Phenothiazine derivatives, their production and use

• US 5,532,171 A
• Filed: 05/23/1994
• Issued: 07/02/1996
• Est. Priority Date: 04/26/1991
• Status: Expired due to Fees

• Current Assignee: Takeda Chemical Industries, Ltd. (Takeda Pharmaceutical Company Limited)
• Original Assignee: Takeda Chemical Industries, Ltd. (Takeda Pharmaceutical Company Limited)
• Inventors: Motsenbocker, Marvin A.
• Primary Examiner(s): Ceperley, Mary E.
• Application Number:
  US08/247,552
• Time in Patent Office: 771 Days
• Field of Search: 436/546, 436/544, 436/533, 436/512, 436/503, 436/811
• US Class Current: 436/533
• CPC Class Codes: C07C 381/02 Thiosulfates
• CPC Class Codes: C07D 207/404 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. succinimide
• CPC Class Codes: C07D 279/18 [b, e]-condensed with two six-membered rings
• CPC Class Codes: C07D 417/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 417/14 containing three or more hetero rings
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: Y10S 436/811 Test for named disease, body condition or organ function

Abstract

There is disclosed a novel phenothiazine derivative of the general formula (I): ##STR1## wherein R₁, R₂, R₃ and R₄ are the same or different and are a C_1-6 alkyl group and at least one of them has as a substituent a group which can react with amino group, thiol group or carboxyl group, and X^- is a counter ion of the phenazathionium. Their production and the intermediates used in the production are also disclosed. The compound (I) is a derivative of methylene blue and is applicable to photodynamic therapy of cancer or immunoassays utilizing chemiluminescence.

Claims

1. A method for determining the concentration of an analyte in a solution, comprising the steps of:

(a) contacting said solution with a conjugate formed by coupling a binding molecule capable of binding specifically to the analyte to a phenothiazine derivative of general formula (I);

##STR12## wherein R₁, R₂, R₃, and R₄ are the same or different and each is a C_1-6 alkyl group, wherein at least one of said alkyl groups is substituted with a substituent so as to react with an amino group, thiol group, or carboxyl group on said binding molecule, and X^- is a counter ion, and

(b) measuring the amount of said conjugate that binds to said analyte.

3. The method as claimed in claim 1, wherein the substituent is selected from the group consisting of a carboxyl group, halogenocarbonyl group, azidocarbonyl group, cyanocarbonyl group, an alkoxycarbonyloxycarbonyl group, a substituted phenoxycarbonyl group, 1-imidazolyl-carbonyl group, 1-benzotriazolyloxycarbonyl group, 5-norbornene-2,3-dicarboximidoxycarbonyl group, succinimidoxycarbonyl group, maleimidoxycarbonyl group, aspartic acid residue, glutamic acid residue, maleimido group, or an aminoalkylcarbamoyl group.

5. The method as claimed in claim 1, wherein the substituent is selected from the group consisting of a carboxyl group, chlorocarbonyl group, azidocarbonyl group, cyanocarbonyl group, ethoxycarbonyloxycarbonyl group, isobutoxycarbonyloxycarbonyl group, p-nitrophenoxycarbonyl group, 2,4-dinitrophenoxycarbonyl group, pentachlorophenoxycarbonyl group, 2,4,5-trichlorophenoxycarbonyl group, pentafluorophenoxycarbonyl group, 1-imidazolyl-carbonyl group, 1-benzotriazolyloxycarbonyl group, 5-norbornene-2,3-dicarboximidoxycarbonyl group, succinimidoxycarbonyl group, maleimidoxycarbonyl group, aspartic acid residue, glutamic acid residue, maleimido group, aminoethylcarbamoyl group, or aminodecylcarbamoyl group.

7. The method as claimed in claim 1, wherein X^- is the counter ion formed by a halogen atom, hydroxyl, perchloric acid, sulfuric acid, nitric acid, trifluoroacetic acid or hydrofluoroboric acid.

9. The method as claimed in claim 1, wherein the phenothiazine derivative is hydrophilic.

11. The method as claimed in claim 1, wherein the phenothiazine derivative is selected from the group consisting of

3-[N-methyl-N-(4-succinimidoxycarbonylbutyl)amino]-7-dimethylaminophenazathionium chloride,

3-[N-methyl-N-(5-succinimidoxycarbonylpentyl)amino]-7-dimethylaminophenazathionium chloride,

3-[N-methyl-N-(3-succinimidoxycarbonylpropyl)amino]-7-dimethylaminophenazathionium chloride,

3-[N-methyl-N-(3-succinimidoxycarbonylpropyl)amino]-7-[N-(3-ethoxycarbonylpropyl)-N-methylamino]phenazathioniumchloride,

3,7-bis[N-methyl-N-(3-succinimidoxycarbonylpropyl)amino]phenazathionium chloride,

3-[N-methyl-N-(3-succinimidoxycarbonylpropyl)amino]-7-[N-(3-carboxypropyl)-N-methylamino]phenazathionium chloride,

3-[N-methyl-N-(3-succinimidoxycarbonylpropyl)amino]-7-[N-[3-(1,3-dicarboxypropylcarbamoyl)-propyl]-N-methylamino]phenazathionium chloride,

3,7-bis[N-methyl-N-(5-succinimidoxycarbonylpentyl)amino]phenazathionium chloride,

3-[N-methyl-N-(5-succinimidoxycarbonylpentyl)amino]-7-[N-(5-carboxypentyl)-N-methylamino]phenazathionium chloride,

3-[N-methyl-N-(5-succinimidoxycarbonylpentyl)amino]-7-[N-(5-(1,3-dicarboxypropylcarbamoyl)-pentyl]-N-methylamino]phenazathionium chloride,

3-[N-(3-maleimidopropyl)-N-methylamino]-7-[N-(3-carboxypropyl]-N-methylaminophenazathionium chloride,

3-[N-(3-maleimidopropyl)-N-methylamino]-7-[N-[3-(1,3-dicarboxypropylcarbamoyl)propyl]-N-methylamino]phenazathionium chloride,

3-[N-(4-maleimidobutyl)-N-methylamino]-7-[N-(4-carboxybutyl)-N-methylamino]phenazathionium chloride, and

3-[N-(4-maleimidobutyl)-N-methylamino]-7-dimethylaminophenazathionium chloride.

2. A method for determining the concentration of an analyte in a solution, comprising the steps of:

(a) contacting said solution with (i) a conjugate formed by coupling a sample of said analyte or an analogue of said analyte to a phenothiazine derivative of general formula (I);

##STR13## wherein R₁, R₂, R₃, and R₄ are the same or different and each is a C_1-6 alkyl group, wherein at least one of said alkyl groups is substituted with a substituent so as to react with an amino group, thiol group, or carboxyl group on said binding molecule, and X^- is a counter ion, and (ii) a binding molecule capable of binding specifically to both said analyte and said conjugate, and

(b) measuring the amount of said conjugate that binds to said binding molecule.

4. The method as claimed in claim 2, wherein the substituent is selected from the group consisting of a carboxyl group, halogenocarbonyl group, azidocarbonyl group, cyanocarbonyl group, an alkoxycarbonyloxycarbonyl group, a substituted phenoxycarbonyl group, 1-imidazolyl-carbonyl group, 1-benzotriazolyloxycarbonyl group, 5-norbornene-2,3-dicarboximidoxycarbonyl group, succinimidoxycarbonyl group, maleimidoxycarbonyl group, aspartic acid residue, glutamic acid residue, maleimido group, or an aminoalkylcarbamoyl group.

6. The method as claimed in claim 2, wherein the substituent is selected from the group consisting of a carboxyl group, chlorocarbonyl group, azidocarbonyl group, cyanocarbonyl group, ethoxycarbonyloxycarbonyl group, isobutoxycarbonyloxycarbonyl group, p-nitrophenoxycarbonyl group, 2,4-dinitrophenoxycarbonyl group, pentachlorophenoxycarbonyl group, 2,4,5-trichlorophenoxycarbonyl group, pentafluorophenoxycarbonyl group, 1-imidazolyl-carbonyl group, 1-benzotriazolyloxycarbonyl group, 5-norbornene-2,3dicarboximidoxycarbonyl group, succinimidoxycarbonyl group, maleimidoxycarbonyl group, aspartic acid residue, glutamic acid residue, maleimido group, aminoethylcarbamoyl group, or aminodecylcarbamoyl group.

8. The method as claimed in claim 2, wherein X^- is the counter ion formed by a halogen atom, hydroxyl, perchloric acid, sulfuric acid, nitric acid, trifluoroacetic acid or hydrofluoroboric acid.

10. The method as claimed in claim 2, wherein the phenothiazine derivative is hydrophilic.

12. The method as claimed in claim 2, wherein the phenothiazine derivative is selected from the group consisting of

3-[N-methyl-N-(4-succinimidoxycarbonylbutyl)amino]-7-dimethylaminophenazathionium chloride,

3-[N-methyl-N-(5-succinimidoxycarbonylpentyl)amino]-7-dimethylaminophenazathionium chloride,

3-[N-methyl-N-(3-succinimidoxycarbonylpropyl)amino]-7-dimethylaminophenazathionium chloride,

3-[N-methyl-N-(3-succinimidoxycarbonylpropyl)amino]-7-[N-(3-ethoxycarbonylpropyl)-N-methylamino]phenazathioniumchloride,

3,7-bis[N-methyl-N-(3-succinimidoxycarbonylpropyl)amino]phenazathionium chloride,

3-[N-methyl-N-(3-succinimidoxycarbonylpropyl)amino]-7-[N(3-carboxypropyl)-N-methylamino]phenazathionium chloride,

3-[N-methyl-N-(3-succinimidoxycarbonylpropyl)amino]-7-[N-[3-(1,3-dicarboxypropylcarbamoyl)-propyl]-N-methylamino]phenazathionium chloride,

3,7-bis[N-methyl-N-(5-succinimidoxycarbonylpentyl)amino]phenazathionium chloride,

3-[N-methyl-N-(5-succinimidoxycarbonylpentyl)amino]-7-[N-(5-carboxypentyl)-N-methylamino]phenazathionium chloride,

3-[N-methyl-N-(5-succinimidoxycarbonylpentyl)amino]-7-[N-(5-(1,3-dicarboxypropylcarbamoyl)-pentyl]-N-methylamino]phenazathionium chloride,

3-[N-(3-maleimidopropyl)-N-methylamino]-7-[N-(3-carboxypropyl]-N-methylaminophenazathionium chloride,

3-[N-(3-maleimidopropyl)-N-methylamino]-7-[N-[3-(1,3-dicarboxypropylcarbamoyl)propyl]-N-methylamino]phenazathionium chloride,

3-[N-(4-maleimidobutyl)-N-methylamino]-7-[N-(4-carboxybutyl)-N-methylamino]phenazathionium chloride, and

3-[N-(4-maleimidobutyl)-N-methylamino]-7-dimethylaminophenazathionium chloride.