A monosubstituted phthalocyanine conjugate of imidazotetrazine group and its preparation method and application

Abstract

The invention discloses a monosubstituted phthalocyanine conjugate of an imidazo tetrazine group and a preparation method and application thereof, wherein 3-nitrophthalonitrile is used as a raw material, 3-(4-carboxymethyl ester phenoxy)phthalonitrile is obtained by a nucleophilic reaction, 1-(4-carboxyphenoxy)phthalocyanine is generated by a ring-forming reaction and an alkaline hydrolysis reaction, and finally a target product is generated by a nucleophilic substitution reaction. The complex can enhance the killing effect on malignant tumors such as melanoma, triple-negative breast cancer and brain glioma, and effectively inhibit the metastasis and invasion of malignant tumor cells, and has an anti-angiogenic effect, providing a new idea for improving photodynamic therapy.

Description

Technical Field

[0001] The invention belongs to the field of medicinal chemistry, and specifically relates to an imidazo tetrazine group monosubstituted phthalocyanine conjugate and a preparation method and application thereof. Background Art

[0002] The current treatment status of malignant tumors is still unsatisfactory. The main reason is that malignant tumors are prone to metastasis and recurrence. Therefore, the task of improving the cure rate and prognosis of malignant tumors is still arduous. For example: breast cancer is the most common cancer among all women, seriously threatening women's lives and health, and is considered to be a breakthrough in China's overall cancer treatment; compared with other types of breast cancer, triple-negative breast cancer, which is negative for estrogen receptors, progesterone receptors, and human epidermal growth factor receptor-2, is the most malignant, with a low age of onset, high malignancy, and early recurrence and metastasis. Although invasive melanoma accounts for only 1% of all skin cancers, it is the main cause of skin cancer-related deaths, making it the fifth most common malignant tumor in men and women. Gliomas, as the most deadly malignant tumor in the brain, have a median survival of only 18 months and a five-year survival of less than 3%. They are difficult to cure, and current treatment methods are resistant and relapses will occur.

[0003] Unlike traditional surgery and chemotherapy, photodynamic therapy has the advantages of high selectivity, broad anti-cancer spectrum, no drug resistance, and protection of appearance and vital organ functions. At the same time, photodynamic therapy can not only kill in situ tumors, but also act on the blood vessels around the tumor and activate anti-tumor immune response. However, the current single photodynamic therapy for the treatment of the above-mentioned malignant tumors still needs to be further improved in activity, especially in anti-malignant tumor metastasis and recurrence. Therefore, enhancing the anti-angiogenesis and anti-tumor metastasis and invasion capabilities of photosensitizer drugs has gradually become the focus of research.

[0004] The imidazotetrazine group is the active domain of temozolomide, a new type of alkylating agent and the main chemotherapy drug for the treatment of melanoma, glioma, etc. In addition to acting on DNA to produce cytotoxicity, the drug has the effect of inhibiting angiogenesis at low and non-toxic doses, thus having the potential to combat the growth and metastasis of vascular-dependent tumors.

[0005] Based on the chemotherapeutic and anti-angiogenic effects of temozolomide, the present invention proposes combining phthalocyanine and the active domain imidazotetrazine group of temozolomide together to improve the killing effect of the phthalocyanine complex on malignant tumors such as triple-negative breast cancer cells, melanoma cells and brain glioma cells, while enhancing the anti-tumor cell metastasis and invasion capabilities. Summary of the invention

[0006] The present invention aims at the problem that triple-negative breast cancer, melanoma and glioma are easy to metastasize and invade and difficult to cure, and provides a mono-substituted phthalocyanine conjugate with an imidazotetrazine group, which can improve the therapeutic effect of phthalocyanine on triple-negative breast cancer, melanoma and glioma, and at the same time enhance the inhibition of metastasis and invasion of triple-negative breast cancer, melanoma and glioma, thereby improving the therapeutic effect.

[0007] In order to achieve the above-mentioned invention object, the present invention adopts the following technical scheme:

[0008] An imidazo tetrazine group monosubstituted phthalocyanine conjugate, the chemical structure of which is:

[0009] .

[0010] A method for preparing a monosubstituted phthalocyanine conjugate of an imidazotetrazine group comprises the following steps:

[0011] 1) 3-nitrophthalonitrile, methyl p-hydroxybenzoate and K2CO3 were dissolved in DMF (dimethylformamide) at a molar ratio of 1:1:2. After reacting at room temperature for 24-48 hours, a certain amount of water was added to the reaction solution to precipitate a white precipitate, which was filtered and dried to obtain compound 1a, whose chemical structure is: ; Wherein, the volume ratio of water to DMF is 5:1;

[0012] 2) Compound 1a and phthalonitrile were dissolved in n-pentanol, heated to 110-130°C, and then a zinc source was added. After stirring, DUB (1,8-diazabicyclo[5.4.0]undec-7-ene) was added, and the mixture was refluxed for 36-48 hours. The n-pentanol was then dried, and an excess of 20 wt% aqueous sodium hydroxide solution was added. The mixture was refluxed for 24-48 hours. After the solution was dried, a 20 wt% aqueous hydrochloric acid solution was added to adjust the pH to about 3.0, and a blue solid was obtained by filtration. The blue solid was passed through a silica gel column, and washed with EA (ethanol)-DMF in a volume ratio of 100:1 as an eluent until there was no phthalocyanine, and then eluted with EA-DMF in a volume ratio of 20:1 to collect the target product compound 2a, whose chemical structure is as follows: ; Wherein, the molar ratio of compound 1a, phthalonitrile, DUB and zinc source is 1:7:8:4, and the zinc source is zinc chloride or zinc acetate;

[0013] 3) Temozolomide, compound 2a, NHS (N-hydroxysuccinimide), and EDCI (carbodiimide) were dissolved in THF (tetrahydrofuran), reacted at room temperature for 48h~72h, and then a diamine compound (hexamethylenediamine) was added to the solution, refluxed for 1 hour, and the solution was spin-dried and passed through a silica gel column, and impurities were eluted with DCM (dichloromethane)-CH3OH in a volume ratio of 100:1, and then eluted and purified with DCM-CH3OH in a volume ratio of 30:1, and the product was collected. The same elution and impurity removal and purification conditions were repeated 2~3 times to obtain a pure target product; wherein the molar ratio of temozolomide, compound 2a, NHS, EDCI, and hexamethylenediamine compound used was 1:1:1:1:1:1.

[0014] Application: The imidazo tetrazine monosubstituted phthalocyanine conjugate can be used to synthesize photodynamic anti-tumor drugs, especially for the preparation of drugs for treating tumors that are prone to invasion and metastasis or difficult to treat, such as melanoma, triple-negative breast cancer and brain glioma.

[0015] The beneficial effects of the present invention are:

[0016] 1) The complex synthesized by the present invention has a stable structure, has no isomers, and is easy to synthesize;

[0017] 2) The introduction of imidazotetrazine groups into the active domain of temozolomide in the present invention can enhance the toxicity of the photosensitizer to tumor cells and inhibit the metastasis and invasion of tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The present invention provides a process route for synthesizing the imidazo tetrazine group monosubstituted phthalocyanine conjugate T-Pc and the control drug C-Pc.

[0019] Figure 2 The figure shows the comparison of the toxicity of T-Pc and C-Pc to 4T1, B16-F10 and U87 cells under light conditions.

[0020] Figure 3 The inhibition of T-Pc and C-Pc on the migration of 4T1 cells and B16-F10 cells.

[0021] Figure 4 The inhibition of T-Pc and C-Pc on the invasion of 4T1 cells and B16-F10 cells.

[0022] Figure 5 This study is to study the activity of T-Pc and C-Pc on Eahy926 cells (vascular endothelial cells). DETAILED DESCRIPTION

[0023] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0024] Example Preparation of T-Pc

[0025] Using 3-nitrophthalonitrile as the raw material, 3-(4-carboxymethyl ester phenoxy)phthalonitrile is obtained through a nucleophilic reaction, and then 1-(4-carboxyphenoxy) phthalocyanine zinc is generated through a cyclization reaction and an alkaline hydrolysis reaction, and then the target product T-Pc is generated through a nucleophilic substitution reaction between 1-(4-carboxyphenoxy) phthalocyanine zinc, hexamethylenediamine and temozolomide.

[0026] The specific steps are as follows:

[0027] 1) 2.00 g (11.6 mmol) 3-nitrophthalonitrile, 1.757 g (11.6 mmol) methyl p-hydroxybenzoate, and 3.202 g (23.2 mmol) K2CO3 were dissolved in 20 ml DMF. After reacting at room temperature for 24 h, 100 mL of water was added to the reaction solution to precipitate a white precipitate, which was filtered and dried to obtain 2.5 g of compound 1a with a yield of 78%. 1 H NMR (400MHz, Chloroform- d ) δ 8.13 (d, J = 8.2 Hz, 2H), 7.64 (t, J = 8.2 Hz, 1H), 7.54(d, J = 7.7 Hz, 1H), 7.16 (dd, J = 17.6, 8.2 Hz, 3H), 3.93 (s, 3H);

[0028] 2) 0.500 g (1.8 mmol) of compound 1a and 1.448 g (12.6 mmol) of phthalonitrile were dissolved in 50 mL of n-pentanol, heated to 130°C, and then 1.318 g (7.2 mmol) of zinc acetate was added. After stirring, 2.189 g (14.4 mmol) of DBU was added. The solution turned green and refluxed for 36 h. After that, the n-pentanol was dried and 20 mL of 20 wt% sodium hydroxide aqueous solution was added. The solution was refluxed for 48 h. After the solution was dried, 50 mL of 20 wt% hydrochloric acid aqueous solution was added to adjust the pH to 3.0. A blue solid was obtained by filtration. 300 mg of the blue solid was loaded onto a silica gel column and washed with EA:DMF = 100:1 (v / v) until there was no zinc phthalocyanine. Then, the column was eluted with EA:DMF = 20:1 (v / v). 200 mg of the target product 2a was collected. The yield was 15%. 1 H NMR (400 MHz, DMSO- d 6) δ 12.59 (s, 1H), 9.12 – 8.76 (m, 6H), 8.49 (s, 1H), 8.26 – 7.69 (m, 10H), 7.40 (s, 2H).

[0029] 3) 100 mg (0.14 mmol) 2a, 16.1 mg (0.14 mmol) N-hydroxysuccinimide, 26.74 mg (0.14 mmol) EDCI, and 27.32 mg (0.14 mmol) temozolomide (TMZ) were dissolved in 20 mL THF. After reacting at room temperature for 48 h, 162.4 mg (0.70 mmol) hexamethylenediamine was added to the solution. The mixture was refluxed for 1 h. The solution was dried by spin drying and passed through a silica gel column. Impurities were removed by elution with DCM:CH3OH = 100:1 (v / v). The product was then purified by elution with DCM:CH3OH = 30:1 (v / v). The product was collected and the same elution and purification conditions were repeated for 3 times to obtain 5 mg of pure product T-Pc with a yield of 3.5%. 1 HNMR (500 MHz, DMSO- d 6) δ 9.16-9.04 (t, 6H), 8.6 (t, 1H), 8.03-7.87 (d, 11H), 7.69(t, 1H), 7.47 (m, 2H), 6.65(s,1H), 3.76(s,3H), 3.35-3.28(d, 4H), 1.3-1.24(d,8H), HRMS (ESI): Calcd C 46 H 28 N10 O8Si[M+Na] + : 899.1753, found: 899.1741.

[0030] Comparative Example Synthesis of C-Pc

[0031] 100 mg (0.14 mmol) 2a, 16.1 mg (0.14 mmol) N-hydroxysuccinimide, and 26.74 mg (0.14 mmol) EDCI were dissolved in 10 mL THF. After reacting at room temperature for 48 h, 14.14 mg (0.14 mmol) n-hexylamine was added to the solution and refluxed for 1 hour. The solution was spin-dried and passed through a silica gel column. Impurities were removed by elution with DCM:CH3OH = 110:1 (v / v), and then purified by elution with DCM:CH3OH = 45:1 (v / v). The product was collected and repeated for 3 times under the same elution and purification conditions to obtain 30 mg of a relatively pure product C-Pc with a yield of 30%. 1 H NMR (500 MHz, DMSO- d 6) δ 9.1-9.01 (m, 6H), 8.57 (t,1H), 8.32 (d,1H), 8.15 – 7.97 (m,9H), 7.82 (d,1H), 7.44(d,2H), 3.18 (m,2H), 1.25 (m,8H), 0.83 (t,3H), HRMS (ESI): Calcd C 45 H 33 N9O2Zn[M+H] + : 796.2121 , found: 796.2083, the chemical structure of C-Pc is as follows:

[0032] .

[0033] Cytotoxicity assay

[0034] The MTT method, also known as the colorimetric method, is a method for detecting the survival status of cells. The succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble formazan and deposit it in the cells, while dead cells cannot reduce MTT to formazan. Dimethyl sulfoxide can dissolve the formazan in the cells, and its wavelength value at 570nm can be measured with an enzyme marker, which can indirectly reflect the number of living cells. Within a certain range of cell numbers, the amount of MTT crystals formed is proportional to the number of cells.

[0035] Under the microscope, 4T1 and B16-F10 cells with good growth status were selected. When the cell density grew to about 90%, they were digested with 1 mL of trypsin (containing EDTA) for 3 minutes (B16-F10 cells were digested for 1 minute), and then 2 mL of culture medium was added to terminate the digestion. The cells were blown evenly, and then counted with a cell counting plate. The cell solution was diluted with DMEM culture medium to 60,000 Cell / mL, and 100 μL of the above cell culture medium solution was added to each well of a 96-well plate. Six replicate wells were set for each concentration of T-Pc and C-Pc drugs, and a cell blank group and a solvent blank group (six replicate wells in each group) were set. The 96-well plate was placed in a 37°C incubator for 24 h.

[0036] Dosing: Use 5% CEL DMSO solution to dilute T-Pc and C-Pc to 7 different concentrations, then use DMEM culture medium to dilute the above 7 concentrations of drugs 100 times, use a spray gun to suck out the old culture medium in the 96-well plate, add 100-fold diluted 5% CEL DMSO solution to the cell control group, add DMFM culture medium to the solvent control group, and add the above-prepared solutions to the remaining drug control groups, and continue to place the 96-well plate in the incubator for 24 hours.

[0037] Phototoxicity: Take out the 96-well plate and carefully aspirate the drug-containing culture medium in the wells with a spray gun. Then wash each well three times with saline to remove the unabsorbed drug. Finally, add 100 μL of culture medium to each well, then irradiate with 670 nm LED light for 2 min, and then put the illuminated plate back into the incubator.

[0038] Test OD value: add 10 μL of 5 mg / mL MTT solution to each well of a 96-well plate, place the 96-well plate in a 37°C incubator for 4 h, then use a dispenser to aspirate the solution from each well, add 100 μL of DMSO solution to each well again, shake on a shaker for 30 min, and test the absorbance at 570 nm using an enzyme-labeled instrument.

[0039] Calculation: The cell survival rate was calculated using the measured absorbance. The calculation formula is as follows:

[0040] Cell viability (%) = (absorbance of sample group - absorbance of solvent blank group) / (absorbance of cell blank group - absorbance of solvent blank group) × 100.

[0041] Figure 2 The toxicity comparison of T-Pc and C-Pc on 4T1 cells, B16-F10 cells and U87 cells under light conditions is shown in the figure. As can be seen from the figure, the T-Pc concentration required to achieve a 50% cell inhibition rate is much lower (about 3-8 times lower) than the C-Pc concentration, indicating the high toxicity of T-Pc. Therefore, the introduction of imidazo tetrazine groups can enhance the toxic effect of T-Pc on tumor cells.

[0042] Cell migration assay:

[0043] (1) Select 4T1 and B16-F10 cells with good growth status and high metastatic potential in the logarithmic growth phase, add trypsin to digest and count, dilute to 300,000 cells / mL with prepared DMEM medium, and inoculate 1 mL per well in a 12-well plate (the well plate has been streaked) and place in a 37°C, 5% carbon dioxide incubator overnight until the fusion rate reaches 100%.

[0044] (2) The next day, use a 100 μL sterile pipette tip to scratch the cell layer perpendicular to the cell plane and at right angles to the previous scratch line (try to use the same pipette tip for different wells).

[0045] (3) After the scratching, wash three times with sterile saline to remove cells that are not attached to the wall and cells floating on the scratch, so that the gap after the scratch is clearly visible, and then replace the drug prepared in the culture medium containing 1% fetal bovine serum.

[0046] (4) Place the cells in an incubator. After 24 hours, take out the 12-well plate, observe the width of the scratch under a microscope and take photos.

[0047] (5) Image J software was used to open and calculate the scratch area, and GraphPad Prism 6.0 statistical analysis software was used to process the data. The experimental results were expressed as Mean ± SD.

[0048] Cell invasion assay:

[0049] (1) One day before the experiment, place the aliquoted matrix gel (16 μL) from -20°C into a 4°C refrigerator overnight. The colloid will change from solid to liquid. Place the chamber under ultraviolet light overnight for sterilization.

[0050] (2) Add 784 μL of serum-free culture medium to every 16 μL of matrix gel, spread the matrix gel in the chamber (100 μL per well), and place it in a 37°C, 5% carbon dioxide incubator for more than 5 hours.

[0051] (3) Select 4T1 and B16-F10 cells that are in good growth condition and in the logarithmic growth phase, add trypsin to digest and count, dilute to 2 million cells / mL with serum-free culture medium, and pipette evenly.

[0052] (4) Take 100 μL of the counted culture medium and 100 μL of the prepared drug and mix them evenly. Pipette 100 μL of the cell suspension and add it to the upper chamber of the chamber. Add 500 μL of culture medium containing 10% fetal bovine serum (FBS) to the 24-well plate (be careful not to create bubbles between the lower culture medium and the chamber). Place the plate in an incubator and culture for 24 hours.

[0053] (5) The next day, remove the 24-well plate, wash the chamber twice with 4°C precooled saline, fix it with methanol for 20 minutes, remove the chamber and let it dry (place it in a fume hood for 10 minutes).

[0054] (6) Add 500 μL of 0.1% crystal violet and stain the chamber at room temperature for 20 minutes. Then use a cotton swab to carefully wipe off the cells on the upper layer of the microporous membrane of the chamber and wash twice with saline.

[0055] (7) Take photos under an inverted microscope. Then, add an aqueous solution containing 33% acetic acid to the 24-well plate to dissolve the crystal violet in the lower chamber. After mixing, pipette the solution into a 96-well plate with 5 replicate wells in each group and 100 μL per well. Measure the OD value at 570 nm and process the data using GraphPad Prism 6.0 software. The experimental results are expressed as Mean ± SD.

[0056] Figure 3 and Figure 4 The figure shows the comparison of the inhibitory ability of T-Pc, C-Pc and TMZ on the metastasis and invasion of 4T1 cells and B16-F10 cells. Figure 3 In the figure, at 0 h, the scratch distance of each group was almost the same. After 24 h, the scratch distance and area of ​​the control group and C-Pc group decreased significantly, indicating that the cells continued to grow and metastasize to the scratch position, while the scratch distance and area of ​​T-Pc and TMZ only decreased slightly, indicating that T-Pc and TMZ can effectively inhibit the growth and metastasis of tumor cells. Figure 4 The number of cells invading the lower chamber after treatment with different drugs is shown. Compared with the control group and C-Pc group, the number of stained cells in the T-Pc and TMZ groups is significantly reduced. This is because T-Pc and TMZ can effectively inhibit the invasion of tumor cells in the upper chamber into the lower chamber. It can be seen that the introduction of imidazotetrazine groups can enhance the ability of T-Pc to inhibit tumor cell metastasis and invasion.

[0057] Figure 5This is a comparison chart of the phototoxicity, dark toxicity and ability to inhibit endothelial cell metastasis of T-Pc and C-Pc on vascular endothelial cells Eahy926. It can be seen from the figure that under light conditions, the concentration of T-Pc that inhibits half of the vascular endothelial cells is significantly lower than that of C-Pc. Under dark conditions, both have no obvious dark toxicity; compared with the control group and C-Pc group, the scratch distance and area of ​​vascular endothelial cells in the T-Pc and TMZ groups did not decrease significantly, indicating that T-Pc and TMZ can effectively inhibit the growth and metastasis of vascular endothelial cells. Therefore, the introduction of azole tetrazine groups is also beneficial to T-Pc inhibiting the growth and metastasis of vascular endothelial cells.

[0058] The above description is only a preferred embodiment of the present invention. Any equivalent structure or equivalent process transformation made by using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. Use of a monosubstituted phthalocyanine conjugate of an imidazo tetrazine group in the preparation of a photodynamic anti-melanoma and brain glioma drug, characterized in that: The chemical structural formula of the imidazo tetrazine monosubstituted phthalocyanine conjugate is: 。 2. A use according to claim 1, characterized in that: The method for preparing the imidazo tetrazine group monosubstituted phthalocyanine conjugate comprises the following steps: 1) 3-nitrophthalonitrile, methyl p-hydroxybenzoate and K2CO3 were dissolved in DMF and reacted at room temperature for 24-48 hours. A certain amount of water was added to the reaction solution to precipitate a white precipitate, which was filtered and dried to obtain compound 1a, whose chemical structure is: ； 2) Compound 1a and phthalonitrile were dissolved in n-pentanol, heated to 110-130°C, and then a zinc source was added. After stirring, DBU was added and the reaction was refluxed for 36-48 hours. After that, the n-pentanol was dried by spin drying, and an excess of 20 wt% sodium hydroxide aqueous solution was added. The reaction was refluxed for 24-48 hours. After the solution was dried by spin drying, a 20 wt% hydrochloric acid aqueous solution was added to adjust the pH to 3.0, and a blue solid was obtained by filtration. The blue solid was passed through a silica gel column, and washed with EA-DMF at a volume ratio of 100:1 as an eluent until there was no phthalocyanine, and then eluted with EA-DMF at a volume ratio of 20:1 to collect the target product compound 2a, whose chemical structure is as follows: ; 3) Temozolomide, compound 2a, NHS and EDCI were dissolved in THF, reacted at room temperature for 48h~72h, and then the diamine compound was added to the solution. The mixture was refluxed for 1h, and then passed through a silica gel column after being spin-dried. The impurities were eluted with DCM-CH3OH in a volume ratio of 100:1, and then eluted with DCM-CH3OH in a volume ratio of 30:

1. The product was collected and the same elution and purification conditions were repeated 2~3 times to obtain the pure target product.

3. The use according to claim 2, characterized in that: The molar ratio of 3-nitrophthalonitrile, methyl p-hydroxybenzoate and K2CO3 used in step 1) is 1:1:

2.

4. The use according to claim 2, characterized in that: The volume ratio of water to DMF used in step 1) is 5:

1.

5. The use according to claim 2, characterized in that: In step 2), the molar ratio of compound 1a, phthalonitrile, DBU and zinc source is 1:7:8:

4.

6. The use according to claim 2, characterized in that: The zinc source is zinc chloride or zinc acetate.

7. The use according to claim 2, characterized in that: In step 3), the diamine compound is hexamethylenediamine.

8. The use according to claim 7, characterized in that: The molar ratio of temozolomide, compound 2a, NHS, EDCI and hexamethylenediamine used in step 3) is 1:1:1:1:1.

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