Patent Application
20240327432
The application claims priority to Chinese patent application No. 2023103346388, filed on Mar. 31, 2023, the entire contents of which are incorporated herein by reference.
The present invention belongs to the technical field of biomedicine, and particularly relates to thiophene-phenothiazine compounds and a synthesis method thereof, as well as application thereof in preparation of phototherapy medicines.
Photodynamic therapy (PDT) has various advantages such as safety, low invasiveness, spatiotemporal accuracy, and strong controllability, and has become an effective and rapidly developing important method for treating benign and malignant diseases. A principle of PDT treatment is that: under light irradiation, a photosensitizer transfers electrons or energy to surrounding molecules through photochemistry, which interact with oxygen to generate a large quantity of highly phototoxic reactive oxygen species (ROS), leading to selective destruction and death of target cells.
At present, photosensitizers commonly used in clinical practice are mainly porphyrin-based photosensitizers which are difficult to prepare and purify, and strong in skin phototoxicity. Phenothiazine photosensitizers are a series of photosensitizers derived from a phenothiazine structure as a parent scaffold. Such photosensitizers have high molar extinction coefficient, good hydrophilicity, good selectivity, and long absorption wavelength, thereby receiving widespread attention of people. Methylene blue (MB) and toluidine blue O (TBO) are two representative phenothiazine photosensitizers that have been clinically applied (structural formulas are shown as below). Further exploration of novel photosensitizers based on the phenothiazine structure is expected to provide novel photosensitive medicines for photodynamic therapy.
In order to overcome shortcomings and deficiencies in the prior art and give full play to the advantages of the phenothiazine structure, a primary objective of the present invention is to provide thiophene-phenothiazine compounds or pharmaceutically acceptable salts thereof.
Another objective of the present invention is to provide a pharmaceutical composition containing the thiophene-phenothiazine compound.
Yet another objective of the present invention is to provide application of the thiophene-phenothiazine compounds or the pharmaceutically acceptable salts thereof in preparation of phototherapy medicines or lead compounds thereof.
The objectives of the present invention are achieved by the following solutions:
Substituted substituents, which are the same or different, are independently selected from hydrogen, halogen, hydroxyl groups, carboxyl groups, carbonyl groups, cyano groups, nitro groups, amino groups, C1-C20 alkyl groups, C1-C20 haloalkyl groups, C3-C20 ester groups, C1-C20 alkenyl groups, C1-C20 alkynyl groups, C6-C20 aryl groups, and C1-C20 hydrocarboxyl groups, respectively.
Further, the thiophene-phenothiazine compound or the pharmaceutically acceptable salt thereof, having the structural formula shown in the formula I, where R1, R2, R4, and R5, which are the same or different, are independently hydrogen, halogen, substituted or unsubstituted C1-C20 linear, branched, or cyclic alkyl chains, substituted or unsubstituted C1-C20 alkoxy groups, substituted or unsubstituted C1-C20 haloalkyl groups, substituted or unsubstituted C3-C20 alkenyl groups, substituted or unsubstituted C3-C20 alkynyl groups, substituted or unsubstituted benzyl groups, substituted or unsubstituted benzyloxy groups, substituted or unsubstituted C1-C20 acyl groups, substituted or unsubstituted C1-C20 sulfonyl groups, substituted or unsubstituted C1-C20 hydrocarboxylcarbonyl groups, substituted or unsubstituted C1-C9 alkylsilyl groups, substituted or unsubstituted phenylsilyl groups, substituted or unsubstituted C6-C20 aryl groups, respectively, or R1 and R2 and N are subjected to cyclization to form substituted or unsubstituted heterocyclic groups or heterocyclic aryl groups, or R4 and R5 and N are subjected to cyclization to form substituted or unsubstituted heterocyclic groups or heterocyclic aryl groups.
Further, the thiophene-phenothiazine compounds or the pharmaceutically acceptable salts thereof, having the structural formula shown in the formula I, where R3, R6 and R7, which are the same or different, are independently selected from hydrogen, halogen, hydroxyl groups, carboxyl groups, carbonyl groups, ester groups, cyano groups, nitro groups, amino groups, C1-C10 alkoxy groups, C1-C10 haloalkyl groups, C1-C10 alkyl groups, C1-C10 alkenyl groups, C1-C10 alkynyl groups, cyclic hydrocarbyl groups, or aryl groups.
Further, the thiophene-phenothiazine compounds or the pharmaceutically acceptable salts thereof, having the structural formula shown in the formula I, where R3, R6 and R7, which are the same or different, are selected from hydrogen, halogen, hydroxyl groups, carboxyl groups, carbonyl groups, ester groups, cyano groups, nitro groups, methoxy groups, amino groups, fluoromethyl groups, methyl groups, ethyl groups, alkenyl groups, alkynyl groups, cyclic hydrocarbyl groups or aryl groups, respectively.
Further, the substituted substituents, which are the same or different, are independently selected from hydrogen, halogen, hydroxyl groups, carboxyl groups, carbonyl groups, cyano groups, nitro groups, amino groups, methyl groups, ethyl groups, propyl groups, methoxy groups, ethoxy groups, fluoromethyl groups, cyclopropyl groups, C1-C20 alkenyl groups, C1-C20 alkynyl groups, and C6-C20 aryl groups, respectively.
Further, one or more hydrogen atoms in R1, R2, R4 and R5, which are the same or different, are substituted with halogen, hydroxyl groups, carboxyl groups, carbonyl groups, ester groups, cyano groups, nitro groups, methoxy groups, amino groups, fluoromethyl groups, methyl groups, ethyl groups, alkenyl groups, alkynyl groups, cyclic hydrocarbyl groups or aryl groups, respectively.
Further, the thiophene-phenothiazine compounds or the pharmaceutically acceptable salts thereof, having a structure shown in a following formula II:
According to the thiophene-phenothiazine compounds or the pharmaceutically acceptable salts thereof of the present invention, a thiophene structure is successfully fused to a phenothiazine framework, providing a novel molecular scaffold. The special structure exhibits excellent photodynamic activity, has strong wavelength absorption and good photostability in a near-infrared region, and can efficiently generate ROS under red light, and therefore, the thiophene-phenothiazine compound or the pharmaceutically acceptable salt thereof can be used as a photosensitizer.
The present invention further provides a pharmaceutical composition, containing the thiophene-phenothiazine compound or the pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.
Further, the excipient may include at least one of a diluent, a wetting agent, a lubricant, a filler and a preservative.
Further, the pharmaceutical composition is made into various pharmaceutical dosage forms by conventional methods, and the dosage forms include: tablets, capsules, oral liquid, oral lozenges, granules, pills, pellets, paste, suspensions, medicinal liquor, tinctures, cataplasm, drops, patches, smearing preparations, gel, sprays, aerosols, mucosal preparations and injections.
The present invention further provides application of the thiophene-phenothiazine compounds or the pharmaceutically acceptable salts thereof in preparation of phototherapy medicines or lead compounds thereof.
Compared with the prior art, the present invention has the following advantages and beneficial effects:
To describe the technical solutions in the examples of the present invention more clearly, the following briefly describes the accompanying drawings required for describing the examples. It should be understood that, the following accompanying drawings show merely some examples of the present invention, and therefore should not be regarded as a limitation on the scope. A person of ordinary skill in the art may still derive other related drawings from these accompanying drawings without creative efforts.
The present invention will be described in further detail below with reference to examples, but implementations of the present invention are not limited thereto. Materials involved in the following examples can be commercially obtained unless otherwise specified. Unless otherwise specified, all methods involved are conventional methods.
In an implementation, a thiophene-phenothiazine compound or pharmaceutically acceptable salt thereof, having a structural formula shown in
Substituted substituents, which are the same or different, are independently selected from hydrogen, halogen, hydroxyl groups, carboxyl groups, carbonyl groups, cyano groups, nitro groups, amino groups, C1-C20 alkyl groups, C1-C20 haloalkyl groups, C3-C20 ester groups, C1-C20 alkenyl groups, C1-C20 alkynyl groups, C6-C20 aryl groups, and C1-C20 hydrocarboxyl groups, respectively.
As a preferred technical solution of the present invention, the thiophene-phenothiazine compound or the pharmaceutically acceptable salt thereof, having the structural formula shown in formula I, where R1, R2, R4, and R5, which are the same or different, are independently hydrogen, halogen, substituted or unsubstituted C1-C20 linear, branched, or cyclic alkyl chains, substituted or unsubstituted C1-C20 alkoxy groups, substituted or unsubstituted C1-C20 haloalkyl groups, substituted or unsubstituted C3-C20 alkenyl groups, substituted or unsubstituted C3-C20 alkynyl groups, substituted or unsubstituted benzyl groups, substituted or unsubstituted benzyloxy groups, substituted or unsubstituted C1-C20 acyl groups, substituted or unsubstituted C1-C20 sulfonyl groups, substituted or unsubstituted C1-C20 hydrocarboxylcarbonyl groups, substituted or unsubstituted C1-C9 alkylsilyl groups, substituted or unsubstituted phenylsilyl groups, substituted or unsubstituted C6-C20 aryl groups, respectively, or R1 and R2 and N are subjected to cyclization to form substituted or unsubstituted heterocyclic groups or heterocyclic aryl groups, or R4 and R5 and N are subjected to cyclization to form substituted or unsubstituted heterocyclic groups or heterocyclic aryl groups.
As a preferred technical solution of the present invention, the thiophene-phenothiazine compound or the pharmaceutically acceptable salt thereof, having the structural formula shown in the formula I, where R3, R6 and R7, which are the same or different, are independently selected from hydrogen, halogen, hydroxyl groups, carboxyl groups, carbonyl groups, ester groups, cyano groups, nitro groups, amino groups, C1-C10 alkoxy groups, C1-C10 haloalkyl groups, C1-C10 alkyl groups, C1-C10 alkenyl groups, C1-C10 alkynyl groups, cyclic hydrocarbyl groups, or aryl groups, respectively.
As a preferred technical solution of the present invention, the thiophene-phenothiazine compound or the pharmaceutically acceptable salt thereof, having the structural formula shown in the formula I, where R3, R6 and R7 which are the same or different are selected from hydrogen, halogen, hydroxyl groups, carboxyl groups, carbonyl groups, ester groups, cyano groups, nitro groups, methoxy groups, amino groups, fluoromethyl groups, methyl groups, ethyl groups, alkenyl groups, alkynyl groups, cyclic hydrocarbyl groups or aryl groups, respectively.
As a preferred technical solution of the present invention, the substituted substituents, which are the same or different, are independently selected from hydrogen, halogen, hydroxyl groups, carboxyl groups, carbonyl groups, cyano groups, nitro groups, amino groups, methyl groups, ethyl groups, propyl groups, methoxy groups, ethoxy groups, fluoromethyl groups, cyclopropyl groups, C1-C20 alkenyl groups, C1-C20 alkynyl groups, and C6-C20 aryl groups, respectively.
As a preferred technical solution of the present invention, one or more hydrogen atoms in R1, R2, R4 and R5 which are the same or different are substituted with halogen, hydroxyl groups, carboxyl groups, carbonyl groups, ester groups, cyano groups, nitro groups, methoxy groups, amino groups, fluoromethyl groups, methyl groups, ethyl groups, alkenyl groups, alkynyl groups, cyclic hydrocarbyl groups or aryl groups, respectively.
As a preferred technical solution of the present invention, the thiophene-phenothiazine compound or the pharmaceutically acceptable salt thereof, having a structure shown in a following formula II:
As a preferred technical solution of the present invention, the thiophene-phenothiazine compound or the pharmaceutically acceptable salt thereof, having one of structures shown below:
According to the thiophene-phenothiazine compound or the pharmaceutically acceptable salt thereof of the present invention, a thiophene structure is successfully fused to a phenothiazine framework, providing a novel molecular scaffold. The special structure exhibits excellent photodynamic activity, has strong wavelength absorption and good photostability in a near-infrared region, and can efficiently generate ROS under red light, and therefore, the thiophene-phenothiazine compound or the pharmaceutically acceptable salt thereof can be used as a photosensitizer.
The present invention also provides a preparation method of the thiophene-phenothiazine compound, including obtaining the thiophene-phenothiazine compound through a reaction of raw materials of an aniline analog (compound A) and a phenylpropanethiophene analog (compound B).
The aniline analog (compound A) can be directly purchased or prepared through conventional techniques in the art, such as referring to a preparation method of CN 107501297B, specifically referring to paragraph [0061].
The phenylpropathiophene analog (compound B) can be directly purchased or prepared using conventional techniques in the art through purchased materials, as shown in compounds B1 and B2.
The present invention further provides a pharmaceutical composition, containing the thiophene-phenothiazine compound or the pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.
Further, the excipient may include at least one of a diluent, a wetting agent, a lubricant, a filler and a preservative.
Further, the pharmaceutical composition is made into various pharmaceutical dosage forms by conventional methods, and the dosage forms include: tablets, capsules, oral liquid, oral lozenges, granules, pills, pellets, paste, suspensions, medicinal liquor, tinctures, cataplasm, drops, patches, smearing preparations, gel, sprays, aerosols, mucosal preparations and injections.
The present invention further provides application of the thiophene-phenothiazine compounds or the pharmaceutically acceptable salts thereof in preparation of phototherapy medicines or lead compounds thereof.
The thiophene-phenothiazine compound of the present invention has strong phototoxicity and high phototherapeutic indexes, exhibits strong photodynamic anti-tumor activity without obvious toxic and side effects, and is expected to become a new generation of clinically applicable novel photosensitizer for preparation of the phototherapy medicines or the lead compounds thereof.
At room temperature, N,N-diethyl-p-phenylenediamine (1.0 g, 6 mmol), 0.6 mL of HCl (10 mol·L−1), and 1 mL of a ZnCl2 solution (6.3 mmol dissolved in water) were added to a mixed solution of water and methanol (12 mL, H2O/MeOH 4:1). Then a reaction mixture was cooled in an ice bath to 0° C., and a newly prepared aqueous solution of sodium thiosulfate (3 mol·L−1, 4 mL) and potassium dichromate (0.5 mol·L−1, 3.6 mL) was added. After stirring at 0° C. for 3 hours, temperature was raised to room temperature, and stirring was continued for 1 hour to obtain a thick precipitation system; and filtration and separation were performed to obtain solid, the solid was washed with a solution of water, dichloromethane, ethyl acetate and methanol (>300 mL), and then drying was performed in a vacuum drying oven to obtain gray solid 2-amino-5-(diethylamino)-benzenesulfoperoxythioic O-acid (compound A1).
4-bromobenzo[b]thiophene (2.74 g, 12.88 mmol) was added to a pressure bottle, and then Cu2O (0.37 g, 2.60 mmol), 28 mL of an ammonia aqueous solution of 28-30% (w/w), and 28 mL of N-methylpyrrolidone (NMP) were added. A mixture was heated at 110° C. in an oil bath to react for 24 hours. Then, the mixture was cooled to room temperature, filtration was performed, 100 mL of cold water was added, and extraction was performed with ethyl acetate (100 mL×3 times). An organic solution was collected, and concentration by rotary evaporation was performed to 100 mL. Washing was performed with a large amount of a NH4Cl solution and saline water (100 mL×3 times) until a washing solution was colorless, and then a remaining organic solvent was spun off. The 4-aminobenzo[b]thiophene (compound B1) was obtained by purification through silica gel column chromatography, with a solvent mixture in which a ratio of n-hexane to dichloromethane was 1:1.
The 2-amino-5-(diethylamino)-benzenesulfoperoxythioic O-acid (compound A1) (0.14 g, 0.5 mmol) and the 4-aminobenzo[b]thiophene (compound B1) (half of the molar quantity of compound A1) were stirred and refluxed in a mixed solution of methanol and ethyl acetate (6 mL, methanol/ethyl acetate=4:1), and silver carbonate (0.14 g, 0.5 mmol) was slowly added. After refluxing for 2 hours, a reaction mixture was cooled to room temperature, 2 g of diatomite was added, then filtration was performed, and filter residues were washed with methanol and dichloromethane alternatively. An organic solvent was collected, then the collected organic solvent was concentrated into blue solid, then the blue solid was dissolved in 5 mL of dichloromethane, and 0.25 mL of HCl (1 mol L−1, methanol) was added. A mixture was gently stirred and concentrated, and purifying was performed by using the silica gel column chromatography to obtain the compound I-1, with a solvent mixture in which a ratio of dichloromethane to methanol was 10:1. 1H NMR (400 MHz, Methanol-d4) δ7.84 (d, J=5.3 Hz, 1H), 7.76 (d, J=9.5 Hz, 1H), 7.71 (d, J=8.2 Hz, 1H), 7.34-7.25 (m, 1H), 7.14 (s, 1H), 6.90 (s, 1H), 3.67 (q, J=7.3 Hz, 4H), 1.38-1.28 (m, 6H). HRMS (ESI): calcd for C18H18ClN3S2[MCl]+340.09421; found, 340.09346.
(1) Synthesis of 2-Amino-5-(Diethylamino)-Benzenesulfoperoxythioic O-Acid (Compound A1) was the Same as that of Example 1.
A compound B1 (0.6 g, 4 mmol), methyl iodide (0.74 mL, 12 mmol), sodium carbonate (0.53 g, 0.5 mmol), and potassium iodide (KI, 66.4 mg) were added to N, N-dimethylformamide (DMF, 10 mL), refluxing was performed at 60° C. for 24 hours, and after reaction mixed liquid was cooled to room temperature, 20 mL of water was added. Extraction was performed with ethyl acetate (20 mL×3 times), washing was performed with saturated NH4Cl and NaCl solutions, and drying was performed with Na2SO4. After reduced-pressure rotary evaporation was performed to remove the solvent, purifying was performed by using silica gel column chromatography (with a solvent mixture in which a ratio of n-hexane to ethyl acetate was 1:10) to obtain a light yellow oily substance, namely N-methyl-4-aminobenzo[b]thiophene (compound B2).
Referring to the method in Example 1, the compound A1 was reacted with the compound B2 to prepare the thiophene-phenothiazine compound (compound I-2). 1H NMR (400 Mhz, Methanol-d4) δ7.85 (d, J=5.4 Hz, 1H), 7.81 (d, J=9.5 Hz, 1H), 7.69 (d, J=5.4 Hz, 1H), 7.33 (dd, J=9.6, 2.5 Hz, 1H), 7.19 (d, J=2.6 Hz, 1H), 7.04 (s, 1H), 3.69 (q, J=7.2 Hz, 4H), 3.19 (s, 3H), 1.34 (t, J=6.4 Hz, 6H). HRMS (ESI): calcd for C19H20ClN3S2[M-Cl]+354.10986; found, 354.10897.
(1) Synthesis of 2-Amino-5-(Diethylamino)-Benzenesulfoperoxythioic O-Acid (Compound A1) was the Same as that of Example 1.
A compound B1 (0.6 g, 4 mmol), bromopropane (0.73 mL, 8 mmol), triethylamine (1.7 mL, 12 mmol), and potassium iodide (KI, 66.4 mg) were added to N, N-dimethylformamide (DMF, 10 mL), refluxing was performed at 70° C. for 48 hours, and after reaction mixed liquid was cooled to room temperature, 20 mL of water was added. Extraction was performed with ethyl acetate (20 mL×3 times), washing was performed with saturated NH4Cl and NaCl solutions, and drying was performed with Na2SO4. After reduced-pressure rotary evaporation was performed to remove a solvent, purifying was performed by using silica gel column chromatography (with a solvent mixture in which a ratio of n-hexane to ethyl acetate was 1:10) to obtain a light yellow oily substance, namely N-propyl-4-aminobenzo[b]thiophene (compound B3).
Referring to the method in Example 1, the compound A1 was reacted with the compound B3 to prepare the thiophene-phenothiazine compound (compound I-3). 1H NMR (400 MHz, Methanol-d4) δ7.88-7.77 (m, 3H), 7.34 (dd, J=9.6, 2.6 Hz, 1H), 7.20 (d, J=2.6 Hz, 1H), 7.14 (s, 1H), 3.69 (q, J=7.5 Hz, 4H), 3.54 (t, J=7.4 Hz, 2H), 1.83 (p, J=7.5 Hz, 2H), 1.34 (t, J=7.1 Hz, 6H), 1.09 (t, J=7.4 Hz, 3H). HRMS (ESI): calcd for C21H24ClN3S2[M-Cl]+382.14116; found, 382.14007.
(1) Synthesis of 2-Amino-5-(Diethylamino)-Benzenesulfoperoxythioic O-Acid (Compound A1) was the Same as that of Example 1.
The compound B1 (0.6 g, 4 mmol), bromopentane (1 mL, 8 mmol), triethylamine (1.7 mL, 12 mmol), and potassium iodide (KI, 66.4 mg) were added to N, N-dimethylformamide (DMF, 10 mL), refluxing was performed at 70° C. for 48 hours, then the mixed liquid was cooled to room temperature, 20 mL of water was added. Extraction was performed with ethyl acetate (20 mL×3 times), washing was performed with saturated NH4Cl and NaCl solutions, and drying was performed with Na2SO4. After reduced-pressure rotary evaporation was performed to remove a solvent, purifying was performed by using silica gel column chromatography (with a solvent mixture in which a ratio of n-hexane to ethyl acetate was 1:10) to obtain a light yellow oily substance, namely N-pentyl-4-aminobenzo[b]thiophene (compound B4).
Referring to the method in Example 1, the compound A1 was reacted with the compound B4 to prepare the thiophene-phenothiazine compound (compound I-4). 1H NMR (400 MHz, Methanol-d4) δ7.90-7.79 (m, 3H), 7.35 (dd, J=9.6, 2.6 Hz, 1H), 7.22 (d, J=2.6 Hz, 1H), 7.16 (s, 1H), 3.69 (q, J=7.2 Hz, 4H), 3.58 (t, J=7.4 Hz, 2H), 1.82 (p, J=7.8 Hz, 2H), 1.47 (dd, J=8.2, 4.5 Hz, 4H), 1.34 (t, J=7.1 Hz, 6H), 0.98 (t, J=7.0 Hz, 3H). HRMS (ESI): calcd for C23H28ClN3S2[MCl]+410.17246; found, 410.17151.
(1) Synthesis of 2-Amino-5-(Diethylamino)-Benzenesulfoperoxythioic O-Acid (Compound A1) was the Same as that of Example 1.
(2) Synthesis of N-Heptyl-4-Aminobenzo [b] Thiophene (Compound B5)
The compound B1 (0.6 g, 4 mmol), bromoheptane (1.26 mL, 8 mmol), triethylamine (1.7 mL, 12 mmol), and potassium iodide (KI, 66.4 mg) were added to N, N-dimethylformamide (DMF, 10 mL), refluxing was performed at 70° C. for 48 hours, and then the mixed liquid was cooled to room temperature, 20 mL of water was added. Extraction was performed with ethyl acetate (20 mL×3 times), washing was performed with saturated NH4Cl and NaCl solutions, and drying was performed with Na2SO4. After reduced-pressure rotary evaporation was performed to remove a solvent, purifying was performed by using silica gel column chromatography (with a solvent mixture in which a ratio of n-hexane to ethyl acetate was 1:10) to obtain a light yellow oily substance, namely N-heptyl-4-aminobenzo[b]thiophene (compound B5).
Referring to the method in Example 1, the compound A1 was reacted with the compound B5 to prepare the thiophene-phenothiazine compound (compound I-5). 1H NMR (400 Mhz, Methanol-d4) δ7.88-7.77 (m, 3H), 7.34 (dd, J=9.7, 2.2 Hz, 1H), 7.20 (d, J=2.2 Hz, 1H), 7.14 (s, 1H), 3.69 (q, J=7.7, 6.9 Hz, 4H), 3.57 (t, J=7.4 Hz, 2H), 1.81 (p, J=7.8, 7.3 Hz, 2H), 1.54-1.40 (m, 4H), 1.33 (t, J=7.0 Hz, 10H), 0.92 (t, J=6.5 Hz, 3H). HRMS (ESI): calcd for C25H32ClN3S2[MCl]+438.20376; found, 438.20283.
Detection of the thiophene-phenothiazine compounds prepared in Examples 1-5: the absorption spectrum of the thiophene-phenothiazine compound in an ethanol solution was measured by using a UV-visible spectrophotometer, and spectral properties of the thiophene-phenothiazine compound were analyzed according to Beer-Lambert law; and a fluorescence emission spectrum of the thiophene-phenothiazine compound in the ethanol solution was measured under excitation of light with a wavelength of 650 nm, and results are shown in Table 1 and
As shown in
As shown in
Good photostability is a prerequisite for ensuring continuous generation of ROS by the photosensitizers under light excitation, which can effectively prevent decomposition of the photosensitizers after illumination, avoiding a decrease in a ROS generation rate with increasing illumination time. An LED light source (630 nm, 106 mW cm−2) was used to continuously irradiate the compound (5 μM), and a UV-visible spectrophotometer was used to measure the absorption spectrum of the compound at 200-800 nm. The photostability of the compound was evaluated by comparing changes in maximum absorption values at 600-800 nm after different irradiation times.
(1) Cell culture: HUVEC cells (human umbilical vein endothelial cells), A549 cells (human lung cancer cells), MCF-7 cells (human breast cancer cells), HeLa cells (human cervical cancer cells) and 4T1 cells (mouse breast cancer cells) were cultured in a DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37° C. and under a condition of a 5% carbon dioxide content. The HUVEC cells, the A549 cells, the MCF-7 cells, the HeLa cells and the 4T1 cells, at a logarithmic growth phase, were taken and inoculated into a 96-well plate, respectively, with approximately 1×104 cells per well, and incubation was performed overnight at 37° C. and under a condition of 5% CO2.
(2) Dark toxicity measurement: the cells were incubated in a dark environment with the compound of the present invention of different concentrations for 24 hours, and cell viability was measured by a CCK-8 method.
(3) Phototoxicity measurement: the cells were incubated in the dark with the compound of the present invention of different concentrations for 1 hour, rinsing was performed with PBS, a fresh culture solution was added, and the cells were irradiated with 630 nm LED light (light dose is 3.2 J cm−2). Then the cells were incubated in the dark environment for 24 hours, and cell viability was measured by the CCK-8 method.
Test results are shown in Table 2-Table 3 and
From
As shown in Tables 2-3, the thiophene-phenothiazine compound of the present invention had excellent photodynamic activity. Where a ratio of the phototoxicity to the dark toxicity is a phototherapeutic index (PI), and the higher the phototherapeutic index, the higher a safety threshold of the photosensitizer; in 4T1 cells, the phototoxicity of the compound of the present invention can reach 74.35 times that of MB by comparing an IC50 value of the compound with that of MB, while a PI value of the compound can reach 24.46 times that of MB. Therefore, the compound of the present invention has a significantly enhanced therapeutic effect compared with MB.
In the HUVEC cells, the dark toxicity IC50 value of the thiophene-phenothiazine compound of the present invention is lower than that in tumor cells, indicating that the thiophene-phenothiazine compound of the present invention has higher selectivity for the tumor cells.
The efficiency of producing singlet oxygen through photosensitization is an important index for evaluating the photosensitizers. In the example, a conventional steady-state method was used, 1,3-diphenylisobenzofuran (DPBF) was used as a scavenger, which irreversibly reacted with the singlet oxygen to cause attenuation of the DPBF. An attenuation rate of the DPBF reflects production efficiency of 1O2. Concentrations of the DPBF in EtOH at multiple irradiation time periods were measured by using a UV/Vis spectrum method. A medicine solution (2 μM in EtOH) was irradiated with a 630 nm LED lamp (106 Mw/cm2), and results were shown in
Dichlorodihydrofluorescein diacetate (DCFH-DA) was used as a scavenger, production of intracellular ROS was detected by a flow cytometer to detect the production efficiency of intracellular reactive oxygen species (ROS) of the thiophene-phenothiazine compounds of the present invention. The results were shown in
7-week-old BALB/c female mice weighing 18 g±1 g (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd) were used to construct a subcutaneous breast cancer (4T1) tumor model. When tumor volumes of the BALB/c mice were approximately 100 mm3, in vivo anti-tumor experiments began. When the tumor volumes of the BALB/c mice were approximately 200 mm3, imaging experiments began.
A solution of 10 mg/L was prepared from the compound I-3 of the present invention by using 1×PBS. The BALB/c mice with the tumor volumes of approximately 200 mm3 were taken, and the solution was injected into tumor bearing mice via tail veins at a dose of 1.6 mg/kg. Imaging photos were taken at different times using Odyssey CLX, with excitation light of 680 nm and detection light of 720 nm. The results were shown in
The BALB/c mice with the tumor volumes of approximately 100 mm3 were taken, and the tumor bearing mice were divided into 4 groups, namely groups A, B, C and D, with 5 mice in each group. The mice in the group A and the group B were injected with 1×PBS 100 μL via tail veins. In addition, the mice in the group C and the group D were injected with compound I-3 via the tail veins at a dose of 1.6 mg/kg; and after 2 hours, the mice in the group B and the group D were irradiated with a light source (655 nm, 300 mW cm2) for 10 minutes. Body weights and tumor volumes of the mice were measured on the 2nd day, 4th day, 8th day, 12th day, 16th day, and 21st day, respectively. On the 21st day, approximately 0.8 mL of mouse blood was collected using an eyeball blood sampling method, of which 0.4 mL of the mouse blood was placed in an EDTA-2K anticoagulant tube, and the remaining 0.4 mL of the mouse blood was centrifuged for serum collection.
As shown in the figure, compared with those in the group A (PBS), the group B (PBS+irradiation), and the group C (compound I-3 alone), growth of tumor volumes in the group D (compound I-3+light irradiation) was significantly inhibited, and the tumor volumes in the mice showed a gradually decreasing trend, while the group of the compound I-3 alone did not show tumor growth inhibition. Therefore, the thiophene-phenothiazine compound of the present invention exhibits enormous potential for photodynamic tumor therapy.
Detection of body weight was performed on the tumor bearing mice in four groups within 21 days, and all results showed a similar increasing trend, indicating that the biological toxicity of the thiophene-phenothiazine compound of the present invention is relatively low. After 21 days of various treatments, blood biochemical analysis was performed on the mice. No significant abnormalities were found in serum, alanine aminotransferase (ALT), blood urea nitrogen (BUN), aspartate aminotransferase (AST), and uric acid (UA) of the mice, and liver and kidney function damage was not significant. Besides, through H&E staining analysis of main organs, normal cell morphology was shown between different groups, indicating that there was no significant toxic damage to internal tissue of the mice. Therefore, the thiophene-phenothiazine compound of the present invention has achieved the effect of successfully inhibiting tumor growth with ultra-low side effects.
The above examples are preferred implementations of the present invention. However, the implementations of the present invention are not limited by the above examples. Any change, modification, replacement combination, and simplification made without departing from the spiritual essence and principle of the present invention should be an equivalent replacement manner, and all are included in the protection scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2023103346388 | Mar 2023 | CN | national |
