CA2270973A1 - New knoevenagel condensation products, method for their production and their use - Google Patents
New knoevenagel condensation products, method for their production and their use Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
- C07F9/576—Six-membered rings
- C07F9/58—Pyridine rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
- C07F9/65583—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system each of the hetero rings containing nitrogen as ring hetero atom
Abstract
The present invention relates to new Knoevenagel condensation products, the method for their production and their use, specially for pharmaceutical and analytical purposes. Further, conjugates of Knoevenagel condensates with biomolecules are disclosed. The invention is characterized by the compound of formula (3).
Description
~'t : ; .' ,i L ."t WO 98l20013 - PCTIEP9'7/06184 -1 New Knoevenagel Condensation Products, Methods of their Production and their Use D a s cripti o:n The concerning invention refers to new Krloevenagel condensation products, methods of their production and their use for pharmaceutical and analytical purposes. Additionally, conjugates of the Knoevenagel condensates with biomolecules are disclosed.
In a publication of Feigl (Z. Anal. Chern. i'4 (1928), 380-386) the use of p-dinzethylaminobenzalrhodanine for the detection of silver is described.
Escobar Godoy and Guiraum Perez (Analyst 111 ~ ( 1986), 1297-1299) describe the use of the pyridoxal analogue (Z)-pyridoxylidenerhoda.nine for the spectrophotometric quantification of silver.
[The European Patent Application EP 0 693 496 A 1 discloses new cephalosporines with interesting pharmacological properties. The European Patent Application EP-A-0 636 630 A 1 discloses N-heteroallyl-substituted propa.namide derivatives which are suitable for the treatment of cardiovascular diseases. V1T~93/12124 discloses substituted benzimidazoles which inhibit the gastric acid secretion in mammals and, .n addition, do not inhibit the iodine import in the thyroid gland. None of the three latent Applications discloses inventions in which a pyridyl group is bound over a ~R-group or a N-atom to a heterocyclic 5-ring. ]
Note: Part [..) was included during the IPE and is not part of the original ~ppl ication) ,he basic problem of the concerning invention was to supply new compounds uitable particularly for pharmaceutical and analytical purposes. This task is irlfilled by the synthesis of the compound (Z)-5'-O-phosphono-WO 98IZOU13 - PCT/EP9'7/06184 - 2 pyridoxylidenerhodanine (3), representing a derivative of the vitamin B6 coenzyme pyridoxal 5'-phosphate ( 1 ).
The preparation of the compound (3) is perfi~rmed by reaction of ( 1 ) with the synthetic heterocyclic compound rhodanine (2) in a Knoevenagel condensation (see also Kesel et aL, Tetrahedron 52 (18.11.1996), 14787-14800).
The compound 3 bears the nomenclature (Z)-5-[[S-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-4-pyridinyl]methylene:]-2-thioxo-4-thiazolidinone. The rhodanine part of this molecule is able to bind 1:o biomolecules like single-stranded nucleic acids. The charged phosphate group enables good water solubility.
The compound 3 undergoes a (Z/E)-stereoisomerism to the (E)-isomer 4. This cis/trans-rearrangement is triggered by e. g. UV-irradiation with the wavelength 254 run or/and 366 nm.
In polar aprotic solvents the originally yellow compound 3 is found as red 3-~yridinolate free acid 5 e. g. in pyridine or dimethylsulfoxide (DMS~).
o N
~s H O , g _ OH O N ~P-0 OH
HO'' ~P O ~~~ + ~ ~S ,~ HO' ~ 10H
OH N CH3 S/~'' - H20 N CH3 Pyridoaal 5'-phosphate (1 ) Rhodanine (2) (Z)-5'-O-Phosphono-pyridoxylidcnerhodanine (3) he yellow compound 3 forms a deep-red nnonosodium salt. Its structure wax ~termined by X-ray crystallography. It was crystallized as hemiheptadecahydratc ~.5 hydrate) and is found as (l)-stereoisomer G. The monosodium salt G, it UV-nrrrr«r... T~...- ....~~ -. _ ___.. ~ .
CA 02270973 1999-OS-06..-WO 98/Z0013 - PCT/EP97~/06184 - 3 irradiated, performes analogously a cis/trans-rearrangement to the red (E)-stereoisomer 7.
Derivatives of the compound 3 can be obtained. with C 12-C 1 g fatty acid chlorides at the OH group.
Furthermore, the concerning invention refers to compounds of the general formula (8).
~ X2 '~X3 Y
R3 ~ , R''-c8~
Therein:
l , X2 and X3 are independently selected from O, S and NRS, respectively;
'isOorS;
is CRS or N;
.1, R2, R3, R4 and RS are independently selected i~rom any substituents, respectively;
~ is selected from possibly substituted hydrocarbon derivatives and O, and n is 0 orl ;
so salts, e. b. salts with alkali metal, alkaline earth or ammonia ions, their hydrates and crec~isomers, with the condition that the compound is not pyridoxylidenerhodanine.
L R6,n WO 98I20013 - PCT/EP9'7/06184 - 4 In the compounds of the general fonnula (8) Xl is preferably O. X2 is preferably NRS, wherein R5 is hydrogen or C 1-C4-alkyl and especially preferred hydrogen.
X3 is preferably S. Y is preferably S. Z is preferably CRS, wherein RS is hydrogen or C 1-C4-alkyl and especially prefer-ed hydrogen.
The substituents R1 to R4 in fact can be any sub:>tituents, if being compatible with the overall structure. If necessary, Rl and RZ or/and R3 and R4 can be bridged with each other. Examples for the sustituents R1 to R4 axe hydrogen, halogen, hydroxyl, amine and also, if necessary, substituted alkyl groups, e.. g. C 1- to Cb-alkyl, aminoalkyl, hydroxyalkyl, alkoxy etc. Especially preferred at .'least one of the substituents R1 to R4 contains an under physiologic conditions negatively charged group, e, g. an acid group like phosphate, carboxylate, sulfonate etc.
Especially preferred the compounds of the general formula (8) represent the (Z)-5-O-phosphono-pyridoxylidenerhodanine (3), also its salts, hydrates and stereoisomers.
A further subject of the concerning invention is a process for the preparation of a ~ompound of the general formula (8), characterized by the Knoevenagel condensation of 3 compound of the formula (9) ~~~3 Y
(9) pith a compound of the formula (10) WO 98n0013 - PC'C/EP9'1/06~84 - S
R3 R;z ~N ~R~
n (lo) wherein X 1, X2, X3, Y, Z, R 1-RS and R6 are defined as for the compounds 8.
The reaction happens preferably in an essentially equimolar ratio of 9 to 10 in a suitable solvent or solvent mixture. The temperature is preferably room temperature to reflux temperature of the solvent.
The compounds of the general formula (8) surprisingly show pharmaceutical effects. So 3 further subj ect of the invention is a pharmaceutical composition containing as effective substance a compound as mentioned before (preferably without respect to the disclaimer n claim 1 ), and also, if necessary, pharmaceutically usual supplemental, supporting, :arrier and diluting substances.
3 first important application of the compounds according to the invention is the reatment of infective diseases, for example viral infections, parasitic diseases, fungal iseases or bacterial diseases. Especially preferred is the use for the treatment of viral ~fections, in particular of such viral infections which are caused by enveloped viruses ke hepadnaviruses, herpesviruses or retroviruses e. g. HIV. Nevertheless, the impounds according to the invention proofed themselves suitable for the treatment of iseases which are caused by other viruses like influenza or papillomaviruses) urlhermore, the compounds according to the invention proofed themselves to be well aitable for the fibhting of parasites like leishmania) plasmodia or trypanosoma. Mostly -eferred arc the compounds in the 1-11V therapy;, for example in combination therapy ith other drugs like AZT or DDI.
CA 02270973 1999-OS-06 w WO 98120013 - PCTIEP9 i'/06184 - 6 Furthermore, the compounds according to the invention are also suitable for the fighting of tumor diseases, for example leukemias, especially T cell leukemias, or of skin tumors like malignant melanoma or Kaposi's sarcoma. Surprisingly, they show a selective cytotoxie action against malignant cells, not against: normal cells.
The compounds 8 also show immunomodulatory actions and, therefore, are suitable for the treatment of autoimmune diseases like multiplE: sclerosis, Alzheimer's disease, lupus ~rythematosus, myasthenia gravis, chronic arthritis, diabetes mellitus type I
etc. and various neurodegenerative diseases.
Furthermore, the compounds 8 are suitable for the treatment of diseases connected with defects of vitamin B metabolism, e. g. as vitamin B6 antagonists.
A further use are diseases connected with defects of enzymatic reactions. The compounds according to the invention serve as e~ffectors, e. g. activators, inhibitors or -nodificators, on enzymatic reactions of the caspase system, and especially as inhibitors ~f nitric oxide synthases.
urthermore, it was found that the compounds 8 ;influence the secretion of TNFa in T
ymphocytes. In addition, they are able to bind to tlhe cellular CD38 receptor, resulting in .n inhibition of the binding of HIV gp4l. In this uray the signal transduction pathway of ~D3 8 is decoupled, resulting in an inhibition of syncytia formation, of NO
liberation, of cell death by apoptosis, and of single cell lysis.
'he compounds according to the invention can be as such used for therapeutical urposes. But they can be additionally used as chelates with polyvalent metal cations, or non-covalent or/and covalent conjugates with biomolecules, e. g. nucleic acids, roteins, lipids, fatty acids etc.
he compounds according io the invention may b<: included in any form, e. g. as cream, ntment) injcctable or orally applicable fluids, liposomal formulations) tablets, coated WO 98I20013 - PCT/EP9'1/06184 - 7 tablets, capsules ete. The application can be made e. g. locally or systemic, topic, orally or by injection. The dosage can be varied in wide ranges, e, g. 0.01 p.g to 1 mg per kg body weight depending on the nature of the disease and application.
Furthermore, the compounds according to the invention are also suitable as analytical reagents. On the one hand they can be used in fonrt of radioactive labelled derivatives, e.
g. 3H~ 14C 32p or 3 S g _ gut preferred is the use in unlabelled form because its strong coloring and fluorescent properties are enabling a simple detection in biological systems.
Therefore, the compounds according to the invention are especially suitable for the detection of biomolecules, e, g. in diagnostic analysis, for example for the detection of single-stranded nucleic acids. In addition, the compounds are suitable for structure determination, e. g. for the sequencing of proteins or nucleic acids.
Furthermore, the compounds according to the invention can be used for eleetrochrornic applications, e. g. as molecular digital switch substances or indicators for pH, =emperature or solvents.
urthermore, the compounds according to the invention and derivatives are suitable as absorber for ions, organic substances etc., or because of their intensive colors and reversible color changes for decorative purposes. F~specially the alkaline earth salts, e. g.
.gig- or Ca-salts of the compounds are to be noted appearing in the form of gels.
also a further field of application is the nanotechnology. l~or example, laser system xcited single fluorescent dye molecules and biomoleeule derivatives can be detected and ~calized by sensitive detection systems, e.g. CCI~ cameras or avalanche detectors and ptical techniques on thin layer plates, in membranes, cells, drops, capillares etc. Further ossibilities of single molecule localization are the optical raster nearfield microscopy on ~inichips, the confocal microscopy in polyacrylamide gels, for example the two-or :ore-dimensional gel electrophoresis of nucleic acids.
WO 98/20013 - PCT/EP97~06184 - 8 Finally, the concerning invention refers to derivatives of compounds (8) which originate ~y hydrolytic degradation and represent for example: the general formulas 11, 12 or 13:
X~ OH
~X3 R ~ ~ ,R2 wNr wR1 Rsln (11) x ' loH
,_ R ~N ~R~
RsJ
n ( 12) R~ ~N ~R~
R6 Jn (13) Herein Xl, X2, X3, Y, Z, Rt-RS and R~ are darned as for the compounds 8.
CA 02270973 1999-OS-06 , ' Furthermore, the invention is explained by the following examples and figures.
It shows:
Fig. 1: the by the compound (3) mediated protection of cells against a HIV-infection.
Fig. 2: an overall figure of hydrolytic side product~~ which originate during synthesis of compound (3);
Examples ! . Preparation Method of 3 'reparation of the Crude Product:
.83 g 2-thioxo-4-thiazolidinone (rhodanine) (M = 133.18 glmol; n = 28.76 mmol) are issolved in 150 ml of absolute ethanol in a 500 ~ml round bottomed flask with reflux ondensor and 7.63 g of pyridoxal 5'-phosphate ma~nohydrate (M = 265.16 g/mol;
In a publication of Feigl (Z. Anal. Chern. i'4 (1928), 380-386) the use of p-dinzethylaminobenzalrhodanine for the detection of silver is described.
Escobar Godoy and Guiraum Perez (Analyst 111 ~ ( 1986), 1297-1299) describe the use of the pyridoxal analogue (Z)-pyridoxylidenerhoda.nine for the spectrophotometric quantification of silver.
[The European Patent Application EP 0 693 496 A 1 discloses new cephalosporines with interesting pharmacological properties. The European Patent Application EP-A-0 636 630 A 1 discloses N-heteroallyl-substituted propa.namide derivatives which are suitable for the treatment of cardiovascular diseases. V1T~93/12124 discloses substituted benzimidazoles which inhibit the gastric acid secretion in mammals and, .n addition, do not inhibit the iodine import in the thyroid gland. None of the three latent Applications discloses inventions in which a pyridyl group is bound over a ~R-group or a N-atom to a heterocyclic 5-ring. ]
Note: Part [..) was included during the IPE and is not part of the original ~ppl ication) ,he basic problem of the concerning invention was to supply new compounds uitable particularly for pharmaceutical and analytical purposes. This task is irlfilled by the synthesis of the compound (Z)-5'-O-phosphono-WO 98IZOU13 - PCT/EP9'7/06184 - 2 pyridoxylidenerhodanine (3), representing a derivative of the vitamin B6 coenzyme pyridoxal 5'-phosphate ( 1 ).
The preparation of the compound (3) is perfi~rmed by reaction of ( 1 ) with the synthetic heterocyclic compound rhodanine (2) in a Knoevenagel condensation (see also Kesel et aL, Tetrahedron 52 (18.11.1996), 14787-14800).
The compound 3 bears the nomenclature (Z)-5-[[S-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-4-pyridinyl]methylene:]-2-thioxo-4-thiazolidinone. The rhodanine part of this molecule is able to bind 1:o biomolecules like single-stranded nucleic acids. The charged phosphate group enables good water solubility.
The compound 3 undergoes a (Z/E)-stereoisomerism to the (E)-isomer 4. This cis/trans-rearrangement is triggered by e. g. UV-irradiation with the wavelength 254 run or/and 366 nm.
In polar aprotic solvents the originally yellow compound 3 is found as red 3-~yridinolate free acid 5 e. g. in pyridine or dimethylsulfoxide (DMS~).
o N
~s H O , g _ OH O N ~P-0 OH
HO'' ~P O ~~~ + ~ ~S ,~ HO' ~ 10H
OH N CH3 S/~'' - H20 N CH3 Pyridoaal 5'-phosphate (1 ) Rhodanine (2) (Z)-5'-O-Phosphono-pyridoxylidcnerhodanine (3) he yellow compound 3 forms a deep-red nnonosodium salt. Its structure wax ~termined by X-ray crystallography. It was crystallized as hemiheptadecahydratc ~.5 hydrate) and is found as (l)-stereoisomer G. The monosodium salt G, it UV-nrrrr«r... T~...- ....~~ -. _ ___.. ~ .
CA 02270973 1999-OS-06..-WO 98/Z0013 - PCT/EP97~/06184 - 3 irradiated, performes analogously a cis/trans-rearrangement to the red (E)-stereoisomer 7.
Derivatives of the compound 3 can be obtained. with C 12-C 1 g fatty acid chlorides at the OH group.
Furthermore, the concerning invention refers to compounds of the general formula (8).
~ X2 '~X3 Y
R3 ~ , R''-c8~
Therein:
l , X2 and X3 are independently selected from O, S and NRS, respectively;
'isOorS;
is CRS or N;
.1, R2, R3, R4 and RS are independently selected i~rom any substituents, respectively;
~ is selected from possibly substituted hydrocarbon derivatives and O, and n is 0 orl ;
so salts, e. b. salts with alkali metal, alkaline earth or ammonia ions, their hydrates and crec~isomers, with the condition that the compound is not pyridoxylidenerhodanine.
L R6,n WO 98I20013 - PCT/EP9'7/06184 - 4 In the compounds of the general fonnula (8) Xl is preferably O. X2 is preferably NRS, wherein R5 is hydrogen or C 1-C4-alkyl and especially preferred hydrogen.
X3 is preferably S. Y is preferably S. Z is preferably CRS, wherein RS is hydrogen or C 1-C4-alkyl and especially prefer-ed hydrogen.
The substituents R1 to R4 in fact can be any sub:>tituents, if being compatible with the overall structure. If necessary, Rl and RZ or/and R3 and R4 can be bridged with each other. Examples for the sustituents R1 to R4 axe hydrogen, halogen, hydroxyl, amine and also, if necessary, substituted alkyl groups, e.. g. C 1- to Cb-alkyl, aminoalkyl, hydroxyalkyl, alkoxy etc. Especially preferred at .'least one of the substituents R1 to R4 contains an under physiologic conditions negatively charged group, e, g. an acid group like phosphate, carboxylate, sulfonate etc.
Especially preferred the compounds of the general formula (8) represent the (Z)-5-O-phosphono-pyridoxylidenerhodanine (3), also its salts, hydrates and stereoisomers.
A further subject of the concerning invention is a process for the preparation of a ~ompound of the general formula (8), characterized by the Knoevenagel condensation of 3 compound of the formula (9) ~~~3 Y
(9) pith a compound of the formula (10) WO 98n0013 - PC'C/EP9'1/06~84 - S
R3 R;z ~N ~R~
n (lo) wherein X 1, X2, X3, Y, Z, R 1-RS and R6 are defined as for the compounds 8.
The reaction happens preferably in an essentially equimolar ratio of 9 to 10 in a suitable solvent or solvent mixture. The temperature is preferably room temperature to reflux temperature of the solvent.
The compounds of the general formula (8) surprisingly show pharmaceutical effects. So 3 further subj ect of the invention is a pharmaceutical composition containing as effective substance a compound as mentioned before (preferably without respect to the disclaimer n claim 1 ), and also, if necessary, pharmaceutically usual supplemental, supporting, :arrier and diluting substances.
3 first important application of the compounds according to the invention is the reatment of infective diseases, for example viral infections, parasitic diseases, fungal iseases or bacterial diseases. Especially preferred is the use for the treatment of viral ~fections, in particular of such viral infections which are caused by enveloped viruses ke hepadnaviruses, herpesviruses or retroviruses e. g. HIV. Nevertheless, the impounds according to the invention proofed themselves suitable for the treatment of iseases which are caused by other viruses like influenza or papillomaviruses) urlhermore, the compounds according to the invention proofed themselves to be well aitable for the fibhting of parasites like leishmania) plasmodia or trypanosoma. Mostly -eferred arc the compounds in the 1-11V therapy;, for example in combination therapy ith other drugs like AZT or DDI.
CA 02270973 1999-OS-06 w WO 98120013 - PCTIEP9 i'/06184 - 6 Furthermore, the compounds according to the invention are also suitable for the fighting of tumor diseases, for example leukemias, especially T cell leukemias, or of skin tumors like malignant melanoma or Kaposi's sarcoma. Surprisingly, they show a selective cytotoxie action against malignant cells, not against: normal cells.
The compounds 8 also show immunomodulatory actions and, therefore, are suitable for the treatment of autoimmune diseases like multiplE: sclerosis, Alzheimer's disease, lupus ~rythematosus, myasthenia gravis, chronic arthritis, diabetes mellitus type I
etc. and various neurodegenerative diseases.
Furthermore, the compounds 8 are suitable for the treatment of diseases connected with defects of vitamin B metabolism, e. g. as vitamin B6 antagonists.
A further use are diseases connected with defects of enzymatic reactions. The compounds according to the invention serve as e~ffectors, e. g. activators, inhibitors or -nodificators, on enzymatic reactions of the caspase system, and especially as inhibitors ~f nitric oxide synthases.
urthermore, it was found that the compounds 8 ;influence the secretion of TNFa in T
ymphocytes. In addition, they are able to bind to tlhe cellular CD38 receptor, resulting in .n inhibition of the binding of HIV gp4l. In this uray the signal transduction pathway of ~D3 8 is decoupled, resulting in an inhibition of syncytia formation, of NO
liberation, of cell death by apoptosis, and of single cell lysis.
'he compounds according to the invention can be as such used for therapeutical urposes. But they can be additionally used as chelates with polyvalent metal cations, or non-covalent or/and covalent conjugates with biomolecules, e. g. nucleic acids, roteins, lipids, fatty acids etc.
he compounds according io the invention may b<: included in any form, e. g. as cream, ntment) injcctable or orally applicable fluids, liposomal formulations) tablets, coated WO 98I20013 - PCT/EP9'1/06184 - 7 tablets, capsules ete. The application can be made e. g. locally or systemic, topic, orally or by injection. The dosage can be varied in wide ranges, e, g. 0.01 p.g to 1 mg per kg body weight depending on the nature of the disease and application.
Furthermore, the compounds according to the invention are also suitable as analytical reagents. On the one hand they can be used in fonrt of radioactive labelled derivatives, e.
g. 3H~ 14C 32p or 3 S g _ gut preferred is the use in unlabelled form because its strong coloring and fluorescent properties are enabling a simple detection in biological systems.
Therefore, the compounds according to the invention are especially suitable for the detection of biomolecules, e, g. in diagnostic analysis, for example for the detection of single-stranded nucleic acids. In addition, the compounds are suitable for structure determination, e. g. for the sequencing of proteins or nucleic acids.
Furthermore, the compounds according to the invention can be used for eleetrochrornic applications, e. g. as molecular digital switch substances or indicators for pH, =emperature or solvents.
urthermore, the compounds according to the invention and derivatives are suitable as absorber for ions, organic substances etc., or because of their intensive colors and reversible color changes for decorative purposes. F~specially the alkaline earth salts, e. g.
.gig- or Ca-salts of the compounds are to be noted appearing in the form of gels.
also a further field of application is the nanotechnology. l~or example, laser system xcited single fluorescent dye molecules and biomoleeule derivatives can be detected and ~calized by sensitive detection systems, e.g. CCI~ cameras or avalanche detectors and ptical techniques on thin layer plates, in membranes, cells, drops, capillares etc. Further ossibilities of single molecule localization are the optical raster nearfield microscopy on ~inichips, the confocal microscopy in polyacrylamide gels, for example the two-or :ore-dimensional gel electrophoresis of nucleic acids.
WO 98/20013 - PCT/EP97~06184 - 8 Finally, the concerning invention refers to derivatives of compounds (8) which originate ~y hydrolytic degradation and represent for example: the general formulas 11, 12 or 13:
X~ OH
~X3 R ~ ~ ,R2 wNr wR1 Rsln (11) x ' loH
,_ R ~N ~R~
RsJ
n ( 12) R~ ~N ~R~
R6 Jn (13) Herein Xl, X2, X3, Y, Z, Rt-RS and R~ are darned as for the compounds 8.
CA 02270973 1999-OS-06 , ' Furthermore, the invention is explained by the following examples and figures.
It shows:
Fig. 1: the by the compound (3) mediated protection of cells against a HIV-infection.
Fig. 2: an overall figure of hydrolytic side product~~ which originate during synthesis of compound (3);
Examples ! . Preparation Method of 3 'reparation of the Crude Product:
.83 g 2-thioxo-4-thiazolidinone (rhodanine) (M = 133.18 glmol; n = 28.76 mmol) are issolved in 150 ml of absolute ethanol in a 500 ~ml round bottomed flask with reflux ondensor and 7.63 g of pyridoxal 5'-phosphate ma~nohydrate (M = 265.16 g/mol;
2$.78 imol) are added. The pyridoxal 5'-phosphate nnonohydrate remains nearly undissolved i the cold.
was refluxed under stirring on the water bath. The pyridoxal 5'-phosphate monohydrate ssolves now slowly and a firstly yellow, then orange, then red suspension emerges.
fter 20 min of refluxing through the condensor l.'>0 ml of water are added because the spension pushes up. It is still heated for 10 min and the red reaction product ecipitates and pushes up. Then it is frozen for no more than 2 h at a temperature of -~°C.
rilication of the Crude Product a orange product is f Itered, collected with the mother Liquor, and washed with l0U ml icccold absolute ethanol. Vacuum dryin6 in the: desiccator over anhydrous calcium WO 98/Z0013 - PCT/EP97/06184 - l0 chloride; yield: 9.8S g orange-yellow crude product (~-5-[[S-Hydroxy-b-methyl-
was refluxed under stirring on the water bath. The pyridoxal 5'-phosphate monohydrate ssolves now slowly and a firstly yellow, then orange, then red suspension emerges.
fter 20 min of refluxing through the condensor l.'>0 ml of water are added because the spension pushes up. It is still heated for 10 min and the red reaction product ecipitates and pushes up. Then it is frozen for no more than 2 h at a temperature of -~°C.
rilication of the Crude Product a orange product is f Itered, collected with the mother Liquor, and washed with l0U ml icccold absolute ethanol. Vacuum dryin6 in the: desiccator over anhydrous calcium WO 98/Z0013 - PCT/EP97/06184 - l0 chloride; yield: 9.8S g orange-yellow crude product (~-5-[[S-Hydroxy-b-methyl-
3-~(phosphonooxy)methyl]-4-pyridinyl]methylene]-2-thioxo-4-thiazolidinone. The product contains eduets, side products and in spite of vacuum drying ethanol and water.
Purification of the Crude Product by Ion-Exchange-Reprecipitation:
9.8S g crude product were suspended in 610 ml of water in a 200D ml round bottomed flask. Under stirring 200 ml of an at room temperature (22°C) saturated solution [B
(NaHC03) = 100 mg/ml] of anhydrous sodium bicarbonate [n (NaHC03) = 23 8) 10 mmol] in several portions was added. This now red solution was titrated with 23 8.10 ml (23 8. I 0 mmol HCl) of a I .0 M hydrochloric acid. '.Che resulting mixture is stirred further for 1 S min. At the end of the titration (pH 2) the color changed from red to yellow and the free acid 3 precipitated. It was collected in a~ filter flask and dried for 24 h at a aressure of 4 mbar and a temperature of 70°C. TJhen it was still dried at a pressure of ).001 mbar in the oil pump vacuum at room temperature; yield: 8.70 g (83 % for overall synthesis) yellow, amorphous powder (~-S-[[S-Hydroxy-b-methyl-3-(phosphonooxy)methyl]-4-pyridinyl]methylene]-2-thioxo-4-thiazolidinone, content w >
~6% ( 1 H-NMR).
the analytics of compound 3 includes ~ H NMR spectroscopy, 13 C NMR
spectroscopy, I P NMR spectroscopy, RP 18 HPLC, fast atom bombardment mass spectrometry (FAB
TS), IR spectroscopy, UV spectrophotometry and fluorescence spectrophotometry.
'here follow typical analytical data of compound 3.
1 I fI I 1 N206P s2~ M = 362.31 g/mol.
p 198-201 °C (dec., uncorr.).
4B MS (M H+ calculated for mlz 362.99): mlz (;rel.lnt.) 3b3.0 ( 12.0%, MH+), 227.4 8. I %), 270.4 (8.7%), 171.2 (8.3%)) 282.4 (7.1 %.), 267. I (2.8%), 283.4 (2.1 %), 26S.1 .S%).
-I NMR (Tl'A-d): Q2.91 (.v, 3 I I, 2'-CH3), S.42 (d; ~3J(3r P,i l~l)~ ~ 8.U
f~lz, 2 I-I, 5'-C1-!2_ ;'031i2). 7.76 (s, 1 1~, 4'-C1-!), 8.47 (.v, 1 H, 6-CI-!).
CA 02270973 1999-OS-06, WO 98/20013 - PCT/EP9710618~ - 11 1H NMR (DMSO-d6): ~ 2.42 (s, 3 H, 2'-CH3), 4.89 (d, (3J(31 P,1 H)~ = 7.SHz, 2 H, S'-CH2-OPO3H2), 7.SS (s, 1 H, 4'-CH), 7.84 (s, 1 H, 6-CH).
1 H NMR (Pyridine-d5): ~ 2.72 (s, 3 H, 2'-CH3), S.62 (d, 3J(31 P,1 H)~ =
8.OHz, 2 H, S'-CH2-OP03H2), 8.36 (s, 1 H, 4'-CH), 8.53 (s, 1 H, 6-CH).
13C NMR, proton-decoupled (T'FA-d): ~ 16.83 (s, 2'-CH3), 65.7S (s, 5'-CH2-OP03H2), l21.04, 122.13 (2 s, C-4'), 133.34 (s, C-6), 136.85, 136.9I (2 s, RR'C-SR's) 138.54 (s, C-5), 142.0S (s, C-4), 147.64 (s, C-2), 153.03 (s, C-:3), 171.93 (s, C=O), 193.39 (s, C=S).
31 p ~ (~A_~: S _ 4.24 (s, R-OP03H2).
FT IR (KBr): 3430 (v N H, m), 1713 (v C=O, s), 1213 (v P=O, s, R-O(HO)P02 ), (v C=S; s, in S-CS-1~, 1024 (v P=O, s, R-O(HO)POz ).
W/VIS (H20): ~,max, 1 = 232 nm [A ( 1%/1 cm) = 373], ~.max, 2 = 308 nm [A (1 %/1 cm) = 271 ], fax, 3 3 3S3 nm (A (1 %/1 cm) = 413], ~;aX, 4 = 454 nm [A (1%/1 cm) =
227].
W/VIS (MeOH): ~.max, 1 = 291 nm [A ( 1 %/1 cm) = 24S], ~,max, 2 = 347 nm [A
~1%/lcm) = 489].
W/VIS (DMSO): ~,max = 51S nm (A (I%/lcm) = 98].
F luorescence excitation (ex) and emission (em) spectra (DMSO): ~m = S7S nrn:
~.ex, nax, 1 = 35S nm, fix, max, 2 = 397 nm, ~.ex, magic, 3 = 4S 1 nm, fix, max, 4 =
467 nm, hex, max, 5 = 483 ~~ hex, max, 6 = 493 nm, ~,,~x, max, 7 = 518 nm; ~x = 490 nm:
hem, max = 575 nm.
luorescence excitation (ex) and emission (em) spectra (Pyridine): ~m = 57S nm:
fix, :sax, 1 = 451 nm, fix, max, 2 = 467 nm, ~.ex, maac, 3 = 471 nm, fix, max, 4 =
483 nm, ~-ex, max, S = 493 nm, ~X, max, 6 = S 18 nm; ~,ex = 490 nm: ~m~ max = 57S nm.
. Preparative Synthesis of 6 .1 mass of 3G3 mg (Z)-S-[[5-1-Iydroxy-b-methyl-3-[(phosphonooxy)methyl]-4-pyridinyl]
ethylene]-2-thioxo-4-thiazolidinone (3) (1.0 mmol) was mixed with 20.0 ml of 0.l0 M
>dium hydroxide solution (2.00 mmol NaOI~). After the mixing with 10.0 ml of ~solutc ethanol the mixture was !'ro~en for 24 h at a temperature af' -1$°C. The fine yslalline needles were filtered; yield: 340 mb (G3%) red (L)-5-[[3-I Iydroxy-2-methyl-5-[(phosphonooxy)methyl]-~-pyridinyl]methylene]~-2-thioxo-4-thiazolidinone monosodium salt (8'/z) hydrate (6). Before elernen~tal analysis this product was dried for two days at a pressure of 0.1 mbar and a temperature of 70°C; yield:
deep red (Z)-5-[[5-Hydroxy-6-methyl-3-[(phosphonooxy)methyl]-4-p;yridinyl]methylene]-2-thioxo-4-thiazolidinone monosodium salt-hemiihydrate (2'/2) hydrate (6).
2.2 8.70 g dry compound 3 (24.00 mmol) were mixed with 240.00 ml of 0.10 M sodium hydroxide solution (24_00 mmol NaOH) and the addition of 124.00 ml of ethanol followed. The mixture was stored 24 h at a temperature of -18°C. The precipitated red crystals were filtered; yield: ?.55 g (73%) of compound 6.
mp 20S-2D8°C (dec., uncorr.).
Anal. C 11 I-Q10 N2 Na 06 P S2 x 2'/i H20, M=429,33 g/mol; C 30.77% H 3.52% N
i.52%; found C 30.97% H 3.43% N 6.35%.
FT IR (KBr): 3277 (v O--H, m, broad), 1700 (w HN-C=O, w), 1229 (v P=O, m, R-'(HO)P02 ), 1198 (v HN-C=S, s), 1090 (v C=S, s~, in S-CS-N), 973 (v P-O--C, m) R-~(HO)P02 ).
~. Detection Method for Single-stranded Nucleic: Acids 'he compounds 3 and 6 can be used in radioactive; labelled form, e. g.
labelled with 3H, 4C, 32P or 35S as probes for the detection of single-stranded nucleic acids.
'he compounds 3/6 can be additionally used for the fluorescence detection of single-randed DNA. The (E)-stereoisomer 4 shows. fluorescence in DMSO, the (G)-ereoisomer in water substantially less. In the case of binding of the (E)-stereoisomer 4 single-stranded DNA fluorescence can be detected after the extraction with DMSO.
ie used excitation wavelenl;th covers the ranl;c; of 420 to 500 nm, the emission in MSO or pyridine is found at 575 nm.
CA 02270973 1999-OS-06.
WO 98/200l3 - PCT/EP97/06184 - 13 The compounds 3, 4 and 6 show a non-covalent association to single-stranded DNA.
This non-covalent binding can be fixated photoche;mically by irradiation with UV light of the wavelength 254 or/and 366 nm. There reacts 3 and 4 with a thymine nucleobase methyl group under splitting of H2S.
~. Inhibition of Isoforms of the Enzyme Nitric O~tide Synthase The substance 6 inhibits the neuronal, endothelial and immunologic/inducible isoforms ~f the nitric oxide synthase (NOS). The inhibition constant (IC50) for nNOS is 61.25 aM. For eNOS and iNOS the inhibition constant (ICSp) is 50 p,M. This values were obtained by measuring the transformation of (3H) arginine to (3H) citrulline with the use ~f recombinant human NOS isoforms from CHO cells.
i. Two-dimensional Nucleic Acid Electrophoresis ~'he subtance 3 can be used in radioactively labelled or unlabelled form for two-iimensional electrophoretic separation of single-stranded nucleic acids. In the first '.irnension there can be a separation after charge and in the second dimension a ~paration after molecule size. The compound 3 can be coupled associative or covalent to ae nucleic acid.
. Therapeutical Use he substance 3 and derivatives of it are effective as therapeutical agents, especially for nuencing proliferative events of infectious agents) e. g. of such emerging in the host ith intermediates of single-stranded DNA like retroviruses, herpesviruses or :patov i ruses.
b.l HIV-Inhibition Myeloic HUT78 cells were exposed to an infection with HIV in absence or presence of 1.87 mM or 0.187 mM of compound 3. The HTV detection is made by photometric evaluation of p24 antigen. In Figure 1 is depicted that in the cells treated according to the invention no p24 was detectable, whereas the control from the 7th day shows a positive p24 test.
6.2 Treatment of Herpes A patient (17, male) with herpes labialis was treated with an olive ail /
water emulsion of he compound 3 (ca. 1 mg/mI). Few hours after local administration on the affected skin portions a total remission was found.
i.3 Treatment of Influenza ~ patient {51, male) with viral influenza was l:reated orally with some drops of a 320/DMSO solution of the compound 3 (ca. 1 ~.g/ml). Over night the disease symptoms lisappeared.
'. Tempersture/Solvent Indicator 'he (Z/E)-stereoisomerism of the compound 3 appears depending on temperature :hermochromism) and depending on solvent (solvatochromism). Therefore, 3 can be red as temperature indicator or/and solvent indicator.
Digital Switch Function ie (Z/C)-stereoisomerism of the compounds 3 and 4 appears depending on applying an ~ctric 'f field (eleclrochromism). Therefore, ~ can be used as di6ital switch etcmcnl :cause of the different iluorcscence spectroscopic properties a6ainst 4 e. i;.
on WO 98I20013 - PC'T/EP9'~/06184 - 15 excitation with laser light of the wavelength 480 nm. Thereby, after applying an electric field on 3 rearrangement to 4 is performed, and it appears fluorescence emission at 575 nm after excitation at 480 nm.
9. Vitamia B6 Antagonism The compound 3 is able to effect in metabolism as antagonist of the coen2ymc pyridoxal 5'-phosphate by binding to enzymes depending on this coenzyme and, therefore, can show tumor suppressing properties.
I0. Proliferation Control The compound 3 can be used in proliferation cont;roUchemotherapy/cytostasis therapy of malignant tumors in local application on skin tumors (malignant melanoma, Kaposi's ~areoma), if necessary, in connection with LJ~~-irradiation, or/and for proliferation control of other skin diseases with increased ee;ll division rate (e. g.
psoriasis). The Binding of compound 3 to replicatively active single-stranded DNA areas can be fixated ~hotochemieally and leads to locally restricted cytotoxieity.
I. Structure Determination 'he compound 3 can be used because of its color and fluorescent properties and its bility to couple to biomolecules, e. g. proteins, for structure determination, e. g. for rhino acid sequence determination of proteins or for DNA sequencing.
2. Detection of Hydrolysis Side Products ie hydrolysis side products which emerge during the synthesis of compound 3 are :amined mass spcctroscopically.
~e concerning compounds are listed in Fig. 2.
Purification of the Crude Product by Ion-Exchange-Reprecipitation:
9.8S g crude product were suspended in 610 ml of water in a 200D ml round bottomed flask. Under stirring 200 ml of an at room temperature (22°C) saturated solution [B
(NaHC03) = 100 mg/ml] of anhydrous sodium bicarbonate [n (NaHC03) = 23 8) 10 mmol] in several portions was added. This now red solution was titrated with 23 8.10 ml (23 8. I 0 mmol HCl) of a I .0 M hydrochloric acid. '.Che resulting mixture is stirred further for 1 S min. At the end of the titration (pH 2) the color changed from red to yellow and the free acid 3 precipitated. It was collected in a~ filter flask and dried for 24 h at a aressure of 4 mbar and a temperature of 70°C. TJhen it was still dried at a pressure of ).001 mbar in the oil pump vacuum at room temperature; yield: 8.70 g (83 % for overall synthesis) yellow, amorphous powder (~-S-[[S-Hydroxy-b-methyl-3-(phosphonooxy)methyl]-4-pyridinyl]methylene]-2-thioxo-4-thiazolidinone, content w >
~6% ( 1 H-NMR).
the analytics of compound 3 includes ~ H NMR spectroscopy, 13 C NMR
spectroscopy, I P NMR spectroscopy, RP 18 HPLC, fast atom bombardment mass spectrometry (FAB
TS), IR spectroscopy, UV spectrophotometry and fluorescence spectrophotometry.
'here follow typical analytical data of compound 3.
1 I fI I 1 N206P s2~ M = 362.31 g/mol.
p 198-201 °C (dec., uncorr.).
4B MS (M H+ calculated for mlz 362.99): mlz (;rel.lnt.) 3b3.0 ( 12.0%, MH+), 227.4 8. I %), 270.4 (8.7%), 171.2 (8.3%)) 282.4 (7.1 %.), 267. I (2.8%), 283.4 (2.1 %), 26S.1 .S%).
-I NMR (Tl'A-d): Q2.91 (.v, 3 I I, 2'-CH3), S.42 (d; ~3J(3r P,i l~l)~ ~ 8.U
f~lz, 2 I-I, 5'-C1-!2_ ;'031i2). 7.76 (s, 1 1~, 4'-C1-!), 8.47 (.v, 1 H, 6-CI-!).
CA 02270973 1999-OS-06, WO 98/20013 - PCT/EP9710618~ - 11 1H NMR (DMSO-d6): ~ 2.42 (s, 3 H, 2'-CH3), 4.89 (d, (3J(31 P,1 H)~ = 7.SHz, 2 H, S'-CH2-OPO3H2), 7.SS (s, 1 H, 4'-CH), 7.84 (s, 1 H, 6-CH).
1 H NMR (Pyridine-d5): ~ 2.72 (s, 3 H, 2'-CH3), S.62 (d, 3J(31 P,1 H)~ =
8.OHz, 2 H, S'-CH2-OP03H2), 8.36 (s, 1 H, 4'-CH), 8.53 (s, 1 H, 6-CH).
13C NMR, proton-decoupled (T'FA-d): ~ 16.83 (s, 2'-CH3), 65.7S (s, 5'-CH2-OP03H2), l21.04, 122.13 (2 s, C-4'), 133.34 (s, C-6), 136.85, 136.9I (2 s, RR'C-SR's) 138.54 (s, C-5), 142.0S (s, C-4), 147.64 (s, C-2), 153.03 (s, C-:3), 171.93 (s, C=O), 193.39 (s, C=S).
31 p ~ (~A_~: S _ 4.24 (s, R-OP03H2).
FT IR (KBr): 3430 (v N H, m), 1713 (v C=O, s), 1213 (v P=O, s, R-O(HO)P02 ), (v C=S; s, in S-CS-1~, 1024 (v P=O, s, R-O(HO)POz ).
W/VIS (H20): ~,max, 1 = 232 nm [A ( 1%/1 cm) = 373], ~.max, 2 = 308 nm [A (1 %/1 cm) = 271 ], fax, 3 3 3S3 nm (A (1 %/1 cm) = 413], ~;aX, 4 = 454 nm [A (1%/1 cm) =
227].
W/VIS (MeOH): ~.max, 1 = 291 nm [A ( 1 %/1 cm) = 24S], ~,max, 2 = 347 nm [A
~1%/lcm) = 489].
W/VIS (DMSO): ~,max = 51S nm (A (I%/lcm) = 98].
F luorescence excitation (ex) and emission (em) spectra (DMSO): ~m = S7S nrn:
~.ex, nax, 1 = 35S nm, fix, max, 2 = 397 nm, ~.ex, magic, 3 = 4S 1 nm, fix, max, 4 =
467 nm, hex, max, 5 = 483 ~~ hex, max, 6 = 493 nm, ~,,~x, max, 7 = 518 nm; ~x = 490 nm:
hem, max = 575 nm.
luorescence excitation (ex) and emission (em) spectra (Pyridine): ~m = 57S nm:
fix, :sax, 1 = 451 nm, fix, max, 2 = 467 nm, ~.ex, maac, 3 = 471 nm, fix, max, 4 =
483 nm, ~-ex, max, S = 493 nm, ~X, max, 6 = S 18 nm; ~,ex = 490 nm: ~m~ max = 57S nm.
. Preparative Synthesis of 6 .1 mass of 3G3 mg (Z)-S-[[5-1-Iydroxy-b-methyl-3-[(phosphonooxy)methyl]-4-pyridinyl]
ethylene]-2-thioxo-4-thiazolidinone (3) (1.0 mmol) was mixed with 20.0 ml of 0.l0 M
>dium hydroxide solution (2.00 mmol NaOI~). After the mixing with 10.0 ml of ~solutc ethanol the mixture was !'ro~en for 24 h at a temperature af' -1$°C. The fine yslalline needles were filtered; yield: 340 mb (G3%) red (L)-5-[[3-I Iydroxy-2-methyl-5-[(phosphonooxy)methyl]-~-pyridinyl]methylene]~-2-thioxo-4-thiazolidinone monosodium salt (8'/z) hydrate (6). Before elernen~tal analysis this product was dried for two days at a pressure of 0.1 mbar and a temperature of 70°C; yield:
deep red (Z)-5-[[5-Hydroxy-6-methyl-3-[(phosphonooxy)methyl]-4-p;yridinyl]methylene]-2-thioxo-4-thiazolidinone monosodium salt-hemiihydrate (2'/2) hydrate (6).
2.2 8.70 g dry compound 3 (24.00 mmol) were mixed with 240.00 ml of 0.10 M sodium hydroxide solution (24_00 mmol NaOH) and the addition of 124.00 ml of ethanol followed. The mixture was stored 24 h at a temperature of -18°C. The precipitated red crystals were filtered; yield: ?.55 g (73%) of compound 6.
mp 20S-2D8°C (dec., uncorr.).
Anal. C 11 I-Q10 N2 Na 06 P S2 x 2'/i H20, M=429,33 g/mol; C 30.77% H 3.52% N
i.52%; found C 30.97% H 3.43% N 6.35%.
FT IR (KBr): 3277 (v O--H, m, broad), 1700 (w HN-C=O, w), 1229 (v P=O, m, R-'(HO)P02 ), 1198 (v HN-C=S, s), 1090 (v C=S, s~, in S-CS-N), 973 (v P-O--C, m) R-~(HO)P02 ).
~. Detection Method for Single-stranded Nucleic: Acids 'he compounds 3 and 6 can be used in radioactive; labelled form, e. g.
labelled with 3H, 4C, 32P or 35S as probes for the detection of single-stranded nucleic acids.
'he compounds 3/6 can be additionally used for the fluorescence detection of single-randed DNA. The (E)-stereoisomer 4 shows. fluorescence in DMSO, the (G)-ereoisomer in water substantially less. In the case of binding of the (E)-stereoisomer 4 single-stranded DNA fluorescence can be detected after the extraction with DMSO.
ie used excitation wavelenl;th covers the ranl;c; of 420 to 500 nm, the emission in MSO or pyridine is found at 575 nm.
CA 02270973 1999-OS-06.
WO 98/200l3 - PCT/EP97/06184 - 13 The compounds 3, 4 and 6 show a non-covalent association to single-stranded DNA.
This non-covalent binding can be fixated photoche;mically by irradiation with UV light of the wavelength 254 or/and 366 nm. There reacts 3 and 4 with a thymine nucleobase methyl group under splitting of H2S.
~. Inhibition of Isoforms of the Enzyme Nitric O~tide Synthase The substance 6 inhibits the neuronal, endothelial and immunologic/inducible isoforms ~f the nitric oxide synthase (NOS). The inhibition constant (IC50) for nNOS is 61.25 aM. For eNOS and iNOS the inhibition constant (ICSp) is 50 p,M. This values were obtained by measuring the transformation of (3H) arginine to (3H) citrulline with the use ~f recombinant human NOS isoforms from CHO cells.
i. Two-dimensional Nucleic Acid Electrophoresis ~'he subtance 3 can be used in radioactively labelled or unlabelled form for two-iimensional electrophoretic separation of single-stranded nucleic acids. In the first '.irnension there can be a separation after charge and in the second dimension a ~paration after molecule size. The compound 3 can be coupled associative or covalent to ae nucleic acid.
. Therapeutical Use he substance 3 and derivatives of it are effective as therapeutical agents, especially for nuencing proliferative events of infectious agents) e. g. of such emerging in the host ith intermediates of single-stranded DNA like retroviruses, herpesviruses or :patov i ruses.
b.l HIV-Inhibition Myeloic HUT78 cells were exposed to an infection with HIV in absence or presence of 1.87 mM or 0.187 mM of compound 3. The HTV detection is made by photometric evaluation of p24 antigen. In Figure 1 is depicted that in the cells treated according to the invention no p24 was detectable, whereas the control from the 7th day shows a positive p24 test.
6.2 Treatment of Herpes A patient (17, male) with herpes labialis was treated with an olive ail /
water emulsion of he compound 3 (ca. 1 mg/mI). Few hours after local administration on the affected skin portions a total remission was found.
i.3 Treatment of Influenza ~ patient {51, male) with viral influenza was l:reated orally with some drops of a 320/DMSO solution of the compound 3 (ca. 1 ~.g/ml). Over night the disease symptoms lisappeared.
'. Tempersture/Solvent Indicator 'he (Z/E)-stereoisomerism of the compound 3 appears depending on temperature :hermochromism) and depending on solvent (solvatochromism). Therefore, 3 can be red as temperature indicator or/and solvent indicator.
Digital Switch Function ie (Z/C)-stereoisomerism of the compounds 3 and 4 appears depending on applying an ~ctric 'f field (eleclrochromism). Therefore, ~ can be used as di6ital switch etcmcnl :cause of the different iluorcscence spectroscopic properties a6ainst 4 e. i;.
on WO 98I20013 - PC'T/EP9'~/06184 - 15 excitation with laser light of the wavelength 480 nm. Thereby, after applying an electric field on 3 rearrangement to 4 is performed, and it appears fluorescence emission at 575 nm after excitation at 480 nm.
9. Vitamia B6 Antagonism The compound 3 is able to effect in metabolism as antagonist of the coen2ymc pyridoxal 5'-phosphate by binding to enzymes depending on this coenzyme and, therefore, can show tumor suppressing properties.
I0. Proliferation Control The compound 3 can be used in proliferation cont;roUchemotherapy/cytostasis therapy of malignant tumors in local application on skin tumors (malignant melanoma, Kaposi's ~areoma), if necessary, in connection with LJ~~-irradiation, or/and for proliferation control of other skin diseases with increased ee;ll division rate (e. g.
psoriasis). The Binding of compound 3 to replicatively active single-stranded DNA areas can be fixated ~hotochemieally and leads to locally restricted cytotoxieity.
I. Structure Determination 'he compound 3 can be used because of its color and fluorescent properties and its bility to couple to biomolecules, e. g. proteins, for structure determination, e. g. for rhino acid sequence determination of proteins or for DNA sequencing.
2. Detection of Hydrolysis Side Products ie hydrolysis side products which emerge during the synthesis of compound 3 are :amined mass spcctroscopically.
~e concerning compounds are listed in Fig. 2.
Claims (24)
1. Compounds of the general formula (8) wherein:
X1, X2 and X3 are independently selected from O, S and NR5, respectively;
Y is O or S;
Z is CR5 or N;
R1, R2, R3, R4 and R5 are independently selected from any substituents, respectively;
R6 is selected from possibly substituted hydrocarbon derivatives and O, and n is 0 or 1;
also salts, their hydrates and stereoisomers, with the condition that the compound is not pyridoxylidenerhodanine.
X1, X2 and X3 are independently selected from O, S and NR5, respectively;
Y is O or S;
Z is CR5 or N;
R1, R2, R3, R4 and R5 are independently selected from any substituents, respectively;
R6 is selected from possibly substituted hydrocarbon derivatives and O, and n is 0 or 1;
also salts, their hydrates and stereoisomers, with the condition that the compound is not pyridoxylidenerhodanine.
Compounds according to claim 1, wherein X1 is O.
3. Compounds according to the claims 1 or 2, wherein X2 is NR5.
4. Compounds according to one of the previous claims, wherein X3 is S.
5. Compounds according to one of the previous claims, wherein Y is S.
6. Compounds according to one of the previous claims, wherein Z is CR5.
7. Compounds according to one of the previous claims, wherein one of the substituents R1-R4 contains an under physiological conditions negatively charged group.
8. Compounds according to one of the previous claims, choosed from (Z)-5-[[5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-4-pyridinyl]methylene]-2-thioxo-4-thiazolidinone of the formula (3) and also salts, hydrates and stereoisomers of it.
9. Processes for synthesis of a compound of the General formula (8), characterized by reacting a compound of the formula (9) ~n a Knoevenagel condensation with a compound of the formula (10) wherein X1, X2, X3, Y, Z, R1- R4, R5 and R6 are defined like in claim 1.
10. Processes for synthesis of a compound according to claim 8, characterized by reacting pyridoxal 5'-phosphate in a Knoevenagel condensation with rhodanine.
~. Pharmaceutical composition, characterized by ~at it contains as effective agent a compound according to the general formula (8), wherein X1, X2, X3, Y, Z, R1-R4, R5 and R6 are defined as in claim 1, and preferred ~e in one of the claims 2 to 8, and also salts, hydrates and stereoisomers of it, and also, ~ necessary, pharmaceutically usual supplemental, supporting, carrier and diluting substances.
12. Use of a compound according to the general formula (8), wherein X1, X2, X3, Y, Z, R1 - R4, R5 and R6 are defined as in claim 1, and preferred like in one of the claims 2 to 8, and also salts, hydrates and stereoisomers of it, for the preparation of a pharmaceutical composition for the treatment of infectious diseases, tumors and autoimmune diseases.
13. Use according to claim 12 for preparation of an anti-virus drug.
14. Use according to claim 13 for the preparation of a drug for the treatment of viral infections caused by hepatoviruses, herpesviruses or retroviruses.
15. Use of a compound according to the general formula (8), wherein X1, X2, X3, Y, Z, R1 - R4, R5 and R6 are defined as in claim 1, and preferred like in one of the claims 2 to 8, and also salts, hydrates and stereoisomers of it, for the preparation of a pharmaceutical composition for the treatment of diseases which stand in connection with defects of the vitamin B metabolism.
16. Use of a compound according to the general formula (8), wherein X1, X2, X3, Y, Z, R1- R4, R5 and R6 are defined as in claim 1, and preferred like in one of the claims 2 to ~ or salts, hydrates and stereoisomers of it, for the preparation of a pharmaceutical composition for the treatment of diseases which stand in connection with defects of the immune system.
17. Use of a compound according to the general formula (8), wherein X1, X2, X3, Y, Z, R1- R4, R5 and R6 are defined as in claim 1, and preferred like in one of the claims 2 to ~ or salts, hydrates and stereoisomers of it, for the preparation of a pharmaceutical composition for the treatment of diseases which stand in connection with defects of enzymatic reactions.
18. Use according to claim 17 as effector of the enzymatic activity of nitric oxide syntases.
19. Use of compounds according to one of the claims 1 to 8 as detecting agents.
20. Use of compounds according to the general formula (8), wherein X1, X2, X3, Y, Z, R1- R4, R5 and R6 are defined as in claim 1, and preferred like in one of the claims 2 to 8, for the detection of biomolecules.
21. Use according to claim 20 for the detection of single-stranded nucleic acids.
22. Use of compounds according to the general formula (8), wherein X1, X2, X3, Y, Z, R1- R4, R5 and R6 are defined as in claim 1, and preferred like in one of the claims 2 to 8, for electrochromic applications.
23. Conjugates of compounds according to the general formula (8), wherein X1, X2, X3, Y, Z, R1 - R4, R5 and R6 are defined as in claim 1, and preferred like in one of the claims 2 to 8, with biomolecules.
24. Derivatives of compounds of the general formula (8) according to the general formulas (11), (12) or (13):
vherein X1, X2, X3, Y, Z, R1- R4, R5 and R6 are defined as in claim 1.
vherein X1, X2, X3, Y, Z, R1- R4, R5 and R6 are defined as in claim 1.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19645974.5 | 1996-11-07 | ||
| DE19645974A DE19645974C1 (en) | 1996-11-07 | 1996-11-07 | (Z) -5 - [[3-Hydroxy-2-methyl-5 - [(phosphonooxy) methyl] -4-pyridinyl] methylene] -2-thioxo-4-thiazolidinone, process for its preparation and use |
| PCT/EP1997/006184 WO1998020013A1 (en) | 1996-11-07 | 1997-11-07 | New knoevenagel condensation products, method for their production and their use |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2270973A1 true CA2270973A1 (en) | 1998-05-14 |
Family
ID=7810958
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002270973A Withdrawn CA2270973A1 (en) | 1996-11-07 | 1997-11-07 | New knoevenagel condensation products, method for their production and their use |
Country Status (17)
| Country | Link |
|---|---|
| EP (1) | EP0937089A1 (en) |
| JP (1) | JP2001503752A (en) |
| KR (1) | KR20000053141A (en) |
| CN (1) | CN1236369A (en) |
| AU (1) | AU728345B2 (en) |
| BR (1) | BR9712930A (en) |
| CA (1) | CA2270973A1 (en) |
| CZ (1) | CZ160999A3 (en) |
| DE (1) | DE19645974C1 (en) |
| HU (1) | HUP9904289A3 (en) |
| IL (1) | IL129826A0 (en) |
| NO (1) | NO992209L (en) |
| NZ (1) | NZ335977A (en) |
| PL (1) | PL333128A1 (en) |
| SK (1) | SK61799A3 (en) |
| TR (1) | TR199901733T2 (en) |
| WO (1) | WO1998020013A1 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2330811A1 (en) | 1998-05-04 | 1999-11-11 | Andreas Johannes Kesel | Monomeric, oligomeric and polymeric knoevenagel condensation products |
| ID27787A (en) * | 1998-08-21 | 2001-04-26 | Viro Pharma Inc | COMPOUND, COMPOSITION AND METHODS FOR TREATMENT OR PREVENTION OF INFECTIONS CAUSED BY VIRUSES AND RELATED DISEASES |
| CA2371653A1 (en) * | 1999-04-30 | 2000-11-09 | Walter Oberthur | Antioxidative vitamin b6 analogs |
| US20030071780A1 (en) * | 2001-10-16 | 2003-04-17 | Vincent Kent D. | High resolution display |
| US6940497B2 (en) | 2001-10-16 | 2005-09-06 | Hewlett-Packard Development Company, L.P. | Portable electronic reading apparatus |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SE9103776D0 (en) * | 1991-12-19 | 1991-12-19 | Astra Ab | NEW COMPOUNDS |
| IT1266570B1 (en) * | 1993-07-30 | 1997-01-09 | Zambon Spa | DERIVATIVES OF PROPANAMIDE N-ETEROARYL SUBSTITUTES USEFUL IN THE TREATMENT OF CARDIOVASCULAR DISEASES |
| FR2722790B1 (en) * | 1994-07-19 | 1996-10-04 | Roussel Uclaf | NOVEL CEPHALOSPORINS COMPRISING IN POSITION 7 A SUBSTITUTED RADICAL BENZYLOXYMINO, THEIR PREPARATION PROCESS AND THEIR APPLICATION AS MEDICAMENTS |
-
1996
- 1996-11-07 DE DE19645974A patent/DE19645974C1/en not_active Expired - Fee Related
-
1997
- 1997-11-07 CN CN97199496A patent/CN1236369A/en active Pending
- 1997-11-07 CA CA002270973A patent/CA2270973A1/en not_active Withdrawn
- 1997-11-07 EP EP97951145A patent/EP0937089A1/en not_active Withdrawn
- 1997-11-07 IL IL12982697A patent/IL129826A0/en unknown
- 1997-11-07 TR TR1999/01733T patent/TR199901733T2/en unknown
- 1997-11-07 SK SK617-99A patent/SK61799A3/en unknown
- 1997-11-07 BR BRPI9712930-5A patent/BR9712930A/en not_active IP Right Cessation
- 1997-11-07 JP JP52107198A patent/JP2001503752A/en active Pending
- 1997-11-07 NZ NZ335977A patent/NZ335977A/en unknown
- 1997-11-07 AU AU54798/98A patent/AU728345B2/en not_active Revoked
- 1997-11-07 WO PCT/EP1997/006184 patent/WO1998020013A1/en not_active Application Discontinuation
- 1997-11-07 PL PL97333128A patent/PL333128A1/en unknown
- 1997-11-07 KR KR1019990704073A patent/KR20000053141A/en not_active Application Discontinuation
- 1997-11-07 CZ CZ991609A patent/CZ160999A3/en unknown
- 1997-11-07 HU HU9904289A patent/HUP9904289A3/en unknown
-
1999
- 1999-05-06 NO NO992209A patent/NO992209L/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| NO992209L (en) | 1999-07-01 |
| DE19645974C1 (en) | 1998-08-13 |
| JP2001503752A (en) | 2001-03-21 |
| KR20000053141A (en) | 2000-08-25 |
| EP0937089A1 (en) | 1999-08-25 |
| HUP9904289A3 (en) | 2001-08-28 |
| NZ335977A (en) | 2000-12-22 |
| NO992209D0 (en) | 1999-05-06 |
| WO1998020013A1 (en) | 1998-05-14 |
| AU728345B2 (en) | 2001-01-04 |
| BR9712930A (en) | 2006-04-18 |
| SK61799A3 (en) | 2000-01-18 |
| PL333128A1 (en) | 1999-11-22 |
| CZ160999A3 (en) | 1999-09-15 |
| HUP9904289A2 (en) | 2001-04-28 |
| IL129826A0 (en) | 2000-02-29 |
| CN1236369A (en) | 1999-11-24 |
| AU5479898A (en) | 1998-05-29 |
| TR199901733T2 (en) | 1999-09-21 |
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