Iridium complex having anti-p. acnes efficacy, and preparation method and use thereof

Abstract

The application belongs to the technical field of metal complex photosensitizer drugs, and discloses an iridium complex with anti-propionibacterium acnes efficacy, a preparation method and application thereof. The iridium complex is an iridium complex formed by a cationic compound with the structure shown in the following formula (I) and anions. The iridium complex provided by the application can achieve the anti-propionibacterium acnes effect at a small drug concentration, and plays a role on propionibacterium acnes through the synergistic effect of dark toxicity and photodynamic effect; the iridium complex can be applied in propionibacterium acnes imaging through its fluorescence.

Description

Technical Field

[0001] This invention belongs to the field of metal complex photosensitizer pharmaceutical technology, and specifically relates to an iridium complex with anti-acne Propionibacterium acnes efficacy, its preparation method and uses. Background Technology

[0002] Acne vulgaris is a chronic inflammatory disease, not a natural part of the hair follicle life cycle as commonly believed, and is one of the most common skin diseases worldwide. Key mechanisms of acne development include alterations in sebum fatty acid composition, hormonal microenvironment dysregulation, excessive keratinization of the hair follicle, and the presence of Propionibacterium acnes (…). C. acnes The infection and subsequent inflammation of *Propionibacterium acnes* are caused by the bacteria. Studies have shown a significant correlation between high colonization of *Propionibacterium acnes* and the severity of acne (mild, moderate, severe). Therefore, acne patients need on-demand treatment targeting both the bacteria and the inflammation. Although various antibiotics and other treatments (such as erythromycin, doxycycline, isotretinoin, benzoyl peroxide, and spironolactone) have been explored to treat acne, these traditional drugs are prone to inducing drug resistance in *Propionibacterium acnes* and may produce harmful side effects such as nausea, vomiting, teratogenicity, and skin irritation.

[0003] Photodynamic therapy (PDT) has garnered significant attention in recent years as a potential treatment for bacterial-associated diseases. It generates cytotoxic reactive oxygen species (ROS) through light irradiation, which can disrupt biofilms and kill both negatively and positively resistant bacteria without inducing drug resistance. Transition metal complex photosensitizers, such as ruthenium (Ru) complexes, platinum (Pt) complexes, and iridium (Ir) complexes, have shown particular interest. Irdium complexes, in particular, possess advantages such as long fluorescence lifetime, good chemical stability, and tunable photophysical properties, making them suitable for designing various highly sensitive PDT photosensitizers. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the existing technology, the primary objective of this invention is to provide an iridium complex with anti-acne Propionibacterium acnes effect.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned iridium complex with anti-acne Propionibacterium acnes effect.

[0006] Another object of the present invention is to provide the application of the above-mentioned iridium complex with anti-acne Propionibacterium acnes effect.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] An iridium complex with anti-Propionibacterium acnes effect, wherein the iridium complex is an iridium complex formed by a cationic compound with the structure shown in formula (I) and an anion;

[0009]

[0010] (I).

[0011] The anion is an inorganic salt anion, specifically ClO4. - Cl - or PF6 - Cl is preferred - or ClO4 - Cl is preferred. - .

[0012] The preparation method of the above-mentioned iridium complex with anti-Propionibacterium acnes effect includes the following steps:

[0013] (1) Weigh 0.75 mmol of 1,10-phenanthroline-5,6-dione, 0.75 mmol of aniline, 0.75 mmol of 6-hydroxy-2-naphthal and 30 mL of glacial acetic acid and mix them in a three-necked flask. Heat to 120°C under argon protection and react for 3-5 h. Then transfer the reaction solution to a container containing deionized water and neutralize the reaction solution with low concentration ammonia water until a yellow precipitate appears. Filter and collect the precipitate. Wash the precipitate with water and ether in sequence to obtain the crude product.

[0014] (2) Dissolve the crude product obtained in step (1) in ethanol, then pack it into a column with 60-100 mesh silica gel, elute with ethanol as the eluent, collect the yellow component, and rotary evaporate to obtain intermediate product 1- PHIPN ;

[0015] (3) Weigh 0.079 mmol of [Ir(pq)2Cl2]·2H2O and 0.158 mmol of the intermediate product 1- obtained in step (2). PHIPN The mixture was stirred, and a 3:1 (v / v) mixture of dichloromethane and methanol was added as the reaction solvent. The mixture was heated to 65°C under argon protection and refluxed for 6–9 h. After cooling to 20–40°C, a saturated solution of anionic salt was added dropwise to produce a precipitate. The precipitate was filtered, evaporated to dryness, and purified by neutral alumina column chromatography. The precipitate was then eluted with a 10:1 (v / v) mixture of acetonitrile and ethanol and dried under vacuum to obtain the iridium complex with anti-Propionibacterium acnes activity.

[0016] The low-concentration ammonia water in step (1) is ammonia water with a concentration of 25wt%; the anionic salt in step (2) is an inorganic salt, specifically a chloride salt, perchlorate or hexafluorophosphate, preferably a chloride salt or perchlorate, and more preferably a chloride salt.

[0017] The synthesis of [Ir(pq)2Cl2]·2H2O in step (2) was carried out according to the following steps: 1.1 mmol IrCl3·3H2O and 2.75 mmol 2-phenylquinoline were weighed and placed in a three-necked flask, and 40 mL of a mixture of ethylene glycol ethyl ether and water with a volume ratio of 3:1 was added. The mixture was heated to 120 °C under argon protection and reacted for 24 h. After cooling to room temperature, distilled water was added and the mixture was filtered. The solid precipitate was collected, dried, and [Ir(pq)2Cl2]·2H2O was obtained.

[0018] The above-mentioned iridium complexes are used in the preparation of drugs against Propionibacterium acnes.

[0019] The above-mentioned iridium complexes are used in the preparation of acne-removing cosmetics.

[0020] The present invention has the following advantages and beneficial effects compared with the prior art:

[0021] (1) The iridium complex provided by the present invention can achieve the effect of anti-Propionibacterium acnes at a relatively low drug concentration.

[0022] (2) The iridium complex provided by the present invention can be used in the imaging of Propionibacterium acnes through its own fluorescence.

[0023] (3) The iridium complex provided by the present invention can exert its effect on Propionibacterium acnes through the synergistic effect of its own dark toxicity and photodynamics. Attached Figure Description

[0024] Figure 1 This is the mass spectrum of the iridium complex in Example 1 of the present invention;

[0025] Figure 2 This is the proton NMR spectrum of the iridium complex in Example 1 of this invention;

[0026] Figure 3 This is the detection of reactive oxygen species (ROS) and singlet oxygen in iridium complexes in Example 2 of the present invention. 1 The results of O2 production are shown in the diagram, with the left diagram showing the results of reactive oxygen species and the right diagram showing the results of singlet oxygen species.

[0027] Figure 4 This is a laser scanning confocal microscope image of the iridium complex against Propionibacterium acnes in Example 4 of the present invention;

[0028] Figure 5 This is a diagram showing the results of the laser scanning confocal microscopy (LSCM) staining experiment on the iridium complex against Propionibacterium acnes in Example 5 of this invention.

[0029] Figure 6 This is a scanning electron microscope (SEM) image showing the results of the iridium complex against Propionibacterium acnes in Example 6 of this invention. Detailed Implementation

[0030] The following specific embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention.

[0031] Example 1: Synthesis of Iridium Complexes

[0032] (1) Synthesis of [Ir(pq)2Cl2]·2H2O (Reference: S. Lamansky, P. Djurovich, D. Murphy, F. Abdel-Razzaq, R. Kwong, I. Tsyba, M. Bortz, B. Mui, R. Bau, METhompson Inorg Chem. 2001) 40 (7), 1704-1711), the specific steps are as follows: Weigh 1.1 mmol IrCl3·3H2O and 2.75 mmol 2-phenylquinoline into a three-necked flask, add 40 mL of a mixture of ethylene glycol ethyl ether and water with a volume ratio of 3:1, heat to 120 °C under argon protection, react for 24 h, cool to room temperature, add distilled water and filter, collect the solid precipitate, dry, and obtain [Ir(pq)2Cl2]·2H2O;

[0033] (2) 1- PHIPN Synthetic method: Weigh 0.75 mmol of 1,10-phenanthroline-5,6-dione, 0.75 mmol of aniline, 0.75 mmol of 6-hydroxy-2-naphthal, and 30 mL of glacial acetic acid and mix them in a three-necked flask. Heat to 120℃ under argon protection and react for 4 h. Transfer the reaction solution to a container containing deionized water and neutralize the reaction solution with a low concentration of 25 wt% ammonia water until a yellow precipitate appears. Filter and collect the precipitate. Wash the precipitate with water and ether sequentially to obtain the crude product. Dissolve the crude product in ethanol and then pack it into an 80-mesh silica gel column. Elute with ethanol as the eluent and collect the yellow component. Rotary evaporation yields the intermediate yellow powder 4-(1-(phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline-2-yl)naphthalene(4 - (1-phenyl-1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)naphthalen -2-ol ligands 1- PHIPN );

[0034] The resulting compound ligand 1-PHIPN Elemental analysis theoretical calculation value: C 29 H 18N4O: C, 79.44; H, 4.14; N, 12.78, elemental analysis experimental measurements: C, 79.40; H, 4.36; N, 12.68.

[0035] The resulting compound ligand 1-PHIPN The mass spectrometry (FAB-MS) of the sample is: m / z = 439 (M+1).

[0036] (3) [Ir(pq)2( 1-PHIPN )] + Synthesis method:

[0037] Weigh 0.079 mmol of [Ir(pq)2Cl2]·2H2O obtained in step (1) and 0.158 mmol of ligand 1 obtained in step (2). PHIPN The product was added to a three-necked flask, followed by a 3:1 (v / v) mixture of dichloromethane and methanol as the reaction solvent. The mixture was heated to 65°C under argon protection and refluxed for 9 h. After cooling to 30°C, a saturated chloride solution was added dropwise to produce a precipitate. The precipitate was filtered, evaporated to dryness, and purified using a neutral alumina column. Elution was performed using a 10:1 (v / v) mixture of acetonitrile and ethanol. The final product, [Ir(pq)2( 1-PHIPN Cl, which is the iridium complex of the present invention that has the effect of fighting Propionibacterium acnes.

[0038] The obtained compound [Ir(pq)2( 1-PHIPN Elemental analysis calculation of Cl: C 59 H 38 IrN6OCl: C, 65.94%; H, 3.56%; N, 7.82%; Elemental analysis results: C, 65.86%; H, 3.64%; N, 7.76%.

[0039] The obtained compound [Ir(pq)2( 1-PHIPN The mass spectra (ES-MS) of Cl are: [CH3CN, m / z]: 1039 ([M–Cl] + )(like Figure 1 (As shown). The proton spectrum is: 1H NMR (400 MHz, DMSO) δ 10.14 (s, 1H), 9.21 (d, J =8.2 Hz, 1H), 8.57 (dt, J = 16.3, 9.0 Hz, 6H), 8.36 (dd, J = 12.8, 7.1 Hz,3H), 8.16 (dd, J = 8.1, 5.3 Hz, 1H), 7.88 – 7.68 (m, 9H), 7.63 – 7.56 (m,2H), 7.47 (dd, J = 15.1, 8.6 Hz, 2H), 7.29 (dd, J = 12.2, 7.1 Hz, 2H), 7.21(t, J = 9.7 Hz, 3H), 7.11 (d, J = 8.8 Hz, 2H), 6.93 – 6.83 (m, 4H), 6.50 (t,J = 7.5 Hz, 2H). (e.g. Figure 2 (As shown).

[0040] The above structural characterization data prove that the compound [Ir(pq)2( 1-PHIPN Cl has the structure shown in the following formula:

[0041] .

[0042] Example 2: Detection of reactive oxygen species (ROS) and singlet oxygen in iridium complexes. 1 O2)

[0043] 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) (1 mM) was hydrolyzed with sodium hydroxide (0.01 mM) at room temperature to obtain a dichlorofluorescein (DCF) solution, which was then neutralized with PBS. An equal volume of the iridium complex solution (10 μM) obtained in Example 1 and the hydrolyzed dichlorofluorescein (DCF) solution was transferred to a 96-well plate. After irradiation at 405 nm, the green fluorescence produced by the oxidative cleavage of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) into dichlorofluorescein (DCF) was measured every 4 s using a microplate reader with Ex = 488 nm and Em = 525 nm. Equal volumes of a singlet oxygen fluorescent probe (SOSG) solution (5 μM) and an iridium complex solution obtained in Example 1 (10 μM) were mixed and transferred to a 96-well plate. After irradiation at 405 nm, the fluorescence generated by the singlet oxygen fluorescent probe (SOSG) was measured every 4 seconds using a microplate reader with Ex = 488 nm and Em = 525 nm. Figure 3As shown in the left figure, under 405 nm illumination, the fluorescence of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) at 525 nm gradually increases with prolonged illumination time, indicating a continuous increase in reactive oxygen species (ROS). Figure 3 As shown in the right figure, at the same wavelength, the fluorescence of the singlet oxygen fluorescent probe (SOSG) also increases with the extension of illumination time, indicating that the production of singlet oxygen (¹O2) increases, suggesting that both reactive oxygen species (ROS) and singlet oxygen (¹O2) are phototime dependent.

[0044] Example 3: Minimum inhibitory concentration (MIC) of iridium complexes against Gram-positive bacteria, Gram-negative bacteria, and fungi.

[0045] Suspensions of several Gram-positive bacteria (Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis, and Propionibacterium acnes), Gram-negative bacteria (Escherichia coli, Proteus mirabilis, Salmonella typhimurium, and Acinetobacter baumannii), and fungi (Candida albicans and Candida parapsilosis) were diluted to 10⁻⁶ using lysozyme broth, modified GAM broth, and Sabouraud dextrose broth. 5 The solution of the iridium complex obtained in Example 1 was diluted from 256 µM to 0.25 µM by half-dilution (CFU / mL); similarly, the vancomycin solution was diluted from 256 µM to 0.25 µM by half-dilution (CFU / mL). The diluted bacterial suspension was mixed with equal volumes of the diluted iridium complex and vancomycin solution in 96-well plates and incubated at 37°C for 18 hours as the sample group. Wells containing only culture medium served as negative controls, and the diluted bacterial suspension (not mixed with the iridium complex solution) served as positive controls, thus determining the minimum dose required to inhibit bacterial growth. The results are shown in Table 1. The iridium complex of this invention exhibited excellent dark toxicity against Gram-positive bacteria (Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis, and Propionibacterium acnes) under dark conditions (MIC = 4-16 μM). This is because the iridium complex of this invention can induce singlet oxygen (…) under light. 1 O2) generation. Under 405 nm light irradiation for 10 minutes, compared to the unilluminated sample group under dark conditions, the minimum inhibitory concentration (MIC) of the iridium complex of this invention decreased to 1-4 μM after light irradiation. The iridium complex of this invention exhibits good antibacterial activity against Gram-positive bacteria, especially against *Propionibacterium acnes*, with significantly better antibacterial activity than other bacteria. However, it has no significant effect on Gram-negative bacteria and fungi under either dark or light conditions. This indicates that the iridium complex of this invention has excellent dark toxicity and photodynamic synergy. Furthermore, compared to the vancomycin sample group, the iridium complex obtained in Example 1 shows superior dark toxicity and photodynamic activity against Gram-positive bacteria.

[0046] Table 1 shows the minimum inhibitory concentrations (MIC (μM)) of iridium complexes and vancomycin against various bacteria.

[0047]

[0048] Example 4: Laser scanning confocal microscopy imaging study of iridium complexes against Propionibacterium acnes

[0049] Take a bacterial culture of Propionibacterium acnes in the logarithmic growth phase and dilute it to a bacterial concentration of 1×10⁻⁶. 6 Up to 1×10 7 The bacterial culture was diluted to CFU / mL, and the resulting iridium complex (10 μM) obtained in Example 1 was added. After incubation for 2 hours, the cells were washed three times with PBS to remove excess complex. The washed cells were then resuspended in PBS, and 10 μL of the resuspended solution was used to prepare a slide for observation under a laser confocal microscope. The results are as follows: Figure 4 As shown: Bright field imaging focuses a laser beam to a single point, creating a bright light field that enables high-resolution imaging of surface details; Fluorescent field imaging uses laser-excited fluorescent markers and a confocal optics system to eliminate out-of-focus interference, achieving high-contrast two-dimensional imaging; Combined field imaging combines bright field and fluorescent field imaging, displaying information from both modes simultaneously in the same image; Figure 4 In the combined field, Propionibacterium acnes showed obvious green fluorescence and could overlap with the bright field image, indicating that the iridium complex obtained in Example 1 can be used for imaging Propionibacterium acnes.

[0050] Example 5: Study on the liveness and death of Propionibacterium acnes using laser scanning confocal microscopy with iridium complexes

[0051] Take a bacterial culture of Propionibacterium acnes in the logarithmic growth phase and dilute it to a bacterial concentration of 1×10⁻⁶. 6 Up to 1×10 7 The diluted bacterial solution was added to the iridium complex (10 μM) obtained in Example 1 at a concentration of CFU / mL as the drug administration group; the bacterial solution of Propionibacterium acnes in the logarithmic growth phase was centrifuged, the supernatant was discarded, the precipitate was separated, and it was resuspended in PBS to obtain a concentration of 1×10⁻⁶ CFU / mL. 6 Up to 1×10 7 CFU / mL bacterial suspension served as the control group. Both the control and treatment groups were further divided into two subgroups: one subgroup was directly incubated in an incubator (groups denoted as Ir, control), and the other subgroup was irradiated at 405 nm for 10 minutes before incubation (groups denoted as Ir / light, control / light). Green fluorescent nucleic acid dye (SYTO 9) and propidium iodide (PI) were added for co-staining under light-protected conditions. After staining, the bacteria were washed three times with PBS and resuspended. 10 μL of the solution was used to prepare slides, and images of bacterial viability and death were obtained using confocal microscopy. Results are as follows: Figure 5As shown: Bright field imaging focuses a laser beam to a single point, forming a bright light field, thereby achieving high-resolution imaging of surface details of an object; SYTO 9 and PI field imaging achieve high-contrast two-dimensional imaging by using a laser to excite fluorescent markers and utilizing a confocal optical system to eliminate out-of-focus light interference; combined field imaging combines bright field and fluorescence field imaging, displaying information from both modes simultaneously in the same image; no red fluorescence was observed in the PI channel in the control group (including control group and control / light-illuminated group), while obvious red fluorescence was observed in the drug-treated group (Ir group and Ir / light-illuminated group), and the red fluorescence in the PI channel of the drug-treated group after light illumination (i.e., Ir / light-illuminated group) was significantly enhanced, indicating that the iridium complex obtained in Example 1 has good dark toxicity and photodynamic synergy.

[0052] Example 6: Scanning electron microscopy study of iridium complexes against Propionibacterium acnes

[0053] Take a bacterial culture of Propionibacterium acnes in the logarithmic growth phase and dilute it to a bacterial concentration of 1×10⁻⁶. 6 Up to 1×10 7 CFU / mL, the drug administration group was prepared by adding the iridium complex obtained in Example 1 to the diluted bacterial solution, with iridium complex concentrations set at 2 μM, 4 μM, and 8 μM; bacterial solutions of Propionibacterium acnes in the logarithmic growth phase were taken, centrifuged, the supernatant was discarded, the precipitate was separated, and resuspended in PBS to obtain a concentration of 1×10⁻⁶ CFU / mL. 6 Up to 1×10 7 The bacterial culture at CFU / mL served as the control group; the control group and each concentration administration group were further divided into two groups, one of which was directly placed in an incubator and incubated at 37°C for 1 hour (at... Figure 6 The group marked "dark" was used as an example. Another group was irradiated with 405nm light for 10 minutes and then incubated at 37°C for 1 hour. Figure 6 (Groups marked "light exposure"); each experimental group was washed with PBS and fixed with 4% glutaraldehyde, dehydrated in a gradient of ethanol, replaced with tert-butanol, and resuspended; samples were mounted on sterile aluminum foil and freeze-dried overnight; subsequently, gold was sputtered and scanning electron microscopy was used to observe bacterial morphological changes. Figure 6 As shown, the morphology of Propionibacterium acnes in all control groups was intact; in all drug-treated groups, the bacterial cells gradually shrank as the drug concentration increased, showing a concentration-dependent effect; and the changes were more significant after light exposure in the drug-treated groups, with bacterial membrane rupture and contents leakage observed at 8 μM, indicating that the iridium complex obtained in Example 1 has good dark toxicity and photodynamic synergistic effect.

[0054] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An iridium complex with anti-Propionibacterium acnes activity, characterized in that: The iridium complex is an iridium complex formed by a cationic compound with the structure shown in formula (Ⅰ) and an anion; （Ⅰ）。 2. The iridium complex with anti-Propionibacterium acnes effect according to claim 1, characterized in that: The anion is an inorganic salt anion.

3. An iridium complex with anti-Propionibacterium acnes effect according to claim 1, characterized in that: The anion is ClO4. - Cl - or PF6 - .

4. An iridium complex with anti-Propionibacterium acnes effect according to claim 1, characterized in that: The anion is Cl. - or ClO4 - .

5. An iridium complex with anti-Propionibacterium acnes effect according to claim 1, characterized in that: The anion is Cl. - .

6. A method for preparing an iridium complex with anti-acne Propionibacterium acnes effect according to any one of claims 1 to 5, characterized in that... The following steps are included: (1) Weigh 0.75 mmol of 1,10-phenanthroline-5,6-dione, 0.75 mmol of aniline, 0.75 mmol of 6-hydroxy-2-naphthal and 30 mL of glacial acetic acid and mix them in a three-necked flask. Heat the mixture to 120°C under argon protection and react for 3-5 h. Then transfer the reaction solution to a container containing deionized water and neutralize the reaction solution with 25 wt% ammonia water until a yellow precipitate appears. Filter the solution and collect the precipitate. Wash the precipitate with water and ether in sequence to obtain the crude product. (2) The crude product obtained in step (1) was dissolved in ethanol, then packed into a column with 60-100 mesh silica gel, and eluted with ethanol as the eluent. The yellow component was collected and rotary evaporated to obtain the intermediate product 1-PHIPN, the structure of which is shown in the following formula: ； (3) Weigh 0.079 mmol [Ir(pq)2Cl2]·2H2O and 0.158 mmol of the intermediate product 1-PHIPN obtained in step (2) and mix them. Then add a mixture of dichloromethane and methanol in a volume ratio of 3:1 as the reaction solvent. Heat to 65°C under argon protection, reflux for 6-9 h, cool to 20-40°C, and then add a saturated solution of anionic salt to produce a precipitate. Filter, evaporate to dryness, and purify by neutral alumina column. Elute with a mixture of acetonitrile and ethanol in a volume ratio of 10:1 and vacuum dry to obtain the iridium complex with anti-acne Propionibacterium acnes effect.

7. The method for preparing an iridium complex with anti-Propionibacterium acnes effect according to claim 6, characterized in that: The anionic salt mentioned in step (3) is an inorganic salt.

8. The method for preparing an iridium complex with anti-Propionibacterium acnes effect according to claim 6, characterized in that: The anionic salt in step (3) is a chloride salt or a perchlorate salt.

9. Use of the iridium complex according to any one of claims 1 to 5 in the preparation of an anti-Propionibacterium acnes drug.

10. Use of the iridium complex according to any one of claims 1 to 5 in the preparation of acne-reducing cosmetics.

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