Use of 10H-benzo[g]pteridine-2,4-dione derivatives

Abstract

Use of 10H-benzo[g]pteridine-2,4-dione derivatives as photosensitizers in the inactivation of microorganisms.

Claims

1. A method for inactivating microorganisms located on or within a subject in need of such inactivation, said method comprising: (i) applying upon said subject a compound having the formula (1): ##STR00022## as a photosensitizer in the inactivation of microorganisms, where A) only 1 R1, R2, R3 or R4 radical is an organic radical of the general formula (2), (3) or (4): ##STR00023## where h is an integer from 1 to 20 and k and l are each independently an integer from 0 to 6, where D and E are each independently hydrogen, halogen, hydroxyl, O—R .sup.(VIII) where R.sup.(VIII) is methyl, ethyl, n-propyl, n-butyl, phenyl or benzyl, O—C (═O)—R.sup.(IX) where R.sup.(IX) is hydrogen, methyl, ethyl, n-propyl, n-butyl, phenyl or benzyl, or thiol, and where X is an organic radical having a) only one uncharged, protonatable nitrogen atom, or b) only one positively charged nitrogen atom, and aryl is a substituted or unsubstituted aromatic system having 5 to 20 carbon atoms or a substituted or unsubstituted heteroaromatic system which does not contain any nitrogen atom and has 4 to 20 carbon atoms, and where the radical having the formula (4) is a heteroaromatic system which is bonded to the isoalloxazine ring via a carbon atom of the heteroaromatic system and which contains a) only one uncharged, protonatable nitrogen atom or b) only one positively charged nitrogen atom or where the radical having the formula (4) is formed from compounds having 5 to 7 ring atoms, these compounds being selected from the group consisting of pyrrolidine, pyrrole, oxazole, oxazoline, oxazolidine, isoxazole, isoxazoline, isoxazolidine, thiazole, thiazoline, thiazolidine, isothiazole, isothiazoline, isothiazolidine, piperidine, pyridine, oxazines, dihydrooxazines and tetrahydrooxazines, where the aforementioned compounds are unsubstituted or may be substituted by at least one radical selected from the group consisting of halogen, phenyl, benzyl, straight-chain and branched alkyl having 1 to 20 carbon atoms and hydroxyl, and where the R1, R2, R3 or R4 radicals which are not an organic radical of the general formula (2), (3) or (4) are the same or different and are each independently hydrogen, halogen, hydroxyl, nitro, carboxylate, aldehyde having 1 to 20 carbon atoms, ketone having 2 to 20 carbon atoms, thiol, O-alkyl having 1 to 20 carbon atoms, S-alkyl having 1 to 20 carbon atoms, O-alkenyl having 2 to 20 carbon atoms, S-alkenyl having 2 to 20 carbon atoms, O-aryl having 5 to 20 carbon atoms, S-aryl having 5 to 20 carbon atoms, ether having 2 to 20 carbon atoms, thioether having 2 to 20 carbon atoms, carboxylic ester having 1 to 20 carbon atoms, carboxamide having 1 to 20 carbon atoms, thioester having 1 to 20 carbon atoms, alkyl having 1 to 20 carbon atoms, alkenyl having 2 to 20 carbon atoms, cycloalkyl having 3 to 20 carbon atoms, cycloalkenyl having 3 to 20 carbon atoms, aryl having 5 to 20 carbon atoms or heteroaryl which does not contain any nitrogen atom and has 4 to 20 carbon atoms, and where each of the R5 or R6 radicals is the same or different and is independently hydrogen, alkyl having 2 to 20 carbon atoms, alkenyl having 2 to 20 carbon atoms, ether having 2 to 20 carbon atoms, thioether having 2 to 20 carbon atoms, cycloalkyl having 3 to 20 carbon atoms, cycloalkenyl having 3 to 20 carbon atoms, aryl having 5 to 20 carbon atoms or heteroaryl which does not contain any nitrogen atom and has 4 to 20 carbon atoms, or where B) only 1 R5 or R6 radical is an organic radical of the general formula (2), (3) or (4): ##STR00024## where h is an integer from 1 to 20 and k and l are each independently an integer from 0 to 6, where D and E are each independently hydrogen, halogen, hydroxyl, O—R.sup.(VIII) where R.sup.(VIII) is methyl, ethyl, n-propyl, n-butyl, phenyl or benzyl, O—C (═O)—R.sup.(IX) where R.sup.(IX) is hydrogen, methyl, ethyl, n-propyl, n-butyl, phenyl or benzyl, or thiol, and where X is an organic radical having a) only one uncharged, protonatable nitrogen atom, or b) only one positively charged nitrogen atom, and aryl is a substituted or unsubstituted aromatic system having 5 to 20 carbon atoms or a substituted or unsubstituted heteroaromatic system which does not contain any nitrogen atom and has 4 to 20 carbon atoms, and where the radical having the formula (4) is a heteroaromatic system which is bonded to the isoalloxazine ring via a carbon atom of the heteroaromatic system and which contains a) only one uncharged, protonatable nitrogen atom or b) only one positively charged nitrogen atom or where the radical having the formula (4) is formed from compounds having 5 to 7 ring atoms, these compounds being selected from the group consisting of pyrrolidine, pyrrole, oxazole, oxazoline, oxazolidine, isoxazole, isoxazoline, isoxazolidine, thiazole, thiazoline, thiazolidine, isothiazole, isothiazoline, isothiazolidine, piperidine, pyridine, oxazines, dihydrooxazines and tetrahydrooxazines, where the aforementioned compounds are unsubstituted or may be substituted by at least one radical selected from the group consisting of halogen, phenyl, benzyl, straight-chain and branched alkyl having 1 to 20 carbon atoms and hydroxyl, and where the R5 or R6 radical which is not an organic radical of the general formula (2), (3) or (4) is hydrogen, alkyl having 1 to 20 carbon atoms, alkenyl having 2 to 20 carbon atoms, ether having 2 to 20 carbon atoms, thioether having 2 to 20 carbon atoms, cycloalkyl having 3 to 20 carbon atoms, cycloalkenyl having 3 to 20 carbon atoms, aryl having 5 to 20 carbon atoms or heteroaryl which does not contain any nitrogen atom and has 4 to 20 carbon atoms, and where the R1 to R4 radicals are the same or different and are each independently hydrogen, halogen, hydroxyl, thiol, nitro, carboxylate, aldehyde having 1 to 20 carbon atoms, ketone having 1 to 20 carbon atoms, O-alkyl having 1 to 20 carbon atoms, S-alkyl having 1 to 20 carbon atoms, O-alkenyl having 2 to 20 carbon atoms, S-alkenyl having 2 to 20 carbon atoms, O-aryl having 5 to 20 carbon atoms, S-aryl having 5 to 20 carbon atoms, ether having 2 to 20 carbon atoms, thioether having 2 to 20 carbon atoms, carboxylic ester having 1 to 20 carbon atoms, carboxamide having 1 to 20 carbon atoms, thioester having 1 to 20 carbon atoms, alkyl having 1 to 20 carbon atoms, alkenyl having 2 to 20 carbon atoms, cycloalkyl having 3 to 20 carbon atoms, cycloalkenyl having 3 to 20 carbon atoms, aryl having 5 to 20 carbon atoms or heteroaryl which does not have any nitrogen atom and has 4 to 20 carbon atoms; wherein X is a radical of formula (5): ##STR00025## and where A is an oxygen or sulfur atom and where n is an integer from 1 to 8 and m is an integer from 0 to 100, and where B is a radical of formula (6a), (6b), (7) or (8): ##STR00026## and where each of the R.sup.(I) and R.sup.(II) radicals is independently selected from hydrogen and C1 -C20 alkyl which may be straight-chain or branched, and where R.sup.(III) is hydrogen and where the radical having the formula (8) ##STR00027## is a substituted or unsubstituted heterocyclic radical having 5 to 7 ring atoms including at least one 1 carbon atom and 1 nitrogen atom and optionally 1 or 2 oxygen or sulfur atoms, where the heterocyclic radical is saturated or unsaturated; and (ii) wherein, following its application, the compound having the formula (I) is irradiated with electromagnetic radiation having a wavelength in a range of from 280 nm to 1000 nm in order to produce reactive oxygen radicals, singlet oxygen or a combination thereof.

2. The method as claimed in claim 1, where the radical of the formula (4): ##STR00028## is selected from the group consisting of radicals of formulae (13a), (13b) and (13c): ##STR00029##

3. The method as claimed in claim 1, where the compound having the formula (1) is selected from compounds having the formulae (14) to (17): ##STR00030##

4. The method as claimed in claim 1, where at least one compound of the formula (1) or a pharmacologically acceptable salt and/or ester and/or complex thereof is present in a pharmaceutical composition together with at least one pharmacologically acceptable excipient.

5. The method of claim 1 wherein said method is a photodynamic therapy.

6. The method as claimed in claim 1, where the microorganisms are selected from the group consisting of viruses, archaea, bacteria, bacterial spores, fungi, fungal spores, protozoa, algae and blood-transmissible parasites.

7. The method as claimed in claim 1, wherein said photosensitizer compound is applied upon said subject in the cleaning of teeth, dentures and/or dental braces and/or treatment of a disorder of the dental tissue and/or of the periodontium.

8. The method as claimed in claim 1, wherein said photosensitizer compound is applied upon said subject in treatment of an infectious skin disease.

9. The method as claimed in claim 1, wherein said halogen is selected from the group consisting of chlorine, bromine, iodine and fluorine.

10. The method as claimed in claim 1, wherein the radical having the formula (4) is formed from a compound selected from the group consisting of morpholine, thiazine, azepines, azepane, and thiazepines.

11. The method as claimed in claim 1, wherein the halogen is selected from the group consisting of chlorine bromine, iodine and fluorine.

12. The method as claimed in claim 1, wherein the electromagnetic radiation has an energy density in a range from 1 μW/cm.sup.2 to 1 kW/cm.sup.2.

Description

(1) FIG. 1 shows the effect of flavin FL-01 (chloride of the compound having the formula (14)) on E. coli and S. aureus.

(2) FIG. 1a: E. coli samples were incubated with various concentrations (0 μM, 1 μM, 10 μM, 50 μM, 100 μM) of flavin FL-01 for 10 min. Subsequently, the samples were either irradiated at 50 mW/cm.sup.2 (210 sec; 10.5 J/cm.sup.2) (dark gray bar) or not irradiated (light gray bar). After 24 h, the surviving colonies were counted (CFU/ml). The black line indicates the dark control reference (0 μM flavin FL-01, no light). The dotted line marked “99.9% killed” indicates a decrease in the CFU/ml by 3 log.sub.10 stages; this corresponds to a decrease by 99.9% (“antibacterial action”). N=3; median CFU/ml±SEM.

(3) FIG. 1b: S. aureus samples were incubated with various concentrations (0 μM, 1 μM, 10 μM, 50 μM, 100 μM) of flavin FL-01 for 10 min. Subsequently, the samples were either irradiated at 50 mW/cm.sup.2 (210 sec; 10.5 J/cm.sup.2) (dark gray bar) or not irradiated (light gray bar). After 24 h, the surviving colonies were counted (CFU/ml). The black line indicates the dark control reference (0 μM flavin FL-01, no light). The dotted line marked “99.9% killed” indicates a decrease in the CFU/ml by 3 log.sub.10 stages; this corresponds to a decrease by 99.9% (“antibacterial action”). N=3; median CFU/ml±SEM.

(4) FIG. 2 shows the effect of flavin FL-03 (chloride of the compound having the formula (16)) on E. coli and S. aureus.

(5) FIG. 2a: E. coli samples were incubated with various concentrations (0 μM, 1 μM, 10 μM, 50 μM, 100 μM) of flavin FL-03 for 10 min. Subsequently, the samples were either irradiated at 50 mW/cm.sup.2 (210 sec; 10.5 J/cm.sup.2) (dark gray bar) or not Irradiated 25 (white bar). After 24 h, the surviving colonies were counted (CFU/ml). The black line indicates the dark control reference (0 μM flavin FL-03, no light). The dotted line marked “99.9% killed” Indicates a decrease in the CFU/ml by 3 log.sub.10 stages; this corresponds to a decrease by 99.9% (“antibacterial action”). N=3; median CFU/ml±SEM.

(6) FIG. 2b: S. aureus samples were incubated with various concentrations (0 μM, 1 μM, 10 μM, 100 μM) of flavin FL-03 for 10 min. Subsequently, the samples were either irradiated at 50 mW/cm.sup.2 (210 sec; 10.5 J/cm.sup.2) (dark gray bar) or not irradiated (white bar). After 24 h, the surviving colonies were counted (CFU/ml). The black line indicates the dark control reference (0 μM flavin FL-03, no light). The dotted line marked “99.9% killed” indicates a decrease in the CFU/ml by 3 log.sub.10 stages; this corresponds to a decrease by 99.9% (“antibacterial action”). N=3; median CFU/ml±SEM.

(7) FIG. 2c: E. coli samples were incubated with various concentrations (0 μM, 1 μM, 10 μM, 100 μM) of flavin FL-03 for 30 min. Subsequently, the samples were either irradiated at 50 mW/cm.sup.2 (210 sec; 10.5 J/cm.sup.2) (dark gray bar) or not irradiated (white bar). After 24 h, the surviving colonies were counted (CFU/ml). The black line indicates the dark control reference (0 μM flavin FL-03, no light). The dotted line marked “99.9% killed” indicates a decrease in the CFU/ml by 3 log.sub.10 stages; this corresponds to a decrease by 99.9% (“antibacterial action”). N=3; median CFU/ml±SEM.

(8) FIG. 2d: S. aureus samples were incubated with various concentrations (0 μM, 1 μM, 10 μM, 100 μM) of flavin FL-03 for 30 min. Subsequently, the samples were either irradiated at 50 mW/cm.sup.2 (210 sec; 10.5 J/cm.sup.2) (dark gray bar) or not irradiated (white bar). After 24 h, the surviving colonies were counted (CFU/ml). The black line indicates the dark control reference (0 μM flavin FL-03, no light). The dotted line marked “99.9% killed” indicates a decrease in the CFU/ml by 3 log.sub.10 stages; this corresponds to a decrease by 99.9% (“antibacterial action”). N=3; median CFU/ml±SEM.

(9) FIG. 3 shows the effect of flavin FL-04 (chloride of the compound having the formula (17)) on E. coli and S. aureus.

(10) FIG. 3a: E. coli samples were incubated with various concentrations (0 μM, 1 μM, 10 μM, 50 μM, 100 μM) of flavin FL-04 for 30 min. Subsequently, the samples were either irradiated at 50 mW/cm.sup.2 (210 sec; 10.5 J/cm.sup.2) (dark gray bar) or not irradiated (light gray bar). After 24 h, the surviving colonies were counted (CFU/ml). The black line indicates the dark control reference (0 μM flavin FL-04, no light). The dotted line marked “99.9% killed” indicates a decrease in the CFU/ml by 3 log.sub.10 stages; this corresponds to a decrease by 99.9% (“antibacterial action”). N=3; median CFU/ml±SEM.

(11) FIG. 3b: S. aureus samples were incubated with various concentrations (0 μM, 1 μM, 10 μM, 50 μM, 100 μM) of flavin FL-04 for 30 min. Subsequently, the samples were either Irradiated at 50 mW/cm.sup.2 (210 sec; 10.5 J/cm.sup.2) (dark gray bar) or not irradiated (light gray bar). After 24 h, the surviving colonies were counted (CFU/ml). The black line indicates the dark control reference (0 μM flavin FL-04, no light). The dotted line marked “99.9% killed” Indicates a decrease in the CFU/ml by 3 log.sub.10 stages; this corresponds to a decrease by 99.9% (“antibacterial action”). N=3; median CFU/ml±SEM.

(12) FIG. 4 shows the effect of flavin FL-05 (chloride of the compound having the formula (15)) on E. coli and S. aureus.

(13) FIG. 4a: E. coli samples were incubated with various concentrations (0 μM, 10 μM, 100 μM) of flavin FL-05 for 10 min. Subsequently, the samples were either Irradiated at 50 mW/cm.sup.2 (210 sec; 10.5 J/cm.sup.2) (dark gray bar) or not irradiated (light gray bar). After 24 h, the surviving colonies were counted (CFU/ml). The black line indicates the dark control reference (0 μM flavin FL-05, no light). The dotted line marked “99.9% killed” Indicates a decrease in the CFU/ml by 3 log.sub.10 stages; this corresponds to a decrease by 99.9% (“antibacterial action”). N=3; median CFU/ml±SEM.

(14) FIG. 4b: S. aureus samples were incubated with various concentrations (0 μM, 10 μM, 100 μM) of flavin FL-05 for 10 min. Subsequently, the samples were either irradiated at 50 mW/cm.sup.2 (210 sec; 10.5 J/cm.sup.2) (dark gray bar) or not irradiated (light gray bar). After 24 h, the surviving colonies were counted (CFU/ml). The black line indicates the dark control reference (0 μM flavin FL-05, no light). The dotted line marked “99.9% killed” Indicates a decrease in the CFU/ml by 3 log.sub.10 stages; this corresponds to a decrease by 99.9% (“antibacterial action”). N=3; median CFU/ml±SEM.

(15) As apparent from FIGS. 1-4, irradiation of the microorganisms used Staphylococcus aureus (S. aureus) and Escherichla coli (E. coli) at a light dose of 10.5 J/cm.sup.2 with blue light (390 nm-500 nm) in the absence of a photosensitizer (0 μM of the respective flavin) has no influence on the number of surviving microorganisms in comparison to the unexposed control.

(16) In addition, the results shown in FIGS. 1-4 show that the incubation (10 min or 30 min) of the respective photosensitizer with the microorganisms without subsequent exposure likewise has no influence on the number of surviving microorganisms.

(17) As apparent from FIGS. 1-4, there is a decrease in the CFU/ml and hence inactivation of E. coli and S. aureus after incubation (10 min or 30 min) of the microorganisms as a function of the concentration used of the respective photosensitizers, and subsequent irradiation with a light dose of 10.5 J/cm.sup.2.

(18) The effectiveness of phototoxicity with respect to bacteria after irradiation was defined according to the following guidelines for hand hygiene in the health sector (Boyce, J. M., and D. Pittet. 2002. Guideline for Hand Hygiene in Health-Care Settings: recommendations of the Healthcare Infection Control Practices Advisory Committee and the HICPAC/SHEA/APIC/IDSA Hand Hygiene Task Force. Infect Control Hosp Epidemiol 23: p. 3-40):

(19) TABLE-US-00001 reduction in the CFU/ml by 1 log.sub.10 stage  90% effectiveness reduction in the CFU/ml by 3 log.sub.10 stages  99.9% effectiveness reduction in the CFU/ml by 5 log.sub.10 stages  99.999% effectiveness

(20) For effective inactivation, the decrease of ≧3 log.sub.10 stages can therefore be adopted, and S. aureus and E. coli were chosen as examples of representatives from the group of the Gram-positive and Gram-negative bacteria (see Boyce J. M and D. Pittet 2009).

(21) The concentration required to achieve a reduction by ≧3 log.sub.10 stages is shown in table 1.

(22) TABLE-US-00002 TABLE 1 Summary of photodynamic inactivation Required concentration [μM] to achieve a reduction by ≧3 log.sub.10 stages (decrease by 99.9%), irradiation at 10.5 J/cm.sup.2 Photosensitizer E. coli S. aureus FL-01 .sup. 10 10 FL-03 >100.sup.(*.sup.) 10 FL-04 >100.sup.(*.sup.) 50 FL-05 >100.sup.(*.sup.) 100 .sup.(*.sup.)to date, only a reduction of less than 3 log.sub.10 stages has been achieved (99%), at a concentration of 100 μM with subsequent irradiation