PHOTOHEXER COMPOUNDS AND PHARMACEUTICAL COMPOSITION AND USE THEREOF

Abstract

The present invention relates to photohexer compounds and a pharmaceutical composition and use thereof. Specifically, the present invention relates to 2(4)-(1-hexyloxy-ethyl)-6,7-bispropionate-1,3,5,8-tetramethyl-4(2)-vinylpor-phyrin and their analogues, which are water soluble, have stable properties, can be used as a photosensitizer, and are suitable for the diagnosis and treatment of malignant tumors, precancerous lesions or benign lesions. The invention also relates to pharmaceutical compositions comprising such novel compounds, their use and preparation methods.

Claims

1. A compound of formula (I) or (II) ##STR00006## wherein R.sub.1 is selected from C.sub.1-8 alkyl; R.sub.2 is selected from C.sub.1-8 alkyl and C.sub.2-8 alkenyl; M, at each occurrence, is independently selected from alkali metal and alkaline earth metal; m is an integer selected from 1 to 6; and n is an integer selected from 1 to 6.

2. The compound of formula (I) or (II) according to claim 1, wherein R.sub.1 is selected from pentyl, hexyl and heptyl; and R.sub.2 is selected from ethyl and vinyl.

3. The compound of formula (I) or (II) according to claim wherein R1 is selected from pentyl, hexyl and heptyl; R2 is selected from ethyl and vinyl; M, at each occurrence, is selected independently from sodium and potassium; m is 2; and n is 2.

4. The compound of formula (I) or (II) according to claim 1, wherein the two Ms are identical.

5. The compound of formula (I) or (II) according to claim 3, wherein R.sub.1 is n-hexyl.

6. The compound of formula (I) or (II) according to claim 1, selected from the following compounds: ##STR00007##

7. A pharmaceutical composition comprising the compound according to claim 1.

8. A method of treating a subject in need thereof, comprising administering to the subject the compound of claim 1, wherein the compound is used as a photosensitizes.

9. A method of treating and/or diagnosing malignant tumors, precancerous lesions or benign lesions in a subject, comprising administering the compound of claim 1 to the subject in a photodynamic therapy.

10. The method of claim 9, wherein the malignant tumors are selected from oral and maxillofacial cancer, nasopharyngeal carcinoma, esophageal cancer, gastric cancer, bile duct cancer, colon cancer, rectal cancer, skin cancer, lung cancer, bronchial carcinoma, breast cancer and subcutaneously metastatic nodules after resection of breast cancer, cervical cancer, liver cancer, bladder cancer, pleural mesothelioma, pancreatic cancer, cancer of the penis, perianal tumor and residual cancer after resection of perianal tumor, Kaposi's sarcoma, prostate cancer, melanoma and brain tumors; the precancerous lesions are selected from Barrett's esophagus and oral leukoplakia; the benign lesions are selected from nevus flammeus, age-related macular degeneration, atherosclerosis, rheumatoid arthritis, skin microvascular malformation, psoriasis, and lupus erythematosus skin lesions.

11. A pharmaceutical composition comprising the compound according to claim 3.

12. A pharmaceutical composition comprising the compound according to claim 6.

13. The method of claim 9, wherein R1 is selected from pentyl, hexyl and heptyl; R2 is selected from ethyl and vinyl; M, at each occurrence, is selected independently from sodium and potassium; m is 2; and n is 2.

14. The method of claim 9, wherein the compound is ##STR00008##

Description

DESCRIPTION OF FIGURES

[0055] FIG. 1: HPLC chromatogram of protoporphyrin dimethylester.

[0056] FIG. 2: HPLC chromatogram of 2(4)-(1-hydroxyethyl)-6,7-bis [2-(methoxycarbonyl)ethyl]-1,3,5,8-tetramethyl-4(2)-vinylporphyrin (HVD-1 and HVD-2).

[0057] FIG. 3: HPLC chromatogram of 2-(1-hydroxyethyl)-6,7-bis [2-(methoxycarbonyl)ethyl]-1,3,5,8-tetramethyl-4-vinylporphyrin (HVD-1).

[0058] FIG. 4: HPLC chromatogram of 4-(1-hydroxyethyl)-6,7-bis[2-(methoxycarbonyl)ethyl]-1,3,5,8-tetramethyl-2-vinylporphyrin (HVD-2).

[0059] FIG. 5: HPLC chromatogram of 2-(1-hexyloxyethyl)-6,7-bis[2-(methoxycarbonyl)ethyl]-1,3,5,8-tetramethyl-4-vinylporphyrin.

[0060] FIG. 6: HPLC chromatogram of 2-(1-hexyloxyethyl)-6,7-di(propionate sodium)-1,3,5,8-tetramethyl-4-vinylporphyrin.

[0061] FIG. 7: HPLC chromatogram of 4-(1-hexyloxyethyl)-6,7-bis[2-(methoxycarbonyl)ethyl]-1,3,5,8-tetramethyl-2-vinylporphyrin.

[0062] FIG. 8: HPLC chromatogram of 4-(1-hexyloxyethyl)-6,7-di(propionate sodium)-1,3,5,8-tetramethyl-2-vinylporphyrin.

[0063] FIG. 9: The killing effect of photohexer-1 (P-1) mediated photodynamic therapy on SGC7901 cells.

[0064] FIG. 10: The killing effect of photohexer-2 (P-2) mediated photodynamic therapy on SGC7901 cells.

[0065] FIG. 11: The killing effect of photohexer-1 (P-1) mediated photodynamic therapy on SW-480 cells.

[0066] FIG. 12: The killing effect of photohexer-2 (P-2) mediated photodynamic therapy on SW-480 cells.

[0067] FIG. 13: The killing effect of photohexer-1 (P-1) mediated photodynamic therapy on SW-620 cells.

[0068] FIG. 14: The killing effect of photohexer-2 (P-2) mediated photodynamic therapy on SW-620 cells.

[0069] FIG. 15: The killing effect of photohexer-1 (P-1) mediated photodynamic therapy on Caco2 cells.

[0070] FIG. 16: The killing effect of photohexer-2 (P-2) mediated photodynamic therapy on Caco2 cells.

[0071] FIG. 17: The killing effect of photohexer-1 (P-1) mediated photodynamic therapy on Eca-109 cells.

[0072] FIG. 18: The killing effect of photohexer-2 (P-2) mediated photodynamic therapy on Eca-109 cells.

[0073] FIG. 19: The killing effect of photohexer-1 (P-1) mediated photodynamic therapy on MDA-MB-231 cells.

[0074] FIG. 20: The killing effect of photohexer-2 (P-2) mediated photodynamic therapy on MDA-MB-231 cells.

[0075] FIG. 21: The killing effect of photohexer-1 (P-1) mediated photodynamic therapy on MCF-7 cells.

[0076] FIG. 22: The killing effect of photohexer-2 (P-2) mediated photodynamic therapy on MCF-7 cells.

[0077] FIG. 23: The killing effect of photohexer-1 (P-1) mediated photodynamic therapy on MCF-7/ADR cells.

[0078] FIG. 24: The killing effect of photohexer-2 (P-2) mediated photodynamic therapy on MCF-7/ADR cells.

[0079] FIG. 25: The killing effect of photohexer-1 (P-1) mediated photodynamic therapy on CT26 cells.

[0080] FIG. 26: The killing effect of photohexer-2 (P-2) mediated photodynamic therapy on CT26 cells.

[0081] FIG. 27: The killing effect of photohexer-1 (P-1) mediated photodynamic therapy on 7721 cells.

[0082] FIG. 28: The killing effect of photohexer-2 (P-2) mediated photodynamic therapy on 7721 cells.

[0083] FIG. 29: Fluorescence microscopy of P-1, P-2 in cells.

[0084] FIG. 30A: The graph of tumor volume after the treatment of P1, P2 combined with light irradiation.

[0085] FIG. 30B: The tumor volume after the treatment of P1 combined with light irradiation.

[0086] FIG. 30C: The tumor volume after the treatment of P2 combined with light irradiation.

[0087] FIG. 31A: The graph of body weight of mice after the treatment of P-1, P-2, combined with light irradiation.

[0088] FIG. 31B: The graph of body weight of mice after the treatment of P-1 combined with light irradiation.

[0089] FIG. 31C: The graph of body weight of mice after the treatment of P-2 combined with light irradiation.

[0090] FIG. 32: Visual picture of 4T1 tumor-bearing mice after the treatment of P-1, P-2 combined with light irradiation.

[0091] FIG. 33A: Visual picture of heart of 4T1 tumor-bearing mice after the treatment of P-1, P-2 combined with light irradiation (including weight).

[0092] FIG. 33B: Visual picture of liver of 4T1 tumor-bearing mice after the treatment of P-1, P-2 combined with light irradiation (including weight).

[0093] FIG. 33C: Visual picture of spleen of 4T1 tumor-bearing mice after the treatment of P-1, P-2 combined with light irradiation (including weight).

[0094] Compared with the blank control mice, there were no obvious changes in the other organs except the spleen had obvious enlargement.

[0095] FIG. 33D: Visual picture of lung of 4T1 tumor-bearing mice after the treatment of P-1, P-2 combined with light irradiation (including pulmonary nodules and weight).

[0096] FIG. 33E: Visual picture of kidney of 4T1 tumor-bearing mice after the treatment of P-1, P-2 combined with light irradiation (including weight).

[0097] FIG. 33F: Visual picture of tumor of 4T1 tumor-bearing mice after the treatment of P-1, P-2 combined with light irradiation (including tumor inhibition rate).

EXAMPLES

[0098] The following examples are used to elaborate the present invention in greater detail, but they should not be misinterpreted as limitations of the scope defined by the claims of the present invention.

Experimental Instruments and HPLC Condition

[0099] 1 Experimental Instruments:

[0100] UV absorption spectrum was determined on Heλiosα type UV spectrophotometer. THERMO SPECTRONIC Co.). Infrared spectrum was recored on Nicolet 5700 Fourier transform infrared spectrometer (KBr tablet) THERMO Co.). The NMR spectra were measured with the AVIIIHD type 600 nuclear magnetic resonance spectrometer (TMS as internal standard) (Bruck Co.). High resolution mass spectrometry and cold spray MS were obtained on Acuu TOF CS type mass spectrometer (JMS-T100CS, JEOL, Japan).

[0101] HPLC analysis was performed with Agillent 1200 Series HPLC analyzer:

[0102] 2HPLC Condition

[0103] Analysis condition: analysis column: Japan Shiseido Capcell C.sub.18 MG 4.6 mm×150×5 μm; detection wavelength: 380 nm; column temperature: 30° C.; sample: the sample was dissolved with methanol, and filtered using 0.45 m nylon microporous membrane before injection; mobile phase: methanol and 1% acetic acid in water; flow rate: 1 mL/min.

TABLE-US-00001 Time (min) A: 1% acetic acid B: methanol (%) in water (%) 0.0 30.0 70.0 30.0 10.0 90.0 45.0 10.0 90.0 60.0 0.0 100.0 70.0 0.0 100.0 70.5 30.0 70.0 100.5 30.0 70.0
(The last 30 minutes is for the equilibrium of column, and ready for the next sample)

[0104] Preparative separation conditions: column chromatography with silica gel H (fineness 200-300 mesh, Qingdao marine chemical plant) is used for the preparative separation of reaction products.

Preparation of Intermediates

Preparation 1. Preparation of 2(4)-(1-hydroxyethyl)-6,7-bis [2-(methoxycarbonyl)ethyl]-1,3,5,8-tetramethyl-4(2)-vinylporphyrin (HVD-1 and HVD-2)

[0105] ##STR00003##

[0106] To 500 ml hydrochloric acid, protoporphyrin dimethylester 100 g was added, stirring to dissolve. The solution was stirred in 25° C. water bath for 6 hours. After that, 20% sodium hydroxide solution (2800 ml) was added at a constant rate and the pH value of the solution should be 13. The solution was allowed to stand for 1 hour. Then 500 ml acetic acid was added until pH was 4-5. After reaction for more 30 min and filtration by suction, the residue was washed with water, dried by suction, and further dried in a dryer. 95 g protoporphyrin dimethylester derivatives were obtained. To protoporphyrin dimethylester derivatives (95 g) a solution of 5% sulfuric acid in methanol (1000 ml) was added, stirred at room temperature for 30 minutes. The reaction solution was neutralized to pH 7 with (NH.sub.4).sub.2CO.sub.3. Then the solution was concentrated under reduced pressure. The concentrated residue was extracted with dichloromethane. The extracted solution was washed with appropriate amount of water and dehydrated by anhydrous sodium sulfate, filtered. Then, dichloromethane was recovered under vacuum to provide the protoporphyrin dimethylester derivatives.rotoporphyrin dimethylester derivatives were dissolved in appropriate amount of acetone. Silica gel 300 g was added and homogenously stirred. Then, acetone was evaporated. The resulting dry silicon gel powder was homogenously added to the top of the prefilled silicon gel chromatographic column (1600 g silicon gel, 200-300 mesh, pre-equilibrated by 0.2% methanol in dichloromethane). Then, 0.2% methanol in dichloromethane was added to separate. Fractions were collected at 200 ml per fraction, at the same time, the total percentage content of 2(4)-(1-hydroxyethyl)-6,7-bis[2-(methoxycarbonyl)ethyl]-1,3,5,8-tetramethyl-4(2)-vinylporphyrin in each fraction was detected by HPLC. For the fractions with less than 90% of purity, after removing solvent under reduced pressure, they were refined with isopropanol and acetone.

Preparation 2. The separation and purification of 2-(1-hydroxyethyl)-6,7-bis[2-(methoxycarbonyl)ethyl]-1,3,5,8-tetramethyl-4-vinylporphyrin} (HVD-1) and 4-(1-hydroxyethyl)-6,7-bis[2-(methoxycarbonyl) ethyl]-1,3,5,8-tetra-methyl-2-vinylporphyrin} (HVD-2)

[0107] 1000 g of silicon gel (Fineness 200-300 mesh) was weighed, to which methylene chloride was added and homogenously stirred. The mixture was filled into glass chromatographic column with 60 mm diameter. Separately, 50 g of 2(4)-(1-hydroxyethyl)-4(2)-vinylpyroporphyrin dimethyl ester was weighed, and an appropriate amount of methylene chloride was added to dissolve. Loading the sample by a wet process, using methylene chloride as mobile phase, fractions were collected and monitored by HPLC, to provide respectively:

[0108] 2-(1-hydroxyethyl)-6,7-bis[2-(methoxycarbonyl)ethyl]-1,3,5,8-tetramethyl-4-vinylporphyrin (HVD-1): .sup.1H NMR (CDC.sub.13) δ10.23, 10.09 (each s, 1H, 2 meso H); 9.89 (s, 2H, 2 meso H); 8.27 (m, 1H, CH═CH.sub.2); 6.35, 6.19 (each d, 1H, CH═CH.sub.2); 6.18 (q, 1H, CH (OH) (CH.sub.3); 4.30 (m, 4H, 2CH.sub.2CH.sub.2CO.sub.2CH.sub.3); 3.67 (s, 6H, 2CO.sub.2CH.sub.3); 3.58, 3.49 (each s, 3H, 2CH.sub.3); 3.51 (s, 6H, 2CH.sub.3); 3.22 (t, 4H, 2CH.sub.2CH.sub.2CO.sub.2CH.sub.3); 2.08 (d, 3H, CH (OH) CH.sub.3). HR-ESI-MS m/z: C.sub.36H.sub.40N.sub.4O.sub.5 calculated value: 609.3136 (M+1), measured value: 609.3163 (M+1). These data were in agreement with those reported in literature.

[0109] 4-(1-hydroxyethyl)-6,7-bis[2-(methoxycarbonyl)ethyl]-1,3,5,8-tetramethyl-2-vinylporphyrin (HVD-2): .sup.1H NMR (CDCl.sub.3) δ10.21, 10.04, 9.96, 9.94 (each s, 1H, meso, H); 8.21 (m, 1H, CH═CH.sub.2); 6.36, 6.18 (each d, 1H, CH═CH.sub.2); 6.23 (q, 1H, CH (OH) CH.sub.3); 4.36 (m, 2H, 2CH.sub.2CH.sub.2CO.sub.2CH.sub.3); 3.68 (s, 6H, 2CO.sub.2CH.sub.3 and 1CH.sub.3); 3.56, 3.53, 3.45 (each s, 3H, 3CH.sub.3); 3.25 (t, 4H, 2CH.sub.2CH.sub.2CO.sub.2CH.sub.3); 2.10 (d, 3H, CH (OH) CH.sub.3). HR-ESI-MS). m/z: C.sub.36H.sub.40N.sub.4O.sub.5 calculated value: 609.3136 (M+1), measured value: 609.3112 (M+1). These data were in agreement with those reported in literature.

[0110] HPLC retention time of HVD-1: 29 min, purity: 94%; HPLC retention time of HVD-2: 31 min, purity: 96%.

Example 1: Preparation of 2-(1-hexyloxyethyl)-6,7-bis(propionate sodium)-1,3,5,8-tetramethyl-4-vinylporphyrin (Photohexer-1) i.e. 2-(1-hexyloxyethyl)-6,7-bis[2-(sodium carbonate)ethyl]-1,3,5,8-tetramethyl-4-vinylporphyrin (Photohexer-1)

[0111] ##STR00004##

Step 1

Synthesis of 2-(1-hexyloxyethyl)-6,7-bis[2-(methoxycarbonyl)ethyl]-1,3,5,8-tetramethyl-4-vinylporphyrin

[0112] Take 2-(1-hydroxyethyl)-6,7-bis[2-(methoxycarbonyl)ethyl]-1,3,5,8-tetramethyl-4-vinylporphyrin (HVD-1, 100 mg), dissolve it in 8 mL of anhydrous dichloromethane. Then to this solution 1.5 ml 1-hexanol was added, subsequently 2.5 ml dichloromethane solution saturated with gaseous hydrogen bromide was added, gently mixed well, plugged tightly with a glass stopper, allowed to stand in the dark at room temperature for 1 hours, when necessary, the reaction time may be prolonged. After that, 20 ml water/dichloromethane (1:1) solution was added and the organic layer was separated, washed with water. After removing water by adding an approprioate amount of anhydrous sodium sulfate, the organic solvent was evaporated under reduced pressure. Then the crude product was separated by silica gel column chromatography, firstly using 0.25% methanol in dichloromethane to elute protoporphyrin dimethyl ester, then using 0.75% methanol in dichloromethane to seperate, to afford dark red powder. Retention time: 62.2 min., purity: 88.9% (by HPLC detection).

Spectral Analysis Detection:

[0113] .sup.1H NMR (CDC.sub.13): 5:10.65, 10.19, 10.09 D, 10.04 (each s, 1H, 4 meso H) 8.30 (m, 1H, CH═CH.sub.2); 6.37, 6.18 (each d, 1H, CH═CH.sub.2); 6.11 (q, 1H, CH.sub.3CHOCH.sub.2C.sub.5H.sub.11); 4.40 (m, 4H, CH.sub.2CH.sub.2CO.sub.2CH.sub.3); 3.73, 3.69 (diester CH.sub.3); 3.65, 3.63) (each d, 6H, 4×CH.sub.3); 3.28 (t, 4; H, CH.sub.2CH.sub.2CO.sub.2CH.sub.3) 2.26 (d, 3H, CH.sub.3CHOCH.sub.2C.sub.5H.sub.11); 0.71-1.79 (m, 11H, CH.sub.3CHOCH.sub.2C.sub.5H.sub.11); −3.67 (s, 2H, NH).

[0114] HR-ESI-MS m/z: C.sub.42H.sub.53N.sub.4O.sub.5 calculated value: 693.4010 (M+1), measured value: 693.4010 (M+1).

[0115] The above measured data were in agreement with the chemical structure of 2-(1-hexyloxyethyl)-6,7-bis[2-(methoxycarbonyl)ethyl]-1,3,5,8-tetra-methyl-4-vinyl-porphyrin.

Step 2: The preparation of 2-(1-hexyloxyethyl)-6,7-bis(propionate sodium)-1,3,5,8-tetramethyl-4-vinylporphyrin (Photohexer-1) i.e. {2-(1-hexyloxyethyl)-6,7-bis[2-(sodium carbonate)ethyl]-1,3,5,8-tetramethyl-4-vinylporphyrin} (Photohexer-1)

[0116] 70 mg 2-(1-hexyloxyethyl)-6,7-bis[2-(methoxycarbonyl)ethyl]-1,3,5,8-tetra-methyl-4-vinylporphyrin was dissolved in 42 ml diethyl ether solution. Under stirring, a solution of sodium hydroxide in isopropanol (50 mg NaOH was dissolved in 15 ml iso-PrOH) was added dropwise. A capillary point sample tube was used to absorb the reaction solution, and the color detection reaction around the precipitate was observed on the filter paper. When the solution around the precipitate on the filter paper is colorless, stop adding. The reaction solution was transferred to a centrifuge tube, and centrifuged for 10 minutes (2500 RPM) in a centrifuge. The supernatant was decanted and the solid in the centrifuge tube was placed in the vacuum dryer for drying, to provide dark red powder. HPLC detection, retention time: 53.3 minutes, purity: 99.6%.

Spectral Analysis Detection:

[0117] UV-vis [CH.sub.3OH, λmax (nm)]: 624, 571, 535, 499, 398, 204;

[0118] IR(KBr): 3398, 3309, 2953, 2927, 2857, 1555, 1414, 1100, 909, 735, 678 cm.sup.−1;

[0119] .sup.1H NMR (CDC.sub.13) δ: 10.65, 10.26, 10.20, 10.17 (each s, 1H, 4 meso H) 8.33 (m, 1; H, CH═CH.sub.2;

[0120] 6.16, 6.14 (each d, 1H, CH═CH.sub.2); 6.14 (q, 1; H, CH.sub.3CHOCH.sub.2C.sub.5H.sub.11) 4.39 (m, 4H. CH.sub.2CH.sub.2CO.sub.2CH.sub.3); 3.76 (q, 2H, CH.sub.3CHOCH.sub.2C.sub.5H.sub.11); 3.74, 3.73, 3.66, 3.64 (each s, 3H, 4×CH.sub.3); 3.08 (t, 4; H, CH.sub.2CH.sub.2CO.sub.2CH.sub.3) 2.21 (d, 3H, CH.sub.3CHOCH.sub.2C.sub.5H.sub.11); 0.58-1.75 (m, 11H, CH.sub.3CHOCH.sub.2C.sub.5H.sub.11).

[0121] CS-MS m/s: 709.39 [M+1].sup.+, 687.39 [M+2H-Na].sup.+. HR-ESI-MS m/z: 709.3356 gives the excimer ion peak [M+H].sup.+ corresponding to the molecular formula C.sub.40H.sub.47N.sub.4O.sub.5Na.sub.2 (calculated value 709.3342), degree of unsaturation is 18.5.

[0122] According to the analytical results of the above spectral data, the final product was in agreement with the chemical structure of 2-(1-hexyloxyethyl)-6,7-bis(propionate sodium)-1,3,5,8-tetramethyl-4-vinyl porphyrin.

Example 2

The preparation of 4-(1-hexyloxyethyl)-6,7-bis(propionate sodium)-1,3,5,8-tetramethyl-2-vinylporphyrin (Photohexer-2) i.e. {4-(1-hexyloxyethyl)-6,7-bis[2-(sodium carbonate)ethyl]-1,3,5,8-tetramethyl-2-vinylporphyrin} (Photohexer-2)

[0123] ##STR00005##

Step 1

Synthesis of 4-(1-hexyloxyethyl)-6,7-bis[2-(methoxycarbonyl)ethyl]-1,3,5,8-tetramethyl-2-vinylporphyrin

[0124] Take 4-(1-hexyloxyethyl)-6,7-bis[2-(methoxycarbonyl)ethyl]-1,3,5,8-tetramethyl-2-vinylporphyrin (HVD-2, 100 mg), dissolve it in 8 mL of anhydrous dichloromethane. To this solution 1.5 ml of 1-hexanol was added, subsequently 2.5 ml dichloromethane solution saturated with gaseous hydrogen bromide was added, gently mixed well, plugged tightly with a glass stopper, allowed to stand in dark at room temperature for 1 hours, when necessary, the reaction time may be prolonged. After that, 20 ml water/dichloromethane (1:1) solution was added and the organic layer was separated, washed with water. After removing water by adding an appropriate amount of anhydrous sodium sulfate, the organic solvent was evaporated under reduced pressure. Then the crude product was separated by silica gel column chromatography, firstly using 0.25% methanol in dichloromethane to elute protoporphyrin dimethyl ester, then using 0.75-1% methanol in dichloromethane to separate, to afford dark red powder, retention time: 62.6 min., purity: 91.8% (by HPLC detection).

Spectral Analysis Detection:

[0125] .sup.1H NMR (CDC.sub.13): 10.61, 10.25, 10.09, 10.07 (each s, 1H, 4 meso H) 8.29 (m, 1H, CH═CH.sub.2); 6.37 6.18 (each d, 1H, CH═CH.sub.2); 6.13 (q, 1H, CH.sub.3CHOCH.sub.2C.sub.5H.sub.11) 4.43 (m, 4H. CH.sub.2CH.sub.2CO.sub.2CH.sub.3); 3.74, 3.69; (diester CH.sub.3) 3.65 (q, 2H, CH.sub.3CHOCH.sub.2C.sub.5H.sub.11); 3.67, 3.66, 3.65, 3.63 (each s, 3H, 4×CH.sub.3); 3.30 (t, 4H, CH.sub.2CH.sub.2CO.sub.2CH.sub.3) 2.27 (d, 3; H, CH.sub.3CHOCH.sub.2C.sub.5H.sub.11; 0.73-1.80 (m, 11H, CH.sub.3CHOCH.sub.2C.sub.5H.sub.11); −3.67 (s, 2H, 2 NH);

[0126] HR-ESI-MS m/z: C.sub.42H.sub.53N.sub.4O.sub.5 calculated value: 693.4010 (M+1), measured value: 693.4046 (M+1).

[0127] The above measured data were in agreement with the chemical structure of 4-(1-hexyloxyethyl)-6,7-bis[2-(methoxycarbonyl)ethyl]-1,3,5,8-tetramethyl-2-vinylporphyrin.

Step 2

The preparation of 4-(1-hexyloxyethyl)-6,7-bis(propionate sodium)-1,3,5,8-tetramethyl-2-vinylporphyrin (Photohexer-2) i.e. {4-(1-hexyloxyethyl)-6,7-bis[2-(sodium carbonate)ethyl]-1,3,5,8-tetramethyl-2-vinylporphyrin} (Photohexer-2)

[0128] 70 mg 4-(1-hexyloxyethyl)-6,7-bis[2-(methoxycarbonyl)ethyl]-1,3,5,8-tetramethyl-2-vinylporphyrin was dissolved in 42 ml diethyl ether solution. Under stirring, a solution of sodium hydroxide in isopropanol (50 mg NaOH was dissolved in 15 ml iso-PrOH) was added dropwise. A capillary point sample tube was used to absorb the reaction solution, and the color detection reaction around the precipitate was observed on the filter paper. When the solution around the precipitate on the filter paper is colorless, stop adding. The reaction solution was transferred to a centrifuge tube, and centrifuged for 10 minutes (2500 RPM) in a centrifuge. The supernatant was decanted and the solid in the centrifuge tube was placed in the vacuum dryer for drying, to provide dark red powder. HPLC detection, retention time: 54.6 minutes, purity: 97.3%.

Spectral Analysis Detection:

[0129] UV-vis [CH.sub.3OH, λmax (nm)]: 624, 571, 535, 499, 398, 286;

[0130] IR(KBr): 3391.6, 3310.4, 2927.7, 2857.5, 1729.1, 1561.0, 1447.4, 1425.0, 1102.2, 838.5, 726.2, 702.8, 678.8 cm.sup.−1;

[0131] .sup.1H NMR (CDCl.sub.3): 10.58, 10.25, 9.92, 9.63 (each s, 1H, 4 meso H) 7.91 (m, 1; H, CH═CH2); 6.06, 6.03 (each d, 1H, CH═CH.sub.2); 6.06 (q, 1H, CH.sub.3CHOCH.sub.2C.sub.5H.sub.11) 4.40 (m, 4H. CH.sub.2CH.sub.2CO.sub.2CH.sub.3; 3.75 (q, 2H, CH.sub.3CHOCH.sub.2C.sub.5H.sub.11); 3.64, 3.61, 3.58, 3.48 (each s, 3H, 4×CH.sub.3); 3.10 (t, 4; H, CH.sub.2CH.sub.2CO.sub.2CH.sub.3) 2.20 (d, 3H, CH.sub.3CHOCH.sub.2C.sub.5H.sub.11); 0.61-1.75 (m, 11H, CH.sub.3CHOCH.sub.2C.sub.5H.sub.11).

[0132] CS-MS m/s: 709.39 [M+1]+, 687.39 [M+2H-Na].sup.+. HR-ESI-MS m/z: 709.3551 gives the excimer ion peak [M+H].sup.+ corresponding to the molecular formula C.sub.40H.sub.47N.sub.4O.sub.5Na.sub.2 (calculated value 709.3342), degree of unsaturation is 18.5.

[0133] According to the analytical results of the above spectral data, the final product was in agreement with the chemical structure of 4-(1-hexyloxyethyl)-6,7-bis(propionate sodium)-1,3,5,8-tetramethyl-2-vinylporphyrin.

[0134] In the above synthesis method, HVD-1 and HVD-2 were separated firstly, then Photohexer-1 and Photohexer-2 were synthesized respectively. Alternatively, a mixture of 2(4)-(1-hydroxyethyl)-6,7-bis[2-(methoxycarbonyl) ethyl]-1,3,5,8-tetramethyl-4(2)-vinylporphyrin was firstly reacted with 1-hexanol in a solution of dichloromethane saturated by hydrogen bromide gas, then the reaction products were separated and purified by silica gel column chromatography to afford 2-(1-hexyloxyethyl)-6,7-bis[2-(methoxy carbonyl)ethyl]-1,3,5,8-tetramethyl-4-vinylporphyrin and 4-(1-hexyloxyethyl)-6,7-bis[2-(methoxycarbonyl)ethyl]-1,3,5,8-tetramethyl-2-vinylporphyrin, which were then made into sodium salt, respectively.

Example 3: Activity of the Compounds of the Present Invention as a Photosensitizer

1. Comparison of Killing Effects of Photodynamic Therapy Mediated by Photohexer-1, Photohexer-2 on Ex Vivo Human Stomach Cancer SGC7901 Cells, Colorectal Cancer Cells (SW-480, SW-620, CT26, and Caco2), Esophageal Cancer Eca-109 Cells, Breast Cancer Cells (MDA-MB-231, MCF-7 Cells and MCF-7/ADR 1) and Liver Cancer 7721 Cells

1.1 Experimental Protocol

[0135] The cells in logarithmic growth phase were collected and inoculated on 24-well culture plates. When reaching 70%-80% confluence, cells were divided randomly into the following groups: control (the parallel control group without photosensitizer and irradiation), irradiation (irradiation treatment alone without photosensitizer); (photosensitizer treatment alone without irradiation), photodynamic treatment (treatment by photosensitizer in combination with irradiation). For the photosensitizer alone treatment group and photodynamic treatment group, an appropriate amount of Photohexer-1 (P-1) or Photohexer-2 (P-2) was respectively added away from light to give a final concentration of 0.8, 1.6, 3.2, 6.4 μM. Each group set 3 replicates. Cells were incubated with the active agent, different treatments were carried out according to the grouping. Three different light intensities were selected for the photodynamic treatment group, which were 2.6, 5.2 and 12.4 J/cm.sup.2, respectively. After treatment and digestion of each group of cells, it was inoculated into a 96-well plate at 100 μL/well, and four wells were set for each treatment. MTT assay was performed after 24 h from inoculation: 20 μL MTT solution was added to each well under the condition away from light, and the culture was continued for 4 hours. 150 μL DMSO was added to each well after discarding the supernatant. After 15 min of dissolution, the absorbance value (OD value) was measured using a microplate reader and the relative survival rate of cells was calculated according to the following formula:

[00001]$\mathrm{Cell}.Math..Math.\mathrm{relative}.Math..Math.\mathrm{survival}.Math..Math.\mathrm{rate}=\frac{\mathrm{The}.Math..Math.\mathrm{OD}.Math..Math.\mathrm{value}.Math..Math.\mathrm{of}.Math..Math.\mathrm{experimental}.Math..Math.\mathrm{group}}{\mathrm{The}.Math..Math.\mathrm{OD}.Math..Math.\mathrm{value}.Math..Math.\mathrm{of}.Math..Math.\mathrm{control}.Math..Math.\mathrm{group}}\times 100.Math.\%$

1.2 Results

[0136] The detailed results were shown in the following Table 1-20 and the FIGS. 9-22:

TABLE-US-00002 TABLE 1 The killing effect of photohexer-1 (P-1) mediated photodynamic therapy on SGC7901 cells. Groups Sample Cell survival rate (%) Control 3 100.00 ± 2.07  Irradiation 3 101.89 ± 1.04  P-1 alone treatment (μM) 0.8 3 100.39 ± 0.20  1.6 3 99.22 ± 1.37 3.2 3 95.80 ± 1.59 6.4 3 89.77 ± 4.99 P-1 at different 0.8 3 90.25 ± 2.16 concentration (μM) 1.6 3 91.19 ± 2.89 combined with 2.6 J/cm.sup.2 3.2 3 88.70 ± 1.24 light irradiation 6.4 3 77.76 ± 0.22 P-1 at different 0.8 3 92.41 ± 0.63 concentration (μM) 1.6 3 92.29 ± 0.15 combined with 5.2 J/cm.sup.2 3.2 3 85.49 ± 0.99 light irradiation 6.4 3 69.94 ± 0.56 P-1 at different 0.8 3 92.26 ± 1.16 concentration (μM) 1.6 3 89.21 ± 0.24 combined with 10.4 J/cm.sup.2 3.2 3 77.02 ± 0.04 light irradiation 6.4 3 38.34 ± 0.13

TABLE-US-00003 TABLE 2 The killing effect of photohexer-2(P-2) mediated photodynamic therapy on SGC7901 cells. Groups Sample Cell survival rate (%) Control 3 100.00 ± 0.78  Irradiation 3 97.67 ± 0.99 P-2 alone treatment (μM) 0.8 3 100.80 ± 0.91  1.6 3 97.25 ± 1.34 3.2 3 82.72 ± 0.54 P-2 at different 0.8 3 83.27 ± 0.82 concentration (μM) 1.6 3 70.77 ± 7.19 combined with 2.6 J/cm.sup.2 3.2 3 52.91 ± 1.96 light irradiation P-2 at different 0.8 3 76.47 ± 1.65 concentration (μM) 1.6 3 60.00 ± 9.82 combined with 5.2 J/cm.sup.2 3.2 3 41.72 ± 0.35 light irradiation P-2 at different 0.8 3 66.13 ± 0.60 concentration (μM) 1.6 3  49.32 ± 12.57 combined with 10.4 J/cm.sup.2 3.2 3 34.58 ± 0.66 light irradiation

TABLE-US-00004 TABLE 3 The killing effect of photohexer-1 (P-1) mediated photodynamic therapy on SW-480 cells. Groups Sample Cell survival rate (%) Control 3 100.00 ± 3.07  Irradiation 3 100.89 ± 2.04  P-1 alone treatment (μM) 0.8 3 101.14 ± 3.20  1.6 3 100.83 ± 2.47  3.2 3 98.02 ± 2.59 6.4 3 91.27 ± 1.09 P-1 at different 0.8 3 99.72 ± 3.16 concentration (μM) 1.6 3 97.08 ± 2.89 combined with 2.6 J/cm.sup.2 3.2 3 96.00 ± 2.24 light irradiation 6.4 3 77.99 ± 1.22 P-1 at different 0.8 3 95.61 ± 3.11 concentration (μM) 1.6 3 94.55 ± 2.77 combined with 5.2 J/cm.sup.2 3.2 3 85.27 ± 1.89 light irradiation 6.4 3 65.38 ± 1.32 P-1 at different 0.8 3 89.28 ± 3.01 concentration (μM) 1.6 3 85.85 ± 2.68 combined with 10.4 J/cm.sup.2 3.2 3 65.70 ± 2.01 light irradiation 6.4 3 57.31 ± 1.03

TABLE-US-00005 TABLE 4 The killing effect of photohexer-2 (P-2) mediated photodynamic therapy on SW-480 cells. Groups Sample Cell survival rate (%) Control 3 100.00 ± 4.07  Irradiation 3 100.24 ± 3.04  P-2 alone treatment (μM) 0.8 3 101.79 ± 4.08  1.6 3 96.97 ± 4.27 3.2 3 68.15 ± 2.78 6.4 3 57.31 ± 2.33 P-2 at different 0.8 3 100.79 ± 3.76  concentration (μM) 1.6 3 91.72 ± 2.89 combined with 2.6 J/cm.sup.2 3.2 3 63.40 ± 2.24 light irradiation 6.4 3 45.36 ± 1.22 P-2 at different 0.8 3 96.70 ± 3.63 concentration (μM) 1.6 3 87.06 ± 2.51 combined with 5.2 J/cm.sup.2 3.2 3 53.01 ± 1.99 light irradiation 6.4 3 42.56 ± 1.06 P-2 at different 0.8 3 94.70 ± 4.16 concentration (μM) 1.6 3 75.11 ± 3.24 combined with 10.4 J/cm.sup.2 3.2 3 43.68 ± 2.54 light irradiation 6.4 3 27.83 ± 1.13

TABLE-US-00006 TABLE 5 The killing effect of photohexer-1 (P-1) mediated photodynamic therapy on SW-620 cells. Groups Sample Cell survival rate (%) Control 3 100.00 ± 4.17  Irradiation 3 101.88 ± 2.99  P-1 alone treatment (μM) 0.8 3 101.33 ± 3.20  1.6 3 100.99 ± 2.47  3.2 3 98.23 ± 1.59 6.4 3 94.83 ± 1.09 P-1 at different 0.8 3 100.74 ± 3.46  concentration (μM) 1.6 3 96.64 ± 2.79 combined with 2.6 J/cm.sup.2 3.2 3 91.39 ± 2.18 light irradiation 6.4 3 86.70 ± 1.07 P-1 at different 0.8 3 99.53 ± 3.71 concentration (μM) 1.6 3 91.84 ± 2.97 combined with 5.2 J/cm.sup.2 3.2 3 87.50 ± 1.69 light irradiation 6.4 3 75.53 ± 1.03 P-1 at different 0.8 3 94.65 ± 4.04 concentration (μM) 1.6 3 87.51 ± 3.16 combined with 10.4 J/cm.sup.2 3.2 3 82.58 ± 2.31 light irradiation 6.4 3 68.33 ± 1.11

TABLE-US-00007 TABLE 6 The killing effect of photohexer-2 (P-2) mediated photodynamic therapy on SW-620 cells. Groups Sample Cell survival rate (%) Control 3 100.00 ± 4.14  Irradiation 3 98.87 ± 3.57 P-2 alone treatment (μM) 0.8 3 101.79 ± 4.27  1.6 3 96.58 ± 3.66 3.2 3 93.16 ± 2.71 6.4 3 85.21 ± 1.03 P-2 at different 0.8 3 97.58 ± 3.86 concentration (μM) 1.6 3 94.77 ± 2.99 combined with 2.6 J/cm.sup.2 3.2 3 89.07 ± 2.34 light irradiation 6.4 3 66.83 ± 1.12 P-2 at different 0.8 3 96.11 ± 4.06 concentration (μM) 1.6 3 90.19 ± 3.18 combined with 5.2 J/cm.sup.2 3.2 3 82.00 ± 2.62 light irradiation 6.4 3 58.46 ± 1.23 P-2 at different 0.8 3 92.99 ± 3.86 concentration (μM) 1.6 3 84.95 ± 3.02 combined with 10.4 J/cm.sup.2 3.2 3 68.54 ± 2.77 light irradiation 6.4 3 47.12 ± 1.04

TABLE-US-00008 TABLE 7 The killing effect of photohexer-1 (P-1) mediated photodynamic therapy on Caco2 cells. Groups Sample Cell survival rate (%) Control 3 100.00 ± 4.37  Irradiation 3 102.03 ± 3.89  P-1 alone treatment (μM) 0.8 3 99.34 ± 3.40 1.6 3 97.36 ± 2.42 3.2 3 95.34 ± 1.67 6.4 3 83.33 ± 1.24 P-1 at different 0.8 3 95.31 ± 3.86 concentration (μM) 1.6 3 91.33 ± 2.69 combined with 2.6 J/cm.sup.2 3.2 3 89.49 ± 2.15 light irradiation 6.4 3 81.65 ± 1.07 P-1 at different 0.8 3 93.10 ± 3.96 concentration (μM) 1.6 3 89.01 ± 2.88 combined with 5.2 J/cm.sup.2 3.2 3 87.46 ± 1.67 light irradiation 6.4 3 74.26 ± 1.14 P-1 at different 0.8 3 88.18 ± 4.24 concentration (μM) 1.6 3 87.63 ± 3.49 combined with 10.4 J/cm .sup.2 3.2 3 84.32 ± 2.76 light irradiation 6.4 3 66.34 ± 1.17

TABLE-US-00009 TABLE 8 The killing effect of photohexer-2 (P-2) mediated photodynamic therapy on Caco2 cells. Groups Sample Cell survival rate (%) Control 3 100.00 ± 4.64  Irradiation 3 100.98 ± 3.97  P-2 alone treatment (μM) 0.8 3 99.09 ± 4.27 1.6 3 98.50 ± 3.63 3.2 3 94.12 ± 2.54 6.4 3 88.45 ± 1.44 P-2 at different 0.8 3 98.47 ± 3.69 concentration (μM) 1.6 3 88.31 ± 2.67 combined with 2.6 J/cm.sup.2 3.2 3 75.55 ± 2.01 light irradiation 6.4 3 64.87 ± 1.13 P-2 at different 0.8 3 98.57 ± 4.26 concentration (μM) 1.6 3 77.26 ± 3.66 combined with 5.2 J/cm.sup.2 3.2 3 63.08 ± 2.35 light irradiation 6.4 3 43.88 ± 1.14 P-2 at different 0.8 3 73.07 ± 3.86 concentration (μM) 1.6 3 58.33 ± 2.60 combined with 10.4 J/cm.sup.2 3.2 3 47.94 ± 2.31 light irradiation 6.4 3 37.06 ± 1.54

TABLE-US-00010 TABLE 9 The killing effect of photohexer-1 (P-1) mediated photodynamic therapy on Eca-109 cells. Groups Sample Cell survival rate (%) Control 3 100.00 ± 4.78  Irradiation 3 101.43 ± 4.34  P-1 alone treatment (μM) 0.8 3 99.32 ± 3.43 1.6 3 97.28 ± 2.48 3.2 3 92.81 ± 1.37 6.4 3 80.68 ± 1.04 P-1 at different 0.8 3 84.64 ± 3.79 concentration (μM) 1.6 3 64.63 ± 2.39 combined with 2.6 J/cm.sup.2 3.2 3 44.02 ± 2.05 light irradiation 6.4 3 42.24 ± 1.01 P-1 at different 0.8 3 74.34 ± 4.23 concentration (μM) 1.6 3 66.40 ± 3.12 combined with 5.2 J/cm.sup.2 3.2 3 41.43 ± 1.27 light irradiation 6.4 3 37.97 ± 1.04 P-1 at different 0.8 3 60.09 ± 4.54 concentration (μM) 1.6 3 41.05 ± 3.47 combined with 10.4 J/cm.sup.2 3.2 3 36.89 ± 2.26 light irradiation 6.4 3 35.04 ± 1.34

TABLE-US-00011 TABLE 10 The killing effect of photohexer-2 (P-2) mediated photodynamic therapy on Eca-109 cells. Groups Sample Cell survival rate (%) Control 3 100.00 ± 4.43  Irradiation 3 102.02 ± 4.07  P-2 alone treatment (μM) 0.8 3 100.38 ± 4.27  1.6 3 95.76 ± 3.16 3.2 3 94.07 ± 2.58 6.4 3 84.49 ± 1.32 P-2 at different 0.8 3 96.96 ± 3.87 concentration (μM) 1.6 3 86.66 ± 2.94 combined with 2.6 J/cm.sup.2 3.2 3 79.33 ± 2.24 light irradiation 6.4 3 83.57 ± 1.12 P-2 at different 0.8 3 75.83 ± 4.22 concentration (μM) 1.6 3 70.66 ± 3.78 combined with 5.2 J/cm.sup.2 3.2 3 56.40 ± 2.33 light irradiation 6.4 3 33.91 ± 1.07 P-2 at different 0.8 3 59.89 ± 4.24 concentration (μM) 1.6 3 51.36 ± 3.67 combined with 10.4 J/cm.sup.2 3.2 3 40.90 ± 2.34 light irradiation 6.4 3 33.91 ± 1.01

TABLE-US-00012 TABLE 11 The killing effect of photohexer-1 (P-1) mediated photodynamic therapy on MDA-MB-231 cells. Groups Sample Cell survival rate (%) Control 3 100.00 ± 4.82  Irradiation 3 98.97 ± 4.24 P-1 alone treatment (μM) 0.8 3 97.82 ± 3.12 1.6 3 91.07 ± 2.02 3.2 3 75.52 ± 1.07 6.4 3 70.10 ± 0.99 P-1 at different 0.8 3 93.29 ± 3.66 concentration (μM) 1.6 3 81.63 ± 2.87 combined with 2.6 J/cm.sup.2 3.2 3 70.24 ± 2.03 light irradiation 6.4 3 62.22 ± 1.04 P-1 at different 0.8 3 90.67 ± 4.08 concentration (μM) 1.6 3 76.33 ± 2.91 combined with 5.2 J/cm.sup.2 3.2 3 68.08 ± 1.43 light irradiation 6.4 3 64.88 ± 1.07 P-1 at different 0.8 3 88.23 ± 4.09 concentration (μM) 1.6 3 71.13 ± 3.79 combined with 10.4 J/cm.sup.2 3.2 3 61.29 ± 2.27 light irradiation 6.4 3 56.27 ± 1.06

TABLE-US-00013 TABLE 12 The killing effect of photohexer-2 (P-2) mediated photodynamic therapy on MDA-MB-231 cells. Groups Sample Cell survival rate (%) Control 3 100.00 ± 3.87  Irradiation 3 101.66 ± 4.09  P-2 alone treatment (μM) 0.8 3 100.17 ± 4.03  1.6 3 97.69 ± 3.63 3.2 3 91.44 ± 2.57 6.4 3 81.55 ± 1.21 P-2 at different 0.8 3 87.82 ± 4.09 concentration (μM) 1.6 3 73.80 ± 3.22 combined with 2.6 J/cm.sup.2 3.2 3 59.92 ± 2.94 light irradiation 6.4 3 49.18 ± 1.33 P-2 at different 0.8 3  71.8 ± 4.16 concentration (μM) 1.6 3 49.35 ± 3.46 combined with 5.2 J/cm.sup.2 3.2 3 46.57 ± 2.43 light irradiation 6.4 3 40.03 ± 1.12 P-2 at different 0.8 3 52.23 ± 4.16 concentration (μM) 1.6 3 45.69 ± 3.16 combined with 10.4 J/cm.sup.2 3.2 3 40.31 ± 2.28 light irradiation 6.4 3 31.15 ± 0.96

TABLE-US-00014 TABLE 13 The killing effect of photohexer-1 (P-1) mediated photodynamic therapy on MCF-7 cells. Groups Sample Cell survival rate (%) Control 3 100.00 ± 4.83  Irradiation 3 100.88 ± 4.24  P-1 alone treatment (μM) 0.8 3 102.94 ± 3.04  1.6 3 82.48 ± 2.24 3.2 3 71.23 ± 1.76 6.4 3 60.81 ± 1.04 P-1 at different 0.8 3 78.02 ± 4.55 concentration (μM) 1.6 3 65.32 ± 2.43 combined with 2.6 J/cm.sup.2 3.2 3 54.13 ± 2.04 light irradiation 6.4 3 50.88 ± 1.02 P-1 at different 0.8 3 68.03 ± 3.97 concentration (μM) 1.6 3 47.92 ± 2.82 combined with 5.2 J/cm.sup.2 3.2 3 45.96 ± 1.76 light irradiation 6.4 3 41.69 ± 1.02 P-1 at different 0.8 3 63.99 ± 4.42 concentration (μM) 1.6 3 49.71 ± 3.94 combined with 10.4 J/cm.sup.2 3.2 3 46.98 ± 2.67 light irradiation 6.4 3 44.28 ± 1.07

TABLE-US-00015 TABLE 14 The killing effect of photohexer-2 (P-2) mediated photodynamic therapy on MCF-7 cells. Groups Sample Cell survival rate (%) Control 3 100.00 ± 4.65  Irradiation 3 99.76 ± 3.27 P-2 alone treatment (μM) 0.8 3 98.46 ± 4.62 1.6 3 84.93 ± 3.16 3.2 3 82.94 ± 2.28 6.4 3 80.23 ± 1.03 P-2 at different 0.8 3 89.08 ± 3.99 concentration (μM) 1.6 3 65.11 ± 2.73 combined with 2.6 J/cm.sup.2 3.2 3 51.79 ± 2.10 light irradiation 6.4 3 40.31 ± 1.02 P-2 at different 0.8 3 76.83 ± 4.26 concentration (μM) 1.6 3 48.15 ± 3.12 combined with 5.2 J/cm.sup.2 3.2 3 44.80 ± 2.22 light irradiation 6.4 3 35.46 ± 1.02 P-2 at different 0.8 3 72.40 ± 4.14 concentration (μM) 1.6 3 41.81 ± 3.77 combined with 10.4 J/cm.sup.2 3.2 3 34.81 ± 2.43 light irradiation 6.4 3 26.14 ± 1.07

TABLE-US-00016 TABLE 15 The killing effect of photohexer-1 (P-1) mediated photodynamic therapy on MCF-7/ADR cells. Groups Sample Cell survival rate (%) Control 3 100.00 ± 4.86  Irradiation 3 102.33 ± 4.51  P-1 alone treatment (μM) 0.8 3 100.36 ± 3.70  1.6 3 93.95 ± 2.47 3.2 3 81.01 ± 1.58 6.4 3 46.84 ± 1.02 P-1 at different 0.8 3 55.89 ± 3.97 concentration (μM) 1.6 3 53.43 ± 2.96 combined with 2.6 J/cm.sup.2 3.2 3 50.99 ± 2.31 light irradiation 6.4 3 45.48 ± 1.04 P-1 at different 0.8 3 41.47 ± 4.88 concentration (μM) 1.6 3 38.42 ± 2.51 combined with 5.2 J/cm.sup.2 3.2 3 36.95 ± 1.89 light irradiation 6.4 3 33.47 ± 1.41 P-1 at different 0.8 3 37.45 ± 4.34 concentration (μM) 1.6 3 32.58 ± 3.54 combined with 10.4 J/cm.sup.2 3.2 3 31.79 ± 2.65 light irradiation 6.4 3 27.27 ± 1.07

TABLE-US-00017 TABLE 16 The killing effect of photohexer-2 (P-2) mediated photodynamic therapy on MCF-7/ADR cells. Groups Sample Cell survival rate (%) Control 3 100.00 ± 4.67  Irradiation 3 101.23 ± 3.79  P-2 alone treatment (μM) 0.8 3 96.93 ± 4.47 1.6 3 92.26 ± 3.26 3.2 3 53.84 ± 2.58 6.4 3 48.88 ± 1.13 P-2 at different 0.8 3 94.88 ± 3.94 concentration (μM) 1.6 3 74.79 ± 2.77 combined with 2.6 J/cm.sup.2 3.2 3 50.37 ± 2.13 light irradiation 6.4 3 43.68 ± 1.06 P-2 at different 0.8 3 92.76 ± 4.32 concentration (μM) 1.6 3 67.82 ± 3.12 combined with 5.2 J/cm.sup.2 3.2 3 48.99 ± 2.16 light irradiation 6.4 3 41.49 ± 0.96 P-2 at different 0.8 3 90.99 ± 4.36 concentration (μM) 1.6 3 54.66 ± 3.43 combined with 10.4 J/cm.sup.2 3.2 3 46.57 ± 2.76 light irradiation 6.4 3 34.75 ± 1.22

TABLE-US-00018 TABLE 17 The killing effect of photohexer-1 (P-1) mediated photodynamic therapy on CT26 cells. Groups Sample Cell survival rate (%) Control 3 100.00 ± 4.18  Irradiation 3 97.89 ± 4.24 P-1 alone treatment (μM) 0.8 3 93.23 ± 3.43 1.6 3 87.15 ± 2.45 3.2 3 56.13 ± 1.69 6.4 3 44.05 ± 1.26 P-1 at different 0.8 3 91.63 ± 3.46 concentration (μM) 1.6 3 62.86 ± 2.29 combined with 2.6 J/cm.sup.2 3.2 3 49.48 ± 2.06 light irradiation 6.4 3 36.03 ± 1.17 P-1 at different 0.8 3 82.12 ± 4.98 concentration (μM) 1.6 3 55.88 ± 2.95 combined with 5.2 J/cm.sup.2 3.2 3 44.86 ± 1.57 light irradiation 6.4 3 30.01 ± 1.07 P-1 at different 0.8 3 82.39 ± 4.63 concentration (μM) 1.6 3 44.00 ± 3.29 combined with 10.4 J/cm.sup.2 3.2 3 41.00 ± 2.66 light irradiation 6.4 3 28.55 ± 1.47

TABLE-US-00019 TABLE 18 The killing effect of photohexer-2 (P-2) mediated photodynamic therapy on CT26 cells. Groups Sample Cell survival rate (%) Control 3 100.00 ± 4.64  Irradiation 3 98.89 ± 3.77 P-2 alone treatment (μM) 0.8 3 94.08 ± 4.27 1.6 3 92.09 ± 3.56 3.2 3 76.30 ± 2.28 6.4 3 50.49 ± 1.03 P-2 at different 0.8 3 83.97 ± 3.96 concentration (μM) 1.6 3 61.29 ± 2.78 combined with 2.6 J/cm.sup.2 3.2 3 44.29 ± 2.41 light irradiation 6.4 3 34.11 ± 1.02 P-2 at different 0.8 3 82.55 ± 4.77 concentration (μM) 1.6 3 49.85 ± 3.55 combined with 5.2 J/cm.sup.2 3.2 3 39.69 ± 2.27 light irradiation 6.4 3 28.61 ± 1.09 P-2 at different 0.8 3 70.66 ± 4.66 concentration (μM) 1.6 3 38.89 ± 3.86 combined with 10.4 J/cm.sup.2 3.2 3 26.01 ± 2.38 light irradiation 6.4 3 18.82 ± 1.01

TABLE-US-00020 TABLE 19 The killing effect of photohexer-1 (P-1) mediated photodynamic therapy on 7721 cells. Groups Sample Cell survival rate (%) Control 3 100.70 ± 4.45  Irradiation 3 101.98 ± 3.89  P-1 alone treatment (μM) 0.8 3 101.56 ± 4.08  1.6 3 100.75 ± 4.14  3.2 3 97.51 ± 3.98 6.4 3 96.52 ± 3.94 P-1 at different 0.8 3 97.88 ± 4.08 concentration (μM) 1.6 3 93.31 ± 3.99 combined with 2.6 J/cm.sup.2 3.2 3 94.92 ± 3.80 light irradiation 6.4 3 94.42 ± 3.87 P-1 at different 0.8 3 95.52 ± 3.42 concentration (μM) 1.6 3 92.36 ± 2.91 combined with 5.2 J/cm.sup.2 3.2 3 92.13 ± 2.77 light irradiation 6.4 3 90.25 ± 4.07 P-1 at different 0.8 3 94.75 ± 4.13 concentration (μM) 1.6 3 91.35 ± 3.59 combined with 10.4 J/cm.sup.2 3.2 3 88.59 ± 2.96 light irradiation 6.4 3 84.98 ± 1.97

TABLE-US-00021 TABLE 20 The killing effect of photohexer-2 (P-2) mediated photodynamic therapy on 7721 cells. Groups Sample Cell survival rate (%) Control 3 100.20 ± 4.14  Irradiation 3 101.89 ± 3.47  P-2 alone treatment (μM) 0.8 3 102.15 ± 4.07  1.6 3 101.37 ± 3.36  3.2 3 100.64 ± 2.98  6.4 3 98.14 ± 2.53 P-2 at different 0.8 3 98.29 ± 4.01 concentration (μM) 1.6 3 94.78 ± 3.88 combined with 2.6 J/cm.sup.2 3.2 3 92.15 ± 3.76 light irradiation 6.4 3 88.02 ± 3.59 P-2 at different 0.8 3 96.77 ± 3.76 concentration (μM) 1.6 3 92.15 ± 3.64 combined with 5.2 J/cm.sup.2 3.2 3 89.23 ± 3.35 light irradiation 6.4 3 82.25 ± 2.79 P-2 at different 0.8 3 95.91 ± 3.95 concentration (μM) 1.6 3 91.44 ± 3.73 combined with 10.4 J/cm.sup.2 3.2 3 78.32 ± 3.19 light irradiation 6.4 3 71.15 ± 2.91

1.3 Conclusions

[0137] In summary, in the selected drug concentration range, Photohexer-1 (P-1) or Photohexer-2 (P-2) alone treatment (i.e., without light irradiation) did not produce significant cytotoxicity on the cell lines, however, the photodynamic therapies mediated by them have significant selective toxic effect on different cell lines. In human gastric cancer SGC7901 cells, P-1/P-2 photodynamic therapies showed an obvious killing effect in drug-dose and light-intensity dependent manner, and the activity of P-2 was better than P-1. In colorectal cancer cells, the activity of P-2 was also better than P-1, the mediated photodynamic therapy had good killing effect on Caco2 cells and showed high dependence on concentration and light intensity. P-2 showed better activity than P-1 in SW-480 cells. While, for the other three colorectal cancer cells (SW-620, Caco2, CT26), there was no large difference between the two photosensitizers. In human esophageal cancer Eca-109 cells, P-1 and P-2 both exerted the photosensitive activity, and P-1 showed significantly stronger toxicity than that of P-2; for human breast cancer MDA-MB-231 cells, P-2 were more sensitive than P-1; in contrast, for MCF-7 cell, P-1 was more sensitive than P-2; P-1 and P-2, especially P-1, showed significant cell toxicity in MCF-7/ADR cells. Similarly, CT26 murine colorectal cancer cell line was sensitive to P-1 or P-2, respectively. Toxic side effects of P-1 were higher than those of P-2, but the photoactivity of P-2 was stronger than that of P-1, and showed strong dependence on the concentration and light intensity. For human liver cancer 7721 cells, either P-1 alone or P-1 in combination with light irradiation, there was no obvious toxic and side effects. P-2 alone showed no significant toxicity, but after combination with a certain amount of light, showed stronger cell toxicity.

[0138] Under the same experimental conditions, the P-2 and P-2 in the cells were observed by fluorescence microscope. It could be inferred that P-2 entered the cell more efficiently than P-1, and relatively more enriched in the cells. P-1 was easy to form aggregates, and distributed in the form of large particles, and mainly localized in the cell membranes.

2. Study on the Tumor-Inhibition Effect of P1 and P2-PDT on 4T1 Xenografted Mice

[0139] Experimental groups: blank control (2 mice), P-1 alone treatment (4 mg/kg, 8 mice), P-2 alone treatment (2 mg/kg, 8 mice), P-1 treatment (2 mg/kg, 4 mg/kg, 8 mice/group) combined with light irradiations, P-2 treatment (1 mg/kg, 2 mg/kg, 4 mg/kg, 8 mice/group) combined with light irradiations, 66 Balb/c mice in total.

[0140] Upon study the inhibitory effect of P1, P2-mediated photodynamic therapy on the growth of 4T1 transplanted tumors, it was found that, among the selected doses of photosensitizers, P1 (4 mg/kg) and P2 (2 mg/kg) alone did not show significant toxic effects. After combination with 50 J/cm.sup.2 irradiation, P1 and P2 showed obvious dose-dependent activity of photosensitizers, especially P2 (the volume of tumor was maintained at about half the size of tumors of control group on the 8.sup.th to 22.sup.nd days after treatment). In addition, this treatment also significantly inhibited the increase of tumor weight. On day 22 after treatment, the tumor inhibition rates of P1-4 mg/kg group and P2-2 mg/kg group and P2-4 mg/kg group combined with light irradiation were 25.6%, 23.35% and 44.68%, respectively, which were significantly higher than those of the control group and the drug alone treatment group. After comparison of the body weights of 4T1 tumor-bearing mice in different treatment groups, it was found that various treatment group has no significant effect on body weight of the mice. In addition, compared with the blank control mice, except for significant swelling of the spleen, the other organs did not significant changes. The results are shown in FIGS. 30A to 33F.

[0141] Note: FIGS. 30A-33F are all the results on 22 day after treatment;

Example 4: Solubility Determination

[0142] The solubility of Photohexer-1 and Photohexer-2 in water was determined by conventional methods in the art. The results showed that at room temperature, each ml of water dissolved about 3.3 mg Photohexer-1, while each ml of water dissolved about 10 mg Photohexer-2.

Example 5: Stability Determination

[0143] The storage stability of Photohexer-1 and Photohexer-2 was determined by conventional methods in the art. The results showed that there was no content change of Photohexer-1 and Photohexer-2 by HPLC test after 3 months of storage in dark at 2-4.

Example 6: Lyophilized Powder for Injection

[0144] Take a certain amount of Photohexer-1 or Photohexer-2, placed in a light-resistant glass container, add water to dissolve to reach a concentration of 5 mg/ml. The solution is filtered by stainless steel sterilization filter under pressure, firstly through pre-filter membrane with 0.45 μm pore size, then through sterilization membrane with 0.2 μm pore size. The solution is quantitatively dispensed in 10 ml vials in an aseptic operation room, the dispension volume is 2 ml or 1 ml. After lyophilizing under vacuum at −20° C. in a freeze-drying machine, lyophilized powder for injection is obtained.