U.S. patent application number 10/376711 was filed with the patent office on 2003-09-11 for methotrexate derivatives.
This patent application is currently assigned to CHUGAI SEIYAKU KABUSHIKI KAISHA. Invention is credited to Kato, Nobuaki, Kuroki, Toshio, Maruyama, Noriaki, Matsuoka, Hiroharu, Nakagomi, Kazuya, Suzuki, Hiroshi, Tsuji, Keiichiro.
Application Number | 20030171376 10/376711 |
Document ID | / |
Family ID | 27462860 |
Filed Date | 2003-09-11 |
United States Patent
Application |
20030171376 |
Kind Code |
A1 |
Matsuoka, Hiroharu ; et
al. |
September 11, 2003 |
Methotrexate derivatives
Abstract
Antirheumatic agent containing as an active ingredient a
compound resented by the following general formula (II): 1 {where W
is a group represented by the general formula: 2 [where R.sub.1 is
a lower alkyl group having 1-4 carbon atoms; R.sub.2 is a lower
alkyl group having 1-4 carbon atoms or a trifluoromethyl group;
R.sub.3 is a hydrogen atom, a lower alkyl group having 1-4 carbon
atoms or a trifluoromethyl group; R.sub.4 is a hydrogen atom or a
lower alkyl group having 1-4 carbon atoms: R.sub.5 is a group
represented by the general formula COOR.sub.6 (where R.sub.6 is a
hydrogen atom or a lower alkyl group having 1-4 carbon atoms) or a
group represented by the formula SO.sub.3H; and n is an integer of
1-4) or the general formula: 3 {where R7 is a lower alkyl group
having 1-4 carbon atoms; R.sub.8 is a hydrogen atom or a lower
alkyl group having 1-4 carbon atoms; R.sub.9 is a group represented
by the general formula COOR.sub.10 (where R.sub.10 is a hydrogen
atom or a lower alkyl group having 1-4 carbon atoms) or a group
represented by the formula SO.sub.3H; and m is an integer of 1-4],
or the general formula: 4 [where R.sub.11 is a hydrogen atom or a
lower alkyl group having 1-4 carbon atoms; R.sub.12 is a hydrogen
atom or a lower alkyl group having 1-4 carbon atoms; R.sub.13 is a
group represent by the general formula COOR.sub.14 (where R.sub.14
is a hydrogen atom or a lower alkyl group having 1-4 carbon atoms)
or a group represented by the formula SO.sub.3H; and l is an
integer of 1 4], or the general formula: 5 [where R.sub.15 is a
hydrogen atom or a lower alkyl group having 1-4 carbon atoms;
R.sub.16 is a hydrogen atom or a lower alkyl group having 1-4
carbon atoms; and k is an integer of 2 or 3]}. All compounds
represented by the general formula (II) 5 are novel except in the
case where k is 2.
Inventors: |
Matsuoka, Hiroharu;
(Gotenba-shi, JP) ; Suzuki, Hiroshi; (Toshima-ku,
JP) ; Kato, Nobuaki; (Gotenba-shi, JP) ;
Tsuji, Keiichiro; (Gotenba-shi, JP) ; Kuroki,
Toshio; (Gotenba-shi, JP) ; Maruyama, Noriaki;
(Gotenba-shi, JP) ; Nakagomi, Kazuya;
(Gotenba-shi, JP) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.
624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Assignee: |
CHUGAI SEIYAKU KABUSHIKI
KAISHA
Tokyo
JP
|
Family ID: |
27462860 |
Appl. No.: |
10/376711 |
Filed: |
March 3, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10376711 |
Mar 3, 2003 |
|
|
|
08256441 |
Jul 12, 1994 |
|
|
|
6559149 |
|
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08256441 |
Jul 12, 1994 |
|
|
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PCT/JP93/00096 |
Jan 27, 1993 |
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Current U.S.
Class: |
514/251 ;
544/260 |
Current CPC
Class: |
C07D 475/08 20130101;
A61P 29/00 20180101 |
Class at
Publication: |
514/251 ;
544/260 |
International
Class: |
A61K 031/525; C07D
475/08 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 27, 1992 |
JP |
53051/1992 |
Feb 13, 1992 |
JP |
75106/1992 |
Mar 16, 1992 |
JP |
108320/1992 |
Mar 24, 1992 |
JP |
115126/1992 |
Claims
What is claimed is:
1. A compound represented by the formula (I) 18wherein W is
represented by 19where R.sub.7 is a lower alkyl group having 1-4
carbon atoms; R.sub.8 is a hydrogen atom or a lower alkyl group
having 1-4 carbon atoms; R.sub.9 is represented by the formula
COOR.sub.10 wherein R.sub.10 is a hydrogen atom or a lower alkyl
group having 1-4 carbon atoms or R.sub.9 is represented by the
formula So.sub.3H; and m is an interger of 1-4, or W is represented
by 20where R.sub.11 is a hydrogen atom or a lower alkyl group
having 1-4 carbon atoms; R.sub.12 is a hydrogen atom or a lower
alkyl group having 1-4 carbon atoms; R.sub.13 is represented by the
formula COOR.sub.14 where R.sub.14 is a hydrogen atom or a lower
alkyl group having 1-4 carbon atoms or R.sub.13 is represented by
the formula SO.sub.3H; and l is an interger of 1-4.
2. A compound according to claim 1 wherein W is 21
3. A compound according to claim 1 wherein W is 22
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a divisional of U.S. Ser. No.
08/256,441 filed Jul. 12, 1994, which is the National Stage of
PCT/JP93/00096 filed Jan. 27, 1993.
TECHNICAL FIELD
[0002] This invention relates to novel methotrexate derivatives,
more particularly to novel methotrexate derivatives that are useful
as antirheumatic agents.
BACKGROUND ART
[0003] Methotrexate had long been used as a therapeutic agent for
leukemia but ever since Guboer et al. reported its effectiveness in
the treatment of rheumatoid arthritis (RA) and psoriasis in 1951,
methotrexate has been used as a therapeutic agent for RA in both
Europe and the United States of America. Fairly recently, detailed
investigations were conducted on the method of administration and
dose of methotrexate to reveal that low-dose methotrexate therapy
causes fairly less side effects and yet exhibit excellent efficacy.
However, the administration of methotrexate causes various side
effects that cannot be ignored, such as hepatopathy and pulmonary
fibrosis and, hence, a strong need exists for the advent of drugs
that cause lesser side effects without compromise in their
efficacy.
[0004] The present inventors conducted intensive studies in search
for methotrexate derivatives that exceed methotrexate in efficacy
as a rheumatism treating agent. The present invention has been
accomplished on the basis of these studies.
DISCLOSURE OF THE INVENTION
[0005] The present invention provides methotrexate derivatives that
are represented by the following general formula (II): 6
[0006] [where W is a group represented by the general formula:
7
[0007] [where R.sub.1 is a lower alkyl group having 1-4 carbon
atoms; R.sub.2 is a lower alkyl group having 1-4 carbon atoms or a
trifluoromethyl group; R.sub.3 is a hydrogen atom, a lower alkyl
group having 1-4 carbon atoms or a trifluoromethyl group; R.sub.4
is a hydrogen atom or a lower alkyl group having 1 carbon atom;
R.sub.5 is a group represented by the general formula COOR.sub.6
(where R.sub.6 is a hydrogen atom or a lower alkyl group having 1-4
carbon atoms) or a group represented by the formula SO.sub.3H; and
n is an integer of 1-4], or the general formula: 8
[0008] [where R.sub.7 is a lower alkyl group having 1-4 carbon
atoms; R.sub.8 is a hydrogen atom or a lower alkyl group having 1-4
carbon atoms; R.sub.9 is a group represented by the general formula
COOR.sub.10 (where R.sub.10 is a hydrogen atom or a lower alkyl
group having 1-4 carbon atoms) or a group represented by the
formula SO.sub.3H; and m is an integer of 1-4], or the general
formula: 9
[0009] [where R.sub.11 is a hydrogen atom or a lower alkyl group
having 1-4 carbon atoms; R.sub.12 is a hydrogen atom or a lower
alkyl group having 1-4 carbon atoms; R.sub.13 is a group
represented by the general formula COOR.sub.14 (where R.sub.14 is a
hydrogen atom or a lower alkyl group having 1-4 carbon atoms) or a
group represented by the formula SO.sub.3H; and l is an integer of
1-4], or the general formula: 10
[0010] [where R.sub.15 is a hydrogen atom or a lower alkyl group
having 1-4 carbon atoms; R.sub.16 is a hydrogen atom or a lower
alkyl group having 1-4 carbon atoms; and k is an integer of 2 or
3].
[0011] The present invention also relates to the use of these
compounds as antirheumatics. The antirheumatics of the present
invention include a known compound, namely, 3'-methylaminopterin
(see Cancer Research, Vol. 20, No. 10, 698 733, 1960); however, the
use of this compound as an anticheumatic agent has not been
known.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIGS. 1. 2 and 3 show the amounts (ratio) of .sup.3H-UdR to
be uptake at a respective concentration of drugs to be tested.
BEST MODE FOR CARRYING OUT THE INVENTION
[0013] The compounds of the present invention may typically be
synthesized by the following methods.
[0014] (Method A) 1112
[0015] (where R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 and n
have the same meanings as defined above; R' is a lower alkyl group
having 1-4 carbon atoms; A.sup.1 is a protective group; and X is a
halogen atom);
[0016] (Method B) 13
[0017] (where R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 and n
have the same meanings as defined above; A.sup.2 is a protective
group; and X is a halogen atom);
[0018] (Method C) 14
[0019] (where R.sub.11, R.sub.12, R.sub.13 and l have the same
meanings as defined above; A.sub.3 is a protective group; and X is
a halogen atom);
[0020] (Method D) 15
[0021] (where R.sub.11, R.sub.12, R.sub.13 and l have the same
meanings as defined above; A.sub.3 is a protective group; and X is
a halogen atom); and
[0022] (Method E) 16
[0023] (where R.sub.15, R.sub.16 and k have the same meanings as
defined above; and X is a halogen atom).
[0024] When implementing method A, the reaction for converting the
compound of the general formula (1) to 10 the compound of the
general formula (2) is carried out by reacting the compound of the
general formula (1) with a halogen of the formula X.sub.2 in a
solvent system composed of a mixture of water/methanol,
water/ethanol, water/propanol or the like in the presence of a
suitable reagent such as sodium hydrogen carbonate, calcium
carbonate or potassium carbonate. Examples of the halogen
represented by the general formula X.sub.2 include fluorine,
chlorine, bromine and iodine. If R.sup.2 or R.sup.3 in the compound
of the general formula (1) is trifluoromethyl, the halogen to be
reacted with is preferably iodine.
[0025] The reaction for converting the compound of the general
formula (2) to the compound of the general formula (3) is carried
out by reacting the compound of the general formula (2) with acetic
anhydride in a solvent such as toluene, benzene, xylene, dioxane or
pyridine. This reaction (acetylation) may be performed prior to the
reaction for converting the compound of the general formula (1) to
the compound of the general formula (2) (halogenation).
[0026] The reaction for converting the compound of the general
formula (3) to the compound of the general formula (4) is carried
out by reacting the compound of the general formula (3) with copper
cyanide in a suitable solvent such 15 as N-methylpyrrolidone,
dimethylformamide, dimethylacetamide, dimethylpropylene urea or
hexamethylphosphoric triamide.
[0027] The reaction for converting the compound of the general
formula (4) to the compound of the general formula (5) is carried
out by reacting the compound of the general formula (4) with a
suitable reagent such as an aqueous solution of 12N HCl or 50%
sodium hydroxide.
[0028] The reaction for producing the compound of the general
formula (7) from the compounds of the general formulas (5) and (6)
is carried out by reacting the two compounds in an alcohol such as
methanol or ethanol in the presence of HCl or H.sub.2SO.sub.4.
Alternatively, the reaction may be performed in an alcohol in the
presence of thionyl chloride. Preferred examples of the alkyl group
represented by R' in formulas (6) and (7) are methyl and ethyl.
[0029] The reaction for producing the compound of the general
formula (9) from the compounds of the general formulas (7) and (8)
is carried out by first dissolving the compound of the general
formula (7) in a solvent such as pyridine or suspending it in
tetrahydrofuran, dimethylformamide, dimethoxyethane, dioxane,
toluene or the like in the presence of sodium hydride, then adding
the compound of the general formula (8) and stirring the mixture.
Examples of the protective group represented by A.sup.1 in general
formulas (8) and (9) include a p-toluenesulfonyl group, a
benzyloxycarbonyl group and an acetyl group.
[0030] The reaction for producing the compound of the general
formula (11) from the compounds of the general formulas (9) and
(10) is carried out by first dissolving the compound of the general
formula (9) in a suitable solvent such as anhydrous
dimethylformamide, tetrahydrofuran, dimethoxyethane or dioxane,
then adding a suitable reagent such as sodium hydride under cooling
with ice, stirring the mixture at 10-120.degree. C., preferably at
room temperature, subsequently adding the compound of the general
formula (10), and stirring the mixture at 10-120.degree. C.,
preferably at room temperature.
[0031] The reaction for converting the compound of the general
formula (11) to the compound of the general formula (12) is carried
out by subjecting the compound of the general formula (11) to
reaction in a solvent such as methanol, ethanol or tetrahydrofuran
in the presence of a reagent such as an aqueous solution of
potassium hydroxide or sodium hydroxide at 10-100.degree. C.,
preferably at room temperature.
[0032] The reaction for converting the compound of the general
formula (12) to the compound of the general formula (13) is carried
out by first suspending the compound of the general formula (12) in
an acid halide former such as thionyl chloride or oxalyl chloride
and then stirring the suspension at room temperature in the
presence of a catalytic amount of a reagent such as
dimethylformamide.
[0033] The reaction for producing the compound of the general
formula (15) from the compounds of the general formulas (13) and
(14) is carried out by first dissolving the compound of the general
formula (13) in a solvent such as dichloromethane, then adding the
solution to an aqueous solution of the compound of the general
formula (14) under cooling with ice or water, and stirring the
mixture at room temperature in the presence of an inorganic base
such as potassium carbonate, sodium hydroxide or sodium hydrogen
carbonate.
[0034] The reaction for converting the compound of the general
formula (15) to the compound of the general formula (16) is carried
out by first dissolving anisole, phenol or the like in a solution
of hydrogen bromide/acetic acid, then adding the compound of the
general formula (15) to the resulting solution, and stirring the
mixture at 10-60.degree. C., preferably at room temperature. If
A.sup.1 is a carbobenzoxy group, the reaction of interest may be
carried out by first dissolving the compound of the general formula
(15) in a solvent such as methanol, ethanol or acetic acid, then
adding palladium-carbon and subsequently stirring the solution in a
hydrogen atmosphere at room temperature.
[0035] The reaction for producing the compound of the general
formula (18) from the compounds of the general formulas (16) and
(17) is carried out by stirring the two reactants in a solvent such
as dimethylacetamide or dimethylformamide at 0-100.degree. C.,
preferably at 50-60.degree. C. In a special case where R.sup.4 is a
hydrogen atom, an aqueous solution of 1N sodium hydroxide may
further be added to a solvent such as methanol, ethanol or
tetrahydrofuran and the mixture is stirred at 0-60.degree. C.,
preferably at 35.degree. C., to yield the end compound.
[0036] When implementing method B, the reaction for producing the
compound of the general formula (20) from the compounds of the
general formulas (5) and (19) is carried out by reacting the
compound of the general formula (5) with the compound of the
general formula (19) in suspension at 0-100.degree. C., preferably
at room temperature. Examples of the protective group represented
by A.sup.2 in formula (19) include a trifluoroacetyl group and an
acetyl group.
[0037] The reaction for producing the compound of the general
formula (22) from the compounds of the general formulas (20) and
(21) is carried out by first preparing an acetone solution of the
two reactants, then adding potassium hydroxide to the solution, and
stirring the mixture at 0-55.degree. C. preferably at 45-53.degree.
C.
[0038] The reaction for converting the compound of the general
formula (22) to the compound of the general formula (23) is carried
out by reacting the compound of the general formula (22) with
potassium hydroxide or sodium hydroxide in water. If desired, the
compound of the general formula (22) may be replaced by the
aforementioned compound of the general formula (11).
[0039] The reaction for producing the compound of the general
formula (24) from the compounds of the general formulas (23) and
(17) is carried out by stirring the two reactants in a solvent such
as dimethylacetamide or dimethylformamide at 0-100.degree. C.,
preferably at 40-55.degree. C.
[0040] The reaction for producing the compound of the general
formula (18) from the compounds of the general formulas (24) and
(14) is carried out by first stirring the compound of general
formula (24) in a solvent such as dimethylformamide,
dimethylacetamide or N-methylpyrrolidinone in the presence of both
diethyl cyanophosphonate or dicyclohexylcarbodiimide and
1-hydroxybenzotriazole or the like to prepare a corresponding
active ester and mixed acid anhydride, then adding the compound of
general formula (14) and stirring the mixture at 0-200.degree. C.,
preferably at 10 -80.degree. C. Alternatively, triethylamine is
added to the compound of general formula (24), then isobutyl
chloroformate is added, and the mixture is stirred in a solvent
such as dimethylformamide, dimethylacetamide or
N-methylpyrrolidinone, followed by adding the compound of general
formula (14) and stirring the mixture at -20 to 50.degree. C.,
preferably at -10 to 0.degree. C. If R.sup.4 is a hydrogen atom, an
aqueous solution of 1N sodium hydroxide is further added to a
solvent such as methanol or ethanol and the mixture is stirred at
0-60.degree. C., preferably at room temperature to yield the end
product.
[0041] When implementing method C, the reaction for converting the
compound of the general formula (25) to the compound of the general
formula (26) is carried out by refluxing the compound of general
formula (25) in acetic anhydride.
[0042] The reaction for converting the compound of the general
formula (26) to the compound of the general formula (27) is
typically carried out by reacting an acetic acid solution of the
compound of general formula (26) with a halogen of the general
formula X.sub.2. Examples of the halogen X.sub.2 include fluorine,
chlorine, bromine and iodine.
[0043] The reaction for converting the compound of the general
formula (27) to the compound of the general formula (28) is carried
out by reacting the compound of general formula (27) with copper
cyanide in a suitable solvent such as N-methylpyrrolidinone,
dimethylformamide, dimethylacetamide, dimethylpropylene urea or
hexamethylphosphoric triamide at 100-250.degree. C., preferably at
200.degree. C.
[0044] The reaction for converting the compound of the general
formula (28) to the compound of the general formula (29) is carried
out by reacting the compound of general formula (28) with a
suitable reagent such as cone. HCl or an aqueous solution of 50%
NaOH.
[0045] The reaction for producing the compound of the general
formula (31) from the compounds of the general formulas (29) and
(30) is typically carried out by first adding the compound of
general formula (29) and ether to an aqueous solution of NaOH and
then adding an ether solution of the compound of general formula
(30) to the mixture. Examples of the protective group represented
by A.sup.3 in formulas (30) and (31) include a p-toluenesulfonyl
group, a benzyloxycarbonyl group and an acetyl group.
[0046] The reaction for converting the compound of the general
formula (31) to the compound of the general formula (32) is carried
out by first suspending the compound of general formula (31) in an
acid halide former such as thionyl chloride or oxalyl chloride and
then stirring the suspension in the presence of a catalytic amount
of dimethylformamide or the like at room temperature.
[0047] The reaction for producing the compound of the general
formula (34) from the compounds of the general formulas (32) and
(33) is carried out by first dissolving the compound of general
formula (32) in a solvent such as dichloromethane, then adding an
aqueous solution of the compound of general formula (33) and
stirring the mixture at room temperature in the presence of an
inorganic base such as potassium carbonate, sodium hydroxide or
sodium hydrogen carbonate.
[0048] The reaction for converting the compound of the general
formula (34) to the compound of the general formula (35) is carried
out by first dissolving anisole, phenol or the like in a solution
of hydrogen bromide/acetic acid, then adding the compound of
general formula (34) to the solution, and stirring the mixture at
10-60.degree. C., preferably at room temperature. If A.sup.3 is a
carbobenzoxy group, the reaction of interest may be carried out by
first dissolving the compound of general formula (34) in a solvent
such as methanol, ethanol or acetic acid, then adding
palladium-carbon to the solution and thereafter stirring the
solution at room temperature in a hydrogen atmosphere.
[0049] The reaction for producing the compound of the general
formula (36) from the compounds of the general formulas (35) and
(17) is carried out by stirring the two reactants in a solvent such
as dimethylacetamide or dimethylformamide at 0-100.degree. C.,
preferably at 50-60.degree. C. If R.sub.12 and R.sub.14 are each a
hydrogen atom, an aqueous solution of 1N NaOH is further added to a
solvent such as methanol, ethanol or tetrahydrofuran and the
mixture is then stirred at 0-60.degree. C., preferably at
35.degree. C., to yield the end product.
[0050] The reaction for converting the compound of the general
formula (36) to the compound of the general formula (37) is carried
out by first dissolving the compound of general formula (36) in a
solvent such as an aqueous solution of sodium hydrogen carbonate
and then stirring the solution together with an aqueous solution of
potassium nitrosodisulfonate. Alternatively, the compound of
general formula (36) may be suspended in dioxane or the like and
the suspension is stirred in the presence of
dichlorodicyanobenzoquinone or the like. If desired, the compound
of general formula (36) may be added to an acetic acid solution of
manganese (III) acetate, followed by stirring the mixture.
[0051] When implementing method D, the reaction for producing the
compound of the general formula (38) from the compounds of the
general formulas (29) and (17) is carried out by stirring the two
reactants in a solvent such as dimethylacetamide or
dimethylformamide at 0-100.degree. C., preferably at 40-55.degree.
C.
[0052] The reaction for producing the compound of the general
formula (36) from the compounds of the general formulas (38) and
(33) is carried out by first stirring the compound of general
formula (38) in a solvent such as dimethylformamide,
dimethylacetamide or N-methylpyrrolidinone in the presence of both
diethyl cyanophosphonate or dicyclohexylcarbodiimide and
1-hydroxybenzotriazole, then adding the compound of general formula
(33), and stirring the mixture at 0-200.degree. C., preferably at
10-80.degree. C. Alternatively, triethylamine is added to the
compound of general formula (38), then isobutyl chloroformate is
added, and the mixture is stirred in a solvent such as
dimethylformamide, dimethylacetamide or N-methylpyrrolidinone;
thereafter, the compound of general formula (33) is added and the
mixture is stirred at -20 to 50.degree. C., preferably at -10 to
0.degree. C. If R.sub.12 is a hydrogen atom, an aqueous solution of
1N NaOH is added to a solvent such as methanol or ethanol and the
mixture is stirred at 0-60.degree. C., preferably at room
temperature to yield the end product.
[0053] When implementing method E, the reaction for converting the
compound of the formula (39) to the compound of the formula (40) is
carried out by suspending the compound of formula (39) in an acid
halide former such as thionyl chloride or oxalyl chloride and then
stirring the suspension at room temperature in the presence of a
catalytic amount of dimethylformamide or the like.
[0054] The reaction for producing the compound of the general
formula (42) from the compound of the formula (40) and the compound
of the general formula (41) is carried out by first dissolving the
compound of formula (40) in a solvent such as dichloromethane, then
adding the compound of general formula (41) to the solution, and
stirring the mixture at room temperature in the presence of both
water and an inorganic base such as potassium carbonate, sodium
hydroxide or sodium hydrogen carbonate.
[0055] The reaction for converting the compound of the general
formula (42) to the compound of the general formula (43) is carried
out by dissolving the compound of general formula (42) in a solvent
such as acetic acid, then adding a zinc powder under cooling with
ice, and stirring the mixture at room temperature.
[0056] The reaction under consideration may be carried out by using
an iron powder in ethanol-HCl or performing catalytic reduction in
a hydrogen atmosphere using palladium-carbon in methanol, ethanol
or tetrahydrofuran.
[0057] The reaction for producing the compound of the general
formula (44) from the compounds of the general formulas (43) and
(17) is carried out by stirring the two reactants in a solvent such
as dimethylacetamide or dimethylformamide at 0-100.degree. C.,
preferably at 50-60.degree. C. Examples of the halogen atom
represented by X in the general formula (17) include chlorine and
bromine. If R.sub.15 and R.sub.16 are each a hydrogen atom, an
aqueous solution of 1N NaOH is further added to a solvent such as
methanol, ethanol or tetrabydrofuran and the mixture is stirred at
0-60.degree. C., preferably at 20-30.degree. C. to yield the end
product.
[0058] The compounds of the general formula (II) thus produced in
accordance with the present invention have antirheumatic action,
which was verified by investigating the ability of the compounds to
inhibit the proliferation of human peripheral blood derived
lymphocytes in the experiment described below.
[0059] (Experiment) Inhibition of the Proliferation of Human
Peripheral Blood Derived Lymphocytes:
[0060] (Method)
[0061] Lymphocytes were separated from human peripheral blood by
means of Ficoll-Paque.RTM.. An adequately diluted drug under test
and 10.sup.5 of the separated lymphocytes were cultivated in a
96-well culture plate for 2 days together with PHA (0.3 .mu.g/ml).
Five hours before the end of cultivation, .sup.3H-UdR (1
.mu.Ci/well) was added and its uptake by the lymphocytes was
measured with a scintillation counter (PHA: phytohaemagglutinin;
UdR: deoxyuridine). The drugs under test were as follows: 17
[0062] (Results and Discussion)
[0063] FIGS. 1, 2 and 3 show the percent uptakes of .sup.3H-UdR by
lymphocytes treated with the drugs under test, with 100% being
assigned to the value of uptake by PHA stimulated lymphocytes in
the absence of drugs. As is clear from FIGS. 1-3, the compounds of
the present invention were found to be more effective in inhibiting
the proliferation of lymphocytes (having better antirheumatic
action) than the control compounds.
[0064] The following examples are provided for the purpose of
further illustrating the present invention but are in no way to be
taken as limiting.
REFERENCE EXAMPLE 1
Synthesis of N-carbobenzoxy-4-amino-3-methylbenzoic Acid
[0065] 4-Amino-3-methylbenzoic acid (5.0 g) was suspended in water
(80 ml) and, thereafter, an ether solution (20 ml) of
carbobenzoxychloride (3.7 g) and sodium hydrogen carbonate (3.6 g)
were alternately added to the suspension under cooling with ice.
Following stirring for 2.5 h at room temperature, an ether solution
(95 ml) of carbobenzoxychioride (3.7 g) and sodium hydrogen
carbonate (7.2 g) were further added alternately under cooling with
ice, and the mixture was stirred for 1.5 h at room temperature.
After rendering the reaction solution acidic with 4N HCl, the
separating insoluble matter was recovered by filtration. The
recovered solids were vacuum-dried to yield the end product (2.8
g).
[0066] .sup.1H-NMR (CD.sub.3COCD.sub.3, .delta.): 2.38(3H,s),
5.20(2H,s), 7.41(5H,m), 7.97(3H,m)
REFERENCE EXAMPLE 2
Synthesis of methyl N-carbobenzoxy-N-methyl-4-amino 5
3-methylbenzoate
[0067] Sodium hydride (1.4 g) was suspended in dimethylformamide
(10 ml) and, thereafter, a dimethylformamide solution (10 ml) of
the compound synthesized in Reference Example 1 (2.8 g) was added
slowly to the suspension at 0.degree. C. After stirring the
reaction solution for 1 h at room temperature, methyl iodide was
added slowly and the mixture was stirred at room temperature for 2
h. Then, the reaction solution was poured into a saturated aqueous
solution of sodium hydrogen carbonate and the mixture was subjected
to extraction with toluene. The organic layer was dried with sodium
sulfate and the solvent was distilled off under vacuum. The
resulting residue was subjected to silica gel column chromatography
and eluted with chloroform to yield the end product (1.8 g).
[0068] .sup.1H-NMR (CDCl.sub.3, .delta.): 2.18(3H,s), 3.22(3H,s),
3.89(3H,s), 5.10(2H,s), 7.21(6H,m), 7.90(2H,m)
REFERENCE EXAMPLE 3
Synthesis of N-carbobenzoxy-N-methyl-4-amino-3 methylbenzoic
Acid
[0069] An aqueous solution (15 ml) of 2N NaOH was added to an
ethanol (15 ml) solution of the compound (1.8 g) synthesized in
Reference Example 2 and the mixture was refluxed for 2 h.
Subsequently, the reaction solution was concentrated to 10 ml under
vacuum. After the concentrate was rendered acidic with 4N HCl, it
was subjected to extraction with chloroform and the extract was
dried with sodium sulfate. The solvent was distilled off under
vacuum to yield the end product (1.5 g).
[0070] .sup.1H-NMR (CDCl.sub.3, .delta.): 2.20(3H,s), 3.24(3H,s),
5.12(2H,s), 6.91-7.56(6H,m), 7.74-8.12(2H,m)
REFERENCE EXAMPLE 4
Synthesis of diethyl N-(N'-carbobenzoxyl-N'-methyl-4
amino-3-methylbenzoyl)-L-glutamate
[0071] The compound (820 mg) synthesized in Reference Example 3 was
added to thionyl chloride (5 ml); thereafter, a catalytic amount of
dimethylformamide was further added and the mixture was stirred for
2 h at room temperature. Subsequently, the reaction solution was
concentrated to dryness under vacuum. The resulting solids was
dissolved in dichloromethane (20 ml) and diethyl glutamate HCl (700
mg) and potassium carbonate (1.4 g) were added to the solution;
after adding water (20 ml) the mixture was stirred vigorously at
room temperature for 12 h. Then, the reaction solution was poured
into water and subjected to extraction with chloroform. The organic
layer was washed with 1N HCl and dried with sodium sulfate. The
solvent was distilled off ,under vacuum. The resulting residue was
subjected to silica gel column chromatography and eluted with a
solvent system composed of chloroform and methanol (100:3) to yield
the end product (700 mg).
[0072] .sup.1H-NMR (CDCl.sub.3, .delta.): 1.1-1.5(6H,m),
2.0-2.7(4H,m), 2.18(3H,s), 3.19(3H,s), 3.9-4.4(4H,m), 4.80(1H,m),
5.10(2H,s), 7.0-7.5(6H,m), 7.63(2H,m)
REFERENCE EXAMPLE 5
Synthesis of diethyl
N-[N'-methyl-(4-amino-3methylbenzoyl)]-L-glutamate
[0073] The compound (700 mg) synthesized in Reference Example 4 was
added to a solution containing anisole (700 mg) in 30% hydrogen
bromide/acetic acid (7 ml) and the mixture was stirred at room
temperature for 4 h. Subsequently, ether (200 ml) was added to the
reaction solution, whereupon a reddish brown oil was precipitated.
With the greater part of the ether layer being removed, the oil was
suspended in chloroform and the resulting suspension was washed
with a saturated solution of sodium hydrogen carbonate to recover
the organic layer by separation. The organic layer was then dried
with sodium sulfate and the solvent was distilled off under vacuum
to yield the end product (360 mg).
[0074] .sup.1H-NMR (CDCl.sub.3, .delta.): 1.0-1.5(6H,m),
2.11(3H,s), 2.90(3H,s), 2.1-2.7(4H,m), 3.9-4.4(4H,m), 4.80(1H,m),
6.54(1H,d,J=8 Hz), 6.80(1H,d,J=7 Hz), 7.61(2H,m)
EXAMPLE 1
Synthesis of diethyl N-{4-[N'-(2.4-diamino-6
Pteridinyl)Methyl-N'-Methylam-
ino]-3-Methylbenzoyl}-L-Glutamate
[0075] The compound (360 mg) synthesized in Reference Example 5 and
an isopropanol adduct of 6-bromomethYl-2,4-diaminopteridine
hydrobromide (407 mg) were suspended in dimethylacetamide (6 ml)
and the suspension was stirred at 55 -60.degree. C. for 4 h. To the
cooled reaction solution, water (30 ml) containing triethylamine
(248 mg) was added and the mixture was stirred, followed by
extraction with chloroform. The chloroform layer was dried with
sodium sulfate and the solvent was distilled off under vacuum. The
resulting residue was subjected to silica gel column chromatography
and eluted with a solvent system consisting of chloroform and
methanol (10:1) to yield the end product (280 mg).
[0076] .sup.1H-NMR (CDCl.sub.3, .delta.): 1.2-1.4(6H,m),
2.0-2.6(4H,m), 2.41(3H,s), 2.73(3H,s), 4.0-4.3(4H,m), 4.32(2H,s),
4.76(1H,m), 7.06(1H,d,J=8.3 Hz), 7.41(1H,d,J=-7.8 Hz), 7.65(2H,m),
8.79(1H,s)
EXAMPLE 2
Synthesis of
N-{4-[N'-(2,4-diamino-6-pteridinyl)methyl-N'-methylamino]-3-m-
ethylbenzoyl}-L-glutamic Acid (Compound 1)
[0077] An aqueous solution of 1N NaOH (1.6 ml) was added to an
ethanol (20 ml) solution of the compound (280 mg) synthesized in
Example 1 and the mixture was stirred at 35.degree. C. for 4 h.
Following stirring at 25.degree. C. for 20 h, water (2 ml) was
added to the reaction solution, which was then evaporated to
dryness under vacuum, with care being taken so that the external
temperature would not exceed 30.degree. C. The resulting yellow
solids were dissolved in water (10 ml) and the solution was
adjusted to pH of 3.7 with 1N HCl, followed by standing in a
refrigerator for 2 h. The separating precipitate was recovered by
filtration to yield the end product (180 mg).
[0078] .sup.1H-NMR (DMSO-d.sub.6, .delta.): 1.9-2.2(2H,m),
2.35(2H,t,J=8.0 Hz), 2.42(3H,s), 2.73(3H,s), 4.34(2H,s),
4.41(1H,m), 7.10(1H,d,J=8.3 Hz), 7.72(2H,m), 8.38(1H,d,J=7.8 Hz),
8.65(1H,s)
REFERENCE EXAMPLE 6
Synthesis of ethyl
N-carbobenzoxy-N-ethyl-4-amino-3-methyl-benzoate
[0079] Sodium hydride (164 mg) was suspended in dimethylformamide
(2 ml) in a nitrogen atmosphere and the temperature was adjusted to
0.degree. C. To the suspension, a dimethylformamide solution (2 ml)
of N-benzyloxycarbonyl-4-amino-3-methylbenzoic acid (650 mg) was
added slowly and, thereafter, ethyl iodine (730 .mu.l) was added
slowly at 0.degree. C. Following stirring at room temperature for
2.5 h, the reaction solution was poured to a saturated solution of
sodium hydrogen carbonate and the mixture was subjected to
extraction with toluene. The toluene layer was dried with magnesium
sulfate and the solvent was distilled off under vacuum to yield the
end product (680 mg).
[0080] .sup.1H-NMR (CDCl.sub.3, .delta.): 0.91-1.63(6H,m),
2.16(3H,s), 3.35-3.90(2H,m), 4.10-4.55(2H,m), 5.07(2H,bs),
6.95-7.35(6H,m), 7.68-7.95 (2H,m)
REFERENCE EXAMPLE 7
Synthesis of N-benzyloxycarbonyl-N-ethyl-4-amino-3-methylbenzoic
Acid
[0081] An aqueous solution (5 ml) of 2N NaOH was added to an
ethanol (5 ml) solution of the compound (680 mg) synthesized in
Reference Example 6 and the mixture was refluxed for 2 h. Then, the
reaction solution was cooled to room temperature and evaporated to
dryness under vacuum. The resulting residue was dissolved in a
small amount of water and the solution was washed with ether,
followed by pH adjustment to 3 with conc. HCl. The separating
precipitate was subjected to extraction with chloroform and dried
with sodium sulfate. The solvent was distilled off under vacuum to
yield the end product (540 mg).
[0082] .sup.1H-NMR (CDCl.sub.3, .delta.): 1.16(3H,t,J=5.4 Hz),
2.20(3H,s), 3.38-3.94(2H,m), 5.12(2H,bs), 6.93-7.44(6H,m),
7.72-8.08(2H,m)
REFERENCE EXAMPLE 8
Synthesis of diethyl
N-[N'-ethyl-3-methyl-(4-aminobenzoyl)]-L-glutamate
[0083] The compound (500 mg) synthesized in Reference Example 7 was
added to thionyl chloride (3 ml) and a catalytic amount of
dimethylformamide was added to the mixture followed by stirring at
room temperature for 2 h. Then, the reaction solution was
concentrated to dryness under vacuum. The resulting solids was
dissolved in dichloromethane (13 ml) and to the solution, diethyl
glutamate ester (382 mg), potassium carbonate (448 mg) and water
(13 ml) were added, followed by vigorous stirring at room
temperature for 12 h. The reaction solution was poured into a
saturated aqueous solution of sodium hydrogen carbonate and the
mixture was subjected to extraction with chloroform, followed by
washing with 1N HCl and drying with sodium sulfate. The solvent was
distilled off under vacuum to yield a colorless oil (830 mg). Then,
a liquid mixture of 30% hydrogen bromide/acetic acid (10 ml) and
anisole (830 mg) was added to the resulting residue, followed by
stirring at room temperature for 3 h. After the reaction, a large
amount of ether was added to the reaction solution, whereupon a
dark brown oil was precipitated. The greater part of the ether
layer was removed and the oil was suspended in chloroform, with the
suspension being washed with a saturated aqueous solution of sodium
hydrogen carbonate. The organic layer was recovered by separation
and dried with magnesium sulfate, followed by distilling off the
solving to yield the end product (360 mg)
[0084] .sup.1H-NMR (CDCl.sub.3, .delta.) 1.03-4.18(9H,m),
1.83-2.62(7H,m), 2.96-3.43(2H,m), 3.83-4.40(4H,m), 4.48-4.98(1H,m),
6.33-6.92(2H,m) 7.34-7.73 (2H,m)
EXAMPLE 3
Synthesis of diethyl N-{4-[N'-(2,4-diamino-6
pteridinyl)methyl-N'-ethylami- no]-3-methylbenzoyll-L-glutamate
[0085] A dimethylacetamide suspension (10 ml) of the compound (360
mg) synthesized in Reference Example 8 and an isopropanol adduct of
6-bromethyl-2,4-diaminopteridine hydrobromide (342 mg) was stirred
at 60.degree. C. for 4 h; an isopropanol adduct of
6-bromomethyl-2,4-diamino- pteridine hydrobromide (342 mg) was
further added and the mixture was stirred at 70.degree. C. for 1.5
h. Then, the reaction solution was poured into a saturated aqueous
solution of sodium hydrogen carbonate and the mixture was subjected
to extraction with chloroform. The chloroform layer was dried with
magnesium sulfate and the solvent was distilled off under vacuum.
The resulting residue was subjected to silica gel column
chromatography and elution was conducted with a solvent system of
chloroform and methanol (10:1) to yield the end product (167
mg).
[0086] .sup.1H-NMR (CDCl.sub.3, .delta.): 1.06(3H,t,J=8.0 Hz),
1.15-1.42(6H,m), 1.76(3H,s), 2.37-2.54(4H,m), 3.08(2H,q,J=8.0 Hz),
4.10(2H,q,J=8.0 Hz), 4.24(2H,q,J=8.0 Hz), 4.38(2H,s),
4.70-4.88(1H,m), 5.32(2H,bs), 6.94(1H,d,J=7.4 Hz), 7.05(1H,d,J=8.8
Hz), 7.56(1H,d,J=5.2 Hz), 7.67(1H,bs), 8.80(1H,s)
EXAMPLE 4
Synthesis of
N-{4-[N'-(2,4-diamino-6-pteridinyl)methyl-N'ethylaminol-3-met-
hylbenzoyl}-L-glutamic Acid (Compound 2)
[0087] An aqueous solution (0.84 ml) of 1N NaOH was added to an
ethanol solution (11 ml) of the compound (150 mg) synthesized in
Example 3 and the mixture was stirred at 35.degree. C. for 4 h.
Following further stirring at 25.degree. C. for 20 h, water (1 ml)
was added to the reaction solution, which was evaporated to dryness
under vacuum, with care being taken so that the external
temperature would not exceed 30.degree. C. The resulting yellow
solids were dissolved in water (5 ml) and the solution was adjusted
to pH of 3.7 with 1N HCl, followed by standing in a refrigerator
for 2 h. The separating precipitate was recovered by filtration to
yield the end product (121 mg).
[0088] .sup.1H-NMR (DMSO-d.sub.6, .delta.): 1.04(3H,t,J=6.8 Hz),
1.85-2.28(2H,m), 2.30-2.48(5H,m), 3.04(2H,q,J=6.8 Hz), 4.39(2H,s),
6.57(2H,bs), 7.10(1H,d,J=8.3 Hz), 7.61(1H,d,J=8.3 Hz), 7.72(1H,s),
8.34(1H,d,J=7.8 Hz), 8.57(1H,s)
REFERENCE EXAMPLE 9
Synthesis of
dimethyl-N-(N'-carbobenzoxy-N'-methyl-4-amino-3-methylbenzoyl-
)-L-2-aminoadipate
[0089] The compound (8.4 g) synthesized in Reference Example 3 was
added to thionyl chloride (29 ml) and a catalytic amount of
dimethylformamide was further added, with the mixture being stirred
at room temperature for 2 h. Then, the reaction solution was
concentrated to dryness under vacuum. The resulting solids were
dissolved in dichloromethane (150 ml) and to the solution, dimethyl
L-2-aminoadipate ester HCl (6.6 g), potassium carbonate (10 g) and
water (150 ml) was added, followed by vigorous stirring at room
temperature for 12 h. The reaction solution was poured into water
and the mixture was subjected to extraction with chloroform,
followed by washing with 1N HCl and drying with sodium sulfate. The
solvent was distilled off under vacuum. The resulting residue was
subjected to silica gel column chromatography and elusion was
conducted with a solvent system of chloroform and methanol (100:3)
to yield the end product (9.7 g).
[0090] .sup.1H-NMR (CDCl.sub.3, .delta.): 1.45-2.55(9H,m),
3.15(3H,s), 3.60(3H,s), 3.72(3H,s), 4.45-4.95(1H,m), 5.05(2H,bs),
6.80-7.35(7H,m), 7.45-7.95(2H,m)
REFERENCE EXAMPLE 10
Synthesis of dimethyl
N-[N'-methyl(4-amino-3-methylbenzoyl)]-L-2-aminoadip- ate
[0091] The compound (9.5 g) synthesized in Reference Example 9 was
added to a solution having anisole (9.5 g) contained in 30%
hydrogen bromide/acetic acid (100 ml) and the mixture was stirred
at room temperature for 4 h. Subsequently, a large amount of ether
was added to the reaction solution, whereupon a reddish brown oil
was precipitated. The greater part of the ether layer was removed
and the oil was suspended in chloroform, with the suspension being
washed with a saturated aqueous solution of sodium hydrogen
carbonate and subjected to extraction with chloroform. The
chloroform layer was dried with sodium sulfate and the solvent was
distilled of under vacuum to yield the end product (4.0 g).
[0092] .sup.1H-NMR (CDCl.sub.3, .delta.): 1.43-2.00(4H,m),
2.03(3H,s), 2.34(2H,t,J=6 Hz), 2.90(3H,s), 3.63(3H,s), 3.73(3H,s),
4.53-5.03(1H,m), 6.33-6.80(2H,m), 7.38-7.68(2H,m)
EXAMPLE 5
Synthesis of dimethyl N{4-[N'-(2,4-diamino-6
pteridinyl)methyl-N'-methylam-
ino]-3-methyl}benzoyl-L-2-aminoadipate
[0093] The compound (3.83 g) synthesized in Reference Example 10
and an isopropanol adduct of 6-bromomethyl-2,4-diaminopteridine
hydrobromide (4.06 g) were suspended in dimethylacetamide (75 ml)
and the suspension was stirred at 55-60.degree. C. for 4 h. After
cooling, the reaction solution was poured into a saturated aqueous
solution of sodium hydrogen carbonate and the mixture was subjected
to extraction with chloroform. The chloroform layer was dried with
sodium sulfate and the solvent was distilled off under vacuum. The
resulting residue was subjected to silica gel column chromatography
and elusion was conducted with a solvent system of chloroform and
methanol (10:1) to yield the end product (3.41 g).
[0094] .sup.1H-NMR (CDCl.sub.3, .delta.): 1.55-1.95(4H,m),
2.26-2.46(5H,m), 2.74(3H,s), 3.67(3H,s), 3.79(3H,s) 4.33(2H,s),
4.70-4.85(1H,m), 5.34(2H,bs), 6.78(1H,d,J=6.8 Hz), 7.05(1H,d,J=8.4
Hz), 7.61(1H,d,J=7.8 Hz), 7.68(1H,s), 8.85(1H,s)
EXAMPLE 6
Synthesis of
N-{4-[N'-(2,4-diamino-6-pteridinyl)methyl-N'-methylamino]-3-m-
ethyl}benzoyl-L-2-aminoadipic Acid (Compound 3)
[0095] An aqueous solution (18 ml) of 1N NaOH was added to an
ethanol (160 ml) solution of the compound (3.0 g) synthesized in
Example 5 and the mixture was stirred at 35.degree. C. for 4 h.
Following further stirring at 25.degree. C. for 20 h, water (20 ml)
was added to the reaction solution, which was evaporated to dryness
under vacuum, with care being taken so that the external
temperature would not exceed 30.degree. C. The resulting yellow
solids was dissolved in water (50 ml) and the solution was adjusted
to pH of 3.7 with 1N HCl, followed by standing in a refrigerator
for 2 h. The separating precipitate was recovered by filtration to
yield the end product (2.3 g).
[0096] .sup.1H-NMR (DMSO-d.sub.6, .delta.): 1.40-1.88(4H,m),
2.24(2H,t,]=7.8 Hz), 2.41(3H,s), 2.72(3H,s), 4.23-4.43(4H,m),
6.64(2H,bs), 7.11(1H,d,J=8.8 Hz), 7.66(1H,d,J=8.3 Hz), 7.74(1H,s),
8.38(1H,d,J=8.2 Hz), 8.64(1H,s)
REFERENCE EXAMPLE 11
Synthesis of
4-2,4-[N'-(diamino-6-pteridinyl)methyl]-N'-methylamino-3-meth-
ylbenzoic Acid
[0097] An isopropanol adduct of 6-bromomethyl-2,4-diaminopteridine
hydrobromide (3.8 g) and 4-methylamino-3-methylbenzoic acid (4.8 g)
were suspended in dimethylacetamide (50 ml) in a nitrogen
atmosphere and the suspension was stirred at 40.degree. C. for 4
days, followed by stirring at 55.degree. C. for 2 days. The
reaction solution was poured into water (500 ml) and the mixture
was adjusted to pH of 5 with 1N NaOH. The resulting precipitate was
recovered by filtration and washed with water and acetone to yield
the end product (3.3 g).
[0098] .sup.1H-NMR (DMSO-d.sub.6, .delta.): 2.40(3H,s) ,
2.74(3H,s), 4.36(2H,s) 7.10(1H,d,J=8.3 Hz), 7.6-7.8(2H,m),
8.64(1H,s)
EXAMPLE 7
Synthesis of
N-{4-[N'-(2,4-diamino-6-pteridinyl)methyl-N'-methylamino]-3-m-
ethyl}benzoyl-L-homocysteic Acid Ammonium Salt
[0099] Triethylamine (2.7 ml) and isobutyl chioroformate (0.85 ml)
were added, in a nitrogen atmosphere, to a dimethylacetamide (250
ml) suspension of the compound (2.4 g) synthesized in Reference
Example 11, and the mixture was stirred for 5 min. Isobutyl
chloroformate (0.12 ml) was further added and the mixture was
stirred for 5 min. To the resulting solution, methyl L-homocystate
HCl (1.5 g) was added and the mixture was stirred for 30 min.
Thereafter, triethylamine (1.35 ml), isobutyl chloroformate (0.42
ml) and methyl L-homocystate HCl (0.75 g) were added in a similar
manner and the mixture was stirred for 30 min. The temperature of
the mixture was raised slowly to room temperature and it was
stirred for 45 min. Thereafter, the solvent was distilled off under
vacuum with care being taken so that the temperature of the bath
would not exceed 40.degree. C., and an aqueous solution of 0.1N
NaOH (400 ml) was added to the residue, with the mixture being
stirred for 2 h at room temperature. Thereafter, an aqueous
solution of 1N NaOH (8 ml) was further added and the mixture was
stirred for 20 min. The reaction solution was freeze-dried and the
resulting residue was subjected to silica gel column chromatography
and elution was conducted with a solvent system of chloroform,
methanol and aqueous ammonia (5:4:1). The resulting crude product
was further loaded on DEAE cellulose column, washed with water and
eluted with an aqueous solution of 3% ammonium bicarbonate,
yielding the end product (1.1 g).
[0100] .sup.1H-NMR (D.sub.2O, .delta.): 2.15-2.25(1H,m),
2.3-2.4(1H,m), 2.4(3H,s), 2.8(3H,s), 2.9-3.1(2H,m), 4.4-4.5(1H,m),
4.5(2H,s), 7.1(1H,d,J=8.5 Hz), 7.6(1H,d,J=8.5 Hz), 7.7(1H,m),
8.6(1H,s)
REFERENCE EXAMPLE 12
Synthesis of methyl N-p-toluenesulfonyl-4-amino-3-ethylbenzoate
[0101] Para-toluenesulfonyl chloride (3 g) was added to a pyridine
(30 ml) solution of methyl 3-ethyl-4-aminobenzoate ester (1.86 g)
and the mixture was stirred at room temperature for 3 h. After the
reaction, the solvent was distilled off under vacuum and both
chloroform and 1N HCl were added to the residue. Following
stirring, the organic layer was recovered by separation. The
organic layer was dried with magnesium sulfate and the solvent was
distilled off under vacuum. The resulting residue was subjected to
silica gel column chromatography and elusion was conducted with a
solvent system of hexane and ethyl acetate (4:1), yielding the end
product (3 g).
[0102] .sup.1H-NMR (CDCl.sub.3, .delta.): 1.10(3H,t), 2.40(3H,s),
2.1-2.6(2H,m), 3.90(3H,s), 6.6-7.9(7H,m)
REFERENCE EXAMPLE 13
Synthesis of methyl N-p-toiuenesulfonyl-N-methyl-4-amino
3-ethylbenzoate
[0103] Sodium hydride (1.3 g) was added, under cooling with ice, to
an anhydrous dimethylformamide (50 ml) solution of the compound (3
g) synthesized in Reference Example 12 and the mixture was stirred
at room temperature for 10 min. Then, methyl iodide (3.8 g) was
added to the reaction solution and the mixture was reverted to room
temperature, followed by stirring for 2 h. After the reaction,
water was added to the reaction solution and the mixture was
subjected to extraction with ethyl acetate. The ethyl acetate layer
was dried with magnesium sulfate and the solvent was distilled off
under vacuum. The resulting residue was subjected to silica gel
column chromatography and elusion was conducted with solvent
chloroform, yielding the end product (1.56 g).
[0104] .sup.1H-NMR (CDCl.sub.3, .delta.): 1.25(3H,t), 2.45(3H,s),
2.7-3.1(2H,m), 3.15(3H,s), 3.90(3H,s), 6.6-8.1(7H,m)
REFERENCE EXAMPLE 14
Synthesis of N-p-toluenesulfonyl-N-methyl-4-amino-3 ethylbenzoic
Acid
[0105] An aqueous solution of 1N NaOH (60 ml) was added to a
methanol (60 ml) solution of the compound (1.56 g) synthesized in
Reference Example 13 and the mixture was stirred under reflux for 2
h. With the temperature of the water bath held below 30.degree. C.,
the solvent was concentrated under vacuum. The reaction solution
was adjusted to pH of 2.5 by addition of 1N HCl and the separating
precipitate was recovered by filtration to yield the end product
(1.33 g).
[0106] .sup.1H-NMR (CDCl.sub.3, .delta.): 1.25(3H,t), 2.45(3H,s),
2.7-3.1(2H,m), 3.15(3H,s), 6.6-8.2(7H,m)
REFERENCE EXAMPLE 15
Synthesis of diethyl
N-(N'-p-toluenesulfonyl-N'-methyl-4-amino-3-ethylbenz-
oyl)-L-glutamate
[0107] The compound (1.33 g) synthesized in Reference Example 14
was added to thionyl chloride (5 ml) and, following the addition of
dimethylformamide in a catalytic amount, the mixture was stirred at
room temperature for 2 h. Subsequently, the reaction solution was
concentrated to dryness under vacuum. The resulting solids was
dissolved in dichloromethane (35 ml) and to the solution, diethyl
glutamate HCl (900 mg), potassium carbonate (1.4 g) and water (15
ml) were added. The mixture was stirred vigorously overnight at
room temperature. The reaction solution was poured into water and
the mixture was subjected to extraction with chloroform. The
chloroform layer was washed with a saturated aqueous solution of
sodium hydrogen carbonate and 1N HCl, then dried with magnesium
sulfate. The solvent was distilled off under vacuum to yield the
end product (1.5 g).
[0108] .sup.1H-NMR (CDCl.sub.3, .delta.): 1.0-1.4(9H,m),
2.0-3.0(6H,m), 2.45(3H,s), 3.10(3H,s), 3.9-4.4(4H,m),
4.5-5.0(1H,m), 6.5-7.9 (7H,m)
REFERENCE EXAMPLE 16
Synthesis of Diethyl
N-(N'-methyl-4-amino-3-ethylbenzoyl)-L-glutamate
[0109] The compound (1.5 g) synthesized in Reference Example 15 was
added to a solution of anisole (1.5 g) in 30% hydrogen
bromide/acetic acid (15 ml) and the mixture was stirred at room
temperature for 4.5 h. Subsequently, ether (300 ml) was added to
the reaction solution, whereupon a reddish brown oil was
precipitated. The greater part of the ether layer was removed and
the oil was suspended in chloroform. The suspension was then washed
with a saturated aqueous solution of sodium hydrogen carbonate and,
thereafter, the organic layer was recovered by separation. The
separated organic layer was dried with magnesium sulfate and the
solvent was distilled off under vacuum to yield the end product
(1.0 g).
[0110] .sup.1H-NMR (CDCl.sub.3, .delta.): 1.0-1.4(9H,m),
2.0-3.0(6H,m), 2.85(3H,s), 3.7-4.3(4H,m), 4.5-4.9(1H,m),
6.4-7.7(3H,m)
EXAMPLE 8
Synthesis of diethyl
N-{4-[N'-(2,4-diamino-6-pteridinyl)methyl-N'-methylam-
ino]-3-ethylbenzoyl}-L-glutamate
[0111] The compound (1.0 g) synthesized in Reference Example 16 and
an isopropanol adduct of 6-bromomethyl-2,4-diaminopteridyl
hydrobromide (1.1 g) were suspended in dimethylacetamide (10 ml)
and the suspension was stirred at 60.degree. C. for 6 h. After
cooling, the reaction solution was poured into a saturated aqueous
solution of sodium hydrogen carbonate and the mixture was subjected
to extraction with chloroform. The organic layer was dried with
magnesium sulfate and the solvent was distilled off under vacuum.
The resulting residue was subjected to silica gel column
chromatography and elusion was conducted with a solvent system of
chloroform and methanol (10:1), yielding the end product (671
mg).
[0112] .sup.1H-NMR (CD.sub.3OD, .delta.): 1.0-1.4(9H,m),
2.0-3.0(6H,m), 2.75(3H,s), 3.9-4.7(7H,m), 7.1-7.8(3H,m),
8.70(1H,s)
EXAMPLE 9
A Synthesis of
N-{4-[N'-(2,4-diamino-6-pteridinyl)methyl-N'-methylaminol-3-
-ethylbenzoyl}-L-glutamic Acid (Compound 4)
[0113] An aqueous solution of 1N NaOH (1.78 ml) was added to an
ethanol (22 ml) solution of the compound (640 mg) synthesized in
Example 8 and the mixture was stirred at 35.degree. C. for 4.5 h.
Following further stirring at 25.degree. C. for 20 h, water (2 ml)
was added to the reaction solution and, then, the reaction solution
was adjusted to pH of 3.7 with 1N HCl, followed by standing
overnight in a cool place. The separating precipitate was recovered
by filtration to yield the end product (340 mg).
[0114] .sup.1H-NMR (DMSO-d.sub.6, .delta.): 1.26(3H,m),
1.9-2.5(4H,m), 2.69(3H,s), 2.80(2H,m), 4.29(2H,s), 4.39(1H,m),
7.22(1H,m), 7.71(2H,m), 8.64(1H,s)
REFERENCE EXAMPLE 17
Synthesis of Ethyl N-carbobenzoxY-4-amino-3,5-dimethylbenzoate
[0115] Ethyl 4-amino-3, 5-dimethylbenzoate (2.0 g) was added to a
tetrahydrofuran (THF) suspension of sodium hydride (0.82 g) in a
nitrogen atmosphere and the mixture was stirred al room temperature
for 30 min. Subsequently, carbobenzoxychloride (4.4 ml) was added
to the suspension and the mixture was stirred overnight. A small
amount of water was added to the reaction solution and the mixture
was poured into ice water, followed by extraction with ethyl
acetate. Following washing and drying (with magnesium sulfate), the
solvent was distilled off under vacuum. The resulting crystal was
recovered by filtration in the presence of ethanol and dried under
vacuum to yield the end product (3.2 g).
[0116] .sup.1H-NMR (CDCl.sub.3, .delta.): 1.39(3H,t,J=7 Hz),
2.05(6H,s), 4.35(2H,q,J=7 Hz), 5.15(2H,s), 7.23(51-1,m),
7.75(2H,s)
REFERENCE EXAMPLE 18
Synthesis of ethyl
N-carbobenzoxy-N-methyl-4-amino-3,5-dimethylbenzoate
[0117] Sodium hydride (1.2 g) was suspended in dimethylformamide
(50 ml) in a nitrogen atmosphere and the suspension was stirred.
The compound (3.2 g) synthesized in Reference Example 17 was added
to the suspension at room temperature, followed by stirring for 30
min. Then, methyl iodide (1.8 ml) was added, followed by further
stirring overnight at room temperature. Subsequently, a few drops
of water were added to the reaction solution under cooling with ice
and the reaction solution was poured into ice water. The mixture
was subjected to extraction with ethyl acetate and dried with
magnesium sulfate. The solvent was distilled off under vacuum and
the resulting residue was subjected to silica gel column
chromatography, followed by elusion with a solvent system of hexane
and ethyl acetate (9:1) to yield the end product (1.47 g).
[0118] .sup.1H-NMR (CDCl.sub.3, .delta.): 1.38(3H,t,J=7 Hz),
2.21(6H,s), 3.17(3H.s), 4.42(2H,q,J=7 Hz), 5.13(2H,s),
7.0-7.6(5H,m), 7.80(2H,s)
REFERENCE EXAMPLE 19
Synthesis of N-carbobenzoxy-N-methyl-4-amino-3,5-dimethylbenzoic
Acid
[0119] An aqueous solution of 1N NaOH (12 ml) was added to a THF
(30 ml) solution of the compound (1.47 g) synthesized in Reference
Example 18 and the mixture was heated at 60.degree. C. for 6 h,
followed by further stirring overnight at room temperature. Then,
the reaction solution was adjusted to pH of ca. 5 with 3N HCl and
concentrated under vacuum. After pH adjustment to ca. 2, the
concentrate was subjected to extraction with THF, followed by
drying with magnesium sulfate. The solvent was distilled off under
vacuum to yield the end product (1.34 g).
[0120] .sup.1H-NMR (CDCl.sub.3, .delta.): 2.18(6H,s), 3.14(3H,s),
5.04(2H.s), 6.9-7.5(5H,m), 7.76(2H,s)
REFERENCE EXAMPLE 20
Synthesis of diethyl
N-(N'-carbobenxzoxy-N'-methyl-4-amino-3,5-dimethylben-
zoyl)-L-glutamate
[0121] The compound (1.34 g) synthesized in Reference Example 19
was added to thionyl chloride (10 ml) and dimethylformamide was
further added in a catalytic amount, followed by stirring at room
temperature for 3 h. Then, the reaction solution was concentrated
to dryness under vacuum. The resulting solids was dissolved in
dichloromethane (25 ml) and to the resulting solution, an aqueous
solution (25 ml) of diethyl glutamate ester HCl (1.0 g) and
potassium carbonate (1.2 g) were added; thereafter, the reaction
solution was adjusted to pH of ca. 10 with potassium carbonate,
followed by vigorous stirring overnight at room temperature. The
dichloromethane layer was recovered by separation, washed with 1N
HCl, a saturated aqueous solution of sodium hydrogen carbonate and
a saturated aqueous solution of NaCl, and dried with sodium
sulfate. The solvent was distilled off under vacuum to yield the
end product (2.14 g).
[0122] .sup.1H-NMR (CDCl.sub.3, .delta.): 1.23(3H,t,J=7 Hz),
1.31(3H,t,J=7 Hz), 2.1-2.7(4H,m), 2.17(6H,s), 3.13(3H,s),
4.13(2H,q,J=7 Hz), 4.25(2H,q,J=7 Hz), 4.73(1H,m), 5.07(2H,s),
6.94(1H,d,J=8 Hz), 7.1-7.5(5H,m), 7.51(2H,s)
REFERENCE EXAMPLE 21
Synthesis of diethyl
N-[N'-methyl-(4-amino-3,5-dimethylbenzoyl)-L-glutamat- e
[0123] A solution of 30% hydrogen bromide/acetic acid (24 ml) 10
was added to an anisole solution (2.4 ml) of the compound (2.4 g)
synthesized in Reference Example 20 and the mixture was stirred at
room temperature for 3 h. Then, ca. 200 ml of ether was added to
the reaction solution, whereupon a reddish brown oil was
precipitated. The greater part of the ether layer was removed and
the oil was suspended in chloroform. The suspension was washed with
a saturated aqueous solution of sodium hydrogen carbonate and the
organic layer was recovered by separation. The separated organic
layer was dried with sodium sulfate and the solvent was distilled
off under vacuum to yield the end product (1.2 g).
[0124] .sup.1H-NMR (CDCl.sub.3, .delta.): 1.21(3H,t,J=7 Hz),
1.29(3H,t,J=7 Hz), 2.0-2.8(4H,m), 2.29(6H,s), 2.87(3H,s), 3.11
(1H,s) , 4.11 (2H,q,J=7 Hz), 4.23(2H,q,J=7 Hz), 4.83(1H,m),
6.83(1H,d,J=8 Hz), 7.43(2H,s)
EXAMPLE 10
Synthesis of diethyl N-(4-[N'-(2,4-diamino-6
pteridinyl)methyl-N'-methylam- ino]-3,5-dimethylbenzoyl}-L
Glutamate
[0125] The compound (1.2 g) synthesized in Reference Example 21 and
an isopropanol adduct of 6-bromomethyl-2,4-diaminopteridine
hydrobromide (1.0 g) were suspended in dimethylacetamide (20 ml)
and the suspension was stirred at 60.degree. C. for 12 h. To the
cooled reaction solution, water was added and the mixture was
stirred, followed by extraction with chloroform. The chloroform
layer was dried with sodium sulfate and the solvent was distilled
off under vacuum. The resulting residue was subjected to silicagel
column chromatography and elusion was conducted with a solvent
system of chloroform and methanol (99:1-95:5), yielding the end
compound (0.64 g).
[0126] .sup.1H-NMR (CDCl.sub.3, +CD.sub.3OD, .delta.)
:1.25(3H,t,J=7 Hz), 1.32(3H,t,J=7 Hz), 2.0-2.7(4H,m), 2.42(6H,s),
2.82(3H,s), 4.18(2H,q,J=7 Hz), 4.28(2H,q,J=7 Hz), 4.42(2H,s),
4.78(1H,m), 7.53(2H,s), 8.89(1H,s,)
EXAMPLE 11
Synthesis of
N-{4-[N'-(2,4-diamino-6-pteridinyl)methyl-N'-methylamino]-3,5-
-dimethylbenzoyl}-L-glutamic Acid (Compound 5)
[0127] The compound (0.59 g) synthesized in Example 10 was
dissolved in a solvent system of ethanol (40 ml) and THF (20 ml);
after adding an aqueous solution of 1N NaOH (3.5 ml), the mixture
was stirred overnight at room temperature. The solvent was
distilled off under vacuum and the resulting yellow solids were
dissolved in water (20 ml) and the solution was adjusted to pH of
3.7 with 1N HCl under cooling with ice. The separating precipitate
was recovered by filtration, washed successively with water and
acetone, and vacuum-dried to yield the end product (0.47 g).
[0128] .sup.1H-NMR (DMSO-d.sub.6, .delta.): 1.8-2.2(2H,m),
2.3-2.5(2H,m), 2.34(6H,s), 2.73(3H,s), 4.3-4.5(1H,m), 4.36(2H,s),
6.66(2H,s), 7.55(2H,s), 8.40(1H,d,J=6 Hz), 8.74(1H,s)
REFERENCE EXAMPLE 22
Synthesis of 4-methylamino-3-trifluoromethylbenzoic Acid
[0129] 4-amino-3-trifluoromethylbenzoic acid (3.6 g) was suspended
in trifluoroacetic anhydride (90 ml) and the suspension was stirred
overnight. The solvent was distilled off under vacuum and the
resulting residue was dissolved in acetone (30 ml). Methyl iodide
(9.9 g) was added to the solution and the mixture was heated to
45.degree. C.; with vigorous stirring, a potassium hydroxide powder
(6.8 g) was added slowly, followed by further stirring for 1 h at
45.degree. C. Then, the solvent was distilled off under vacuum and
the resulting residue was dissolved in water (50 ml). The solution
was heated under reflux for 2 h. The reaction solution was then
adjusted to pH of 3.5 with 1N HCl under cooling with ice. The white
precipitate was recovered by filtration and washed with a small
amount of water and vacuum-dried to yield the end product (2.2
g).
[0130] .sup.1H-NMR (CDCl.sub.3: CD3OD=1:1, .delta.): 2.95(3H,s),
6.74(1H,d,J=9.OHz), 7.96-8.12(2H,m)
REFERENCE EXAMPLE 23
Synthesis of methyl 4-methylamino-3-trifluoromethylbenzoate
[0131] Hydrochloric acid gas was blown, under cooling with ice,
into a methanol solution (25 ml) of the compound (2.2 g)
synthesized in Reference Example 22 and stirring was continued at
room temperature for 4 h. The solvent was distilled off under
vacuum and the resulting residue was dissolved in ethyl acetate.
The solution was washed with a saturated aqueous solution of sodium
hydrogen carbonate and dried with anhydrous magnesium sulfate,
followed by distilling off the solvent under vacuum. The resulting
residue was subjected to silica gel column chromatography and
elusion was conducted with a solvent system of ethyl acetate and
n-hexane (1:3), yielding the end product (1.3 g).
[0132] .sup.1H-NMR (CDCl.sub.3, .delta.): 2.91(3H,s), 3.83(3H,s),
4.63(1H,bs), 6.65(1H,d,J=9.0 Hz), 7.92-8.99(2H,m)
REFERENCE EXAMPLE 24
Synthesis of
4-[N-(2,4-diamino-6-pteridinyl)methyl-N-methyl]amino-3-triflu-
oromethylbenzoic Acid
[0133] Sodium hydride (0.26 g) was added, under cooling with ice in
a nitrogen armosphere, to a hexamethylphosphoric triamide solution
(20 ml) of the compound (1.3 g) synthesized in Reference Example
23, and the mixture was stirred for 30 min. A 1/3 isopropanol
adduct of 6-bromomethyl-2,4-diaminopteridine hydrobromide (2.8 g)
was added to the reaction solution and the mixture was stirred for
30 min, followed by further stirring at 70.degree. C. for 6 h and
at room temperature for 2 days. The reaction solution was poured
into dilute HCl and neutralized with a saturated aqueous solution
of sodium hydrogen carbonate; the resulting brown precipitate was
recovered by filtration. The recovered precipitate was vacuum
dried, suspended in a solvent system of chloroform and methanol
(1:1) and heated under reflux for 1 h. The insoluble matter was
filtered off and the filtrate was concentrated to dryness under
vacuum. The resulting residue was dissolved in methanol (50 ml)
and, following addition of an aqueous solution of 1N NaOH (20 ml),
the mixture was stirred at 60.degree. C. for 1 h. Methanol was
distilled off under vacuum and the insoluble matter was recovered
by filtration. Under cooling with ice, 1N HCl was added for pH
adjustment to 4. The orange precipitate was recovered by
filtration, vacuum dried and subjected to silica gel column
chromatography. Elution was conducted first using ethyl acetate as
a solvent, then using a solvent system of chloroform, methanol and
28% aqueous ammonia (5:4:1). The desired fractions were
concentrated to dryness under vacuum and the resulting residue was
dissolved in water. Following pH adjustment to 4 with 1N HCl under
cooling with ice, the resulting yellow precipitate was recovered by
filtration and vacuum dried to yield the end product (102 mg).
[0134] .sup.1H-NMR (DMSO-d.sub.6:CDCl.sub.3=7:1, .delta.):
2.79(3H,s), 4.42(2H,s), 7.61(1H,d,J=8.3 Hz), 8.11-8.17(2H,m),
8.69(1H,s)
EXAMPLE 12
Synthesis of N-{4-[N'-(2,4-diamino-6-pteridinyl)methyl
N'-methyl]amino-3-trifluoromethylbenzoyl}-L-2-aminoadipic Acid
[0135] (Synthesis of L-2-{4-[N'-(2,4-diamino-6-pteridinyl)methyl
N'-methyl]amino-3-trifluoromethylbenzoyl}aminoadipic Acid)
[0136] Hydroxybenzotriazole (36 mg) and dicyclohexylcarbodiimide
(55 mg) were added, under cooling with ice in a nitrogen
atmosphere, to a hexamethylphosphoric triamide solution (2 ml) of
the compound (88 mg) synthesized in Reference Example 24, and the
mixture was stirred for 30 min. Subsequently, dimethyl
L-2-aminoadipate ester HCl (63 ma) and N-methylmorpholine (34 mg)
were added and the mixture was slowly reverted to room temperature,
followed by stirring for 2 days. The reaction solution was
subjected to silica gel column chromatography and purified by
elusion first using chloroform as a solvent, then using a solvent
system of chloroform and methanol (19:1). The desired fractions
were concentrated to dryness under vacuum and dissolved in methanol
(2 ml). An Aqueous solution of 1N NaOH (0.4 ml) was added under
cooling with ice in a nitrogen atmosphere and the mixture was
stirred for 5 h. The solvent was distilled off under vacuum and the
resulting residue was dissolved in water. The insoluble matter was
filtered off and the filtrate was adjusted to pH of 3.5 with 1N
HCl. The resulting yellow Precipitate was recovered by filtration,
washed with a small amount of water and vacuum dried. The crude
product was subjected to preparative thin-layer chromatography (on
silica gel) and separation was effected by elusion with a solvent
system of chloroform, methanol and 28% aqueous ammonia (5:4:1). The
effluent was concentrated to dryness under vacuum and dissolved in
an aqueous solution of sodium hydrogen carbonate. After filtering
off the insoluble matter, 1N HCl was added for pH adjustment to
3.5. The resulting pale yellow precipitate was recovered by
filtration and vacuum dried to yield the end product (18 mg).
[0137] .sup.1H-NMR (DMSO-d.sub.6:CDCl.sub.3=7:1, .delta.):
1.53-1.93(4H,m), 2.25(2H,t,J=7.3 Hz), 2.75(3H,s), 4.34-4.45(3H,m),
6.91(2H,bs), 7.63-7.67(2H,m), 7.95(1H,bs), 8.16(1H,d,J=8.3 Hz),
8.22(1H,s), 8.72(1H,s), 8.79 (1H, d, J=7.8 Hz)
REFERENCE EXAMPLE 25
Synthesis of 1-acetyl-7-methylindoline
[0138] A solution of 7-methylindoline (10.2 g) in acetic anhydride
(25 ml) was refluxed for 30 min. After cooling, the reaction
solution was poured into ice water (250 ml) and the resulting
crystal was filtered by means of suction, heated and vacuum dried
to yield the end product (11.7 g).
[0139] .sup.1H-NMR (CDCl.sub.3, .delta.): 2.24(3H,s), 2.25(3H,s),
2.99(2H,t,J=7 Hz), 4.06(2H,t,J=7 Hz), 7.01(2H,s)
REFERENCE EXAMPLE 26
Synthesis of 5-bromo-1-acetyl-7-methylindoline
[0140] Bromine (3.1 ml) was added dropwise at room temperature to
an acetic acid (70 ml) solution of the compound (11.5 g)
synthesized in Reference Example 25, and the mixture was stirred
for 1 h. The reaction solution was poured into ice water (600 ml)
and discolored by addition of Na.sub.2S.sub.2O.sub.3. The deposit
was filtered by means of suction, dissolved in hot methanol and
subjected to recrystallization, yielding the end product (10.8
g).
[0141] .sup.1H-NMR (CDCl.sub.3, .delta.): 2.24(6H,s), 2.98(2H,t,J=7
Hz), 4.06(2H,t,J=7 Hz), 7.14(2H,s) Reference
EXAMPLE 27
Synthesis of 5-cyano-1-acetyl-7-methylindoline
[0142] The compound (10.8 g) synthesized in Reference Example 26
and copper cyanide (5.5 g) were suspended in N-methylpyrrolidinone
(50 ml) and the suspension was heated in a nitrogen atmosphere at
cat 200.degree. C. for 4 h. After cooling, the reaction solution
was poured into cooled aqueous ammonia (100 ml) and the deposit was
filtered by means of suction. The deposit was washed with aqueous
ammonia until the blue color disappeared and, thereafter, it was
dissolved in hot chloroform (50 ml). After filtration by means of
suction, the filtrate was washed with water and an aqueous solution
of 1N NaOH, dried with magnesium sulfate and concentrated to give
the crude product. The crude product was recrystallized with
acetonitrile to yield the end product (4.9 g).
[0143] .sup.1H-NMR (CDCl.sub.3, .delta.): 2.24(3H,s), 2.27(3H,s),
3.05(2H,t,J=7 Hz), 4.08(2H,t,J=7 Hz), 7.30(2H,s)
REFERENCE EXAMPLE 28
Synthesis of 7-methylindoline-5-carboxylic Acid
[0144] A mixture of the compound (4.9 g) synthesized in Reference
Example 27 and conc. HCl (60 ml) was refluxed for 3 h. After
cooling, the mixture was poured into ice water (200 ml) and the pH
was adjusted to higher than 12 with an aqueous solution of sodium
hydroxide, followed by filtration by means of suction. The filtrate
was adjusted to pH of ca. 4 with HCl under cooling with ice. The
deposit was recovered by filtration, washed, heated and vacuum
dried to yield the end product (3.4 g).
[0145] .sup.1H-NMR (DMSO-d.sub.6, .delta.): 2.05(3H,s),
2.96(2H,t,J=8 Hz), 3.54(2H,t,J=8 Hz), 7.42(2H,s), 11.83(1H,br
s)
REFERENCE EXAMPLE 29
Synthesis of N-benzyloxycarbonyl-7-methylindoline-5-carboxylic
Acid
[0146] The compound (3.3 g) synthesized in Reference Example 28 was
added to an aqueous solution of sodium hydroxide (0.84 g) in a
nitrogen atmosphere. Following the addition of ether (60 ml). The
mixture was cooled with ice. Then, an aqueous solution (15 ml) of
sodium hydroxide (3.3 g) and an ether solution of benzyloxycarbonyl
chloride (6.8 ml) were added dropwise simultaneously and the
mixture was stirred at room temperature for 4 h. The ether layer
was separated and the aqueous layer was washed with ether, followed
by pH adjustment to 1-2 with 3N HCl under cooling with ice. The
separating crystal was filtered by means of suction, washed with
water and dried by heating to yield the end product (5.3 g).
[0147] .sup.1H-NMR (DMSO-d.sub.6, .delta.): 2.22(3H,s),
3.02(2H,t,J=7 Hz), 4.10(2H,t,J=7 Hz), 5.16(2H,s), 7.32(5H,s),
7.55(2H,s)
REFERENCE EXAMPLE 30
Synthesis of dimethyl
N-(1-benzyloxycarbonyl-7-methylindoline-5-carbonyl)--
L-.alpha.-aminoadipate
[0148] Thionyl chloride (15 ml) was suspended in the compound (2.6
g) synthesized in Reference Example 29. To the suspension, a
catalytic amount of dimethylformamide was added and the mixture was
stirred at room temperature for 2 h. Then, the reaction solution
was concentrated to dryness under vacuum. The resulting solids was
dissolved in dichloromethane (50 ml); an aqueous solution (50 ml)
of dimethyl L-.alpha.-aminoadipate HCl (2.4 g) and potassium
carbonate (2.3 g) were added to the solution; the pH of the
reaction solution was adjusted to ca. 10 with potassium carbonate,
followed by vigorous stirring overnight at room temperature. The
dichloromethane layer was recovered by separation, washed
successively with 1N HCl, a saturated solution of sodium hydrogen
carbonate and a saturated aqueous solution of sodium chloride, and
dried with sodium sulfate. The solvent was distilled off under
vacuum to yield the end product (4.2 g).
[0149] .sup.1H-NMR (CDCl.sub.3, .delta.): 1.5-2.1(4H,m),
2.2-2.6(2H,m), 2.29(3H,s), 3.00(2H,t,J=7 Hz), 3.64(3H,s),
3.76(3H,s), 4.14(2H,t,J=7 Hz), 4.5-5.0(1H,m), 5.18(2H,s),
6.67(1H,d,J=8 Hz), 7.32(5H,s), 7.44(2H,s)
REFERENCE EXAMPLE 31
Synthesis of dimethyl
N-(7-methylindoline-5-carbonyl)-L-.alpha.-aminoadipa- te
[0150] A solution of 30% hydrogen bromide/acetic acid (42 ml) was
added to an anisole (4.2 ml) solution of the compound 4.2 g)
synthesized in Reference Example 30 and the mixture was stirred at
room temperature for 4 h. Then, ca. 400 ml of ether was added to
the reaction solution, whereupon a reddish brown oil was
precipitated. The greater part of the ether layer was removed and
the oil was suspended in chloroform; the suspension was washed with
a saturated solution of a sodium hydrogen carbonate and the end
product was extracted with chloroform. The chloroform layer was
dried with sodium sulfate and the solvent was distilled off under
vacuum to yield the end product (2.6 g).
[0151] .sup.1H-NMR (CD.sub.3OD, .alpha.): 1.5-2.1(4H,m),
2.15(3H,s), 2.38(2H,t,J=7 Hz), 3.05(2H,t,J=7 Hz), 3.60(2H,t,J=7
Hz), 3.66(3H,s), 3.73(3H,s), 4.3-4.8(1H,m), 7.46(2H,s)
EXAMPLE 13
Synthesis of dimethyl N-{1-[(2,4-diamino-6
pteridinyl)methyl]-7-methylindo-
line-5-carbonyl}-L-.alpha.-aminoadipate
[0152] The compound (2.6 g) synthesized in Reference Example 31 and
in isopropanol adduct of 6-bromomethyl-2,4 diaminopteridine
hydrobromide (3.0 g) were suspended in dimethylacetamide (40 ml)
and the suspension was stirred at 60.degree. C. for 12 h. After
cooling the reaction solution, water was added and the mixture was
stirred; the pH of the mixture was adjusted to ca. 8 with sodium
hydrogen carbonate and the insoluble matter was recovered by
filtration. The chloroform layers were combined and dried with
sodium sulfate; the solvent was distilled off under vacuum and the
resulting residue was subjected to silica gel column
chromatography, followed by elusion with a solvent system of
chloroform and methanol (99:1-95:5) to yield the end product (2.6
g).
[0153] .sup.1H-NMR (CDCl.sub.3+CD.sub.3OD, .delta.): 1.5-2.2(4H,m),
2.3-2.7(2H,m), 2.40(3H,s), 2.8-3.9(4H,m), 3.69(3H,s), 3.78(3H,s),
4.6-5.1(1H,m), 4.77(2H,s), 7.46(2H,s), 8.75(1H,s)
EXAMPLE 14
Synthesis of
N-{1-[(2,4-diamino-6-pteridinyl)methyl]-7-methylindoline-5-ca-
rbonyl}-L-.alpha.-aminoadipic Acid
[0154] The compound (2.6 g) synthesized in Example 13 was dissolved
in a solvent system of ethanol (400 ml) and tetrahydrofuran (500
ml): after addition of an aqueous solution of 1N NaOH (15 ml), the
mixture was stirred overnight at room temperature. The solvent was
distilled off under vacuum and the resulting yellow solids was
dissolved in water (50 ml), followed by treatment with activated
carbon and filtration through Celite. The filtrate was adjusted to
pH of 2 with 1N HCl under cooling with ice and the separating
precipitate was recovered by filtration. Following washing with
water and acetone, the precipitate was vacuum dried to yield the
end product (1.4 g).
[0155] .sup.1H-NMR (DMSO-d.sub.6, .delta.): 1.4-2.0(4H,m),
2.23(2H,t,J=7 Hz), 2.34(3H,s), 2.97(2H,t,J=8 Hz), 3.56(2H,t,J=8
Hz), 4.32(1H,m), 4.72(2H,s), 6.87(2H,s), 7.44(1H,s), 7.49(1H,s),
8.14(1H,d,J=8 Hz), 8.72(1H,s)
EXAMPLE 15
Synthesis of
N-(1-[(2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl}-L-.-
alpha.-aminoadipic Acid
[0156] (Process 1)
[0157] An aqueous saturated sodium hydrogen carbonate solution (1.0
ml) containing potassium nitrodisulfonate (Fremy's salt) (51.2 mg)
was added dropwise, under cooling with ice, to an aqueous saturated
sodium hydrogen carbonate solution (1.0 ml) of N-{1-[(2,4-diamino-6
pteridinyl)methyl]indoline-5-carbonyl-}-L-.alpha.-aminoadipic acid
(11.0 mg) and the mixture was stirred at room temperature for 5 h.
Subsequently, an aqueous saturated sodium hydrogen carbonate
solution (1.0 ml) was added to the reaction solution and the
mixture was subjected to high-performance liquid chromatography.
The column was of YMC Pack A-322 ODS type and the elusion solvent
was a 25:75 mixture of methanol and 0.1M ammonium formate buffered
solution (pH, 4.5). The end product (3.9 mg) was recovered by
separation under the following conditions: flow rate, 2.5 ml/min;
column temperature, RT; detection wavelength, 303 nm; injection
volume, 200 .mu.l.
[0158] .sup.1H-NMR (DMSO-d.sub.6, .delta.): 1.58 (2H,m), 1.74
(1H,m) , 1.83(1H,m) 2.21(2H,t,J=7.8 Hz), 4.19(1H,m), 5.57(2H,s),
6.62(1H,d), 7.6-7.8(3H,m), 8.12(1H,s), 8.17(1H.d,J=6.0 Hz),
8.57(1H,s)
[0159] (Process 2)
[0160] Dichlorodicyanobenzoquinone (18 mg) was added to a dioxane
suspension (20 ml) of
N-{1-[(2,4-diamino-6-pteridinyl)methyl]indoline-5-c-
arbonyl}-L-.alpha.-aminoadipic acid (20 mg) and the mixture was
stirred at 80.degree. C. for 3 h. The solvent was distilled off
under vacuum and an aqueous solution of saturated sodium hydrogen
carbonate (5.0 ml) was added to the residue. The mixture was
subjected to high-performance liquid chromatography under the same
conditions as in Process 1, thereby yielding the end produce (7.7
mg), while was recovered by separation.
[0161] (Process 3)
[0162] An acetic acid solution (10 ml) of
N-{1-[(2,4-diamino-6-pteridinyl)-
methyl]indoline-5-carbonyl}-L-.alpha.-aminoadipic acid (20 mg) was
added dropwise to an acetic acid solution (10 ml) of manganese
(III) acetate (1.5 mg) and the mixture was stirred at 80.degree. C.
for 1 h. Acetic acid was distilled off under vacuum and an aqueous
solution of saturated sodium hydrogen carbonate (7.0 ml) was added
to the residue. Then, the mixture was subjected to high-performance
liquid chromatography under the same conditions as in Process 1,
yielding the end product (0.8 mg), which was recovered by
separation.
EXAMPLE 16
Synthesis of
N-{1-[(2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl}-L-h-
omocysteic Acid Ammonium Salt
[0163] The procedure of Process 1 in Example 15 was repeated,
except that
N-{1-[(2,4-diamino-6-pteridinyl)methyl]indoline-5-carbonyl}-L-.alpha.-ami-
noadipic acid was replaced by
N-{1[82,4-diamino-6-pteridinyl)methyl]indoli-
ne-5-carbonyl}-L-homocysteic acid ammonium salt (10.0 mg). As a
result, the end product was yielded (4.4 mg).
[0164] .sup.1H-NMR (DMSO-d.sub.6, .delta.): 2.0-2.2(2H,m),
2.56(2H,t,J=7.3 Hz), 4.35(1H,m), 5.56(2H,s), 6.61(1H,m),
7.6-7.8(3H,m), 8.17(1H,s), 8.58(1H,s), 8.82(1H,m)
EXAMPLE 17
Synthesis of
N-{1-[(2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl}-L-g-
lutamic Acid (Compound 6)
[0165] The procedure of Process 1 in Example 15 was repeated,
except that
N-{1-[(2,4-diamino-6-pteridinyl)methyl]indoline-5-carbonyl}-L-aminoadipic
acid was replaced by
N-{1-[(2,4-diamino-6-pteridinyl)methyl]indoline-5-ca-
rbonyl}-L-glutamic acid (10.4 mg). As a result, the end product was
yielded (3.1 mg).
[0166] .sup.1H-NMR (DMSO-d.sub.6, .delta.): 1.9-2.0(2H,m),
2.2-2.5(2H,m), 4.38(1H,m), 5.57(2H,s), 6.62(1H,d), 7.6-7.8(3H,m),
8.14(1H,s), 8.25(1H,m), 8.56(1H,s)
EXAMPLE 18
Synthesis of
N-{1-[(2,4-diamino-6-pteridinyl)methyl]-7-methylindole-5-carb-
onyl}-L-aminoadipic Acid
[0167] Using N-{1-[(2,4-diamino-6-pteridinyl)methyl]-7
methyl-indoline-5-carbonyl}-L-aminoadipic acid (20 mg), the
procedure of process 1 in Example 15 was repeated to yield the end
product (8.4 mg).
[0168] .sup.1H-NMR (DMSO-d.sub.6, .delta.): 1.5-1.9(4H,m),
2.17(2H,m), 2.62(3H,s), 4.05(1H,m), 5.78(2H,s), 6.66(1H,d,J=3.4
Hz), 7.33(1H,s), 7.59(1H,d,J=3.4 Hz), 7.89(1H,m), 7.92(1H,s),
8.44(1H,s)
REFERENCE EXAMPLE 32
Synthesis of diethyl N-(4-nitro-3-methylbenzoyl)-L Glutamate
[0169] 4-Nitro-3-methylbenzoic acid (10 g) was suspended in thionyl
chloride (30 ml); thereafter, a catalytic amount of
dimethylformamide was added and the mixture was refluxed for 2 h.
After cooling, the reaction solution was evaporated to dryness
under vacuum. The resulting solids were dissolved in
dichloromethane (250 ml); to the solution, diethyl L-glutamate HCl
(13.4 g), potassium carbonate (30 g) and water (250 ml) were added
and the mixture was stirred vigorously at room temperature for 12
h. The reaction solution was poured into water and, following
extraction with chloroform, the solution was washed with 1N HCl and
dried with sodium sulfate. The solvent was distilled off under
vacuum. The resulting residue was subjected to silica gel column
chromatography and elusion was conducted with a solvent system of
chloroform and methanol (100:1) to yield the end product (10.0
g).
[0170] .sup.1H-NMR (CDCl.sub.3, .delta.): 1.2-1.4(6H,m),
2.0-2.4(2H,m), 2.4-2.6(2H,m), 2.62(3H,s), 4.14(2H,q,J=7.3 Hz),
4.25(2H,q,J=7.2 Hz), 4.76(1H,m), 7.44(1H,d,J=7.3 Hz), 7.76(1H,m),
7.81(1H,s), 7.98 (1H,d, J=8.3 Hz)
REFERENCE EXAMPLE 33
Synthesis of diethyl N-(4-amino-3-methylbenzoyl)-L-glutamate
[0171] The compound (10.0 g) synthesized in Reference Example 32
was dissolved in acetic acid (160 ml) and a zinc powder (18 g) was
added slowly to the solution under cooling with ice. After stirring
at room temperature for 2 h, the mixture was filtered and the
filtrate was concentrated under vacuum. The resulting concentrate
was dissolved in chloroform (200 ml) and washed with a saturated
aqueous solution of sodium hydrogen carbonate. The organic layer
was dried with sodium sulfate and the solvent was distilled off
under vacuum to yield the end product (7.8 g).
[0172] .sup.1H-NMR (CDCl.sub.3, .delta.): 1.1-1.4(6H.m),
2.0-2.4(4H,m), 2.15(3H,s), 4.0-4.3(4H,m), 4.77(1H,m),
6.62(1H,d,J=8.3 Hz), 6.81(1H,d,J=7.3 Hz), 7.4-7.6(2H,m)
EXAMPLE 19
Synthesis of diethyl
N-{4-[N'-(2,4-diamino-6-pteridinyl)methylamino]-3-met-
hyl}benzoyl-L-glutamate
[0173] The compound (1.9 g) synthesized in Reference Example 33 and
an isopropanol adduct of 6-bromomethyl-2,4 diaminopteridine
hydrobromide (1.0 g) were suspended in dimethylacetamide (16 ml)
and the mixture was stirred at 55-60.degree. C. for 5 h. After
cooling, the reaction solution was poured into a saturated aqueous
solution of sodium hydrogen carbonate and subjected to extraction
with a solvent system of chloroform and methanol (1:1). The organic
layer was dried with sodium sulfate and the solvent was distilled
off under vacuum. The resulting residue was subjected to silica gel
column chromatography and elusion was conducted with a solvent
system of chloroform and methanol (10:1) to yield the end product
(580 mg).
[0174] .sup.1H-NMR (CDCl.sub.3+CD.sub.3OD, .delta.): 1.2-1.4(6H,m),
2.0-2.5(4H,m), 2.30(3H,s), 4.0-4.3(4H,m), 4.67(3H,m),
6.61(1H,d,J=7.8 Hz), 7.59(3H,m), 8.75(1H,s)
EXAMPLE 20
Synthesis of
N-{4-[N'-(2,4-diamino-6-pteridinyl)methylamino]-3-methyl}benz-
oyl-L-glutamic Acid (Compound 7)
[0175] An aqueous solution of 1N NaOH (3.5 ml) was added to an
ethanol (40 ml) suspension of the compound (580 ma) synthesized in
Example 19 and the mixture was stirred at 35.degree. C. for 4 h.
Following further stirring at 25.degree. C. for 20 h, water (5 ml)
was added to the reaction solution, which was then evaporated to
dryness under vacuum, with care being taken so that the external
temperature would not exceed 30.degree. C. The resulting yellow
solids was dissolved in water (10 ml) and the solution was adjusted
to pH of 3.7 with 1N HCl, followed by standing in a refrigerator
for 2 h. The deposit was precipitated by centrifuge (2000
rpm.times.15 min) and the supernatent was removed. The same
procedure was followed five times and the resulting precipitate was
dried to yield end produce (331 mg).
[0176] .sup.1H-NMR (DMSO-d.sub.6, .delta.): 1.8-2.4(4H,m),
2.25(3H,s), 4.40(1H,m), 4.60(2H,d,J=4.9 Hz), 6.18(1H,m),
6.56(1H,d,J=8.3 Hz), 7.56(2H,m). 8.08(1H,d,J=7.8 Hz),
8.71(1H,s)
REFERENCE EXAMPLE 34
Synthesis of dimethyl N-(3-methyl-4-nitrobenzoyl)-L-2
aminoadipate
[0177] 3-Methyl-4-nitrobenzoic acid (3.4 g) was suspended in
thionyl chloride (20 ml) and, thereafter, a catalytic amount of
dimethylformamide was added, followed by refluxing of the mixture
for 2 h. After cooling, the reaction was evaporated to dryness
under vacuum. The resulting solids were dissolved in
dichloromethane (80 ml); thereafter, dimethyl L-2-aminoadipate HCl
(4.2 g), potassium carbonate (10 g) and water (80 ml) were added to
the solution and the mixture was stirred vigorously at room
temperature for 12 h. The reaction solution was poured into water
and subjected to extraction with chloroform, followed by washing
with 1N HCl and drying with sodium sulfate. The solvent was
distilled off under vacuum. The resulting residue was subjected to
silica gel column chromatography and elusion was conducted with a
solvent system of chloroform and methanol (100:1) to yield the end
product (6.0 g).
[0178] .sup.1H-NMR (CDCl.sub.3, .delta.): 1.6-2.4(4H,m),
2.39(2H,m), 2.62(3H,s), 3.68(3H,s), 3.80(3H,s), 4.79(1H,m),
7.13(1H,m), 7.79(2H,m), 7.98(1H,d,J=8.3 Hz)
REFERENCE EXAMPLE 35
Synthesis of dimethyl
N-(4-amino-3-methylbenzoyl)-L-2-aminoadipate
[0179] The compound (5.9 g) synthesized in Reference Example 34 was
dissolved in acetic acid (100 ml) and a zinc powder (11 g) was
added slowly to the solution under cooling with ice. Following
stirring at room temperature for 2 h, the mixture was filtered and
the filtrate was concentrated under vacuum. The resulting
concentrate was dissolved in chloroform (100 ml) and washed with a
saturated aqueous solution of sodium hydrogen carbonate. The
organic layer was drieid with sodium sulfate and the solvent was
distilled off under vacuum to yield the end product (7.8 g).
[0180] .sup.1H-NMR (CDCl.sub.3, .delta.) 1.6-2.1(4H,m), 2.19(3H,s),
2.36(2H,m), 3.66(3H,s), 3.77(3H,s), 4.7s(1H,m), 6.63(2H,m),
7.55(2H,m)
EXAMPLE 21
Synthesis of dimethyl
N-({4-[N'-(2,4-diamino-6-pteridinyl)methylamino]-3-m-
ethyl}benzoyl-L-2-aminoadipate
[0181] The compound (2.73 g) synthesized in Reference Example 35
and an isopropanol adduct of 6-bromomethyl-2,4-diaminopteridine
hydrobromide (1.51 g) were suspended in dimethylacetamide (25 ml)
and the suspension was stirred at 55-60.degree. C. for 5 h. After
cooling, the reaction solution was poured into a saturated aqueous
solution of sodium hydrogen carbonate and subjected to extraction
with a solvent system of chloroform and methanol (1:1). The organic
layer was dried with sodium sulfate and the solvent was distilled
off under vacuum. The resulting residue was subjected to silica gel
column chromatography and elution was conducted with a solvent
system of chloroform and methanol (10:1) to yield the end product
(760 mg).
[0182] .sup.1H-NMR (CDCl.sub.3+CD.sub.3OD, .delta.): 1.6-2.0(4H,m),
2.29(3H,s), 2.39(2H,m), 3.68(3H,s), 3.77(3H,s), 4.65(2H,s),
4.74(1H,m), 6.62 (1H,d,J=9.3 Hz), 7.18(1H,d,J=7.3 Hz), 7.60(2H,m),
8.77(1H,s)
EXAMPLE 22
Synthesis of
N-{4-[N'-(2,4-diamino-6-pteridinyl)methylamino]-3-methyl}benz-
oyl-L-2-aminoadipic Acid
[0183] An aqueous solution of 1N NaOH (4.6 ml) was added to an
ethanol (100 ml) suspension of the compound (760 mg) synthesized in
Example 21, and the mixture was stirred at 35.degree. C. for 4 h.
Following further stirring at 25.degree. C. for 20 h, water (10 ml)
was added to the reaction solution and the mixture was evaporated
to dryness under vacuum, with care being taken so that the external
temperature would not exceed 30.degree. C. The resulting yellow
solids were dissolved in water (20 ml) and the solution was
adjusted to pH of 3.7 with 1N HCl, followed by standing in a
refrigerator for 2 h. The deposit was precipitated by centrifuge
(2000 rpm.times.15 min) and the supernatant was removed. The same
procedure was followed five times and the resulting precipitate was
dried to yield the end product (400 mg).
[0184] .sup.1H-NMR (DMSO-d.sub.6, .delta.): 1.5-1.9(4H,m),
2.24(5H,m), 4.34(1H,m), 4.58(2H,m), 6.21(1H,m), 6.56(1H,d,J=8.8
Hz), 7.59(2H,m), 8.05(1H,d,J=7.3 Hz), 8.70(1H,s)
[0185] Industrial Applicability:
[0186] The methotrexate derivatives of the present invention
exhibit better lymphocyte proliferation inhibiting action than
heretofore known compounds and, hence, they are very useful as
rheumatism treating agents.
* * * * *