobenzoxy-L-glutamic acid .alpha.-benzyl ester was reacted with 
5.8 g (0.03 mole) of n-propyl p-aminophenylacetate to give 14.05 (88% 
yield) of an intermediate. 
The intermediate was dissolved in 500 ml of tetrahydrofuran, 200 ml of 
methanol and 100 ml of water. To the solution was added 0.5 g of palladium 
black and the mixture was subjected to hydrogenation at atmospheric 
pressure to give 6.09 g (79% yield) of 
N-(4-n-propyloxycarbonylmethylphenyl)-L-glutamine, m.p. 
177.6.degree.-178.9.degree. C. 
______________________________________ 
Elementary analysis (wt. %) 
C H N 
______________________________________ 
Calc. for C.sub.16 H.sub.22 N.sub.2 O.sub.5 : 
59.61 6.88 8.69 
Found: 59.88 6.99 8.52 
______________________________________ 
[.alpha.].sub.D.sup.25 =+22.8.degree. (c=1 2 N HCl) 
The IR spectrum of the product is shown in FIG. 7. 
EXAMPLE 5 
N-(4-n-Butyloxycarbonylmethylphenyl)-L-glutamine: Compound (5) 
Following the procedure described in (a) of Example 1, 11.14 g (0.03 mole) 
of N-carbobenzoxy-L-glutamic acid .alpha.-benzyl ester was reacted with 
6.21 g (0.03 mole) of n-butyl p-aminophenylacetate to give 15.0 g (92% 
yield) of an intermediate. 
The intermediate was dissolved in 500 ml of tetrahydrofuran, 200 ml of 
methanol and 100 ml of water. To the solution was added 0.5 g of palladium 
black and the mixture was subjected to hydrogenation at atmospheric 
pressure to give 5.72 g (62% yield) of 
N-(4-n-butyloxycarbonylmethylphenyl)-L-glutamine, m.p. 
177.0.degree.-179.2.degree. C. 
______________________________________ 
Elementary analysis (wt. %) 
C H N 
______________________________________ 
Calc. for C.sub.17 H.sub.24 N.sub.2 O.sub.5 : 
60.70 7.19 8.23 
Found: 60.61 7.11 8.44 
______________________________________ 
[.alpha.].sub.D.sup.25 =+25.4.degree. (c=1 2 N HCl) 
The NMR and IR spectra of the product are shown in FIGS. 8 and 9, 
respectively. 
EXAMPLE 6 
N-(3-Ethoxycarbonylmethylphenyl)-L-glutamine: Compound (6) 
Following the procedure described in (a) of Example 1, 11.14 g (0.03 mole) 
of N-carbobenzoxy-L-glutamic acid .alpha.-benzyl ester was reacted with 
4.96 g (0.03 mole) of ethyl m-aminophenylacetate to give 12.95 g (81% 
yield) of an intermediate. 
The intermediate was dissolved in 500 ml of tetrahydrofuran, 200 ml of 
ethanol and 100 ml of water. To the solution was added 0.3 g of palladium 
black and the mixture was subjected to hydrogenation at atmospheric 
pressure to give 6.58 g (89% yield) of 
N-(3-ethoxycarbonylmethylphenyl)-L-glutamine, m.p. 
175.5.degree.-176.2.degree. C. 
______________________________________ 
Elementary analysis (wt. %) 
C H N 
______________________________________ 
Calc. for C.sub.15 H.sub.20 N.sub.2 O.sub.5 : 
58.43 6.54 9.09 
Found: 58.41 6.46 9.01 
______________________________________ 
[.alpha.].sub.D.sup.25 =+26.4.degree. (c=1 2 N HCl) 
The IR spectrum of the product is shown in FIG. 10. 
EXAMPLE 7 
N-(2-Ethoxycarbonylmethylphenyl)-L-glutamine: Compound (7) 
Following the procedure described in (a) of Example 1, 11.14 g (0.03 mole) 
of N-carbobenzoxy-L-glutamic acid .alpha.-benzyl ester was reacted with 
4.96 g (0.03 mole) of ethyl o-aminophenylacetate to give 14.19 g (88% 
yield) of an intermediate. 
The intermediate was dissolved in 200 ml of tetrahydrofuran, 200 ml of 
ethanol and 100 ml of water. To the solution was added 0.3 g of palladium 
black and the mixture was subjected to hydrogenation at atmospheric 
pressure to give 6.85 g (84% yield) of 
N-(2-ethoxycarbonylmethylphenyl)-L-glutamine, m.p. 
171.2.degree.-171.8.degree. C. 
______________________________________ 
Elementary analysis (wt. %) 
C H N 
______________________________________ 
Calc. for C.sub.15 H.sub.20 N.sub.2 O.sub.5 : 
58.43 6.54 9.09 
Found: 58.16 6.31 8.99 
______________________________________ 
[.alpha.].sub.D.sup.25 =+21.6.degree. (c=1 2 N HCl) 
The IR spectrum of the product is shown in FIG. 11. 
EXAMPLE 8 
N-[4-(2-Ethoxycarbonylethyl)phenyl]-L-glutamine: Compound (8) 
Following the procedure described in (a) of Example 1, 11.14 g (0.03 mole) 
of N-carbobenzoxy-L-glutamic acid .alpha.-benzyl ester was reacted with 
5.8 g (0.03 mole) of ethyl p-aminophenylpropionate to give 12.8 g (78% 
yield) of an intermediate. 
The intermediate was dissolved in 200 ml of tetrahydrofuran, 200 ml of 
ethanol and 100 ml of water. To the solution was added 0.3 g of palladium 
black and the mixture was subjected to hydrogenation at atmospheric 
pressure to give 7.09 g (92% yield) of 
N-[4-(2-ethoxycarbonylethyl)phenyl]-L-glutamine, m.p. 
179.5.degree.-180.6.degree. C. 
______________________________________ 
Elementary analysis (wt. %) 
C H N 
______________________________________ 
Calc. for C.sub.16 H.sub.22 N.sub.2 O.sub.5 : 
59.71 6.88 8.69 
Found: 59.63 6.68 8.62 
______________________________________ 
[.alpha.].sub.D.sup.25 =+25.8.degree. (c=1 2 N HCl) 
The IR spectrum of the product is shown in FIG. 12. 
EXAMPLE 9 
N-[4-(3-Ethoxycarbonyl-n-propyl)phenyl]-L-glutamine: Compound (9) 
Following the procedure described in (a) of Example 1, 11.14 g (0.03 mole) 
of N-carbobenzoxy-L-glutamic acid .alpha.-benzyl ester was reacted with 
6.22 g (0.03 mole) of ethyl p-aminophenylbutyrate to give 14.7 g (87% 
yield) of an intermediate. 
The intermediate was dissolved in 200 ml of tetrahydrofuran, 200 ml of 
ethanol and 100 ml of water. To the solution was added 0.3 g of palladium 
black and the mixture was subjected to hydrogenation at atmospheric 
pressure to give 8.6 g (98% yield) of 
N-[4-(3-ethoxycarbonyl-n-propyl)phenyl]-L-glutamine, m.p. 
179.0.degree.-180.1.degree. C. 
______________________________________ 
Elementary analysis (wt. %) 
C H N 
______________________________________ 
Calc. for C.sub.17 H.sub.24 N.sub.2 O.sub.5 : 
60.70 7.19 8.33 
Found: 60.37 6.99 8.55 
______________________________________ 
[.alpha.].sub.D.sup.25 =+24.0.degree. (c=1 2 N HCl) 
The NMR and IR spectra of the product are shown in FIGS. 13 and 14, 
respectively. 
EXAMPLE 10 
N-[4-(2-Ethoxycarbonylvinyl)phenyl]-L-glutamine: Compound (10) 
Following the procedure described in (c) of Example 1, 25.9 g (0.1 mole) of 
N-phthalyl-L-glutamic acid anhydride was reacted with ethyl 
p-aminocinnamate and then treated with hydrazine to give 11.3 g (35% 
yield) of N-[4-(2-ethoxycarbonylvinyl)phenyl]-L-glutamine, m.p. 
192.5.degree.-193.3.degree. C. 
______________________________________ 
Elementary analysis (wt. %) 
C H N 
______________________________________ 
Calc. for C.sub.16 H.sub.20 N.sub.2 O.sub.5 : 
59.99 6.29 8.75 
Found: 59.71 6.14 8.91 
______________________________________ 
[.alpha.].sub.D.sup.25 =+30.3.degree. (c=1 2 N HCl) 
The NMR and IR spectra of the product are shown in FIGS. 15 and 16, 
respectively. 
EXAMPLE 11 
N-(4-Ethoxycarbonylmethylphenyl)-L-glutamine: Compound (11) hydrochloride 
A mixture of 5.0 g (0.016 mole) of 
N-(4-ethoxycarbonylmethylphenyl)-L-glutamine in 100 ml of water and 200 ml 
of ethanol was heated until dissolution was completed and 10 ml of 21 wt. 
% hydrogen chloride-ethanol solution was added. The solvent was then 
distilled off in vacuo and the residue was taken up in ethanol. After 
treatment of the solution with activated charcoal, ether was added thereto 
for crystallization. The precipitated crystals were sucked off and dried 
in vacuo to give 2.6 g (0.0075 mole, 47% yield) of 
N-(4-ethoxycarbonylmethylphenyl)-L-glutamine hydrochloride, m.p. 
154.8.degree.-155.6.degree. C. 
______________________________________ 
Elementary analysis (wt. %) 
C H N Cl 
______________________________________ 
Calc. for C.sub.15 H.sub.21 N.sub.2 O.sub.5 Cl.sub.1 : 
52.25 6.14 8.12 10.28 
Found: 54.42 5.60 7.81 10.94 
______________________________________ 
EXAMPLE 12 
N-(4-Ethoxycarbonylmethylphenyl)-D-glutamine: Compound 12 
Following the procedure described in (c) of Example 1, 12.96 g (0.05 mole) 
of N-phthalyl-D-glutamic acid anhydride was reacted with 8.96 g (0.05 
mole) of ethyl p-aminophenylacetate and then treated with hydrazine to 
give 8.79 g (57% yield) of N-(4-ethoxycarbonylmethylphenyl)-D-glutamine, 
m.p. 176.2.degree.-177.2.degree. C. 
______________________________________ 
Elementary analysis (wt. %) 
C H N 
______________________________________ 
Calc. for C.sub.15 H.sub.20 N.sub.2 O.sub.5 : 
58.43 6.54 9.09 
Found: 58.21 6.44 9.27 
______________________________________ 
[.alpha.].sub.D.sup.26 =-28.0.degree. (c=1 2 N HCl) 
The NMR and IR spectra of the product are shown in FIGS. 17 and 18, 
respectively. 
PREATION 2 
Ethyl-2-(4-aminophenyl)-n-butyrate 
In 100 ml of N,N-dimethylformamide was suspended 11 g of 50% sodium hydride 
which had been washed with n-hexane and the suspension was stirred under 
ice cooling. Thereafter, 37.6 g (0.2 mole) of diethyl ethylmalonate was 
added dropwise thereto and stirring was continued until evolution of 
hydrogen ceased. Subsequently, a solution of 31.51 g (0.2 mole) of 
p-chloronitrobenzene in 50 ml of N,N-dimethylformamide was added dropwise. 
At the end of the dropwise addition, the mixture was heated on an oil bath 
at 100.degree. C. for 9 hours and N,N-dimethylformamide was distilled off 
in vacuo. The residue was extracted with ethyl acetate and the ethyl 
acetate layer was washed with 5% hydrochloric acid and saturated sodium 
chloride solution and then dried over magnesium sulfate. The ethyl acetate 
was distilled off in vacuo and the residue was subjected to chromatography 
on silica gel eluting with a mixture of benzene and n-hexane (1:1) to give 
48.64 g (0.157 mole, 79% yield) of diethyl 
2-ethyl-2-(4-nitrophenyl)malonate. 
In 150 ml of ethanol was dissolved 37.18 g (0.12 mole) of diethyl 
2-ethyl-2-(4-nitrophenyl)malonate and a solution of 30.41 g (0.76 mole) of 
sodium hydroxide in 100 ml of water was added and then heated under reflux 
for 3 hours on an oil bath. After the solvent was distilled off in vacuo, 
the residue was dissolved in 200 ml of water and then extracted with 300 
ml of ether. The ether layer was removed and the water layer was acidified 
with conc. hydrochloric acid and extracted again with ether. The ether 
layer was separated, washed with saturated sodium chloride solution and 
dried over magnesium sulfate. The ether was distilled off in vacuo to give 
17.25 g (0.083 mole, 69% yield) of 2-(4-nitrophenyl)-n-butyric acid. 
In 250 ml of ethanol was dissolved 16.71 g (0.08 mole) of 
2-(4-nitrophenyl)-n-butyric acid and 15 ml of conc. sulfuric acid was then 
added and heated under reflux for 3.5 hours on an oil bath. After the 
ethanol was distilled off in vacuo, the residue was neutralized with 
saturated sodium bicarbonate solution and extracted with ethyl acetate. 
The ethyl acetate layer was washed with saturated sodium chloride solution 
and dried over sodium sulfate. After the ethyl acetate was distilled off 
in vacuo, the residue was subjected to silica gel chromatography eluting 
with a mixture of benzene and n-hexane (1:1) to give 9.13 g (0.0385 mole, 
48% yield) of ethyl 2-(4-nitrophenyl)-n-butyrate. 
A solution of 9.13 g (0.0385 mole) of ethyl 2-(4-nitrophenyl)-n-butyrate in 
150 ml of ethanol was subjected to hydrogenation in the presence of 
palladium black catalyst to give 7.51 g (0.0363 mole, 94% yield) of ethyl 
2-(4-aminophenyl)-n-butyrate. 
PREATION 3 
Ethyl 2-(4-aminophenyl)propionate 
Following the procedure described in Preparation 2, 35.28 g (0.1196 mole) 
of diethyl 2-methyl-2-(4-nitrophenyl)malonate was obtained from 25.0 g 
(0.159 mole) of p-chloronitrobenzene and 29.08 g (0.167 mole) of diethyl 
methylmalonate (75% yield), and this intermediate was then subjected to 
hydrolysis, decarboxylation, esterification with ethanol-sulfuric acid and 
reduction with palladium catalyst in the same way as in Preparation 2 to 
give 7.86 g (0.041 mole, 26% yield) of ethyl 2-(4-aminophenyl)propionate. 
PREATION 4 
Ethyl 2-(4-aminophenyl)-2-methylpropionate 
In 100 ml of N,N-dimethylformamide was suspended 10.56 g (equivalent to 
0.22 mole) of 50% sodium hydride which had been washed with n-hexane and 
the suspension was stirred under ice cooling. A solution of 20.9 g (0.1 
mole) of ethyl 4-nitrophenylacetate in 100 ml of N,N-dimethylformamide was 
then added dropwise and stirring was continued for 1 hour under ice 
cooling and for 2 hours at room temperature. After the 
N,N-dimethylformamide was distilled off in vacuo, the residue was 
extracted with ethyl acetate and the ethyl acetate layer was washed with 
5% hydrochloric acid and saturated sodium chloride solution and dried over 
sodium sulfate. The ethyl acetate was then distilled off in vacuo to give 
20.33 g (0.086 mole, 86% yield) of ethyl 
2-methyl-2-(4-nitrophenyl)propionate. 
In 120 ml of ethanol, 20.33 g of the ester obtained above was hydrogenated 
with 0.3 g of palladium black catalyst. The palladium catalyst was 
filtered off and the ethanol was distilled off in vacuo. The residue was 
taken up in 5% hydrochloric acid and washed with ethyl acetate. The 
separated water layer was neutralized with sodium carbonate and extracted 
with ethyl acetate. After the ethyl acetate layer was dried over sodium 
sulfate, the ethyl acetate was distilled off in vacuo and the residue was 
subjected to silica gel chromatography using chloroform as a solvent to 
give 8.31 g (0.04 mole, 47% yield) of ethyl 
2-(4-aminophenyl)-2-methylpropionate. 
EXAMPLE 13 
N-[4-(1-Ethoxycarbonyl-n-propyl)phenyl]-L-glutamine: Compound (13) 
To 150 ml of tetrahydrofuran were added 13.23 g (0.0357 mole) of 
N-carbobenzoxy-L-glutamic acid .alpha.-benzyl ester and 5 ml of 
triethylamine and the mixture was stirred under ice cooling. After 4.7 ml 
of isobutyl chlorocarbonate was added dropwise, the mixture was stirred 
under ice cooling for another 30 minutes. A solution of 7.38 g (0.0357 
mole) of ethyl 2-(4-aminophenyl)butyrate in 10 ml of tetrahydrofuran was 
then added dropwise and stirring was continued for 1 hour under ice 
cooling and for 18 hours at room temperature. After the tetrahydrofuran 
was distilled off in vacuo, the residue was extracted with ethyl acetate 
and the ethyl acetate layer was washed with saturated sodium bicarbonate 
solution, 5% hydrochloric acid and saturated sodium chloride solution and 
then dried over sodium sulfate. The ethyl acetate was distilled off in 
vacuo and the residue was recrystallized from ethyl acetate-n-hexane to 
give 12.99 g (0.0232 mole, 65% yield) of an intermediate. 
This intermediate (12.99 g) was dissolved in 200 ml of ethanol and 
hydrogenated with addition of 0.3 g of palladium black. The palladium 
catalyst was then filtered off and the ethanol was distilled off in vacuo. 
The residue was recrystallized from ethanol-water to give 4.59 g (0.0136 
mole, 38% yield) of N-[4-(1-ethoxycarbonyl-n-propyl)phenyl]-L-glutamine, 
m.p. 156.8.degree.-157.0.degree. C. 
______________________________________ 
Elementary analysis 
C H N 
______________________________________ 
Calc. for C.sub.17 H.sub.24 N.sub.2 O.sub.5 : 
60.70 7.19 8.33 
Found: 60.42 7.07 8.45 
______________________________________ 
[.alpha.].sub.D.sup.27.degree. =+25.0.degree. (2 N-HCl) 
The IR spectrum (KBr) and NMR spectrum (CF.sub.3 COOH) are shown in FIGS. 
19 and 20, respectively. 
EXAMPLE 14 
N-[4-(1-Ethoxycarbonyl-1-methylethyl)phenyl]-L-glutamine: Compound (14) 
Following the procedure described in Example 13, 4.84 g (0.0086 mole) of an 
intermediate was obtained from 5.91 g (0.0159 mole) of 
N-carbobenzoxy-L-glutamic acid .alpha.-benzyl ester and 3.3 g (0.0159 
mole) of ethyl 2-methyl-2-(4-aminophenyl)propionate (54% yield), and it 
was hydrogenated with palladium black catalyst in the same manner as in 
Example 13 to give 1.05 g (0.0031 mole, 36% yield) of 
N-[4-(1-ethoxycarbonyl-1-methylethyl)phenyl]-L-glutamine, m.p. 
146.6.degree.-148.5.degree. C. 
______________________________________ 
Elementary analysis 
C H N 
______________________________________ 
Calc. for C.sub.17 H.sub.24 N.sub.2 O.sub.5 : 
60.70 7.19 8.33 
Found: 60.40 6.60 8.59 
______________________________________ 
[.alpha.].sub.D.sup.27.degree. =+25.7.degree. (2 N-HCl) 
The IR spectrum (KBr) is shown in FIG. 21. 
EXAMPLE 15 
N-[4-(1-Ethoxycarbonylethyl)phenyl]-L-glutamine: Compound (15) 
Following the procedure described in Example 13, 7.88 g (0.0144 mole) of an 
intermediate was obtained from 7.13 g (0.0192 mole) of 
N-carbobenzoxy-L-glutamic acid .alpha.-benzyl ester and 4.4 g (0.0192 
mole) of ethyl 2-(4-aminophenyl)propionate hydrochloride (75% yield), and 
it was hydrogenated with palladium black catalyst in the same manner as in 
Example 13 to give 2.7 g (0.0084 mole, 44% yield) of 
N-[4-(1-ethoxycarbonylethyl)phenyl]-L-glutamine, m.p. 
157.4.degree.-157.9.degree. C. 
______________________________________ 
Elementary analysis 
C H N 
______________________________________ 
Calc. for C.sub.16 H.sub.22 N.sub.2 O.sub.5 : 
59.62 6.88 8.69 
Found: 59.31 6.86 8.57 
______________________________________ 
[.alpha.].sub.D.sup.27.degree. =+27.9.degree. (2 N HCl) 
The IR spectrum (KBr) is shown in FIG. 22. 
EXAMPLE 16 
N-[4-(1-Carboxy-n-propyl)phenyl]-L-glutamine: Compound (16) 
In 10 ml of methanol was suspended 1.68 g (0.005 mole) of 
N-[4-(1-ethoxycarbonyl-n-propyl)phenyl]-L-glutamine and a solution of 0.42 
g of sodium hydroxide in 20 ml of water was added thereto and stirred for 
1.5 hours at room temperature. The solvent was then distilled off in vacuo 
to about a half volume and the remaining solution was acidified to pH 4 
with 5% hydrochloric acid. The precipitated crystals were collected by 
filtration, washed with cold water and dried in vacuo to give 1.16 g 
(0.0038 mole, 75% yield) of N-[4-(1-carboxy-n-propyl)phenyl]-L-glutamine, 
m.p. 166.5.degree.-167.0.degree. C. 
______________________________________ 
Elementary analysis 
C H N 
______________________________________ 
Calc. for C.sub.15 H.sub.20 N.sub.2 O.sub.5 : 
58.43 6.54 9.09 
Found: 57.67 6.33 9.00 
______________________________________ 
[.alpha.].sub.D.sup.27.degree. =+24.0.degree. (2 N-HCl) 
The IR spectrum (KBr) is shown in FIG 23. 
TEST 1 ACUTE TOXICITY 
The test drug was suspended in aqueous 50% solution of Tween 80 and 
administered to ddY mice weighing 20-25 g orally or intraperitoneally in 
doses indicated in Table 1 below. The number of medicated mice that died 
during 7 days after the administration is shown in Table 1. 
TABLE 1 
______________________________________ 
Dose No. of dead mice/ 
Drug (mg/kg) Route No. of test animals 
______________________________________ 
Compound (1) 
1,000 IP 0/5 
5,000 PO 0/5 
Compound (2) 
1,000 IP 0/5 
5,000 PO 0/5 
Compound (13) 
1,000 IP 0/5 
5,000 PO 0/5 
______________________________________ 
TEST 2 EFFECT ON PLAQUE FORMING CELLS IN THE SPLEEN OF MICE WHEN INOCULATED 
WITH SHEEP RED BLOOD CELLS 
Mice were inoculated by intravascular or intraperitoneal administration of 
1.times.10.sup.8 sheep cells per animal. Each group consisted of five 
mice. The test drug was administered orally or intraperitoneally for four 
consecutive days from the day when the sheep cells were administered. Four 
days later, the mice were sacrificed and the number of plaque forming 
cells (PFC) in the spleen based on the sheep red blood cells was 
determined according to the Fujiwara et al. method ["Procedure of Immune 
Experiments", Vol. 5, p. 1475, Japan, (1976)] which is a modification of 
the Canninghum method. 
The results are shown in Table 2. 
TABLE 2 
__________________________________________________________________________ 
Immunization 
Run 
Strain 
Weekly 
route of Dose PFC .times. 2,500/spleen 
No. 
of mouse 
age sheep cells 
Drug (mg/kg) 
Route 
(Mean .+-. standard 
__________________________________________________________________________ 
error) 
1 Balb/c 
4 Intravascular 
Physiological 
-- P.O. 
81.4 .+-. 6.16 
saline 
" " Azathioprine 
50 " 22.8 .+-. 2.40 
" " Compound (2) 
30 " 99.0 .+-. 4.60 
" " " 100 " 91.8 .+-. 18.6 
" " " 300 " 73.4 .+-. 12.7 
2 C3H/He 
7 I.P. Physiological 
-- P.O. 
76.5 .+-. 26.3 
saline 
" " Azathioprine 
50 " 46.7 .+-. 1.2 
" " Compound (2) 
30 " 113.0 .+-. 20.4 
" " " 100 " 160.0 .+-. 20.6 
" " " 300 " 152.4 .+-. 11.4 
__________________________________________________________________________ 
TEST 3 EFFECTS ON DELAYED HYPERSENSITIBITY 
Into the palms of the right rear limbs of mice (ddY strain, weighing 25-30 
g) was injected 40 .mu.l of sheep red blood cells the concentration of 
which was adjusted to 1.times.10.sup.7 /40 .mu.l. The test drug was 
administered intraperitoneally or orally for four consecutive days 
inclusive of the day of sheep red blood cell administration. 
Three days after the day of sheep red blood cell administration, 40 .mu.l 
of sheep red blood cells which were adjusted to have a concentration of 
5.times.10.sup.8 /40 .mu.l were administered into the palms of the left 
rear limbs. 
After 24 hours, the thickness of the palms of the right and left rear limbs 
was measured. The edema was expressed as the difference in thickness 
between the palms of the left and right rear limbs. The results are shown 
in FIG. 3, in which the edema in the medicated group is expressed as 
percent control which means the percentage of the edema found in the group 
in which physiological saline is applied (control group). 
TABLE 3 
______________________________________ 
Drug Dose (mg/kg) Route % Control 
______________________________________ 
Compound (1) 
3 I.P. 85.2 
10 " 78.2 
30 " 91.7 
30 P.O. 85.7 
100 " 76.4 
300 " 74.5 
Compound (2) 
3 I.P. 69.7 
10 " 65.9 
30 " 62.3 
30 P.O. 78.8 
100 " 66.2 
300 " 65.0 
Compound (4) 
3 I.P. 70.4 
10 " 80.2 
30 " 91.7 
30 P.O. 91.7 
100 " 91.9 
300 " 79.6 
Compound (5) 
3 I.P. 91.6 
10 " 95.7 
30 " 72.2 
30 P.O. 94.5 
100 " 84.7 
300 " 82.2 
Compound (6) 
3 I.P. 94.6 
10 " 83.2 
30 " 84.3 
30 P.O. 81.1 
100 " 82.8 
300 " 83.1 
Compound (7) 
3 I.P. 92.4 
10 " 91.1 
30 " 86.8 
30 P.O. 92.8 
100 " 84.8 
300 " 82.3 
Compound (8) 
3 I.P. 76.2 
10 " 76.7 
30 " 81.0 
30 P.O. 85.0 
100 " 80.6 
300 " 92.3 
Compound (9) 
3 I.P. 91.3 
10 " 80.2 
30 " 100.6 
30 P.O. 85.3 
100 " 96.7 
300 " 88.9 
Compound (10) 
3 I.P. 100 
10 " 101 
30 " 85.8 
30 P.O. 81.3 
100 " 109.9 
300 " 82.6 
Compound (11) 
3 I.P. 77.7 
10 " 67.3 
30 " 84.1 
30 P.O. 89.8 
100 " 80.8 
300 " 83.7 
Hydrochloride 
3 I.P. 79.6 
Compound (2) 
10 " 90.7 
30 " 90.0 
30 P.O. 81.8 
100 " 75.7 
300 " 67.2 
Compound (12) 
3 I.P. 76.1 
10 " 107.9 
30 " 89.1 
30 P.O. 85.4 
100 " 81.7 
300 " 80.7 
Compound (15) 
3 I.P. 78.0 
10 " 62.6 
30 " 67.6 
Compound (14) 
3 I.P. 77.3 
10 " 72.6 
30 " 71.9 
Compound (13) 
3 I.P. 82.1 
10 " 70.5 
30 " 64.2 
Compound (13) 
30 P.O. 89.6 
100 " 87.4 
300 " 75.7 
Azathioprine 
30 I.P. 61.0 
100 P.O. 58.2 
______________________________________ 
TEST 4 EFFECT ON RAT ADJUVANT ARTHRITIS 
Test Method 
The test animals were Sprague-Dawley rats at the age of 8 weeks and each 
group consisted of ten animals. An intracutaneous injection of 0.05 ml of 
a suspension of 0.5 mg of Mycobacterium butyricum in liquid paraffin was 
applied to each animal at the paw of the right rear limb. For 27 
consecutive days from the day before the adjuvant injection, Compound (2) 
was orally administered at doses of 3, 10 and 30 mg/kg with measurement of 
the volume of the paw. 
RESULTS 
About 10 days after the adjuvant injection, so-called secondary 
inflammations were developed. They were observed as swelling in the 
injected and non-injected limbs and tubercles in the ear, tail, limbs and 
the like. Compound (2) exerted an inhibitory action on these secondary 
inflammations in every dose. However, any definite dose dependency was not 
appreciated. In the rats affected with adjuvant arthritis, the development 
of secondary inflammations was accompanied by a decrease in body weight. 
With respect to the decrease in body weight an improving tendency, was 
noted in the group to which Compound (2) was applied. 
The inhibitory action of Compound (2) on primary inflammations caused by 
direct reaction of the adjuvant was very slight. Having now fully 
described the invention, it will be apparent to one of ordinary skill in 
the art that many changes and modifications can be made thereto without 
departing from the spirit of the invention as set forth herein.