WO1998018763A1 - Tetrahydroisoquinoline derivatives - Google Patents

Abstract

Medicinal compositions containing as the active ingredient compounds having an inhibitory effect on dipeptidyl-peptidase IV and having the tetrahydroisoquinoline skeleton, for example, tetrahydroisoquinoline derivatives represented by general formula (I) and the tetrahydroisoquinoline derivatives, wherein R1 represents (1) a group having the structure of an amino acid with optionally substituted amino from which the hydroxy atomic group in the carboxy atomic group has been eliminated, or (2) an amino protective group; R2 represents (1) optionally protected hydroxy, (2) having the structure of an amino acid with an optionally protected carboxy atomic group from which a hydrogen atom in the amino atomic group has been eliminated, or (3) a group having the stuctures of a primary or secondary amine or ammonia from which a hydrogen atom on the nitrogen atom has been eliminated; and R?3, R4, R5 and R6¿ are the same or different and each represents hydrogen hydroxy or lower alkoxy.

Description

 Description Tetrahydroisoquinoline derivative

Technical field

 The present invention relates to a pharmaceutical composition comprising a compound having a dipeptidyl peptidase IV inhibitory activity and having a tetrahydroisoquinoline skeleton as an active ingredient, a novel tetrahydroisoquinoline derivative, and a method for producing the same.

Background art

 Deptidyl peptidase IV is a serine protease that specifically hydrolyzes X--Pr0 (X is any amino acid) from the free N-terminus of the polypeptide chain. In immune system cells, expression is induced by activation of T cells, and plays an important role in T cell activation and proliferation (Yoichi Pian 'Cyanal' Ob Immunorossi, Eu ropean Journa 1 of Immunology, 17, 1821-1826, 1987; Noological 'chemistry' Hoppe Issailer (Bi 0 10 gica 1

ChememistryHoppe—Seylyr), 371 1, 699—705, p. 1990). That is, blocking dipeptidyl peptidase IV with an antibody or an inhibitor suppresses the activation of T cells. In addition, there is interest in the relationship between this enzyme and pathological conditions in disorders of collagen metabolism and immune disorders. For example, in rheumatoid patients, the percentage of dipeptidyl peptidase IV positive in peripheral blood T cells is increased, and high dipeptidyl peptidase IV activity is detected in urine of nephritis patients.

Examples of known dipeptidyl peptidase IV inhibitors include triptides. Certain diprotin A (L-isoleucyl-L-prolyl-L-isoleucine), diprotin B (L-valylol L-prolyl-L-mouth isine) and diprotin C (L-valyl-L-prolyl-1: L-isoleucine) (JP 59-Patent _{25366), A 1 a- P r} o- two Torobe emission zone I le hydroxyl § Min (journal O Bed 'Enzaimu-Inhihishiyon, jounal 0 t En z yme I nhibition), 2 vol. Pp. 129-142, 1988), A1a-boroPro and Pro-boroPro, where boroPro is a compound in which the carboxyl group of proline is substituted by a B (OH) ₂ group. (Procedings of the National Aka Demi 1 of the Sciences of the United States, the United States of America, the United States of America, the United States (Procedingso I the National)

Ac a d emy o f S c i e n c e s o f h e Un i t e d

S ttesof America), 88, 1556–1559, 1991) and Lys— (Z (NO ₂ ))-thiazolidine (where Z (N〇 ₂ ) is 4-nitrobenzene) (Indicating a xycarbonyl group) (Biological 'chemistry' hoppe-se-fu (Biological and hemem istry hoppe—

 S e y 1 er), vol. 372, p. 305—311, p. 1991) are known. However, a dipeptidyl peptidase IV inhibitor having a tetrahydroisoquinoline skeleton is not known.

Disclosure of the invention

 The present invention provides a compound having excellent dipeptidyl peptidase IV inhibitory activity, and a pharmaceutical composition comprising the compound as an active ingredient.

The present inventors have been examining cultures of microorganisms mainly isolated from soil, and show that they exhibit dipeptidyl peptidase IV inhibitory activity in cultures of Aspergillus fungi. Compounds were found to be produced. These compounds were isolated and purified from the culture solution, and their physicochemical properties were examined to determine their chemical structures. As a result, they were found to be novel compounds.

 On the other hand, the present inventors have found that inhibition of dipeptidyl peptidase IV can specifically suppress the activation of T cells, and immunological disorders involving activation of T cells such as rheumatoid arthritis As a result of intensive studies, we believe that a series of compounds having a tetrahydroisoquinoline skeleton, including the aforementioned microbial-derived compounds, may have an inhibitory effect on dipeptidyl peptidase IV. They have obtained new knowledge that they are effective in preventing and treating autoimmune diseases (arthritis, rheumatoid arthritis, etc.), and have completed the present invention based on the knowledge.

 That is, the present invention provides a pharmaceutical composition comprising a compound having a dipeptidyl peptidase IV inhibitory activity and having a tetrahydroisoquinoline skeleton or a pharmaceutically acceptable salt thereof as an active ingredient. is there.

 Further, the present invention provides a compound represented by the general formula [I]:

(Wherein, R 1 is (1) a group having a structure obtained by removing a hydroxy group of a lipoxy group from an amino acid in which an amino group may be protected or (2) a protecting group of an amino group, and R ² is (1) a hydroxyl group which may be protected, (2) a group having a structure in which one hydrogen atom of an amino group has been removed from an amino acid whose carboxy group may be protected, or (3) a primary or secondary group One hydrogen atom on a nitrogen atom from amine or ammonia R 3, R ⁴ , R 5 and R 6 are the same or different and represent a hydrogen atom, a hydroxyl group or a lower alkoxy group.

The present invention provides a novel tetrahydroisoquinoline derivative represented by or a pharmaceutically acceptable salt thereof, and a method for producing these compounds.

 Further, the present invention provides the following general formula [II]:

(Wherein one of R ⁷ and R ⁸ represents a hydroxyl group and the other represents a hydrogen atom or a hydroxyl group) or a pharmacologically acceptable salt thereof. In the above general formula [II], a compound in which R ⁷ and R ⁸ are a hydroxyl group is hereinafter referred to as TMC ^ (: − 28 and a compound in which R ⁷ is a hydroxyl group and R ⁸ is a hydrogen atom. A compound in which R 7 is a hydrogen atom and R 8 is a hydroxyl group is referred to as TMC-2C hereinafter.

 The present invention also provides a method for producing TMC-2A, TMC-2B and TMC-2C using microorganisms.

BRIEF DESCRIPTION OF THE FIGURES

 Figure 1 shows the UV spectrum of TMC-2A.

 Figure 2 shows the UV spectrum of TMC-2B.

 Figure 3 shows the UV spectrum of TMC-2C.

 Figure 4 shows the IR spectrum of TMC-2A.

Figure 5 shows the IR spectrum of TMC-2B. Figure 6 shows the IR spectrum of TMC-2C.

 FIG. 7 is the 1 H-NMR spectrum of TMC-2A.

 FIG. 8 is a 1 H-NMR spectrum of TMC-2B.

Figure 9 is the ¹ H-NMR spectrum of TMC-2C.

FIG. 10 is a ¹³ C-NMR spectrum of TMC-2A.

 Figure 11 shows the 13 C-NMR spectrum of TMC-2B.

 FIG. 12 is a 13 C-NMR spectrum of TMC-2 C.

 FIG. 13 is a diagram showing the inhibitory effect of cho1 ^ (:-28 on rat alkyldiamine-induced arthritis.

 C 0 n t (-): control group not receiving alkyldiamine

 C 0 n t (+): control group to which alkyldiamine was administered

 FIG. 14 is a diagram showing the inhibitory effects of TMC-2A and the compound of Example 13 on rat adjuvant-induced arthritis.

 C 0 n t (-): control group to which heat-killed Mycobacterium tuberculosis was not administered

 C 0 n t (+): control group to which heat-killed Mycobacterium tuberculosis was administered

The best shape bear for invention

 Examples of the pharmaceutical composition of the present invention include a pharmaceutical composition having a dipeptidyl peptidase IV inhibitory activity and having, as an active ingredient, a compound having a tetrahydroisoquinoline skeleton or a pharmaceutically acceptable salt thereof. Specifically, it has a diptidyl peptidase IV inhibitory activity, and has the formula:

And a pharmacologically acceptable salt thereof as an active ingredient. Further, a pharmaceutical composition comprising a tetrahydridoisoquinoline derivative represented by the general formula [I] or a pharmaceutically acceptable salt thereof as an active ingredient can be mentioned.

 The pharmaceutical composition of the present invention is useful as a dipeptidyl peptidase IV inhibitor, a prophylactic / therapeutic agent for autoimmune diseases, especially a prophylactic / therapeutic agent for arthritis, and a prophylactic / therapeutic agent for rheumatoid arthritis. .

 Examples of the tetrahydroisoquinoline derivative of the present invention include a compound represented by the general formula [I].

 Further, the tetrahydroisoquinoline derivative [I] of the present invention or a pharmacologically acceptable salt thereof has excellent dipeptidyl peptidase IV inhibitory activity, and is therefore useful as a dipeptidyl peptidase IV inhibitor. Furthermore, the tetrahydroisoquinoline derivative [I] or a pharmacologically acceptable salt thereof has an excellent preventive / therapeutic action for autoimmune diseases, and is a prophylactic / therapeutic agent for autoimmune diseases, especially for the prevention / treatment of arthritis. It is useful as an agent for the prevention and treatment of rheumatoid arthritis.

Among the compounds represented by the general formula [I], preferred compounds are those in which R 1 is (1) aryloxycarbonyl group, aryl substituted lower alkoxycarbonyl group or lower alkoxycarbonyl group, and is an amino group. A group having a structure in which a hydroxy group of a carboxy group is removed from an amino acid which may be protected, or (2) an aryloxycarbonyl group, an aryl group-substituted lower alkoxycarbonyl group or a lower alkoxycarbonyl group, and R ² is (1) a hydrogen atom of an amino group which may be substituted with an aryl group-substituted lower alkyl group, or (2) an amino acid whose carboxy group may be protected with a lower alkyl or an aryl group-substituted lower alkyl group. 1 group having the removed structure or (3) lower alkyl or aryl substituted lower alkyl An amino group optionally substituted with one or two groups selected from the group consisting of R 3, R 4 and R

⁵ and R ⁶ may be the same or different and include a hydrogen atom, a hydroxyl group or a lower alkoxy group.

Ό o

Of these, more preferred compounds include those in which R ¹ is (1) a benzyloxycarbonyl group or a tert-butoxycarbonyl group whose amino atom group is protected, such as a retryptofil group, a lysyl group, a phenylalanyl group, or 2) benzyloxycarbonyl group or tert-butoxycarbonyl group, 1 ^ ² ^^) hydroxyl group optionally protected by benzyl group, (2) carboxy group protected by methyl group or benzyl group. May be, alanine, valine, leucine, isoleucine, proline, fenylalanine, tryptophan, methionine, glycine, serine, 0-benzyl-serine, threonine, cysteine, glutamine, asparagine, tyrosin, lysine, arginine, histidine, Aspartic acid, glutamic acid and Formula:

(Wherein, R ⁷ and R ⁸ have the same meanings as above)

A group having a structure obtained by removing one hydrogen atom from the _ff -amino group of α-amino acid selected from the group consisting of (3) a tert-butyl group and a benzyl group; an amino group, R ³ force? hydrogen atom or a lower alkoxy group, R ⁴ is a hydrogen atom or a hydroxyl group, R ^{5 months?} hydrogen atom or a lower alkoxy group, R ⁶ is hydrogen atom, water group or a lower alkoxy group Is raised.

Furthermore, as compounds with favorable medicinal properties, R 1 is a tributophyl group, R ² is (1) a hydroxyl group, (2) alanine, norin, leucine, isoleucine, proline, phenyla. One of the hydrogen atoms of the amino group is selected from the amino acids selected from lanin, tributofan, methionine, glycine, serine, threonine, cystine, glutamine, asparagine, tyrosine, lysine, arginine, histidine, aspartic acid and glutamic acid Examples include the group having the removed structure or (3) an amino group, and a compound in which R ³ , R ⁴ , R 5 and R ⁶ are hydrogen atoms.

 Among these compounds, more preferable compounds from the viewpoint of medicinal effect are as follows: R 1 is a tributyl group, 1 ^ 2 is (1) a hydroxyl group, (2) glutamine, serine, aspartic acid, glutamic acid, alanine, cysteine, arginine Α-amino acids selected from methionine and asparagine, a group having a structure in which one hydrogen atom of an amino group has been removed from an α-amino acid or (3) an amino group, R 3, R 4, R 5 and R 6 Compounds that are hydrogen atoms are mentioned.

 Furthermore, a particularly preferable compound in terms of pharmacological effect is 2-L-tributophyl-1,2,3,4-tetrahydroisoquinolyl-13-carboxylic acid.

 Further, another compound that is preferable in terms of efficacy is TMC-2A, TMC-2B or TMC-2C, that is, a compound represented by the general formula [II]:

(Wherein, R ⁷ and R ⁸ have the same meanings as above)

The compound shown by these is mentioned.

In the present specification, amino acids include both L-form, D-form and mixtures thereof, and include, for example, alanine, norin, leucine, isoleucine, proline, phenyla Protein components such as lanine, tryptophan, methionine, glycine, serine, threonine, cystine, glutamine, asparagine, tyrosine, lysine, arginine, histidine, aspartic acid and glutamic acid α-amino acid, norleucine, α-aminobutyric acid, y-amino Aliphatic monoaminocarboxylic acids such as butyric acid, monoaminoisobutyric acid, β-rylanine, homoserine, trimethyl-serine, dibenzyl-serine, 0-potassium rubamyl-serine, and hydroxy-1-oxo-norvaline; Monoaminodicarboxylic acids such as aminoadipic acid, theanine, alphamethyleneglutamic acid and y-methylglutamic acid, orditin, -lysine, diaminomonocarboxylic acids such as α, —diaminopropionic acid and α, y-diaminobutyric acid, and dia Diaminodicarboxylic acids such as nopimellinic acid, sulfonic acid-containing amino acids such as cysteinic acid, aromatic amino acids such as thyronine, kynurenine and 3,4-dioxyphenyl-l-alanine, aziridine-1,2,3-dicarboxylic acid, 2-amino- Heterocyclic amino acids such as 3- (isosazozolin-5-one--4-yl) propionic acid and anticapsin; bases such as 4-oxalidine, 4-oxolidinine and 3,6-diamino-15-hydroxyhexanoic acid Sulfur-containing amino acids such as sex amino acids, cystatione, lanthonin and S-methyl-cysteine; cyclic amino acids such as pipecolic acid, azetidine-12-carboxylic acid and 2-aminocyclopentane-11-carboxylic acid; and citrulline, alanosine and Special functional group-substituted amino acids such as azaserine, etc.

(Wherein, R ⁷ and R ⁸ have the same meanings as above)

Are shown. Of these, preferred are, for example, alanine, norin, leucine, iso-isocyanate, proline, feniralanine, tryptophan, methionine, glycine, serine, 0-benzyl-serine, threonine, cysteine, glutamine, asparagine, tyrosine Amino acids and formulas such as lysine, lysine, arginine, histidine, aspartic acid and glutamic acid;

(Wherein, R ⁷ and R ⁸ have the same meanings as above)

Are shown.

In the present specification, examples of the “group having a structure in which a hydroxy group of a carboxy atom group has been removed from an amino acid” in R ¹ include, for example, alanine, norin, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, A group having a structure obtained by removing the hydroxy group of α-carboxy group from a monoamino acid such as glycine, serine, threonine, cysteine, glutamine, asparagine, tyrosine, lysine, arginine, histidine, aspartic acid, glutamic acid, etc. Aranyl group, s-rifle group, leucyl group, isoleucyl group, prolyl group, phenylaralanyl group, tributyl group, methionyl group, glycyl group, seryl group, threonyl group, cystinyl group, glutamyl group, aspara Examples include a ginyl group, a tyrosyl group, a lysyl group, an arginyl group, a histidyl group, an aspartyl group and a glutaminyl group. Of these, preferred examples include a tributophyl group, a lysyl group, and a phenylalanyl group, and particularly preferred examples include a tributophyl group. In addition, the carboxy group in R ² may be replaced with an amino acid which may be protected. Examples of the group having a structure in which one hydrogen atom of the amino group has been removed include, but are not limited to, alanine, norin, leucine, isoleucine, proline, phenylalanine, triptophan, methionine, glycine, serine, threonine, and cysteine. , Glutamine, asparagine, tyrosine, lysine, arginine, histidine, aspartic acid, glutamic acid, isoleucine methyl ester, benzyl lucerin benzyl ester, and the formula:

(Wherein, R ⁷ and R ⁸ have the same meanings as above)

And a group having a structure in which one hydrogen atom of an α-amino group has been removed from an amino acid, such as the amino acid represented by Of these, preferred are groups having a structure in which one hydrogen atom of the _α -amino group of glutamine, serine, aspartic acid, glutamic acid, alanine, cysteine, arginine, methionine and asparagine has been removed, and more preferred examples are Is a group having a structure obtained by removing one hydrogen atom from the amino group of glutamine and serine. Another good example is the expression:

ヽ

(Wherein, R ⁷ and R ⁸ have the same meanings as above)

And the group represented by Examples of the “group having a structure in which one hydrogen atom on a nitrogen atom has been removed from a primary or secondary amine or ammonia” for R 2 include, for example, one group selected from lower alkyl or aryl-substituted lower alkyl or An amino group which may be substituted by two is exemplified. Of these, an amino group which may be substituted by one or two groups selected from a tert-butyl group and a benzyl group is preferred. More specific examples thereof include a tert-butylamino group, a benzylamino group, an amino group and the like, and a more preferred example is an amino group.

 The target compound [I] of the present invention includes any one of the tetrahydroisoquinoline skeleton moiety having the R-configuration at the 3-position, the S-configuration, and a mixture thereof. Are preferred. When compound [I] further has an asymmetric carbon atom, any stereoisomer based on the asymmetric carbon atom or a mixture thereof is also included in the present invention.

 The compound [I], which is the active ingredient of the present invention, can be prepared by a conventional method for peptide synthesis, for example, “peptide synthesis” (Synthetic Chemistry Series, published by Maruzen Co., Ltd., 1979) and “ Experiments ”(published by Maruzen Co., Ltd., 1985) or a method analogous thereto can be produced by either a liquid phase method or a solid phase method.

 Among the compounds [I], the general formula [I-a]:

(Wherein, R ^{2 2} is (1) group or a (2-carboxy atomic group having one obtained by removing the structure of the hydrogen atom of Amino atomic group from amino acids which may optionally be protected) primary or secondary amine emissions or A group with a structure in which one hydrogen atom on a nitrogen atom has been removed from ammonia, R R3, R4, R5 and R6 have the same meaning as above)

The compound represented by the general formula [I I I]:

(Wherein, R 1 is (1) a group having a structure in which at least an amino group protected by a s ′ protected amino acid is surrounded by a hydroxy group of a carboxy group or (2) a protecting group for an amino group , R ³ , R ⁴ , R 5 and R 6 have the same meaning as above)

Or a reactive derivative at the carboxy group thereof and a general formula [IV]:

 H-R 21 [IV]

(Wherein, R ² 1 is (1) group or (2) a primary or secondary Amin or ammonia with one obtained by removing the structure of the hydrogen atom of at least amino acids Karaa Mino atomic group carboxy atomic group is protected Represents a group having a structure obtained by removing one hydrogen atom on a nitrogen atom from

By condensation with a compound of the formula [V]:

(Wherein, RH, R21, R3, R4, R5 and R6 have the same meanings as described above), and if necessary, the protective group may be removed to produce the compound. In addition, among the compounds [I], the general formula [I—b]

[I b]

(Wherein, R 1 ³ is a group having the structure Amino atomic group obtained by removing a hydroxy atomic group of a carboxy group of atoms from amino acids which may be ^{substituted, R 2, R 3, R} 4, R 5及Pi R ⁶ Has the same meaning as above)

The compound represented by the general formula [VI]:

(Wherein, R ²³ represents (1) a protected hydroxyl group, (2) a group having a structure in which at least one hydrogen atom of an amino group has been removed from an amino acid having at least a protected carboxy group, or (3) A group having a structure in which one hydrogen atom on a nitrogen atom has been removed from primary or secondary amine or ammonia, and R ³ , R ⁴ , R 5 and R 6 have the same meaning as described above.)

And a compound of the general formula [VI I]:

R ¹² 2-OH [VI I]

(Wherein, R 1 ² denotes a group having a structure obtained by removing a hydroxy atomic carboxy atomic group of amino acids at least Amino atomic group is protected)

Condensed with the amino acid represented by By the general formula [VIII]:

(Wherein R 12, R 23, R 3, R 4, R 5 and R 6 have the same meanings as described above), and, if desired, by removing the protecting group There is a monkey.

 Examples of more specific production methods are shown in the following (A) to (C).

(A) —General formula [I X]:

(In the formula, R 14 is a protecting group for amino group, and R 31, R 41, R 51, and R 61 are the same or different and represent a hydrogen atom, a hydroxyl group, or a lower alkoxy group.)

Or a reactive derivative at the carboxy group thereof and a general formula [X]:

 H-R 2 [X]

(Wherein R ²⁴ represents (1) a group having a structure in which at least one hydrogen atom of an amino group is removed from an amino acid in which at least a carboxy group is protected, or (2) a primary or secondary amine or ammonia Represents a group having a structure in which one hydrogen atom on a nitrogen atom has been removed) The compound represented by the general formula [XI] is condensed in a suitable solvent at a temperature of from 30 ° C. to room temperature using a suitable condensing agent.

(Wherein, R 14, R 24, R 31, R 41, R 51 and R 61 have the same meanings as described above), and if necessary, the protecting group is removed by a conventional method. Thus, the corresponding target compound [I] can be produced.

 In addition, if desired, the general formula [XI] obtained by removing the protecting group R14 at the N-terminal of the compound [XI] [

X I I]:

 Wherein R 24, R 31, R 41, R 51 and R 61 have the same meaning as described above, and a general formula [XI 11]:

 R 15-OH [X I I I]

(Wherein, R 1 ⁵ represents a group having a structure obtained by removing a hydroxy atomic carboxy atomic group of amino acids at least amino atomic group is protected)

Is condensed with an appropriate condensing agent in an appropriate solvent under ice-cooling to room temperature to obtain a compound of the general formula [XIV]:

 (Wherein, R 15, R 24, R 31, R 41, R 51 and R 61 have the same meanings as described above). The corresponding target compound [I] can be produced by removing the compound according to the above.

In the above compounds [IX], [X], [XI], [XII], [XIII] and [XIV], preferred examples of R ⁴ include an aryl group-substituted lower alkoxycarbonyl group. Preferred examples of ⁵ include a group having a structure in which a hydroxy atom group of an α-carboxy atom group is removed from an α-amino acid in which an amino group is protected by a lower alkoxycarbonyl group, and a preferred example of R ²⁴ Is selected from a group having a structure in which one hydrogen atom of an a-amino group is removed from an α-amino acid in which a carboxy group is protected by a lower alkyl group, or a lower alkyl or aryl substituted lower alkyl. Examples of the amino group which may be substituted with one or two groups include R 3 i, R ⁴ i, R 51 and R 6 i, which may be the same or different and include a hydrogen atom or a lower alkoxy group. can give.

 (B) —General formula [XV]:

(Wherein, R 25 represents a hydroxyl group having a protecting group; R 32, R 42, R 52 and R 62 are the same or different and represent a hydrogen atom, a hydroxyl group or a lower alkoxy group)

And a compound represented by the general formula [XVI]:

 R 16—OH [XV I]

(Wherein, R ¹⁶ represents a group having a structure in which at least the amino group is protected and the hydroxy group of the carboxy group is removed from the amino acid)

Is condensed with an amino acid or a reactive derivative at the carboxy group thereof in an appropriate solvent in a suitable solvent at a temperature of from water to room temperature to obtain a compound of the general formula [XVI I]:

(Wherein, R 16, R 25, R 32, R 42, R 52 and R 62 have the same meanings as described above). The corresponding target compound [I] can be produced by removal according to the method.

In the above compounds [XV], [XVI] and [XVII], preferred examples of R 16 include an α-amino acid having an amino group substituted by a lower alkoxycarbonyl group to a hydroxy of an α-carboxy atom group. A group having a structure from which an atomic group has been removed is mentioned. A preferred example of R ²⁵ is a lower alkoxy group substituted with an aryl group. A preferred example of R ³² , R ⁴² , R ⁵² and R ⁶² is hydrogen. Atoms.

(C) Among the compounds obtained in the above (B), a compound represented by the general formula “XV III”:

 Wherein R 16, R 32, R 42, R 52 and R 62 have the same meaning as described above, or a reactive derivative at the carboxy group thereof and a general formula [XIX]:

 H-R 26 [X I X]

(In the formula, R ²⁶ represents a group having a structure in which at least one hydrogen atom of an amino group has been removed from an amino acid in which at least a carboxy group is protected, or (2) a group having a primary or secondary amine or ammonia. Represents a group having a structure in which one hydrogen atom on a nitrogen atom has been removed)

Is condensed with an appropriate condensing agent in an appropriate solvent at —30 ° C. to room temperature to obtain a compound of the general formula [XX]:

(Wherein, R 26, R 16, R 32, R 42, R 52 and R 62 have the same meanings as described above). The corresponding target compound can be prepared by removing according to the following.

In the above compounds [XV III], [XIX] and [XX], a preferable example of R 16 is a << amino which is protected with an amino group by a lower alkoxycarbonyl group. From acid. Motogaa Gerare having a structure obtained by removing a hydroxy group of atoms one carboxy group of atoms, preferred examples of R ^{2 6} is carboxy sheet atomic in Ariru substituted lower alkyl groups are protected "alpha from single amino acid —A group having a structure in which one hydrogen atom of an amino group has been removed or an amino group, and examples of R 32, R 42, R 52 and R 62 include a hydrogen atom .

 The protecting group for the carboxy group (carboxyl group) and the amino group (amino group) may be any group that does not participate in the condensation reaction and can be easily removed by a conventional method. It is usually used as a protecting group for amino acids in peptide synthesis. Can be used. Examples of the protecting group for the carboxy atom group include a lower alkyl group and an aryl group-substituted lower alkyl group, and specifically, a methyl group, an ethyl group, a benzyl group, and the like. Among them, preferred is an aryl group-substituted lower alkyl group, such as a benzyl group. Examples of the protecting group for the amino group include a substituted and unsubstituted lower alkoxycarbonyl group. Specific examples include a benzyloxycarbonyl group, a 4-methoxybenzyloxycarbonyl group, and a 9-fluorenyl. Examples include a methyloxycarbonyl group, a tert-butoxycarbonyl group, and a 2,2,2-trichloroethyloxycarbonyl group. Among them, preferred are an aryl group-substituted lower alkoxycarbonyl group and an unsubstituted lower alkoxycarbonyl group, for example, a benzyloxycarbonyl group and a tert-butoxycarbonyl group. The protecting groups for the carboxy and amino groups can be easily removed by a known method, for example, a conventional method of peptide chemistry.

Examples of the reactive derivative at the carboxyl group of the amino acid include its active ester, such as succinimide ester and benzotriazole ester. As a suitable condensing agent, 1,3-dicyclohexylcarbodiimide and the like are used. Condensation reaction can also be performed with a combination of an ester activator and a condensing agent. For example, 1-hydroxybenzotriazole (monohydrate) and 1-ethyl-3- (3-dimethylaminopropyl) carboxydiimidide Hydrochloride, N-hydroxysuccinimide and 1,3-dicyclohexylcarpoimide can be used. Of these, a combination of 1-hydroxybenzotriazole (monohydrate) and 1-ethyl-13- (3-dimethylaminopropyl) carboxydiimide hydrochloride is preferred.

 Suitable solvents may be any inert solvents that do not participate in the condensation reaction, such as dimethylformamide, dimethylsulfoxide, dichloromethane, dichloroethane, chloroform, tetrahydrofuran, ethyl acetate and N-methylpyrrolidone. And dimethylformamide is preferred.

 When the compound to be subjected to the condensation reaction has a reactive functional group other than a carboxy atom group and an amino atom group, the functional group is protected in advance and then subjected to the condensation reaction according to a conventional method. Deprotection is preferred.

 The target compound [I] of the present invention is subjected to condensation and deprotection of a carrier obtained by binding a desired starting amino acid to a resin and a corresponding starting amino acid derivative by using a commercially available automatic synthesizer. After removing the resin, the peptide may be purified by a means for separating the peptide, for example, extraction, distribution, reprecipitation, crystallization, recrystallization, various types of chromatography, high-performance chromatography, or the like.

As the resin, any resin can be used as long as the target substance can be finally cut out in the form of an amide. For example, Ν— — 9-fluorenylmethoxycarbonyl-super-acid labile polyethylene glycol handle polystyrene (Product name: F moc—Ν—SAL—PEGR esin; Watanabe Chemical), (4 1 2 ', 4'-Dimethoxyphenyl 1-Hiichi 9-Fluorenylmethoxycarbonyl monoaminomethyl) Phenoxy resin (trade name: Fmo c— c—SAL

Resin; manufactured by Watanabe Chemical) and (4-2 ', 4'-dimethoxyphenyl-Ν-"-19-fluorenylmethoxycarbonyl-aminomethyl) -phenoxyacetamide-norleucine-ρ-methyl-benzhydroxyamine resin (Trade name: Fmoc—NH-SAL-MBHA Resin; Watanabe Chemical).

 Further, among the compounds which are the active ingredients of the present invention, general formula [II]:

[I 门

(Wherein, R ⁷ and R ⁸ have the same meanings as above)

The TMC-2A, TMC-2B or TMC-2C represented by the following formula, ie, TMC-2A, TMC-2B or TMC-2C, can also be obtained by culturing a mold belonging to the genus Aspergillus and isolating from the culture.

 Hereinafter, a method for producing TMC-2A, TMC-2B and TMC-2C by a mold belonging to the genus Aspergillus will be described in more detail.

 An example of a strain producing TMC-2A, TMC-2B and TMC-2C is A374 strain isolated from soil in Kochi City, Kochi Prefecture. The mycological properties of this strain are as follows.

Table 1 shows the growth of A374 strain in various media after culturing at 25 ° C for 7 days. The color tone was determined according to the JIS standard color table (Z 872 1). Table 1 Physicochemical properties of TMC-2A, 2B and 2C Property TMC-2A TMC-2B TMC-2C Shape White powder White powder White powder Melting point (° c) 166 168 166 168 175 175 181 Solubility Water , Soluble in methanol, soluble in methanol, soluble in methanol,

: Bath, bath η b π b +; ^ no ノ ΐ A, g UU nolem UU <g no 1 V / | ^N 0- insoluble insoluble

Specific rotation ¹ ) + 2.39 ° (C 0.2, + 11.42 ° (C 0.1, -17.5 ° (C 0.1,

 (¾0) ¾0) MeOH)

FAB-MS 571 (M + H) + 555 (Μ + Η) 10 555 (M + H) +

HR FAB- S 571.2331 (M + H) + ― ―

Molecular formula ^C 28¾4 ^N 4 ° 9 ^C 28¾4 ^N 4 ° 8 C ₂₈ H ₃₄ N ₄ 0 ₈

U V 212 272 (sh) 212 272 (sh) 212 272 (sh) MAX (MeOH) nm 280, 288 280, 288 280 '288

IR y max (KBr) cm- ¹ 3380, 1650 3380, 1650 3300, 1650 Color reaction Ninhydrin Ninhydrin Ninhydrin Sili force gel (1) 0.45 (1) 0.51 (1) 0.49

TLC Rf 2) (2) 0.30 (2) 0.58 (2) 0.52

 HPLC (1) 3.27 (1) 3.82 (1) 3.83

Rt (min) 3) (2) 12.17 (2) 12.83 (2) 12.90

1) TMC-2A and TMC-2B were measured as aqueous solutions, and TMC-2C was measured as a methanol solution at 20 ° C.

2) Developing solvent: (1) CH ₂ Cl ₂ : Me OH: EtOH: H ₂₀ (10: 4: 4: 2)

(2) E t OA c: Me OH: H 2 0 (1 0: 1 0: 2)

3) Elution solvent: (1) 20% CH ₃ CN—80% H ₂₀ (for 1.2 ml)

(2) Linear gradient: 10% CH ₃ CN—90% H ₂₀ (0 min) —35% CH ₃ CN-6 5% H ₂₀ (25 min) (1.2 m 1 min) The growth temperature range of A374 strain is 15-45 ° C, and the optimal temperature range is 20-40 ° C. The growth pH range is pH 2 to 13, and the optimal pH range is pH 3 to 11.

 Hyphae have smooth surfaces and partition walls. Conidia (6.7-8.0 zmX 23-1 100 m) with podocytes (foot-cell; 6.7-8.7 ^ mX 37-57 m) are formed from a part of the hypha, However, some club-shaped apical sac (17-24 1 «19-27 m) is observed. From the upper semicircular part of the apical sac, there is a pin-shaped one-stage incisor (pri marysteri gma ta; 2.3 to 3.3 mX 13 m), from which there is no spherical conidium (4.0 to 6.7 m) It is formed into a bundle in a chain. The conidium head is mainly cylindrical (50-100 mX16-200 m), but some conidium heads have a globular shape and a spherical shape (33-60 111 20-47 111). Is recognized.

 From the above characteristics, it was found that the A374 strain belongs to the genus Aspergillus (Aspergii11us). The As pergilluss p. A 374 strain was deposited on May 18, 1995 with the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry (1-3 1-3 Higashi, Tsukuba, Ibaraki, Japan) under the accession number FERM. Deposited as P-14934, and then transferred to the Institute on September 1.9, 1997 as Accession No. FERM BP-6113.

 In order to produce TMC-2A, TMC-2B, and TMC-2C by the method of the present invention, a TMC-2A, TMC-2B and TMC-2C-producing bacterium belonging to the genus Aspergillus is contained as a nutrient source. The medium is inoculated and grown aerobically. This results in a culture containing TMC-12A, TMC-2B and TMC-2C.

As a nutrient source, a carbon source and a nitrogen source that can be used as a nutrient source for microorganisms can be used. For example, peptone, meat extract, corn 'steep' rice, Nitrogen sources such as cottonseed flour, peanut flour, soy flour, yeast extract, NZ-amine, casein hydrolyzate, ammonium nitrate, ammonium sulfate, and starch, glycerin, sucrose, glucose, galactose, mannose, molasses Carbon sources such as carbohydrates or fats can be used. In addition, inorganic salts such as salt, calcium carbonate, phosphate, magnesium sulfate and the like can be added. These can be used as long as they are used by the producing bacteria and are useful for producing TMC-2A, TMC-2B and TMC-2C. Liquid culture is preferred for culturing the above-mentioned bacteria producing TMC-2A, TMC-2B and TMC-2C. The cultivation temperature can be used in a range in which the producing bacteria grow and produce a desired substance, and is usually 20 to 35 ° C. The cultivation can be carried out by appropriately selecting from the above conditions depending on the properties of the producing bacteria to be used. The desired TMC-2A, TMC-2B and TMC-2C are produced in culture. Isolation and purification of those products can be carried out by a method known per se, for example, ion exchange chromatography, partition chromatography, reverse phase chromatography, etc., as appropriate.

The physicochemical properties of the dipeptidyl peptidase IV inhibitors TMC-2A, TMC-2B and TMC-2C of the present invention are shown in Table 2 below.

Table 2 Growth status of A3 7 4 strains in various media Colony Front surface Back surface Medium diameter

 (Average) 31 elephant parts except the margin

 The whole surface is velvet and double colored and the outside is

Malt flake flat and thin irregular

 No wrinkles

7 to 76 Light yellow (5Y6.3 / 8.5) powdery, dark gray at center

Sugar culture light yellow

 (74) Greenish yellow (6.3 Y / 6/8) powdery, central (Ν8.5)

 Light gray (N8.0) fluffy on ground (2.5Y8 / 4)

Potato deer 6 ~ / () ■ LM LM5ΐJ t Beef ¾, enter ^ · ^ ζ_ Jim 1 Lesma \ Jutan specialty piece; Wrinkleless Kist (68) Leafy yellow-green (10Y5 / 6) powder In the center, a light gray dull yellow loin cold light gray (Ν8.0) fluffy and pine needle (N8.5) (5Y8 / 3) sky medium (10Y5 / 4) powdery Dotted

 52a 52 odmm ~ Γ¾Η ι · ヽ ヽ; ¾a Wrinkle-free, lace-like white, yellow on top

 Ku agar culture (53) Light gray Dyed yellow ground (2.5Υ7.5.12)

 (N8.5) (2.5Y8 / 6)

68 The entire surface is lace-like and very light yellow (5Υ9 / 2) Flat, thin and without wrinkles

 ^ n f- V seedling ^ Tomoe & I, ^ Ο Vϊ¾δ // I 丄 (Re 1, No sword ^ J x> v · / Αj?

 There are long mycelium scattered in it, bordered by (N8.5) (7.5YR9 / 3) Very pale yellow (2Y9 / 2) and powdery on agar medium

 Is there

 o 68 recitation 3 n2 ^ iten ヽ φ; ¾Ξ¾rule¾Ξ¾ |! Radial wrinkles Auto Radial wrinkles, powdery

 Gray yellow with correct white (N9) Meal cold (68) Yellowish green (10Υ6 / 6), bright gray in center

 (7.5YR6 / 4) Sky medium Color (Ν8.0) Fluffy spots

 To

 0/60 orchids Flat and regular Radial wrinkles The whole surface is velvety and double colored and the outside is

 Yp S s Cold (58) Dark white (N9)

 Large medium is taken

 (7.5.8 / 2) fluff

 (10 chars. 5/3)

76 78 Thigh White (N9) with slightly radial wrinkles

MY 20 cold (77) The whole surface is velvety and bright yellow Irregularly stripped leg culture medium (2.5Y7 / 10) with many powdery horns

 (2.5Y8 / 4)

74 77 orchid White (N9), slightly radial wrinkles 40 Y cold (76) Velvet, dull yellow-green (5Υ6 / 8) Irregularly bordered leg natural medium Many powdery Exists horn color

(2.5Y8 / 4) The UV and IR spectra of TMC-2A, TMC-2B and TMC-2C are shown in FIGS. 1 to 3 and 4 to 6, respectively. The UV spectrum was analyzed for a 50 gZm1 methanol solution of each sample. IR spectra were analyzed on potassium bromide tablets containing 1% (w / w) of each sample.

 Further, the 400 MHz proton nuclear magnetic resonance spectra of TMC-2A, TMC-2B and TMC-2C are shown in FIGS. 7 to 9 respectively. TMC-2A and TMC-2B were measured in heavy water using TSP (sodium trimethylsilylpropylsulfonate) as an internal standard. TMC-2C was measured in heavy methanol using TMS (tetramethylsilane) as an internal standard. The chemical shifts (ppm) are described below.

 TMC- 2 A: 7.55 (1H, d), 7.46 (1H, d), 7.34 (1H, s), 7.13 (1H, t), 7.07 (1H, t), 6.02 ( 1H, s), 4.82 (1H, d), 4.50 (1H, dd), 4.11 (1H, dd), 3.77 (1H, d), 3.73 (4H, m), 3.49 Up to 3.28 (5H, m), 3.19 (1H, dd), 2.35 (1H, dd), 1.68 (1H, ddd), 1.44 (1H, ddd), 1.14 (1H, dd), 0.7 1 (1 H, m)

 TMC-2B: 7.58 (1 H, dd), 7.52 (1 H, d), 7.38 (1 H, s), 7.22 (1 H, dd), 7.16 (1 H, dd), 6.09 (1H, s), 4.80 (1H, dd), 4.50 (1H, dd), 4.04 (1H, dd), 3.83 (1H, d), 3.77 (1H, m), 3.73 (3 H, s), 3.50 (1 H, dd), 3.43 (1 H, dd), 3.08 (2 H, d), 2.39 (1 H, dd), 1.64 (1 H, ddd), 1.20 (2 H, m), 0.63 (4 H, m)

TMC- 2.C: 7.53 (1 H, d), 7.40 (1 H, d), 7.24 (1 H, s), 7.12 (1 H, dd), 7.07 (1 H, dd), 6.09 ( 1 H, s), 5.06 (1 H, d ), 4.23 (1H, dd), 4.22 (1H, dd), 3.73 (3H, s), 3.70 (1H, d), 3.60 (1H, dd), 3.36 (2H, dd) ), 3.12 (2 H, d), 2.37 (1 H, dd), 1.45 (2 H, dd), 1.29 (1 H, m), 0.58 (1 H, m), 0.50 ( 3 H, d)

 The 10 OMHz carbon nuclear magnetic resonance spectra of TMC-2A, TMC-2B and TMC-2C are shown in FIGS. 10 to 12, respectively. TMC-2A and TMC-2B were measured in heavy water using dioxane as an internal standard. TMC-2C was measured in heavy methanol using TMS as an internal standard. The chemical shifts (ppm) are described below.

 TMC-2A: 181.4, 174.1, 173.7, 151.1, 148.6, 139.6, 137.1, 132.0, 129.2, 128.0, 125.2, 1 22.6, 1 20.7, 1 14.9, 1 13.6, 109.9, 109.1, 65.6, 63.5, 62.7, 59.3, 55.5, 54.8, 41.6, 41.4, 33.1, 32.2, 3 0.5

 TMC-2B: 182.0, 174.4, 174.2, 151.5, 149.1, 139.5, 137.7, 132.4, 129.6, 128.5, 125.7, 123. 1, 1 2 1.2, 1 15.4, 1 14.0, 1 10.4, 109.1, 67.6, 64.0, 59.7, 56.3, 55.2, 42.1, 37.7, 34.4, 33.4, 30.9, 20 . 1

TMC-2C: 179.6, 171.9, 171.4, 150.8, 148.0, 138.1, 135.8, 130.0, 128.1, 125.6, 123.2, 120.6, 119.3, 112.7, 111.4, 108.2, 107.7, 68.6, 60.9, 58.2, 53.6, 53.5, 39.9, 36.9, 33.1, 31.7, 29.5, 15.5 Protons bonded to nitrogen and oxygen atoms were prepared by preparing acetylated form of D-(-28), and measuring the proton and carbon nuclear magnetic resonance spectra in the double-mouthed form. I confirmed.

 Based on the above results, it was determined that TMC-2A, TMC-2B and TMC-2C had the structure of the aforementioned general formula [II]. No compound with this chemical structure has been reported so far, and TMC_2A, TMC-2B and TMC-2C are new substances.

 Whether a compound has an inhibitory effect on dipeptidyl peptidase IV is determined, for example, by determining whether the compound has L-Gly-L-Pro-p-nitroanilide by dipeptidyl peptidase IV. Judgment can be made based on whether or not the reaction that is hydrolyzed by L-Gly-L-Pro and p-nitroaline is inhibited.

 The compounds of the present invention can be used for pharmaceutical use either in free form or in the form of a pharmaceutically acceptable salt. Such pharmacologically acceptable salts may be any conventional non-toxic salts, for example, inorganic salts such as hydrochloride, hydrobromide, sulfate or phosphate, and formate. , Acetate, trifluoroacetate, oxalate, maleate, fumarate, tartrate, metasulfonate, organic salts such as benzenesulfonate or toluenesulfonate, sodium salt, potassium salt, etc. And alkaline earth metal salts such as calcium salts and the like, and salts with amino acids such as arginine salts, aspartate and glutamate.

 In addition, the target compound of the present invention and the pharmaceutically acceptable salts thereof should be construed as including any of inner salts, adducts, solvates or hydrates thereof, etc.

When a pharmaceutical composition comprising the compound of the present invention and a pharmaceutically acceptable salt thereof as an active ingredient is used as a drug, an aerosol, tablet, pill, powder, granule, troche Preparations, liquids, suspensions, emulsions, capsules, microcapsules, suppositories, injections, plasters, ointments, syrups, cataplasms, liniments, lotions, and other conventional pharmaceutical preparations. It can be administered orally or parenterally (intravenously, intramuscularly, intradermally, subcutaneously, intraperitoneally or rectally, etc.).

 Additives for these drugs may be used as long as they do not impair the therapeutic effect of each drug and are harmless at the dose of the drug. Any conventional additives can be used. For example, stabilizers, buffers, flavoring agents, suspending agents, emulsifiers, fragrances, preservatives, solubilizing agents, excipients, coloring agents, binders, disintegrants, sweeteners, thickeners, wetting agents , A solvent and the like can be used.

 The amount of the active ingredient in the pharmaceutical preparation may be an amount sufficient to produce the desired therapeutic effect. For example, oral or parenteral administration is 0.0 lmg Z kg to: LOO mg Z kg, preferably 1 mg Z kg to 3 O mg Z kg.

In the present specification, examples of the protecting group for the amino group (protecting group for the amino group) in the “amino acid in which the amino group may be protected” include, for example, an acyl group, an akanoyl group, an aroyl group, an aralkylcarbonyl group. Groups, an aryloxycarbonyl group, an aryl group-substituted lower alkoxycarbonyl group or a lower alkoxycarbonyl group, and the like, and especially an aryloxycarbonyl group, an aryl group-substituted lower alkoxycarbonyl group or a lower alkoxycarbonyl group. The amino acid having a protected carboxy group in the “amino acid whose carboxy group may be protected” is a compound in which the carboxy group of an amino acid is esterified (amino acid ester) or the carboxy atom of an amino acid. And the like. Examples of such compounds include amino acids in which the carboxy group is protected by lower alkyl, amino acids in which the carboxy group is protected by aryl-substituted lower alkyl, and di-lower alkylamines. And an amino acid having a protected carboxy atom group. Further, when the “amino acid” has a reactive residue other than the amino group and the carboxy group (for example, a hydroxyl group in serine), it may be used in the field of peptide synthesis depending on the type of the reactive residue. It may be protected by a commonly used protecting group (for example, a benzyl group for a hydroxyl group).

Examples of the "aryloxycarbonyl group" include a phenoxycarbonyl group and a naphthyloxycarbonyl group. Examples of the "aryl group-substituted lower alkoxycarbonyl group" include a benzyloxycarbonyl group and a phenethyloxy group. Examples thereof include a carbonyl group and a naphthylmethyloxy group, and preferably include a benzyloxycarbonyl group. The “lower alkoxycarbonyl group” includes a methoxycarbonyl group, an ethoxycarbonyl group, an n-propoxycarbonyl group, an isopropoxycarbonyl group, an n-butoxycarbonyl group, an isobutoxycarbonyl group, a tert-butoxycarbonyl group, sec - butoxide deer Lupo sulfonyl group, n- pentyl group, isopentyl group, sec - pentyl group, tert - pentyl group, _n - hexyl group, a cyclohexyl group isohexyl, hexyl group and tert into sec- One hexylcarbonyl group and the like are preferable, and a tert-butoxycarbonyl group is preferable. Examples of the “aryl substituted lower alkyl” include a benzyl group, a phenethyl group and a naphthylmethyl group, and preferably a benzyl group.

 In the present specification, aryl means phenyl, naphthyl and the like. In addition, lower alkyl and lower alkoxy represent a branched or straight chain having 1 to 6 carbon atoms, and preferably a branched or straight chain having 1 to 4 carbon atoms.

 The present invention will be described more specifically with reference to the following experimental examples and examples, but the present invention is not limited thereto.

Experimental example 1 The reaction was performed at 37 ° C using a 96-well flat-bottom plate according to the method of Nagata et al. (Analytical Iochemistry, Vol. 74, pp. 466-476, 1976). I went in. Leis is rat kidney prepared by the method of Ciddle et al. (Preparative Biochemistry), Vol. 21 (2-3), 141-150, 1991. Dipeptidyl peptidase IV enzyme (25 mU / m 1) solution 51, water 30 and 2 mM dimethyl sulfoxide solution 5 μl of test compound were mixed, pre-incubated for 10 minutes, then 71 OmM G 1 yNaOH (pH 8.7) buffer solution 10 mM 3 mM L-Gly-L-Pr0-p troanilide (G1y-Pr0-pNA; Sigma) 50 μl aqueous solution was added. The amount of ρ-nitroaniline produced is measured using a plate reader (THERMOmax; manufactured by Molecular Devices) to measure the increase in absorbance (Δ〇D) at a wavelength of 405 nm to determine the activity of dipeptidyl peptidase IV enzyme. The inhibition rate (%) is determined by the following equation 1. However, 1 U of dipeptidyl peptidase IV enzyme activity is defined as the amount of enzyme that produces 1 mol of p-troalinerin per minute. The results are shown in Table 3. l-(AOD -AOD _blank )

 Inhibition rate (%) = X 100

△ ^0E tr _0l-

△ instead of AOD A ^OD control = analyte in OD _blar ,, = di peptidyl peptidase no addition, AOD upon addition of DMSO Table 3

 *) The product obtained in the examples described later was used as an analyte compound in the examples.

Experimental example 2

As in Experimental Example 1, 3 mM G 1 y—Pr 0—p NA501 and sample 101 were collected. After addition and incubation at 37 ° C for 15 minutes, add 70 μl of OmM glycine buffer (pH 8.7) to 10 μm distilled water and 25 μl of distilled water and dipeptidyl peptidase from Lewis rat kidney. 5 μl of an IV enzyme (5 OmU / m 1) solution was added and mixed and reacted at 37 ° C., and the inhibition rate (%) of the dipeptidyl peptidase IV enzyme activity was determined from the above equation (1). As a result, 4.6 g / m1 of TMC-2A, 9.5 g / m1 of TMC-2B or 11 μg / 1 of TMC-2C showed dipeptidyl peptidase IV activity. Was inhibited by 50%.

TMC-2A, TMC-2B and TMC-2C were purified from rat kidney, as well as rat spleen, human peripheral blood mononuclear cells and human colon cancer cell line C. ac 0 di peptidyl peptidase was prepared from 2 peptidase IV was also confirmed inhibited child and power ^s.

 Furthermore, the effects of TMC-2A, TMC-2B and TMC-2C on other peptidases were examined. That is, the effects of TMC-2A, TMC-2B and TMC-2C on prolyl endopeptidase, subtilisin, trypsin, cathepsin C, leucine aminopeptidase and proline aminopeptidase at a concentration of 100 g / 1 were examined. did. As a result, TMC-2A and TMC-2B had no effect on all tested and tested peptidases. TMC-2C, on the other hand, showed weak inhibition of oral lendopeptidase and proline aminopeptidase, but not other peptidases. Therefore, TMC-2A, TMC-1B and TMC-2C were found to be highly specific inhibitors of dipeptidyl peptidase IV.

Experiment 3

Arthritis was induced by administering 35 mg / kg of alkyldiamine to the ridge of F344 / Jc1 rats (five weeks old, female). Ding 1 ^ 〇 ー 28 is in saline It was dissolved and administered once daily subcutaneously to the back for 3 weeks from the day of alkyldiamine administration to the end of the test. The doses were 30, 10, 3, and 1 mgZkg.

 FIG. 13 shows how much the foot swelled at the end of the test in each test group (percentage of the foot swelled based on the volume of the foot before administration of alkyldiamine in each individual). The volume of the foot is measured by a foot volume measurement device.

 The measurement was performed using Plethy smome ter (manufactured by Unicom). As is evident from Fig. 13, Ding ^^. -28 suppressed the onset and progression of alkyldiamine-induced arthritis in a dose-dependent manner. In particular, in the groups to which 3 Omg / kg and 1 OmgZkg were administered, they were significantly suppressed as compared with the control group (the physiological saline administration group).

Experiment 4

 Arthritis by administering 0.6mgZ kg of heat-killed M. tuberculosis H37Ra strain (Difco) suspended in liquid paraffin to the ridge of Lewis strain rats (6 weeks old, female) The test compound was dissolved in physiological saline and administered subcutaneously to the back once a day for 18 days from the administration of the heat-killed M. tuberculosis bacteria to the end of the test. Was 1 OmgZkg, and that of the compound of Example 17 was 30 and 1 Omg / kg.

 FIG. 14 shows how much the foot swelled at the end of the test in each test group (percentage of the foot swelling based on the volume of the foot before administration of the heat-killed M. tuberculosis in each individual). The volume of the foot is measured by a foot volume measurement device.

It was measured using a Plethy smometer (manufactured by Unicom). As is evident from FIG. 14, TMC-2A and the compound of Example 13 suppressed the onset and progression of adjuvant-induced arthritis in a dose-dependent manner. In particular, in the group in which TMC-2A was administered at 1 Omg / kg and the compound of Example 13 was administered at 3 Omg / kg, the suppression was significantly suppressed as compared with the control group (group administered with physiological saline). Example 1

 (3S) —1,2,3,4-tetrahydroisoquinoline-13-carboxylic acid'benzyl ester 7.3 g, N-tert-butyloxycarbonyl-1-L_tryptophan 4.56 g, 1-hydroxybenzotriazole (monohydrate Dissolve 2.76 g) and 3.45 g of 1-ethyl 3- (3-dimethylaminopropyl) rubodimide hydrochloride in 50 ml of dimethylformamide under ice-cooling and then under water-cooling for 2 hours. After stirring at room temperature for 16 hours, the reaction solution was concentrated under reduced pressure. The residue was extracted with ethyl acetate, washed, dried, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate: n-hexane = 1: 2) to obtain amorphous (3S) -2- (N-tert-butyloxycarbonyl-1-L-tryptofil) -1, 2, 8.07 g of 3,4-tetrahydroisoquinoline-3-carboxylic acid 'benzyl ester was obtained.

IR (KB r, cm- ¹ ): 3320, 2975, 1730, 1700, 1645,

1450, 1430, 1 170, 745

MS (S I MS): 554 (M + 1)

Examples 2 to 6

1,2,3,4-Tetrahydroisoquinoline-l-carboxylic acid benzyl ester and N-tert-butoxycarbonyl-l-amino acid derivative were treated in the same manner as in Example 1 to obtain the compounds shown in Table 4 below. Was. Table 4

In the table, Bn- represents a benzyl group, Boc- represents a tert-butoxycarbonyl group, and Z- represents a benzyloxycarbonyl group. Example 7

 7. O g of the compound obtained in Example 1 was dissolved in 50 ml of methanol, a catalytic amount of palladium-carbon was added, and the solution was hydrogenated at room temperature under balloon pressure for 3 hours. The catalyst was removed by filtration, and the filtrate was concentrated. The residue was crystallized from ethyl acetate-n-hexane solvent to give colorless crystals of (3S) -2- (N-tert-butyloxycarbonyl-L-tryptophyll) -1,2,3,4- Tetrahydroisoquinoline 3-butyric acid 5.

25 g were obtained.

m.p .: 143. . (Disassembly)

 I R (KB r, cm-": 3340, 2980, 1720, 1700, 1630,

1440, 1 1 70, 740

MS (S I MS): 464 (M + 1)

Example 8: I 1

The compounds obtained in Examples 2 to 5 were treated in the same manner as in Example 7 to obtain the compounds shown in Table 5 below.

Table 5

 However, Boc- in the table represents a tert-butoxycarbonyl group.

Example 1 2

2.977 g of the compound obtained in Example 6 was dissolved in 20 ml of methanol, and 5 ml of a 1 M aqueous sodium hydroxide solution was added thereto, followed by stirring at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, the residue was washed with ether, and acidified with an aqueous solution of potassium hydrogen sulfate. Extracted with ethyl. The extract is washed, dried and concentrated, and the amorphous (3S) _2- | Ν (α) -tert-butyloxycarbonyl-1- (ω) -benzyloxycarbonyl L-lysyl1-1 2.62 g of 1,2,3,4-tetrahydroisoquinoline-13-carboxylic acid was obtained.

 I R (KB r, cm-": 3340, 2975, 1705, 1625, 1520, 1440, 1240, 1165

MS (S I MS): 540 (M + 1)

Example 13

 4.0 g of the compound obtained in Example 7 was dissolved in 50 ml of a 4 M hydrochloric acid-dioxane solution, and the mixture was stirred at room temperature under a nitrogen atmosphere for 30 minutes. The reaction solution was concentrated under reduced pressure, the residue was dissolved in water, and lyophilized to give a pale red powder (3S) -2- (L-tributophyl) -1,2,3,4-tetrahydroisoquinoline-3 3.3 g of carboxylic acid 'hydrochloride were obtained. m. p .: 1 58 ° C (decomposition)

 I R (KB r, cm-": 3400, 29 10, 1 720, 1 645, 1490,

1460, 1205, 1 120, 745

MS (S IMS): 364 (M + 1)

Example 14-: I 8.

The compounds obtained in Examples 8 to 12 were treated in the same manner as in Example 13 to obtain the compounds shown in Table 6 below. Table 6 (Part 1)

COOH

Example Physical constants, etc.

 \ IL L · I

 No.

 * ェ Ϊ

 Saotono ^ ^ Noreno, Ma 'J-lr ^

 IR (KBr, cm-R: 3400, 2920, 1720, 1650, 1490, 1460, 1360, 1110, 870, 740

IH - Anonymous _{(DMS0-d 6, d)} : 11.0 (d-

14 D-Trp S

 like, J = 33 Hz, 1H), 8.5-8.2 (br, 2H), 7.6-6.9 (m, 9H), 5.1-4.0 (m, 4H), 3.5-2.3 (m, 4H)

 MS (SIMS): 364 (M + 1)

 Lyophilized amorphous material

 IR (KBr, cm-R: 3400, 2920,

15 L-Trp R 1740, 1645, 1480, 1460, 1120,

 870, 745

 MS (SIMS): 364 (M + 1)

 Amorphous material

 IR (KBr, cm-i): 3410, 1650, 1460,1330, 740

_{1H-NMR (DMS0-d 6} , d): 11.0

16 S

 (br, 1H), 7.5—6.9 (ra, 9H),

H 5.5-5.3 (dd-like, 2H), 4.5- 4.1 (m, 4H), 3.3-2.8 (m, 5H) MS (SIMS): 374 (M-1) Table 6 (Part 2)

 Here, z- in the table represents a benzyloxycarbonyl group.

Example 19

463 mg of the compound obtained in Example 7, 0-benzyl-L-serine-benzylester 410 mg, 1-hydroxybenzotriazole (monohydrate) 184 mg, 1-ethyl-3- (3-dimethylamino) 23 Omg of propyl) carposimid hydrochloride was dissolved in 20 ml of dimethylformamide under water cooling, and the mixture was stirred for 2 hours under ice cooling and further at room temperature for 16 hours, and then concentrated under reduced pressure. The residue was extracted with ethyl acetate, washed, dried, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate: n-hexane = 1: 1), and amorphous N— | (3S) —2— (N-tert-butyloxycarbonyl 1 L) 1,3-, 3-, 4-tetrahydroisoquinoline-3-tryptofil) 1-, 0-benzyl-l-serine-benzyl ester 670 mg was obtained.

 I R (KB r, cm-i): 3305, 2975, 1735, 1660, 1640, 1480, 1450, 1170, 745

MS (S IMS): 73 1 (M + 1)

Example 20

 The compound obtained in Example 7 and L-one-isocyanate'benzyl ester were treated in the same manner as in Example 19 to give N- | (3S) -2- (N-tert-butyloxycarbonyl-1-L-tryptofil. 1) 1,2,3,4-Tetrahydroisoquinoline-l-carbonyl-l-L isocyanate benzyl ester was obtained.

m. p .: 99—100 ° C (decomposition)

MS (S I MS): 667 (M + 1)

Example 2 1

 The compound obtained in Example 19 was treated in the same manner as in Example 7, and the product was separated by silica gel chromatography (chloroform: methanol = 20: 1) to give (1) an amorphous form. N—K3 S) —2— (N-tert-butyloxycarbonyl L-tryptofil) -1-1,2,3,4-tetrahydroisoquinoline-3-carbonyl-1—L-serine and (2) amofus N- 1 (3 S)-2- (N-tert-butyloxycarbonyl-L-tryptofil)-1,2,3,4-tetrahydroisoquinoline- 3-carbonyl-1-0-benzyl-L-serine Was.

(1)

 I R (KB r, cm-": 33 15, 2960, 1 680, 1640, 1 500, 1450, 1 1 65, 745

MS (S IMS): 64 1 (M + 1)

(2) IR (KB r, cm-": 3335, 2960, 1680, 163, 1510, 1450, 1430, 1165, 745

MS (S I MS): 551 (M + 1)

Example 22

 1.16 g of the compound obtained in Example 20 was dissolved in 20 ml of a 4 M hydrochloric acid-dioxane solution, and the mixture was stirred at room temperature for 30 minutes under a nitrogen atmosphere. The reaction solution is concentrated under reduced pressure, and the residue is crystallized from ethanol to give a colorless powdery N-K3S) -2-L-tributophyl-1,1,2,3,4-tetrahydroisoquinoline-13-capillonyl 1-L one-port isine '990 mg of benzyl ester were obtained.

m.p .: 132-134 ° C

MS (S IMS): 567 (M + 1)

Example 23

 N-1 (3S) -2- (N-tert-butyloxycarbonyl L-tributyl) -1,2,3,4-tetrahydroisoquinoline-13-carbonyl} 1 L-serine Amorphous N-1 (3S) -2-L-tributofyl-1,2,3,4-tetrahydroisoquinoline-3-carbonyl 1-L-serine / hydrochloric acid treated in the same manner as in Example 13 Salt was obtained. .

 I R (KB r, cm- ": 3400, 2940, 1725, 1650, 1450, 745

 MS (S IMS): 45 1 (M + 1)

Example 24

After 750 mg of the compound obtained in Example 22 was dissolved in 50 ml of methanol and a catalytic amount of palladium-carbon was added, the solution was hydrogenated at room temperature under balloon pressure for 3 hours. The catalyst was filtered off, and the filtrate was concentrated under reduced pressure to obtain a residue. Crystallize the residue from ethanol Then, colorless crystals of N— | (3S) —2—L-tributofyl-1,2,3,4-tetrahydroisoquinoline-3-carbonyl-1—L-leucine hydrochloride were obtained in an amount of 65 mg. mp: 1 55. C (decomposition)

 MS (S IMS): 477 (M + 1)

Example 25

 (3S) -2-benzyloxycarbonyl-1,5,8-dimethoxy-1,2,3,4-tetrahydroisoquinoline-13-carboxylic acid 4.5 g, L-leucine methylester hydrochloride 1.82 g, 1-Hydroxybenzotriazole (monohydrate) 1 · 95 g, 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride 2.44 g, Triethylamine 1.7 ml in ice-cooled dimethylformamide 35 The mixture was dissolved in m1, stirred for 2 hours under ice-cooling, and further stirred at room temperature for 16 hours, and then concentrated under reduced pressure. The residue was extracted with ethyl acetate, washed, dried and concentrated. The residue was purified by silica gel column chromatography (chloroform: ethyl acetate = 9: 1) to give a colorless oil of N-1 (3S) -12-benzyloxycarbonyl 2,8-dimethyl. Toxin-1,2,3,4-tetrahydridoisoquinoline-13_carbonyl 1 —L-leucine methyl ester 5.0 g was obtained.

 I R (KB r, cm-: 3400, 2955, 1740, 1 670, 1485, 1260, 1 120, 1085

MS (S IMS): 499 (M + 1)

Example 26

700 mg of the compound obtained in Example 25 was dissolved in 10 ml of methanol, and 2.8 ml of a 1 M aqueous sodium hydroxide solution was added thereto, followed by stirring at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, and the residue was washed with ether. The mixture was acidified with aqueous sodium hydrogen sulfate solution and extracted with ethyl acetate. The extract is washed, dried and concentrated to obtain amorphous N- 1,470 mg of 1 (3 S) -2-benzyloxycarbonyl 5,8-dimethoxy-1,2,3,4-tetrahydroisoquinoline-l-capillonyl 1-L isocyanate was obtained. IR (KB r, cm-R: 3400, 2960, 1685, 1485, 1260, 1085

 MS (S IMS): 485 (M + 1)

Example 27

 4.0 g of the compound obtained in Example 25 was dissolved in 40 ml of methanol, and after adding a catalytic amount of palladium-carbon, the solution was hydrogenated at room temperature under balloon pressure for 3 hours. The catalyst was filtered off, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (chloroform: ethyl acetate = 2: 1), and the obtained colorless oil was crystallized from isopropyl ether to give N-((3S) -5,8) as colorless crystals. -Dimethoxy-1,2,3,4-tetrahydroisoquinoline-l-hexylcarbonyl-1 L-leucine 'methyl ester 2.02 g was obtained.

Melting point: 101-115 ° C

 I R (KB r, cm- ": 3360, 2955, 1735, 1665, 1480, 1255, 1075

MS (S I S): 36.5 (M + 1)

Example 28

1.5 g of the compound obtained in Example 27, 1.39 g of N-benzyloxycarbonyl mono-L-tributylamine, 662 mg of 1-hydroxybenzotriazole (monohydrate), and 1-ethyl-3- (3-dimethyl) After dissolving 82 mg of aminopropyl) carbodiimide hydrochloride (82 mg) in 15 ml of dimethylformamide under cooling with water, stirring for 2 hours with cooling with water and further at room temperature for 16 hours, the reaction solution was concentrated under reduced pressure. The residue was extracted with ethyl acetate, washed, dried and concentrated. The crystalline residue was crystallized from ethyl acetate to give N— | ( 3 S) — 2- (N-benzyloxycarbonyl-L-tryptofil) -1,5,8-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carbonyl} —L-leucine methyl ester 2.20 g were obtained.

m.p .: 1 30-149 ° C

 I R (KB r, cm- "): 33 15, 2955, 1740, 1660, 1530, 1485, 1260, 1085, 745

MS (S I MS): 685 (M + 1)

Example 29

 350 mg of the compound obtained in Example 28 was dissolved in 15 ml of methanol, a catalytic amount of palladium-carbon was added, and the solution was hydrogenated under a balloon pressure at room temperature for 3 hours. The catalyst was filtered off, and the filtrate was concentrated under reduced pressure to obtain a crystalline residue. The crystalline residue was washed with isopropyl ether, and amorphous N-1 (3S) -2-L-tryptofil 5,8-dimethoxy-1,2,3,4-tetrahydroisoquinoline 1-3-carbonate L-leucine methyl ester 17 Omg was obtained.

 I R (KB r, cm-": 3295, 2955, 1740, 1645, 1485, 1260, 1090, 745

MS (S IMS): 551 (M + 1)

Example 30

1.3 g of the compound obtained in Example 28 was dissolved in 30 ml of methanol, 3.8 ml of a 1 M aqueous solution of sodium hydroxide was added, and the mixture was stirred at room temperature for 6 hours. The reaction solution was concentrated under reduced pressure, the residue was washed with ether, neutralized with an aqueous solution of potassium hydrogen sulfate, and extracted with ethyl acetate. The extract was washed, dried and concentrated to obtain a crystalline residue. The crystalline residue was washed with isopropyl ether to give a colorless powder of N-1 (33) -2 (^ 1-benzyloxycarbonyl-L-tryptofil) -1,5,8-dimethoxy-1,2,3 , 1.11 g of 4-tetrahydroisoquinoline-1- (3-carbonylyl) -1-L-leucine was obtained.

m.p .: 1 52-166 ° C

 I R (KB r, cm-": 33 10, 2960, 1700, 1 675, 1 520, 1485, 1260, 1085, 745

MS (S IMS): 67 1 (M + 1)

Example 3 1

 600 mg of the compound obtained in Example 30 was dissolved in 15 ml of methanol, and after adding a catalytic amount of palladium-carbon, the solution was hydrogenated at room temperature under balloon pressure for 3 hours. The catalyst was filtered off, and the filtrate was concentrated under reduced pressure to obtain a crystalline residue. The crystalline residue was washed with isopropyl ether, and amorphous N—j (3S) —2-L-tributyryl-1,5,8-dimethoxy-1,1,2,3,4-tetrahydroisoquinoline-3 —Carbon} —L-leucine 345 mg was obtained.

IR (KB r, cm- ¹ ): 3400, 2955, 1655, 1480, 1260,

1 085, 745

 MS (S IMS): 537 (M + 1)

Examples 32 to 51

 Peptide synthesis was performed using the automated solid phase method peptide synthesizer P S SM-8 (manufactured by Shimadzu Corporation) according to the following procedure. A carrier (Fmoc-Amino) in which the corresponding starting amino acid is bonded to a benzoxybenzyl alcohol type resin

Acid—Resin) 100 mg, and benzotriazole-1-yloxystris (pyrrolidino) phosphonium hexafluorophosphate, 1-hydroxybenzotriazole, N-methyl Morpholine system as deprotection system 20% piperidine / N, N-dimethylformamide as condensed amino acid N-α-9-Fluorenylcarbonyl 1,2-, 3,4-tetrahydroisoquinolin-13-carboxylic acid and N-tert-butyloxycarbonyl L-tryptophan The synthesis was performed using the equipped standard protocol. After the synthesis, the mixture was treated with 1 ml of trifluoroacetic acid: water: thioanisole: ethanedithiol (75: 10: 10: 5) for 3 hours, deprotected, and cleaved from the resin. The reaction solution was filtered to remove the resin, and the filtrate was precipitated by adding anhydrous getyl ether, or concentrated to obtain the desired crude peptide. The resulting crude peptide was dissolved in water, purified through a short reverse-phase column, and then lyophilized to give a colorless powder of N-1 (3S) -2-L-tributophyl-1,1 as described in Table 7 2,3,4-Tetrahydroisoquinoline-3-carbonyl) -l-amino acid was obtained.

Table 7

Example No. R2 Physical constant, etc.

 32 L-Ala MS (SIMS): 435 (M + 1)

33 L-Arg MS (SIMS): 520 (M + 1)

34 L-Asn MS (SIMS): 478 (M + 1)

35 L-Asp MS (SIMS): 479 (M + 1)

36 L-Glu MS (SIMS): 493 (M + 1)

37 Les Gin MS (SIMS): 492 (M + 1)

38 L-Gly MS (SIMS): 421 (M + 1)

39 L-His MS (SIMS): 501 (M + 1)

40 L-lie MS (SIMS): 477 (M + 1)

41 L-Leu MS (SIMS): 477 (M + 1)

42 L-Lys MS (SIMS): 492 (M + 1)

43 L-Met MS (SIMS): 495 (M + 1)

44 L-Phe MS (SIMS): 511 (M + 1)

45 L-Pro MS (SIMS): 461 (M + 1)

46 L-Ser MS (SIMS): 451 (M + 1)

47 L-Thr MS (SIMS): 465 (M + 1)

48 L-Trp MS (SIMS): 550 (M + 1)

49 L-Tyr MS (SIMS): 527 (M + 1)

50 L-Val MS (SIMS): 463 (M + 1)

51 L-Cys MS (SIMS): 467 (M + 1) Example 52

 The compound obtained in Example 1 was treated in the same manner as in Example 13 except that trifluoroacetic acid was used instead of the hydrochloric acid-dioxane solution, to give amorphous (3S) -2-L-tributyrophil 1,2,3,3 4-Tetrahydroisoquinoline-3-carboxylic acid · benzylester ′ trifluoroacetate was obtained.

 I R (KB r, cm- ": 3420, 3035, 1730, 1 675, 1460,

1 205, 745

 MS (S I MS): 454 (M + 1)

Example 53

 After dissolving 2.3 g of the compound obtained in Example 7 and 607 mg of N-methylmorpholine in 20 ml of tetrahydrofuran, −19 mg of isobutoxycarbonyl chloride was added dropwise at −15 ° C. The mixture was stirred at the same temperature for 10 minutes. After 2 Oml of a saturated ammonia 'tetrahydrofuran solution was added dropwise, and the mixture was stirred at 115 ° C for 1 hour, the reaction solution was concentrated under reduced pressure. The residue was extracted with chloroform, the extract was washed, dried, and concentrated. The residue was purified by silica gel chromatography (chloroform: methanol = 20: 1) to give an amorphous (3 S) — 2— (N-tert-butyloxycarbonyl) L-tryptofile) 1,2,3 ,,, 4-tetrahydroisoquinoline-13-carboxamide 1.6

2 g were obtained.

 I R (KB r, cm-i): 3340, 2980, 1700, 1 675, 1 630,

1440, 1 1 70, 740

MS (S IMS): 463 (M + 1)

Example 54

1.6 g of the compound obtained in Example 5.3 was treated in the same manner as in Example 13 to give a pale red powdery (3S) -2- (L-tryptofile) -1-1,2,3,4-tetrahydrofuran. Droisokinori 1.3 g of 3-hydroxylpoxamide hydrochloride was obtained.

IR (KB r, cm " ¹ ): 3425, 2900, 1675, 1630, 1480, 1460, 1360, 1 120, 745

MS (S IMS): 363 (M + 1)

Example 55

 The compound obtained in Example 53 was treated in the same manner as in Example 13 using trifluoroacetic acid instead of the hydrochloric acid-dioxane solution, to give a pale red powdery (3S) -2- (L-tribute file). There was obtained 1,1,2,3,4-tetrahydroisoquinoline-13-carboxamide 'trifluoroacetate.

 MS (S IMS): 363 (M + 1)

Example 56

 (1) 0.5% glucose (manufactured by Nakarai Co., Ltd.), 2% Soyaflower A (manufactured by Nisshin Oil Co., Ltd.), 296 glycerol (manufactured by Nakarai Co., Ltd.), 0.2% yeast extract (manufactured by Asahi Breweries, Ltd.), 0.25% A spergi 11 ussp. A374 strain (100 ml) in a liquid medium (pH 7.0) composed of sodium salt sodium chloride (manufactured by Nacalai Co., Ltd.) and 0.4% calcium carbonate (manufactured by Nitto Powder Chemical Co., Ltd.) was cultured on an agar slope. One loopful of FE RM BP-6 13 13) was inoculated and cultured with shaking at 27 ° C for 3 days.

 The obtained culture was used as a seed culture. One hundred ml of a seed culture solution was inoculated into 100 Erlenmeyer flasks containing 100 ml of a liquid medium having the above-mentioned composition, and cultured with shaking at 27 ° C for 5 days.

(2) The cells were removed by filtration from 85 L of the culture solution obtained by the above method (1), and the culture solution was passed through a 3 L DIAION HP-20 (manufactured by Mitsubishi Chemical Corporation) column. Then, TMC-2A, TMC-2B and TMC-2C were adsorbed on the column. 5 L each of distilled water, 20%, 5096 and 100% methanol L, 75 L, and 30 L were allowed to flow down. The 50% methanol fraction exhibiting dipeptidyl peptidase IV inhibitory activity was concentrated under reduced pressure to obtain a crude substance. This crude material was chromatographed on silica gel (Kogel C-300, manufactured by Wako Pure Chemical Industries, Ltd., 60 × 900 mm). The elution was performed by first mixing 5 L of a mixed solution of dichloromethane: methanol: ethanol (10: 4: 4) and then mixing a mixed solution of dichloromethane: methanol: ethanol: water in the following order with a ratio of ^ 7j. That is, a mixture of dichloromethane: methanol: ethanol: water was mixed with a 10: 4: 4: 0.1 solution 5 L, a 10: 4: 4: 0.2 solution 5 L, and a 10: 4: 4: 0.5 solution, respectively. Elution was carried out with 10 L of a 0: 4: 4: 1 solution and finally with 10 L of a 10: 4: 4: 2 solution. Fractions showing dipeptidyl peptidase IV inhibitory activity were collected and concentrated under reduced pressure to obtain a crude substance.

 This crude substance was subjected to chromatography using a reverse-phase silica gel column (ODS A60, manufactured by JMc Co., Ltd., 60 × 900 mm). Elution was performed with 20% acetonitrile-80% water. Each eluted fraction was analyzed by high performance liquid chromatography, fractions containing only TMC-2A were collected, concentrated under reduced pressure, and lyophilized. The high performance liquid chromatography for analysis was performed using a YMC_Pack AM_301-3 (manufactured by Jemushi Corporation) 4.6 x 100 mm column with a linear gradient of 1096 to 35% from acetonitrile for 15 minutes (flow rate 1.2m 1 Zm in ). The detection was based on the absorbance at 210 nm and 254 nm. By the above operation, about 1.6 g of pure TMC-2A was obtained.

Purification of TMC-2B and TMC-2C was performed as follows. Fractions containing TMC-2B and TMC-2C in the above reverse phase chromatography were collected and concentrated under reduced pressure. TMC-2B and TMC-2C were fractionated from the concentrate using silica gel chromatography (Co-gel C-300, Wako Pure Chemical Industries, Ltd., 22 × 500 mm). For elution, flow 500 ml of a mixture of dichloromethane: methanol: ethanol (10: 4: 4). After that, the mixed solution of dichloromethane: methanol: ethanol: water was 200 ml for a 10: 4: 4: 0.1 solution, 200 ml for a 10: 4: 4: 0.2 solution, and 200 ml for a 10: 4: 4: 0.5 solution. 500 ml of a 10: 4: 4: 1 solution and 500 ml of a 10: 4: 4: 2 solution were successively dropped. Each eluted fraction was analyzed by silica gel TLC (silica gel; Merck N 0.575, developing solvent: dichloromethane: methanol: ethanol: water = 10: 4: 4: 2). Fractions containing only TMC-2B or TMC-2C were concentrated to dryness, respectively. By the above operations, pure TMC-2B and TMC-2C were obtained in 5.4 mg and 2 lmg, respectively.

 Commercially available

 The tetrahydroisoquinoline derivative of the present invention selectively inhibits dipeptidyl peptidase IV at a low concentration, and prevents and treats autoimmune diseases (immune disorders and immunodeficiencies) such as arthritis and rheumatoid arthritis. Useful as an agent.

Claims

Scope of Claim 1. A pharmaceutical composition comprising a compound having a dipeptidyl peptidase IV inhibitory activity and having a tetrahydroisoquinoline skeleton or a pharmaceutically acceptable salt thereof as an active ingredient.

 2. It has a dipeptidyl peptidase IV inhibitory action, and has the formula:

A pharmaceutical composition comprising, as an active ingredient, a compound having a partial structure represented by or a pharmacologically acceptable salt thereof.

 3. General formula [1]:

(Wherein, R ¹ is (1) a group having a structure in which a hydroxy group of a carboxy group is removed from an amino acid in which an amino group may be protected or (2) a protecting group of an amino group, and R ² is ( 1) a hydroxyl group which may be protected, (2) a group having a structure in which one hydrogen atom of an amino group has been removed from an amino acid wherein a carboxy group may be protected, or (3) a primary or secondary group R ³ , R ⁴ , R 5, and R ⁶ are the same or different hydrogen radicals, with a structure in which one hydrogen atom on a nitrogen atom has been removed from an amine or ammonia. Represents a hydroxyl, hydroxyl or lower alkoxy group)

A pharmaceutical composition comprising, as an active ingredient, a tetrahydroisoquinoline derivative represented by the formula: or a pharmaceutically acceptable salt thereof.

4. R 1 is a hydroxy group of a carboxy group from an amino acid whose amino group may be protected by (1) an aryloxycarbonyl group, an aryl group-substituted lower alkoxycarbonyl group or a lower alkoxycarbonyl group. A group having a captured structure or (2) an aryloxycarbonyl group, an aryl group-substituted lower alkoxy group, even if R ² is substituted with (1) an aryl group-substituted lower alkyl group; A good hydroxyl group, (2) a lower alkyl or aryl group, a group having a structure in which one hydrogen atom of an amino group has been removed from an amino acid whose carboxy group may be protected by substituted lower alkyl, or (3) a lower alkyl or aryl group. A group selected from reel-substituted lower alkyl optionally substituted with one or two amino ^4. The pharmaceutical composition according to claim ³ , wherein the groups R ³ , R ⁴ , R ⁵ and R ⁶ are the same or different and are a hydrogen atom, a hydroxyl group or a lower alkoxy group. '

5. R 1 is a (1) benzyloxycarbonyl group or a tert-butoxycarbonyl group, a tributophyl group, whose amino group may be protected, a lysyl group or a phenylalanyl group or a (2) benzyloxycarbo group. Alanine wherein a carboxy group may be protected by a methyl group or a benzyl group; (2) a hydroxyl group in which R ² is protected by (1) a benzyl group, or a tert-butoxycarbonyl group; , Norin, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionin, Glycine, Serine, 0-Venzyluserin, Threonine, Cystine, Glutamin, Asparagine, Tyrosine, Lysine, Arginine, Histargine, Asparagine And formula:

Wherein one of R ⁷ and R ⁸ represents a hydroxyl group and the other represents a hydrogen atom or a hydroxyl group. —A group having a structure in which one hydrogen atom of the α-amino group of an amino acid has been removed or (3) an amino group which may be substituted with one or two groups selected from a tert-butyl group or a benzyl group, R ³ is a hydrogen atom or a lower alkoxy group, R ⁴ is a hydrogen atom or a hydroxyl group, R ⁵ is a hydrogen atom or 5. The pharmaceutical composition according to claim 4, wherein the lower alkoxy group and R ⁶ are a hydrogen atom, a hydroxyl group or a lower alkoxy group.

6. R 1 s, Tributophyl group, R 2 is (1) hydroxyl group, (2) alanine, valine, leucine, isoleucine, proline, fenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, glutamin , Asparagine, tyrosine, lysine, arginine, histidine, aspartic acid and glutamic acid, a group having a structure having one hydrogen atom of one amino group consisting of an amino acid or (3) an amino group, R ³ 6. The pharmaceutical composition according to claim 5, wherein R ⁴ , R 5 and R 6 are hydrogen atoms.

7. R 1 is a tributophyl group, R ² is (l) hydroxyl group, (2) glutamine, serine, aspartic acid, glutamic acid, alanine, cystine, arginine, α-amino acid selected from methionine and asparagine; 7. The pharmaceutical composition according to claim 6, wherein the group having a structure obtained by removing one hydrogen atom from the group or (3) an amino group, R 3, R 4, R 5 and R 6 is a hydrogen atom.

8. A structure in which one hydrogen atom of the amino acid group consisting of one amino acid is selected from the group consisting of (1) a hydroxyl group, (2) glutamine and serine. 8. The pharmaceutical composition according to claim 7, wherein the group having a structure or (3) an amino group, R ³ , R ⁴ , R ⁵ and R ⁶ are hydrogen atoms.

 9.2. A pharmaceutical composition comprising, as an active ingredient, 2-L-tributofil-1,2,3,4-tetrahydroisoquinolyl-13-hydroxycarboxylic acid and a pharmaceutically acceptable salt thereof.

 10. General formula [I I]:

(Wherein one of R ⁷ and R ⁸ represents a hydroxyl group and the other represents a hydrogen atom or a hydroxyl group) or a pharmaceutically acceptable salt thereof as an active ingredient.

 1 1. Claim 3, 4, 5, 6, which is a dipeptidyl peptidase IV inhibitor.

7. The pharmaceutical composition according to 7, 89 or 10.

 12 Claims 1, 2, 3, 4, 5, 6, which are preventive and therapeutic agents for autoimmune diseases

11. The pharmaceutical composition according to 7, 89 or 10.

 13. The pharmaceutical composition according to claim 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, which is an agent for preventing or treating arthritis.

 14. The pharmaceutical composition according to claim 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 which is an agent for preventing or treating rheumatoid arthritis.

1 5. —General formula [1]:

(Wherein, R i is (1) a group having a structure in which a hydroxy group of a carboxy group is removed from an amino acid whose amino group may be protected, or (2) a protecting group of an amino group, and R ² is ( 1) a hydroxyl group which may be protected, (2) a group having a structure in which one hydrogen atom of an amino group has been removed from an amino acid wherein a carboxy atom group may be protected, or (3) a primary or secondary amine. Or a group having a structure in which one hydrogen atom on a nitrogen atom has been removed from ammonia; R ³ , R ⁴ , R 5 and R ⁶ are the same or different and represent a hydrogen atom, a hydroxyl group or a lower alkoxy group)

Or a pharmacologically acceptable salt thereof.

16. RI converts (1) an amino group whose amino group may be protected with an aryloxycarbonyl group, an aryloxy-substituted lower alkoxycarbonyl group or a lower alkoxycarbonyl group to a hydroxy group of a carboxy group. group or having removed the structure (2) § reel O alkoxycarbonyl group, Ariru substituted lower alkoxy force Ruponiru group or a lower alkoxycarbonyl group, optionally substituted with R ² force? (1) Ariru substituted lower alkyl group A good hydroxyl group; (2) a lower alkyl or aryl group; a group having a structure in which one hydrogen atom of an amino group has been removed from an amino acid whose carboxy group may be protected by substituted lower alkyl; or (3) a lower alkyl or aryl group. Amyl which may be substituted with one or two groups selected from reel-substituted lower alkyl Group, R ^3, R ^4, R ⁵ and R ⁶ are the same or different and each represents a hydrogen atom, tetrahydroisoquinolinyl emissions derivative or a drug according to claim 1 5, wherein a hydroxyl group or a lower alkoxy group Physically acceptable salts.

17. R 1 is a (1) benzyloxycarbonyl group or a tert-butoxycarbonyl group, and a tributofyl group, an amino group of which may be protected, a lysyl group or a phenylalanyl group or a (2) benzyl group. A carboxy group may be protected by a (1) a benzyl group which may be protected by (1) a benzyl group, or (2) a methyl group or a benzyl group, wherein R ² is a carbonyl group or a tert-butoxycarbonyl group. , Alanine, norin, leucine, isoleucine, proline, fenylalanine, triptophan, methionine, glycine, serine, 0-benzyl-serine, threonine, cystine, glutamine, asparagine, tyrosine, lysine, arginine, histarginine, histargine , Glutamic acid and formula:

Wherein one of R ⁷ and R ⁸ represents a hydroxyl group and the other represents a hydrogen atom or a hydroxyl group, or a group having a structure obtained by removing one hydrogen atom from the amino group ) text—an amino group which may be substituted with one or two groups selected from a butyl group or a benzyl group, R ³ is a hydrogen atom or a lower alkoxy group, R ⁴ is a hydrogen atom or a hydroxyl group, and R ⁵ is a hydrogen atom 17. The tetrahydroisoquinoline derivative according to claim ¹⁶ , wherein R ⁶ is a hydrogen atom, a hydroxyl group or a lower alkoxy group, or a lower alkoxy group, or a pharmaceutically acceptable salt thereof.

1 8. R 1 force ^s Toributofiru group, R 2 is (1) hydroxyl group, (2) Aranin, valine, leucine, isoleucine, proline, Fuweniruaranin, tryptophan, Mechio Nin, glycine, serine, threonine, cysteine, glutamine, Asuparagin, Selected from tyrosine, lysine, arginine, histidine, aspartic acid and glutamic acid, a group having a structure in which one hydrogen atom of the amino group has been removed from an amino acid or (3) an amino group, R ³ , R ⁴ , 18. The tetrahydroisoquinoline derivative or the pharmaceutically acceptable salt thereof according to claim ^17, wherein R ⁵ and R 6 are a hydrogen atom.

1 9. R ¹ force Tryptophyll group, R 2 is (1) hydroxyl group, (2) glutamine, serine, aspartic acid, glutamic acid, alanine, cysteine, arginine, methionine and asparagine. 19. The tetrahydroisoquinoline derivative according to claim 18, wherein the group having a structure obtained by removing one hydrogen atom of the atomic group or (3) an amino group, R 3, R 4, R 5 and R 6 is a hydrogen atom. A salt that is acceptable.

20. R 1 is selected from the group of tributofyl, R 2 is selected from (1) hydroxyl group, (2) glutamine and serine. 10. The group according to claim 19, wherein the group having a structure in which one hydrogen atom of the α-amino atomic group is removed from the amino acid or (3) the amino group, R ³ , R ⁴ , R ⁵ and R ⁶ are hydrogen atoms. A tetrahydroisoquinoline derivative or a pharmaceutically acceptable salt thereof.

 2.1.2-L-tryptophyll-1,2,3,4-tetrahydroisoquinolyl-13-caprolubonic acid and pharmaceutically acceptable salts thereof.

 22. —General formula [I I]:

[I 门

Wherein one of R ⁷ and R ⁸ represents a hydroxyl group and the other represents a hydrogen atom or a hydroxyl group, or a pharmaceutically acceptable salt _c thereof.

23. —General formula [I I]:

Wherein one of R ⁷ and R ⁸ represents a hydroxyl group and the other represents a hydrogen atom or a hydroxyl group, and a carbon source capable of assimilating power belonging to the genus Aspergillus; A method for producing a tetrahydroisoquinoline derivative, comprising culturing in a nutrient medium containing a nitrogen source, and isolating Compound [II] from the culture.

 24. General formula [I 1 1]:

(Wherein Ri 1 is (1) a group having a structure in which at least an amino group in which an amino group is protected is removed from a hydroxy group of a carboxy group, or (2) a protecting group of an amino group, R ³ , R ⁴ , R ⁵ and R ⁶ are the same or different and represent a hydrogen atom, a hydroxyl group or a lower alkoxy group)

Or a reactive derivative at the carboxy group thereof and a general formula [IV]:

 H-R 2 1 [I V]

(Wherein amino R ² 1 are protected at least carboxy atomic group (1) Karaa A group having a structure in which one hydrogen atom of the amino group has been removed, or (2) a group having a structure in which one hydrogen atom on a nitrogen atom has been removed from primary or secondary amine or ammonia)

By condensation with a compound of the general formula [V]:

Wherein RH, R 21, R 3, R 4, R 5 and R 6 have the same meanings as described above, and optionally removing a protecting group. Expression [

I-a]:

(Wherein, R 1 is (1) group or (2 Amino atomic groups from amino acids which may be protected has a Torisa One structure hydroxy atomic group carboxy atomic group) protecting group for the amino group, R ^{2 2} Is a group having a structure obtained by removing one hydrogen atom of an amino group from an amino acid whose carboxy group may be protected or (2) a hydrogen atom on a nitrogen atom from a primary or secondary amine or ammonia. R ³ , R ⁴ , R 5, and R 6 have the same meaning as above, with one atom removed.

A method for producing a compound represented by the formula:

25. General formula [VI]:

(Wherein, R ²³ is (1) a protected hydroxyl group, (2) a group having a structure in which at least one hydrogen atom of an amino group has been removed from an amino acid having at least a protected carboxy group, or (3) A group having a structure in which one hydrogen atom on a nitrogen atom has been removed from primary or secondary amine or ammonia; R ³ , R ⁴ , R ⁵ and R ⁶ may be the same or different and are a hydrogen atom, a hydroxyl group or a lower alkoxy group; Represents)

And a compound represented by the general formula [V 11]:

 R 12 100H [VI I]

(Wherein, R 1 ² represents a group having a One Torisa hydroxy atomic group carboxy atomic structure of amino acids at least amino atomic group is protected)

By condensation with an amino acid represented by the formula or a reactive derivative at the carboxy group thereof:

Wherein R 12, R 23, R 3, R 4, R 5 and R 6 have the same meaning as described above, and optionally removing the protecting group. General formula [

I -b]: [I-I b]

(Wherein, R i ³ is a group having a structure obtained by removing a hydroxy group of a carboxy group from an amino acid which may be substituted with an amino group, R ² is (1) a hydroxyl group which may be protected, 2) a group having a structure in which one hydrogen atom of an amino group has been removed from an amino acid which may have a protected propyloxy group, or (3) a hydrogen atom on a nitrogen atom from a primary or secondary amine or ammonia RR ³ , R ⁴ , R 5 and R 6 have the same meaning as described above.

A method for producing a compound represented by the formula:

 26. Autoimmunity characterized by administering to an autoimmune disease patient an effective amount of a compound having an inhibitory effect on dipeptidyl peptidase IV and having a tetrahydroisoquinoline skeleton or a pharmaceutically acceptable salt thereof. A method for preventing or treating a disease.

 27. The compound or a pharmaceutically acceptable salt thereof has the formula:

27. The method for preventing or treating an autoimmune disease according to claim 26, which is a compound having a partial structure represented by or a pharmacologically acceptable salt thereof.

28. For patients with autoimmune disease, an effective amount of the general formula [I]:

(Wherein R or (1) a group having a structure in which a hydroxy group of a carboxy group is removed from an amino acid in which an amino group may be protected, or (2) a protecting group of an amino group, R ² is (1) ) A hydroxyl group which may be protected, (2) a group having a structure in which one hydrogen atom of an amino group has been removed from an amino acid wherein a carboxy atom group may be protected, or (3) a primary or secondary amine or R ³ , R ⁴ , R 5 and R 6 are the same or different and represent a hydrogen atom, a hydroxyl group or a lower alkoxy group.

A method for preventing or treating an autoimmune disease, which comprises administering a tetrahydroisoquinoline derivative or a pharmaceutically acceptable salt thereof represented by the formula:

 29. The method according to claim 26, 27 or 28, which is a method for preventing and treating arthritis.

 30. The method according to claim 26, 27 or 28, which is a method for preventing or treating rheumatoid arthritis.

 31. Use of a compound having a dipeptidyl peptidase IV inhibitory activity and having a tetrahydroisoquinoline skeleton or a pharmaceutically acceptable salt thereof for preventing or treating an autoimmune disease.

32. The compound or a pharmaceutically acceptable salt thereof has a dipeptidyl peptidase IV inhibitory action, and a compound represented by the formula:

Including the partial structure shown by

The use according to claim 31, which is a pharmacologically acceptable salt.

 33. General formula [I]:

(Wherein, R 1 is (1) a group having a structure in which a hydroxy group of a carboxy group is removed from an amino acid whose amino group may be protected, or (2) a protecting group of an amino group, and R ² is ( 1) a hydroxyl group which may be protected, (2) a group having a structure in which one hydrogen atom of an amino group has been removed from an amino acid wherein a carboxy group may be protected, or (3) a primary or secondary amine Or a group having a structure in which one hydrogen atom on a nitrogen atom has been removed from ammonia; R ³ , R ⁴ , R ⁵ and R ⁶ are the same or different and represent a hydrogen atom, a hydroxyl group or a lower alkoxy group)

Or a pharmacologically acceptable salt thereof for use in the prevention or treatment of an autoimmune disease.

 34. Use according to claim 31, 32 or 33 for use in the prevention or treatment of arthritis.

 35. The use according to claim 31, 32 or 33 for use in the prevention or treatment of rheumatoid arthritis.