NON-PERIPHERAL QUATERNARY AMMONIUM-MODIFIED ZINC PHTHALOCYANINE AND PREPARATION METHOD AND USE THEREOF

Abstract

The present disclosure provides a non-peripheral quaternary ammonium-modified zinc phthalocyanine and a preparation method and use thereof, belonging to the field of preparation of photodynamic drugs or photosensitizers. The non-peripheral quaternary ammonium-modified zinc phthalocyanine can be used as a photosensitizer for a photodynamic therapy (PDT) and photodynamic diagnosis, and can also be used for a photodynamic-immune synergistic therapy. Due to a unique structure, the non-peripheral quaternary ammonium-modified zinc phthalocyanine can be combined with an immune checkpoint blocker to achieve an excellent synergistic anti-tumor effect, which has a significant prospect for use in treating a metastatic tumor.

Claims

1. A non-peripheral quaternary ammonium-modified zinc phthalocyanine, having a structural formula shown as follows: ##STR00007##

2. A preparation method of the non-peripheral quaternary ammonium-modified zinc phthalocyanine according to claim 1, comprising the following steps: conducting a reaction on 1-[4-(aminoethyl)phenoxy] zinc phthalocyanine and methyl iodide as reactants in N,N-dimethylformamide as a solvent under the protection of nitrogen at 0° C. to a room temperature for 5 h to 50 h; and removing impurities by solvent cleaning and column chromatography isolation to obtain 1-[4-(N,N,N-trimethyl-2-aminoethyl)phenoxy] zinc phthalocyanine iodide.

3. The preparation method according to claim 2, wherein the 1-[4-(aminoethyl)phenoxy] zinc phthalocyanine and the methyl iodide have a mass ratio of 1:(50-200).

4. The preparation method according to claim 2, wherein 1 mg of the 1-[4-(aminoethyl)phenoxy] zinc phthalocyanine requires 0.3 mL to 3 mL of the N,N-dimethylformamide.

5. A preparation method of a photodynamic drug or a photosensitizer comprising using the non-peripheral quaternary ammonium-modified zinc phthalocyanine according to claim 1.

6. The preparation method according to claim 5, wherein the non-peripheral quaternary ammonium-modified zinc phthalocyanine is capable of inhibiting a distal tumor in combination with an immune checkpoint blocker PD-L1 antibody.

7. The preparation method according to claim 5, wherein the photosensitizer or the photodynamic drug prepared by the non-peripheral quaternary ammonium-modified zinc phthalocyanine is capable of treating a tumor in combination with an immune checkpoint blocker.

8. The preparation method according to claim 5, wherein the photodynamic drug or the photosensitizer is used for a photodynamic therapy (PDT), photodynamic diagnosis, or photodynamic disinfection.

9. The preparation method according to claim 8, wherein the PDT comprises a PDT of a malignant tumor, a PDT of a benign tumor, an in vitro photodynamic purification therapy of leukemia, or a PDT of a non-cancer disease.

10. The preparation method according to claim 9, wherein the non-cancer disease comprises a bacterial infection, an oral disease, a macular degeneration-caused eye disease, arteriosclerosis, a traumatic infection, a skin disease, or a viral infection.

11. The preparation method according to claim 8, wherein the photodynamic disinfection comprises photodynamic sterilization and purification of blood or a blood derivative, photodynamic sterilization and disinfection of water, and photodynamic disinfection of a medical or household appliance.

12-20. (canceled)

21. The preparation method according to claim 7, wherein the photodynamic drug or the photosensitizer is used for a photodynamic therapy (PDT), photodynamic diagnosis, or photodynamic disinfection.

22. The preparation method according to claim 21, wherein the PDT comprises a PDT of a malignant tumor, a PDT of a benign tumor, an in vitro photodynamic purification therapy of leukemia, or a PDT of a non-cancer disease.

23. The preparation method according to claim 22, wherein the non-cancer disease comprises a bacterial infection, an oral disease, a macular degeneration-caused eye disease, arteriosclerosis, a traumatic infection, a skin disease, or a viral infection.

24. The preparation method according to claim 21, wherein the photodynamic disinfection comprises photodynamic sterilization and purification of blood or a blood derivative, photodynamic sterilization and disinfection of water, and photodynamic disinfection of a medical or household appliance.

Description

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] The present disclosure is further described below with reference to the accompanying drawings and examples.

Example 1

[0034] A non-peripheral quaternary ammonium-modified zinc phthalocyanine (1-[4-(N,N,N-trimethyl-2-aminoethyl)phenoxy] zinc phthalocyanine iodide) had a structure shown in the following formula:

##STR00003##

[0035] 20 mg of 1-[4-(aminoethyl)phenoxy] zinc phthalocyanine (28.5 μmol) and K.sub.2CO.sub.3 (168.28 μmol) were dissolved in a single-neck round-bottomed flask containing 10 mL of anhydrous DMF by ultrasonication; a mixture was cooled to 0° C., added with 2,000 mg of CH.sub.3I slowly, stirred for 30 min, and reacted at room temperature. TLC spot plate was conducted, and terminated after 24 h, a reaction solvent was spin-dried, and a reactant was dissolved with 5 mL of DMF and filtered with a 0.22 μm syringe filter membrane to remove insoluble matters. The solvent was spin-dried by vacuumizing, a remaining product was dissolved with 1 mL of the DMF, passed through an S-X1 gel column, and blue-green components at the forefront were collected using the DMF as an eluent. The solvent was spin-dried by vacuumizing, an obtained product was dissolved in EA and passed through a silica gel column of 100 mesh to 200 mesh (an eluent was EA: DMF=100:1) to remove a yellow component at the forefront, and blue-green bands were collected using EA: DMF=10:1. The solvent was spin-dried by vacuumizing, a remaining product was passed through the S-X1 gel column (an eluent was DMF), and a blue component was collected. The blue component was precipitated in a large amount of solution including n-hexane: DCM=2:1, and dried in an oven at 45° C. to obtain a blue-green solid. The blue-green solid weighed 9.8 mg had a yield of 39.8%. The product had a maximum absorption peak at 674 nm in the DMF and a maximum absorption wavelength at 679 nm in the aqueous solution.

[0036] A structural characterization data of the product are as follows: .sup.1H NMR (400 MHz, DMSO) δ 9.23 (d, J=23.3 Hz, 6H), 8.83 (s, 1H), 8.16 (s, 6H), 7.77 (d, J=6.2 Hz, 1H), 7.44 (s, 2H), 7.37 (s, 2H), 7.09 (s, 1H), 3.90 (s, 2H), 2.74 (s, 2H), 1.50 (s, 2H), 1.26 (s, 4H), 0.84 (s, 3H). HRMS (ESI) m/z calcd for C.sub.43H.sub.32N.sub.9OZ.sub.n [M-I].sup.+: 754.2016; found: 754.2042. HPLC (674 nm): >95%.

Example 2

[0037] The reaction solvent of Example 1 was replaced with 6 mL or 60 mL of anhydrous DMF, other conditions remained unchanged, and a target product could also be obtained. A structural characterization data of the product are as follows: .sup.1H NMR (400 MHz, DMSO) δ 9.23 (d, J=23.3 Hz, 6H), 8.83 (s, 1H), 8.16 (s, 6H), 7.77 (d, J=6.2 Hz, 1H), 7.44 (s, 2H), 7.37 (s, 2H), 7.09 (s, 1H), 3.90 (s, 2H), 2.74 (s, 2H), 1.50 (s, 2H), 1.26 (s, 4H), 0.84 (s, 3H). HRMS (ESI) m/z calcd for C.sub.43H.sub.32N.sub.9OZ.sub.n [M-I].sup.+: 754.2016; found: 754.2042. HPLC (674 nm): >95%.

Example 3

[0038] 2,000 mg of the CH.sub.3I in Example 1 was replaced with 1,000 mg of the CH.sub.3I or 4,000 mg of the CH.sub.3I, other conditions remained unchanged, and a target product could also be obtained. A structural characterization data of the product are as follows: .sup.1H NMR (400 MHz, DMSO) δ 9.23 (d, J=23.3 Hz, 6H), 8.83 (s, 1H), 8.16 (s, 6H), 7.77 (d, J=6.2 Hz, 1H), 7.44 (s, 2H), 7.37 (s, 2H), 7.09 (s, 1H), 3.90 (s, 2H), 2.74 (s, 2H), 1.50 (s, 2H), 1.26 (s, 4H), 0.84 (s, 3H). HRMS (ESI) m/z calcd for C.sub.43H.sub.32N.sub.9OZ.sub.n [M-I].sup.+: 754.2016; found: 754.2042. HPLC (674 nm): >95%.

Example 4

[0039] The reaction time of Example 1 was changed to 5 h or 50 h, other conditions remained unchanged, and a target product could also be obtained. A structural characterization data of the product are as follows: .sup.1H NMR (400 MHz, DMSO) δ 9.23 (d, J=23.3 Hz, 6H), 8.83 (s, 1H), 8.16 (s, 6H), 7.77 (d, J=6.2 Hz, 1H), 7.44 (s, 2H), 7.37 (s, 2H), 7.09 (s, 1H), 3.90 (s, 2H), 2.74 (s, 2H), 1.50 (s, 2H), 1.26 (s, 4H), 0.84 (s, 3H). HRMS (ESI) m/z calcd for C.sub.43H.sub.32N.sub.9OZ.sub.n [M-I]*: 754.2016; found: 754.2042. HPLC (674 nm): >95%.

Comparative Example 1

[0040] 1-[4-(aminoethyl)phenoxy] zinc phthalocyanine was synthesized with reference to Chinese patent ZL201711099145.1 (a structure was shown in the following formula).

##STR00004##

Comparative Example 2

[0041] Other phthalocyanine compounds (structures shown below) were synthesized with reference to published papers (Bioorg. Med. Chem. Lett. 2015, 25: 2386-2389; Chem. Sci., 2018, 9: 2098-2104; Angew. Chem. Int. Ed. 2018, 57: 9885-9890; J. Am. Chem. Soc. 2019, 141: 1366-1372; Theranostics 2019, 9, 6412-6423)

##STR00005## ##STR00006##

Example 5

[0042] The non-peripheral quaternary ammonium-modified zinc phthalocyanine prepared in Example 1 was dissolved in a 1% castor oil derivative (Cremophor EL, wt %) aqueous solution to prepare a 0.1 mM photosensitizer. The dark toxicity and photodynamic activity of the photosensitizer were tested on human cervical cancer cells Hela.

[0043] 0.1 mM or 0.2 mM of the photosensitizer was diluted into a cell medium to prepare a cell medium containing zinc phthalocyanine complexes at different concentrations. The cancer cells were cultured in the medium containing different concentrations of zinc phthalocyanine complexes for 2 h; after staining, the medium was discarded; and after washing with PBS, the cells were added into a new medium (without zinc phthalocyanine complexes). Illumination experiment group: the cells were irradiated with red light for 30 min at a power of irradiated light at 15 mW cm.sup.−2 using red light with a wavelength greater than 600 nm as an excitation light source; non-illumination control group: the cells were placed in the dark for 30 min. After illumination or non-illumination, a cell viability was examined by MTT. The specific experimental steps referred to “Bioorganic & Medicinal Chemistry Letters, 2006, 16, 2450-2453”.

[0044] The red light with a wavelength greater than 610 nm was provided by a 500 W halogen lamp connected to an insulated water tank and an optical filter greater than 610 nm.

[0045] The results show that when being diluted to a concentration of 4 μM (4×10.sup.−6 mol/L), the non-peripheral quaternary ammonium-modified zinc phthalocyanine solution does not kill and inhibit the growth of human cervical cancer cells Hela without irradiation, indicating that the zinc phthalocyanine has no dark toxicity; however, if red light irradiation exists, the zinc phthalocyanine can kill 100% of the cancer cells. By examining a dosage-effect relationship between the concentration of non-peripheral quaternary ammonium-modified zinc phthalocyanine and the cell viability, a half-maximal inhibitory concentration (IC.sub.50, a drug concentration required to kill 50% of the cancer cells) under light conditions of 0.9 μM was obtained (the non-peripheral quaternary ammonium-modified zinc phthalocyanine in Example 1). The lower IC.sub.50 indicates that the non-peripheral quaternary ammonium-modified zinc phthalocyanine has a relatively high photodynamic activity.

[0046] The above 1% castor oil derivative (Cremophor EL, wt %) in water was replaced with 1% castor oil derivative (Cremophor EL, wt %) in PBS or 0.5% castor oil derivative (Cremophor EL, wt %) in water, and same experimental results could also be obtained.

Example 6

[0047] A bilateral tumor-bearing mouse model of melanoma cells B16-F10 (proximal and distal) was established. During PDT, the proximal referred to an illuminated side, and the distal referred to a non-illuminated side. The distribution and metabolism of photosensitizers in the tumor-bearing mouse were investigated by a small animal fluorescence imager and a tissue extraction method, and PDT was conducted under optimal conditions. The following 5 groups of experiments were set up, with 5 mice in each group: a PBS control group; an anti-PD-L1 antibody treatment group; a simple phthalocyanine (no illumination) treatment group; a phthalocyanine+illumination treatment group, namely a PDT group; and a phthalocyanine+illumination+anti-PD-L1 antibody treatment group, namely a combination therapy group. The anti-PD-L1 antibody was purchased from BioX cell.

[0048] In the combination therapy group, the PDT group and the simple photosensitizer group, 100 μL of a phthalocyanine compound (at a concentration of 200 μM, diluted with 0.5% CEL) was administered to each mouse. The combination therapy group and the PDT group were irradiated by a laser with a wavelength of 685 nm (at an irradiation power of 15 mW/cm.sup.2 for 5 min) on the right tumor (proximal tumor) 8 h to 12 h after the administration. The combination therapy group was administered at 50 μg of PD-L1 antibody/mouse immediately after laser treatment, and the antibody group was administered 50 μg of PD-L1 antibody/mouse at the same time. One treatment was given on the first and fourth day separately.

[0049] From the first treatment, a body weight and a tumor volume of all mice were measured every other day, and a tumor size was calculated according to a formula: tumor volume=tumor length×width×height×π/6; after 14 consecutive days of observation, a tumor inhibition rate of each experimental group was calculated.

[0050] The experimental results show that for the PDT group (phthalocyanine+illumination treatment group), the non-peripheral quaternary ammonium-modified zinc phthalocyanine in Example 1, the non-peripheral amine-modified zinc phthalocyanine in Comparative Example 1, and the other phthalocyanine photosensitizers in Comparative Example 2 have tumor inhibition rates of 51%, 69%, and 50% to 65% on proximal tumors (illuminated tumors) in the B16-F10 tumor-bearing mice, respectively. However, these three groups have almost no inhibitory effect (the tumor inhibition rate was less than 2.5%) on distal tumors (unilluminated tumors), indicating that the photodynamic therapy using phthalocyanine alone cannot inhibit tumor metastasis and metastatic tumors. On the other hand, the PD-L1 antibody therapy alone has a very limited inhibitory effect on proximal and distal tumors, with tumor inhibition rates of 12% and 13%, respectively. Although a photodynamic tumor-inhibitory effect of the zinc phthalocyanine in Example 1 alone is not particularly prominent, the zinc phthalocyanine exhibits an unexpected and significant ability to inhibit distal tumors in synergy with the PD-L1 antibody. In Example 1, the zinc phthalocyanine+illumination+anti-PD-L1 antibody treatment group has a tumor inhibition rate of up to 90% on the distal tumors (unilluminated tumors) of B16-F10 tumor-bearing mice, which was significantly higher than that of the phthalocyanine photosensitizers in Comparative Examples 1 to 2 (under same conditions, the combination anti-PD-L1 antibody treatment has a tumor inhibition rate on distal tumors of 40% to 70%).

[0051] More importantly, the mice treated with a combination of the zinc phthalocyanine in Example 1 and the PD-L1 antibody have an immune memory effect, which can effectively prevent tumor recurrence. On a 7th day after the combination therapy of zinc phthalocyanine in Example 1 and PD-L1 antibody, 1×10.sup.6 B16-F10 cells were subcutaneously injected into a left ventral side of each ICR mouse (5 mice in each group), and the mice were continued to be observed for 21 d. The studies have found that in the combination therapy group of zinc phthalocyanine in Example 1 and PD-L1 antibody, only one of the five mice has developed tumors again, indicating an effective rate of 80% in preventing recurrence. However, the mice in remaining control groups are observed to have obvious melanoma regrowth, and no ability is observed to prevent tumor recurrence. It can be seen that compared with other phthalocyanine photosensitizers, the combination of phthalocyanine-mediated PDT in Example 1 and PD-L1 antibody has significantly enhanced the duration of immune response, successfully stimulated the host immune system, promoted immune memory, and inhibited tumor recurrence.

[0052] The above description of examples is merely provided to help illustrate the method of the present disclosure and a core idea thereof. It should be noted that several improvements and modifications may be made by persons of ordinary skill in the art without departing from the principle of the present disclosure, and these improvements and modifications should also fall within the protection scope of the present disclosure. Various amendments to these embodiments are apparent to those of professional skill in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present disclosure. Thus, the present disclosure is not limited to the examples shown herein but falls within the widest scope consistent with the principles and novel features disclosed herein.