CN114409663A - Mcl-1 enzyme targeted zinc phthalocyanine 3-chloro-6 methylbenzo [ b ] thiophene-2-carboxylic acid conjugates and methods of making the same - Google Patents
Mcl-1 enzyme targeted zinc phthalocyanine 3-chloro-6 methylbenzo [ b ] thiophene-2-carboxylic acid conjugates and methods of making the same Download PDFInfo
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Abstract
The invention discloses a zinc phthalocyanine 3-chloro-6-methylbenzo [ b ] thiophene-2-carboxylic acid conjugate targeting Mcl-1 enzyme and a preparation method thereof, belonging to the technical field of pharmaceutical chemistry. 3-nitrophthalonitrile is used as an original raw material, 3- (4-carboxymethylester phenoxy) phthalonitrile is obtained through nucleophilic reaction, 1- (4-carboxyphenoxy) phthalocyanine is generated through cyclization reaction and nucleophilic reaction, and finally the target product zinc phthalocyanine 3-chloro-6-methylbenzo [ b ] thiophene-2-carboxylic acid conjugate is generated through nucleophilic substitution reaction. The conjugate can enhance the killing effect on triple negative breast cancer cells and provides a new idea for improving photodynamic therapy.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a zinc phthalocyanine 3-chloro-6-methylbenzo [ b ] thiophene-2-carboxylic acid conjugate targeting Mcl-1 enzyme and a preparation method thereof.
Background
Cancer is a major disease that endangers human health. The existing cancer treatment means include chemotherapy, radiotherapy, surgical excision and photodynamic therapy, wherein the photodynamic therapy has greater potential due to selectivity, minimal invasion and low toxicity. The photosensitizer used in photodynamic therapy mainly comprises porphyrin and phthalocyanine, the phthalocyanine is a macrocyclic planar structure, the self-targeting performance is insufficient, the effect of photodynamic therapy can be seriously reduced, and the improvement of the targeting performance of the phthalocyanine is an urgent problem to be solved.
The Mcl-1 enzyme is highly expressed in some tumors, such as triple negative breast cancer, but is hardly expressed in normal tissues. Clinical data indicate that the Mcl-1 enzyme is involved throughout the tumor, from its initial stage to metastasis and spread, and thus the Mcl-1 enzyme is a potential target. 3-chloro-6-methylbenzo [ b ] thiophene-2-carboxylic acid is an inhibitor of Mcl-1 kinase, which can be targeted to the Mcl-1 enzyme.
Based on the important role of Mcl-1 enzyme in tumor development, the invention provides that the active structural domains of phthalocyanine and 3-chloro-6-methylbenzo [ b ] thiophene-2-carboxylic acid are combined together, and the targeting of the 3-chloro-6-methylbenzo [ b ] thiophene-2-carboxylic acid to the Mcl-1 enzyme is utilized to enhance the killing effect of the conjugate on triple-negative breast cancer cells.
Disclosure of Invention
The invention provides a zinc phthalocyanine-3-chloro-6-methylbenzo [ b ] thiophene-2-carboxylic acid conjugate targeting Mcl-1 enzyme and a preparation method thereof, aiming at the problem that phthalocyanine is easy to aggregate. The targeting of 3-chloro-6-methylbenzo [ b ] thiophene-2-carboxylic acid to Mcl-1 enzyme is utilized, the targeting of phthalocyanine is enhanced by combining the phthalocyanine and the enzyme, the singlet oxygen yield is improved, and the treatment efficiency is further improved.
In order to achieve the purpose, the invention adopts the following technical scheme:
a zinc phthalocyanine-3-chloro-6 methylbenzo [ b ] thiophene-2-carboxylic acid conjugate that targets the Mcl-1 enzyme, having the chemical structure:
a method of making the Mcl-1 enzyme targeted zinc phthalocyanine-5-bromo-1-benzofuran-2-carboxylic acid conjugate comprising the steps of:
1) 3-nitrophthalonitrile, methyl p-hydroxybenzoate and K2CO3Dissolving the materials in DMF (dimethyl formamide) according to a molar ratio of 1:1:2, reacting at normal temperature for 24-48 h, adding a certain amount of water into the reaction solution to separate out white precipitate, filtering and drying to obtain a compound 1a, wherein the chemical structural formula is as follows:
(ii) a Wherein the volume ratio of the water to the DMF is 5: 1;
2) dissolving the compound 1a and phthalonitrile in n-amyl alcohol, heating to 110-130 ℃, adding a zinc source or an aluminum source, uniformly stirring, and then adding DUB (1, 8-diazabicyclo [5.4.0 ]]Undec-7-ene), performing reflux reaction for 36-48 h, then spin-drying n-amyl alcohol, adding an excessive 20wt% sodium hydroxide aqueous solution, performing reflux reaction for 24-48 h, adding a 20wt% hydrochloric acid aqueous solution after spin-drying the solution, adjusting the pH to about 3.0, filtering to obtain a blue solid, putting the blue solid on a silica gel column, washing the blue solid until phthalocyanine is not generated by using an eluent with the volume ratio of EA (ethanol) -DMF being 100:1, then eluting with the volume ratio of EA-DMF being 20:1, and collecting a target product 2a, wherein the chemical structural formula is as follows:
(ii) a Wherein the molar ratio of the compound 1a, phthalonitrile, DUB and a zinc source is 1:7:8:4, and the zinc source is zinc chloride or zinc acetate;
3) reacting 3-chloro-6-methylbenzo [ b ]]Dissolving thiophene-2-carboxylic acid, NHS (N-hydroxysuccinimide), DCC (N, N-dicyclohexylcarbodiimide) with DCM (dichloromethane), reacting at normal temperature for 24-48 h, then spin-drying, passing the product through a neutral silica gel column, and collecting a white solid 3a by taking DCM as an eluent, wherein the chemical structural formula is as follows:
(ii) a Wherein 3-chloro-6-methylbenzo [ b ] is used]The molar ratio of the thiophene-2-carboxylic acid to the NHS to the DCC is 1:1: 1;
4) the products 2a, NDissolving HS and EDCI (carbodiimide) in THF (tetrahydrofuran), reacting at normal temperature for 48-72 h, adding a diamine compound into the solution, refluxing for 1h, drying the solution, adding water into a round-bottom flask, shaking up, pouring off the water, drying, adding a white solid 3a and DMF, reacting at 100-120 ℃ for 2h, passing through a silica gel column after drying, and adding DCM-CH3Eluting OH with a volume ratio of 100:1 to remove impurities, and adding DCM-CH3Eluting and purifying OH at a volume ratio of 30:1, collecting the product, and repeatedly passing through the column for 2-3 times under the same elution, impurity removal and purification conditions to obtain a pure target product; wherein the molar ratio of the product 2a, NHS, EDCI, the hexamethylenediamine compound and the yellow solid 3a is 1:1:1:5: 1.
The zinc phthalocyanine-3-chloro-6-methylbenzo [ b ] thiophene-2-carboxylic acid conjugate targeting Mcl-1 enzyme is applied to synthesis of photodynamic antitumor drugs.
The invention has the beneficial effects that:
1) the conjugate synthesized by the invention has stable structure, no isomer and easy synthesis;
2) the introduction of the active structural domain of the 3-chloro-6-methylbenzo [ b ] thiophene-2-carboxylic acid can enhance the uptake of the conjugate by tumor cells, and the active structural domain of the 3-chloro-6-methylbenzo [ b ] thiophene-2-carboxylic acid can target Mcl-1 enzyme, so that the killing effect of the conjugate on triple-negative breast cancer cells is enhanced, and the effect of photodynamic therapy is enhanced.
Drawings
FIG. 1 is a process scheme of the present invention for synthesizing phthalocyanine-3-chloro-6-methylbenzo [ b ] thiophene-2-carboxylic acid conjugate BC-Pc.
FIG. 2 is a process scheme for the comparative example synthesis of C6-Pc.
FIG. 3 shows the UV-VIS absorption spectra of BC-Pc (A) and C6-Pc (B) in DMF synthesized in example.
FIG. 4 is a graph comparing the toxicity of BC-Pc and C6-Pc in light for MDA-MB-231 cells and HELF cells.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
EXAMPLE 1 preparation of Zinc phthalocyanine 3-chloro-6-methylbenzo [ b ] thiophene-2-carboxylic acid conjugate (BC-Pc)
3-nitrophthalonitrile is used as an original raw material, 3- (4-carboxymethylphenoxy) phthalonitrile is obtained through nucleophilic reaction, 1- (4-carboxyphenoxy) zinc phthalocyanine is generated through cyclization reaction and alkaline hydrolysis reaction, and a target product of zinc phthalocyanine 3-chloro-6-methylbenzo [ b ] thiophene-2-carboxylic acid conjugate (3-chloro-6-methyl-N- (6- (4- (phenylyamine-1-yloxy) benzamido) hexyl) benzol [ b ] thiophene-2-carboxylic acid, BC-Pc, is generated through nucleophilic substitution reaction among 1- (4-carboxyphenoxy) zinc phthalocyanine, hexamethylenediamine and 3-chloro-6-methylbenzo [ b ] thiophene-2-carboxylic acid active ester. The synthetic process route for the zinc phthalocyanine 3-chloro-6 methylbenzo [ b ] thiophene-2-carboxylic acid conjugate is shown in figure 1.
The method comprises the following specific steps:
1) 2.00g (11.6 mmol) of 3-nitrophthalic acid, 1.757g (11.6 mmol) of methyl p-hydroxybenzoate, 3.202g (23.2 mmol) of K2CO3Dissolving in 20ml of Dimethylformamide (DMF), reacting at room temperature for 24h, adding 100ml of water to the reaction solution to precipitate a white precipitate, filtering and drying to obtain 2.5 g of compound 1a with a yield of 78%,1H NMR (400 MHz, 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)；
the chemical structure of compound 1a is:
；
2) 0.500g (1.8 mmol) of the compound 1a and 1.448g (12.6 mmol) of phthalonitrile are dissolved in 50ml of n-pentanol, after heating to 130 ℃ 1.318g (7.2 mmol) of zinc acetate are added, after stirring homogeneously 2.189g (14.4 mmol) of DBU are added, the solution turns green in color, the reaction is then refluxed for 36h, then n-pentanol is dried, 20ml of 20wt% aqueous sodium hydroxide solution is added, the reaction is refluxed for 48h, and after the solution is dried, the solution is added to the inside50ml of a 20% by weight aqueous hydrochloric acid solution to adjust the pH to 3.0, and filtered to obtain a blue solid; applying 300 mg of blue solid to a silica gel column, washing with EA: DMF =100:1 (v/v) until no zinc phthalocyanine is present, eluting with EA: DMF =20:1 (v/v), collecting 200 mg of the target product 2a with a yield of 15%,1H 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)。
the chemical structural formula of the target product 2a is as follows:
；
3) 0.633g (2.8 mmol) of 3-chloro-6-methylbenzo [ b ]]Thiophene-2-carboxylic acid, 0.327g (2.8 mmol) N-hydroxysuccinimide (NHS), 0.576g (2.8 mmol) N, N-Dicyclohexylcarbodiimide (DCC) were dissolved in 20mL DCM, reacted at room temperature for 24h and then spin-dried, passed through a neutral silica gel column using DCM as an eluent, collected to give 0.6 g white solid 3a with a yield of 50%,1h NMR (500 MHz,) δ 7.89 (d, J = 8.4 Hz, 1H), 7.65-7.63 (m, 1H), 7.37-7.33 (m, 1H), 2.91 (s, 4H), 2.53 (s, 3H), the chemical structural formula of white solid 3a is:
；
4) 100mg (0.14 mmol) of the desired product 2a, 16.1mg (0.14 mmol) of N-hydroxysuccinimide, 26.74mg (0.14 mmol) of carbodiimide (EDCI) were dissolved in 20ml of THF (tetrahydrofuran), reacted at room temperature for 48 hours, 81.2mg (0.70 mmol) of hexamethylenediamine was added to the solution, refluxed for 1 hour, the solution was dried, 50ml of water was added to the round-bottomed flask, slightly shaken for several hours, then the water was poured off, dried, 45.22 mg (0.14 mmol) of white solid 3a and 10ml of DMF were added to the flask, reacted at 100 ℃ for 2 hours, the DMF was dried, and then DCM (dichloromethane): CH was used3OH (methanol) =100:1 (v/v) eluting to remove impurities, and then DCM: CH3OH =30:1 (v/v), collecting the product, repeating the column for 3 times by using the same elution, impurity removal and purification conditions to obtain 20mg of pure product zinc phthalocyanine 3-chloro-6 methylbenzene[b]Thiophene-2-carboxylic acid conjugate (BC-Pc) in a yield of 7%,1H NMR (500 MHz, DMSO-d 6) δ 9.10 – 8.96 (m, 6H), 8.54 (d, J = 7.4 Hz, 1H), 8.30 (t, J = 5.7 Hz, 1H), 8.22 (t, J = 5.7 Hz, 1H), 8.14 – 7.93 (m, 9H), 7.81 (t, J = 8.8 Hz, 1H), 7.75 (d, J = 0.6 Hz, 1H), 7.60 (d, J = 8.3 Hz, 1H), 7.42 – 7.38 (m, 2H), 7.27 (dd, J = 8.3, 0.9 Hz, 1H), 3.18 (tt, J = 14.2, 7.2 Hz, 4H), 2.37 (s, 3H), 1.36 – 1.23 (m, 8H). HRMS (ESI): Calcd C55H39ClN10O3SZn[M+H]+: 1019.1980 , found: 1019.1950.
the chemical structural formula of the zinc phthalocyanine 3-chloro-6-methylbenzo [ b ] thiophene-2-carboxylic acid conjugate is as follows:
example 2
3-nitrophthalonitrile is used as an original raw material, 3- (4-carboxy methyl ester phenoxy) phthalonitrile is obtained through nucleophilic reaction, 1- (4-carboxy phenoxy) zinc phthalocyanine is generated through cyclization reaction and alkaline hydrolysis reaction, and a target product of BC-Pc is generated through nucleophilic substitution reaction among 1- (4-carboxy phenoxy) zinc phthalocyanine, hexamethylene diamine and 3-chloro-6-methylbenzo [ b ] thiophene-2-carboxylic acid active ester.
The method comprises the following specific steps:
1) 2.00g (11.6 mmol) of 3-nitrophthalic acid, 1.757g (11.6 mmol) of methyl p-hydroxybenzoate, 3.202g (23.2 mmol) of K2CO3Dissolving in 20ml Dimethylformamide (DMF), reacting at room temperature for 48h, adding 100ml water into the reaction solution to precipitate white precipitate, filtering, oven drying to obtain 2.5 g compound 1a with yield of 80%,1H NMR (400 MHz, 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)；
the chemical structure of compound 1a is:
；
2) dissolving 0.500g (1.8 mmol) of the compound 1a and 1.448g (12.6 mmol) of phthalonitrile in 50ml of n-amyl alcohol, heating to 110 ℃, adding 0.982g (7.2 mmol) of zinc chloride, stirring uniformly, adding 2.189g (14.4 mmol) of DBU, wherein the solution turns green, then refluxing for 48 hours, then spin-drying n-amyl alcohol, adding 20ml of 20wt% sodium hydroxide aqueous solution, refluxing for 36 hours, adding 50ml of 20wt% hydrochloric acid aqueous solution into the solution after spin-drying to adjust the pH to 3.0, and filtering to obtain a blue solid; applying 300 mg of blue solid to a silica gel column, washing with EA: DMF =100:1 (v/v) until no zinc phthalocyanine is present, eluting with EA: DMF =20:1 (v/v), collecting 200 mg of the target product 2a with a yield of 16%,1H 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)。
the chemical structural formula of the target product 2a is as follows:
；
3) 0.633g (2.8 mmol) of 3-chloro-6-methylbenzo [ b ]]Thiophene-2-carboxylic acid, 0.327g (2.8 mmol) N-hydroxysuccinimide (NHS), 0.576g (2.8 mmol) N, N-Dicyclohexylcarbodiimide (DCC) were dissolved in 20mL DCM, reacted at room temperature for 48h and then spin-dried, passed through a neutral silica gel column using DCM as an eluent, collected to give 0.6 g white solid 3a with a yield of 55%,1h NMR (500 MHz,) δ 7.89 (d, J = 8.4 Hz, 1H), 7.65-7.63 (m, 1H), 7.37-7.33 (m, 1H), 2.91 (s, 4H), 2.53 (s, 3H), the chemical structural formula of white solid 3a is:
；
4) after 100mg (0.14 mmol) of the desired product 2a, 16.1mg (0.14 mmol) of N-hydroxysuccinimide, 26.74mg (0.14 mmol) of carbodiimide (EDCI) were dissolved in 20ml of THF (tetrahydrofuran) and reacted at room temperature for 72 hours, 81.2mg (0.70 mmol) of hexamethylenediamine was added to the solution, and the mixture was refluxed for 1 hour and then revolvedDry solution, adding 50ml water into round bottom flask, slightly shaking, pouring off water, spinning, adding 45.22 mg (0.14 mmol) white solid 3a and 10ml DMF, reacting at 120 deg.C for 2h, spinning off DMF, adding DCM (dichloromethane): CH3OH (methanol) =100:1 (v/v) eluting to remove impurities, and then DCM: CH3OH =30:1 (v/v), collecting the product, repeating the column for 2 times by using the same elution, impurity removal and purification conditions to obtain 20mg of pure product zinc phthalocyanine 3-chloro-6 methylbenzo [ b]Thiophene-2-carboxylic acid conjugate (BC-Pc) in a yield of 9%,1H NMR (500 MHz, DMSO-d 6) δ 9.10 – 8.96 (m, 6H), 8.54 (d, J = 7.4 Hz, 1H), 8.30 (t, J = 5.7 Hz, 1H), 8.22 (t, J = 5.7 Hz, 1H), 8.14 – 7.93 (m, 9H), 7.81 (t, J = 8.8 Hz, 1H), 7.75 (d, J = 0.6 Hz, 1H), 7.60 (d, J = 8.3 Hz, 1H), 7.42 – 7.38 (m, 2H), 7.27 (dd, J = 8.3, 0.9 Hz, 1H), 3.18 (tt, J = 14.2, 7.2 Hz, 4H), 2.37 (s, 3H), 1.36 – 1.23 (m, 8H). HRMS (ESI): Calcd C55H39ClN10O3SZn[M+H]+: 1019.1980 , found: 1019.1950.
the chemical structural formula of the zinc phthalocyanine 3-chloro-6-methylbenzo [ b ] thiophene-2-carboxylic acid conjugate is as follows:
example 3
3-nitrophthalonitrile is used as an original raw material, 3- (4-carboxy methyl ester phenoxy) phthalonitrile is obtained through nucleophilic reaction, 1- (4-carboxy phenoxy) zinc phthalocyanine is generated through cyclization reaction and alkaline hydrolysis reaction, and a target product of BC-Pc is generated through nucleophilic substitution reaction among 1- (4-carboxy phenoxy) zinc phthalocyanine, hexamethylene diamine and 3-chloro-6-methylbenzo [ b ] thiophene-2-carboxylic acid active ester.
The method comprises the following specific steps:
1) 2.00g (11.6 mmol) of 3-nitrophthalic acid, 1.757g (11.6 mmol) of methyl p-hydroxybenzoate, 3.202g (23.2 mmol) of K2CO3Dissolving in 20ml of dimethyl formamide (DMF), reacting for 36 hours at normal temperature,adding 100ml of water to the reaction solution to precipitate a white precipitate, filtering and drying to obtain 2.5 g of compound 1a with a yield of 80%,1H NMR (400 MHz, 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)；
the chemical structure of compound 1a is:
；
2) dissolving 0.500g (1.8 mmol) of the compound 1a and 1.448g (12.6 mmol) of phthalonitrile in 50ml of n-amyl alcohol, heating to 120 ℃, adding 1.318g (7.2 mmol) of zinc acetate, stirring uniformly, adding 2.189g (14.4 mmol) of DBU, wherein the solution turns green, then refluxing for 40h, then spin-drying n-amyl alcohol, adding 20ml of 20wt% sodium hydroxide aqueous solution, refluxing for 36h, adding 50ml of 20wt% hydrochloric acid aqueous solution after the solution is spin-dried to adjust the pH to 3.0, and filtering to obtain a blue solid; applying 300 mg of blue solid to a silica gel column, washing with EA: DMF =100:1 (v/v) until no zinc phthalocyanine is present, eluting with EA: DMF =20:1 (v/v), collecting 200 mg of the target product 2a with a yield of 16%,1H 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)。
the chemical structural formula of the target product 2a is as follows:
；
3) 0.633g (2.8 mmol) of 3-chloro-6-methylbenzo [ b ]]Thiophene-2-carboxylic acid, 0.327g (2.8 mmol) N-hydroxysuccinimide (NHS), 0.576g (2.8 mmol) N, N-Dicyclohexylcarbodiimide (DCC) were dissolved in 20mL DCM, reacted at room temperature for 36h and then spin-dried, passed through a neutral silica gel column using DCM as an eluent, collected to give 0.6 g white solid 3a with a yield of 55%,1h NMR (500 MHz,) δ 7.89 (d, J = 8.4 Hz, 1H), 7.65-7.63 (m, 1H), 7.37-7.33 (m, 1H), 2.91 (s, 4H), 2.53 (s, 3H), white solidThe chemical structure of body 3a is:
；
4) 100mg (0.14 mmol) of the desired product 2a, 16.1mg (0.14 mmol) of N-hydroxysuccinimide, 26.74mg (0.14 mmol) of carbodiimide (EDCI) were dissolved in 20ml of THF (tetrahydrofuran), and after reaction at room temperature for 60 hours, 81.2mg (0.70 mmol) of hexamethylenediamine was added to the solution, which was refluxed for 1 hour, the solution was dried, 50ml of water was added to the round-bottomed flask, after shaking slightly, the water was poured off, after which the solution was dried, 45.22 mg (0.14 mmol) of white solid 3a and 10ml of DMF were added to the flask, and after reaction at 110 ℃ for 2 hours, the DMF was dried, and after drying, DCM (dichloromethane): CH was used3OH (methanol) =100:1 (v/v) eluting to remove impurities, and then DCM: CH3OH =30:1 (v/v), collecting the product, repeating the column for 3 times by using the same elution, impurity removal and purification conditions to obtain 20mg of pure product zinc phthalocyanine 3-chloro-6 methylbenzo [ b]Thiophene-2-carboxylic acid conjugate (BC-Pc) in a yield of 9%,1H NMR (500 MHz, DMSO-d 6) δ 9.10 – 8.96 (m, 6H), 8.54 (d, J = 7.4 Hz, 1H), 8.30 (t, J = 5.7 Hz, 1H), 8.22 (t, J = 5.7 Hz, 1H), 8.14 – 7.93 (m, 9H), 7.81 (t, J = 8.8 Hz, 1H), 7.75 (d, J = 0.6 Hz, 1H), 7.60 (d, J = 8.3 Hz, 1H), 7.42 – 7.38 (m, 2H), 7.27 (dd, J = 8.3, 0.9 Hz, 1H), 3.18 (tt, J = 14.2, 7.2 Hz, 4H), 2.37 (s, 3H), 1.36 – 1.23 (m, 8H). HRMS (ESI): Calcd C55H39ClN10O3SZn[M+H]+: 1019.1980 , found: 1019.1950.
the chemical structural formula of the zinc phthalocyanine 3-chloro-6-methylbenzo [ b ] thiophene-2-carboxylic acid conjugate is as follows:
comparative example Synthesis of N-hexyl-4- (Zinc phthalocyanine-1-oxy) benzamide
The process scheme of N-hexyl-4- (zinc phthalocyanine-1-oxyl) benzamide (N-hexyl-4- (phthalocyanin-1-yloxy) benzamide, C6-Pc) is shown in FIG. 2. The method comprises the following specific steps:
after dissolving 100mg (0.14 mmol) of 2a, 16.1mg (0.14 mmol) of N-hydroxysuccinimide, 26.74mg (0.14 mmol) of EDCI in 10ml of THF and reacting at room temperature for 48 hours, 14.14mg (0.14 mmol) of N-hexylamine was added to the solution, refluxing for 1 hour, and spin-drying the solution with DCM: CH3OH =110:1 (v/v) to remove impurities, and DCM: CH3OH =45:1 (v/v), collecting the product, repeating the column for 3 times by using the same elution, impurity removal and purification conditions to obtain 30mg of purer product H-Pc with the yield of 30%,1H NMR (400 MHz, Chloroform-d) δ 9.15 (d, J = 44.7 Hz, 6H), 8.79 (s, 1H), 8.31 – 7.70 (m, 10H), 7.63 (s, 2H), 3.54 (s, 2H), 1.40 (s, 9H), 0.98 (s, 3H)，HRMS (ESI): Calcd C45H33N9O2Zn[M+H]+ 796.2121, found 796.2083 and C6-Pc have the following chemical structural formula:
application example 1 cytotoxicity test
The MTT method is also called colorimetric method, and is a method for detecting the survival state of cells. Succinate dehydrogenase in the mitochondria of living cells can enable exogenous MTT to be reduced into water-insoluble formazan and deposited in the cells, and dead cells can not enable MTT to be reduced into the formazan. Dimethyl sulfoxide can dissolve formazan in cells, the wavelength value of the formazan at 570nm is measured by an enzyme labeling instrument, and the number of living cells can be indirectly reflected. Within a certain range of cell number, MTT crystals are formed in an amount proportional to the cell number.
Selecting cells MDA-MB-231 and HELF with good growth state under a microscope, digesting for 3 minutes by 1ml of pancreatin (containing EDTA) when the Cell density is about 90 percent (wherein the Hela cells are digested for 1 minute), then adding 2ml of culture medium to stop digestion, blowing uniformly, counting by using a Cell counting plate, diluting the Cell solution to 6 ten thousand cells/ml by using a DMEM culture medium, adding 100 mu L of the Cell culture medium solution into each hole of a 96-well plate, setting 6 duplicate holes for each concentration of BF-Pc and H-Pc medicines, setting a Cell blank group and a solvent blank group (6 duplicate holes for each group), and placing the 96-well plate in an incubator at 37 ℃ for 24 hours.
Adding medicine: BC-Pc was diluted to 200.0, 100.0, 50.0, 25.0, 12.5, 6.2, 3.1. mu.M with 5vol% castor oil (CEL) in DMSO, C6-Pc was diluted to 25, 12.5, 6.2, 3.1, 1.6, 0.8, 0.4. mu.M with 5vol% castor oil in DMSO, respectively, and then the above 7 concentrations of drugs were diluted 100-fold with DMEM medium, the old medium in the 96 well plate was aspirated out with a drain gun, 5vol% CEL in DMSO diluted 100-fold was added to the cell control group, DMFM in the solvent control group, the remaining drug control group was added to the above prepared solution, respectively, and the 96 well plate was put in the incubator for 24 hours.
Light toxicity: taking out the 96-well plate, sucking out the medicated culture medium in the hole with a gun, washing each hole with physiological saline for 3 times to remove the medicine not taken up, adding 100 μ L culture medium into each hole, irradiating with 670nm LED lamp for 2min, and returning the irradiated plate to the incubator.
And (3) testing OD value: mu.l of 5mg/ml MTT solution was added to each well of a 96-well plate, the 96-well plate was placed in an incubator at 37 ℃ for 4 hours, and then the solution in each well was aspirated by a pipette, and 100. mu.l of DMSO solution was added to each well again, shaken on a shaker for 30 minutes, and then absorbance at 570nm was measured by a microplate reader.
And (3) calculating: the cell viability was calculated using the measured absorbance by the following formula:
cell survival (%) = (sample group absorbance-solvent blank group absorbance)/(cell blank group absorbance-solvent blank group absorbance) × 100.
FIG. 3 shows the UV-visible absorption spectrum of BC-Pc (A) and C6-Pc (B) in DMF, and it can be seen from FIG. 3 that BC-Pc and C6-Pc are both in the substantially non-aggregated state in DMF.
FIG. 4 is a graph comparing the toxicity of BC-Pc and C6-Pc in light for MDA-MB-231 cells and HELF cells. As can be seen from the figure, the introduction of 3-chloro-6-methylbenzo [ b ] thiophene-2-carboxylic acid can enhance the toxic effect of BC-Pc on tumor cells.
The above description is only a preferred embodiment of the present invention, and all modifications, equivalents, and flow changes made by using the contents of the present invention or directly or indirectly applied to other related technical fields are included in the scope of the present invention.
Claims (7)
1. A zinc phthalocyanine 3-chloro-6 methylbenzo [ b ] thiophene-2-carboxylic acid conjugate that targets the Mcl-1 enzyme, characterized in that: the chemical structural formula is as follows:
2. a method of preparing a zinc phthalocyanine 3-chloro-6 methylbenzo [ b ] thiophene-2-carboxylic acid conjugate targeting the Mcl-1 enzyme of claim 1, wherein: the method comprises the following steps:
1) 3-nitrophthalonitrile, methyl p-hydroxybenzoate and K2CO3Dissolving in DMF, reacting for 24-48 h at normal temperature, adding a certain amount of water into the reaction solution to separate out white precipitate, filtering and drying to obtain a compound 1a, wherein the chemical structural formula is as follows:
；
2) dissolving a compound 1a and phthalonitrile in n-amyl alcohol, heating to 110-130 ℃, adding a zinc source, uniformly stirring, adding DBU, carrying out reflux reaction for 36-48 h, then carrying out spin-drying on the n-amyl alcohol, adding an excessive 20wt% sodium hydroxide aqueous solution, carrying out reflux reaction for 24-48 h, adding a 20wt% hydrochloric acid aqueous solution after the solution is spin-dried, adjusting the pH to 3.0, filtering to obtain a blue solid, passing the blue solid through a silica gel column, washing until phthalocyanine is not generated by taking the volume ratio of EA-DMF as an eluent to be 100:1, eluting by taking the volume ratio of EA-DMF as 20:1, and collecting a target product 2a, wherein the chemical structural formula is as follows:
；
3) reacting 3-chloro-6-methylbenzo [ b ]]Thiophene-2-carboxylic acidDissolving acid, NHS and DCC by using DCM, reacting for 24-48 h at normal temperature, then spin-drying, passing the product through a neutral silica gel column, taking DCM as an eluent, and collecting a white solid 3a, wherein the chemical structural formula is as follows:
；
4) dissolving the products 2a, NHS and EDCI with THF, reacting at normal temperature for 48-72 h, adding a diamine compound into the solution, carrying out reflux reaction for 1h, spin-drying the solution, adding water into a round-bottom flask, shaking up, pouring off the water, spin-drying, adding a yellow solid 3a and DMF, reacting at 100-120 ℃ for 2h, passing through a silica gel column after spin-drying, and reacting with DCM-CH3Eluting OH with a volume ratio of 100:1 to remove impurities, and adding DCM-CH3Eluting OH at the volume ratio of 30:1, collecting the product, and repeatedly passing through the column for 2-3 times under the same elution, impurity removal and purification conditions to obtain a pure target product.
3. Mcl-1 enzyme targeted zinc phthalocyanine 3-chloro-6 methylbenzo [ b ] according to claim 2]A process for the preparation of a thiophene-2-carboxylic acid conjugate, characterized in that: 3-nitrophthalonitrile, methyl p-hydroxybenzoate and K used in step 1)2CO3In a molar ratio of 1:1:2, the volume ratio of water to DMF being 5: 1.
4. The method of preparing a zinc phthalocyanine 3-chloro-6 methylbenzo [ b ] thiophene-2-carboxylic acid conjugate targeting an Mcl-1 enzyme according to claim 2, wherein: in the step 2), the molar ratio of the compound 1a, the phthalonitrile, the DBU and the zinc source is 1:7:8: 4; the zinc source is zinc chloride or zinc acetate.
5. The method of preparing a zinc phthalocyanine 3-chloro-6 methylbenzo [ b ] thiophene-2-carboxylic acid conjugate targeting an Mcl-1 enzyme according to claim 2, wherein: the molar ratio of the 3-chloro-6-methylbenzo [ b ] thiophene-2-carboxylic acid, NHS and DCC used in the step 3) is 1:1: 1.
6. The method of preparing a zinc phthalocyanine 3-chloro-6 methylbenzo [ b ] thiophene-2-carboxylic acid conjugate targeting an Mcl-1 enzyme according to claim 2, wherein: the molar ratio of product 2a, NHS, EDCI, hexamethylenediamine and white solid 3a used in step 4) was 1:1:1:5: 1.
7. Use of a zinc phthalocyanine 3-chloro-6 methylbenzo [ b ] thiophene-2-carboxylic acid conjugate targeting Mcl-1 enzyme according to claim 1, wherein: is used for synthesizing photodynamic anti-tumor drugs.
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