I-type two-photon photosensitizer without heavy atoms and with aggregation-induced emission capability as well as preparation method and application of I-type two-photon photosensitizer

Abstract

The invention discloses a heavy-atom-free I-type two-photon photosensitizer with aggregation-induced emission capability as well as a preparation method and application of the I-type two-photon photosensitizer. The I-type two-photon photosensitizer provided by the invention can realize single-photon or two-photon fluorescence imaging. Meanwhile, superoxide anions and hydroxyl radicals can be generated after single-photon or two-photon laser irradiation, tumor cells can be effectively killed under the condition of normal oxygen or oxygen deficiency, and dark toxicity is low. Therefore, the I-type two-photon photosensitizer can be used for preparing tumor imaging reagents and antitumor drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a type-I two-photon photosensitizer without heavy atoms and having aggregation-induced emission ability, and a preparation method and application thereof, especially its pharmaceutical use for single-photon or two-photon fluorescence imaging and photodynamic therapy, and its application in the preparation of tumor treatment drugs. Background Art

[0002] As a non-invasive treatment method, photodynamic therapy (PDT) has become an alternative technology for cancer treatment. In PDT, a non-toxic photosensitizer (PS) generates reactive oxygen species (ROS) under the action of light and oxygen molecules, destroying the proteins, nucleic acids, organelles, etc. of cells, thereby killing tumors. Compared with traditional therapies such as surgery, chemotherapy (CT), and radiotherapy (RT), PDT has the advantages of high selectivity for tumor destruction, non-invasiveness, and no obvious drug resistance. It should be noted that the PS absorbs a photon with an appropriate wavelength and transitions from its ground state (S0) to the first singlet excited state (S1) with a lifetime of nanoseconds, and then undergoes intersystem crossing (ISC) to form a stable excited triplet state (T1) with a lifetime of microseconds. T1 usually undergoes a series of photochemical reactions to transfer energy to O2 to generate highly reactive 1 O2 (type-II PDT). Due to the relatively low excitation energy for forming 1 O2, most of the reactions in PDT are mainly type-II reactions. However, the oxygen-dependent type-II PDT still has an "Achilles' heel", that is, it aggravates hypoxia. Different from type-II PDT, T1 can also react with intracellular substrates such as proteins and lipids through an electron transfer mechanism to form free radicals, such as OH· and O2· - , which is non-oxygen-dependent type-I PDT. Therefore, it is of great significance to develop type-I photosensitizers for improving the treatment effect of hypoxic tumors.

[0003] Although photosensitizers based on porphyrin and phthalocyanine (Pc) have achieved clinical success, most of them are type-II photosensitizers, and their main absorption bands are located in the ultraviolet-visible (UV-vis) range, with poor light penetration ability, so their clinical applications are limited, and PDT is only applied to superficial tissues such as bladder cancer, esophageal cancer, and skin diseases. Therefore, a large number of near-infrared (NIR) light-triggered PSs have been developed because the penetration depth of near-infrared light is greater than that of UV-vis and the phototoxicity to normal tissues is lower.

[0004] Currently, most organic photosensitizers (PS) with strong absorbance in the PDT optical window (700 - 1000 nm) usually exhibit an aggregated quenched state due to their hydrophobicity, resulting in a decrease in fluorescence quantum yield and ROS generation in aqueous solutions. To overcome this limitation, two - photon excitation (TPE) - based PDT technology has gradually developed, with better light penetration ability to destroy deeper tumors, providing more precise phototherapy and higher spatial resolution for fluorescence imaging.

[0005] As a promising therapy, TPE PDT requires PS to have a large two - photon absorption cross - section (TPA) and a high ROS generation efficiency. Currently, developed two - photon photosensitizers mainly include metal complexes based on Ir(III) and Ru(II), as well as triphenylamine derivatives. Although metal complexes have shown good anti - tumor activity, PS containing heavy metal atoms cannot be widely used clinically due to their high intrinsic cytotoxicity and poor degradation. Moreover, currently available two - photon photosensitizers mainly generate 1 O2, playing a type II PDT mechanism, while there are fewer two - photon photosensitizers for type I PDT.

[0006] Therefore, the development of heavy - atom - free type I two - photon photosensitizers is of great significance for the practical application and clinical translation of future PS. Summary of the Invention

[0007] Object of the Invention: The object of the present invention is to provide a heavy - atom - free type I two - photon photosensitizer with aggregation - induced emission ability, its preparation method and application.

[0008] The present invention has developed a heavy - atom - free type I photosensitizer based on triphenylamine, which can be used for two - photon PDT of cancer. The introduction of the triphenylamine skeleton endows the type I photosensitizer with aggregation - induced emission properties, increasing the brightness of the photosensitizer in the aggregated state and having a more efficient ROS generation efficiency. Under irradiation at 520 nm and 808 nm, the type I photosensitizer generates O2 ·- and ·OH through a type I photochemical process. Therefore, the type I photosensitizer can also generate destructive ROS under hypoxic conditions, overcoming the problem of endogenous cell hypoxia. As one of the effective PS working under hypoxic conditions, it will expand the clinical translation perspective of PS in cancer treatment.

[0009] Technical Solution: The object of the present invention is achieved by the following technical solutions:

[0010] The present invention provides a class of heavy - atom - free type I two - photon photosensitizers with aggregation - induced emission ability having the structure shown in General Formula I:

[0011]

[0012] Wherein,

[0013] Each R is independently selected from one of H, halogen, hydroxyl, nitro, alkyl, alkoxy, alkylthio, alkenyl, alkynyl, cycloalkyl, cycloalkyloxy, cycloalkylthio, acyl, ester, amide, aryl, heterocyclic group, heteroaryl, heterocycloalkyl, monoalkylamino or dialkylamino;

[0014] n = 1 or 2.

[0015] A preferred embodiment of the present invention is that R is selected from H, -CH3, -F, -Cl, -Br, -I, -OH, tert-butyl or methoxy.

[0016] A preferred embodiment of the present invention is that the type I two-photon photosensitizer preferably has the following compounds, and their structural formulas are shown in Table 1:

[0017] Table 1 Compound codes and corresponding structures of general formula I

[0018]

[0019]

[0020] I1: (E)-4-(5-((2,2-difluoro-5-(4-fluorophenyl)-2,3-dihydro-1,3λ 4 ,4,2λ 4 -oxadiazol-1-ylidene)methyl)thiophen-2-yl)-N,N-diphenylaniline;

[0021] I2: (E)-4-(5-((5-(4-chlorophenyl)-2,2-difluoro-2,3-dihydro-1,3λ 4 ,4,2λ 4 -oxadiazol-1-ylidene)methyl)thiophen-2-yl)-N,N-diphenylaniline;

[0022] I3: (E)-4-(5-((5-(4-bromophenyl)-2,2-difluoro-2,3-dihydro-1,3λ 4 ,4,2λ 4 -oxadiazol-1-ylidene)methyl)thiophen-2-yl)-N,N-diphenylaniline;

[0023] I4: (E)-4-(5-((2,2-difluoro-5-(4-iodophenyl)-2,3-dihydro-1,3λ 4 ,4,2λ 4 -oxadiazol-1-ylidene)methyl)thiophen-2-yl)-N,N-diphenylaniline;

[0024] I5: (E)-4-(5-((2,2-difluoro-5-phenyl-2,3-dihydro-1,3λ 4 ,4,2λ 4 -diazoxol-1-ylidene)methyl)thiophen-2-yl)-N,N-diphenylamine;

[0025] I6: (E)-4-(5-((2,2-difluoro-5-(p-tolyl)-2,3-dihydro-1,3λ 4 ,4,2λ 4 -diazoxol-1-ylidene)methyl)thiophen-2-yl)-N,N-diphenylaniline;

[0026] I7: (E)-4-(5-((5-(4-(tert-butyl)phenyl)-2,2-difluoro-2,3-dihydro-1,3λ 4 ,4,2λ 4 -diazoxol-1-ylidene)methyl)thiophen-2-yl)-N,N-diphenylaniline;

[0027] I8: (E)-4-(3-((5-(4-(diphenylamino)phenyl)thiophen-2-yl)methylene)-2,2-difluoro-2,3-dihydro-1,3λ 4 ,4,2λ 4 -diazoxol-1-yl)phenol;

[0028] I9: (E)-4-(5-((5-(3,4-dichlorophenyl)-2,2-difluoro-2,3-dihydro-1,3λ 4 ,4,2λ 4 -diazoxol-1-ylidene)methyl)thiophen-2-yl)-N,N-diphenylaniline;

[0029] I 10 : (E)-4-(5-((2,2-difluoro-5-(4-methoxyphenyl)-2,3-dihydro-1,3λ 4 ,4,2λ 4 -diazoxol-1-ylidene)methyl)thiophen-2-yl)-N,N-diphenylaniline;

[0030] I 11 : (E)-4-(5-((5-(3,5-dimethoxyphenyl)-2,2-difluoro-2,3-dihydro-1,3λ 4 ,4,2λ 4 -diazoxol-1-ylidene)methyl)thiophen-2-yl)-N,N-diphenylaniline;

[0031] I 12:(E)-4-(5-((5-(2,6-difluorophenyl)-2,2-difluoro-2,3-dihydro-1,3λ 4 ,4,2λ 4 -oxadiazol-1-ylidene)methyl)thiophen-2-yl)-N,N-diphenylaniline.

[0032] The present invention also provides a preparation method of a type I two-photon photosensitizer represented by general formula I, comprising the following steps:

[0033]

[0034] (1) Condense 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde with benzohydrazide containing different substituents R under heating conditions;

[0035] (2) The condensation product is then complexed with boron trifluoride diethyl ether solution to obtain a type I two-photon photosensitizer represented by general formula I;

[0036] wherein, R is as defined in general formula I.

[0037] Preferably, in step (1), 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde is refluxed overnight with benzohydrazide containing different substituents R in anhydrous acetonitrile or ethanol until the reaction is complete, and the solid is obtained by filtration, which is the condensation product; wherein, R is as defined in general formula I.

[0038] Preferably, in step (2), the condensation product and boron trifluoride diethyl ether solution are refluxed overnight in toluene until the reaction ends, and a type I two-photon photosensitizer represented by general formula I is obtained by recrystallization.

[0039] The compounds of general formula I of the present invention can all be prepared by the above or similar preparation methods, and the corresponding starting materials can be selected according to the different substituents and the different positions of the substituents. Those skilled in the art should recognize that the above route helps to understand the present invention, but does not limit the content of the present invention. Unless otherwise specified, the variables are defined as mentioned in general formula I.

[0040] The present invention also provides a pharmaceutical composition, which comprises a type I two-photon photosensitizer of general formula I and a pharmaceutically acceptable carrier or excipient.

[0041] The pharmaceutical compositions of the present invention can be administered in various known ways, such as orally, parenterally, or via an implanted reservoir. The pharmaceutical compositions of the present invention can be administered alone or in combination with other drugs. Oral compositions can be any orally acceptable dosage form, including but not limited to tablets, capsules, emulsions, and suspensions, dispersions, and solutions. Commonly used pharmaceutically acceptable carriers or excipients include stabilizers, diluents, surfactants, lubricants, antioxidants, binders, colorants, fillers, emulsifiers, etc.

[0042] Sterile injectable compositions can be formulated using suitable dispersing or wetting agents and suspending agents according to techniques known in the art. Pharmaceutically acceptable carriers and solvents that can be used include water, mannitol, sodium chloride solution, etc.

[0043] The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration and that is non-toxic to the patient. The selected dosage level depends on a variety of factors, including the activity of the specific compound of the present invention employed, the route of administration, the time of administration, the excretion rate of the specific composition employed, the duration of the treatment, other drugs, compounds, and / or materials used in combination with the specific composition employed, the age, sex, weight, general health status, and medical history of the patient being treated, and similar factors well known in the medical arts.

[0044] The present invention also provides the use of the type I two-photon photosensitizer described above in the preparation of tumor imaging reagents.

[0045] The type I two-photon photosensitizer enables single-photon or two-photon fluorescence imaging in vitro and in vivo.

[0046] The present invention also provides the use of the type I two-photon photosensitizer described above in the preparation of anti-tumor drugs.

[0047] The type I two-photon photosensitizer has a photodynamic therapy effect and can generate superoxide anions and hydroxyl radicals after single-photon or two-photon laser irradiation.

[0048] The type I two-photon photosensitizer has low dark toxicity and can effectively ablate tumor cells after single-photon or two-photon laser irradiation under normoxic or hypoxic conditions.

[0049] Beneficial effects:

[0050] The type-I two-photon photosensitizer provided by the present invention has a high two-photon absorption cross-section, can achieve single-photon or two-photon fluorescence imaging, and has a high tissue penetration depth. At the same time, it can effectively generate superoxide anions and hydroxyl radicals after single-photon or two-photon laser irradiation, can effectively kill tumor cells under both normoxic and hypoxic conditions, and has low dark toxicity. Therefore, the type-I two-photon photosensitizer of the present invention can be used to prepare tumor imaging reagents and anti-tumor drugs, and has broad application prospects in the treatment of deep hypoxic tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 It is the fluorescence spectrogram of the compound (10 μM) of the present invention in water / DMSO solutions with different ratios (0%, 99%);

[0052] Among them, Figure 1 A is the fluorescence spectrum of compound I1 in water / DMSO solutions with different ratios (0%, 99%); Figure 1 B is the fluorescence spectrum of compound I2 in water / DMSO solutions with different ratios (0%, 99%); Figure 1 C is the fluorescence spectrum of compound I3 in water / DMSO solutions with different ratios (0%, 99%); Figure 1 D is the fluorescence spectrum of compound I9 in water / DMSO solutions with different ratios (0%, 99%); Figure 1 E is the fluorescence spectrum of compound I 12 in water / DMSO solutions with different ratios (0%, 99%).

[0053] Figure 2 It is the two-photon fluorescence emission spectrogram of the compound (10 μM) of the present invention in THF solution under the powers of 200 mW and 500 mW of 808 nm laser;

[0054] Among them, Figure 2 A is the two-photon fluorescence spectrum of compound I1 in THF solution; Figure 2 B is the two-photon fluorescence spectrum of compound I2 in THF solution; Figure 2 C is the two-photon fluorescence spectrum of compound I3 in THF solution; Figure 2 D is the two-photon fluorescence spectrum of compound I9 in THF solution; Figure 2 E is the two-photon fluorescence spectrum of compound I 12 in THF solution; the excitation wavelength is 808 nm (200 mW cm -2 / 500 mW cm -2 ).

[0055] Figure 3Results of the total amount and types of ROS generated after irradiation of Compound I1 (10 μM) with 520 nm and 808 nm light

[0056] Among them, Figure 3 A is the time-dependent change in the fluorescence intensity (I - I0) of DCFH (5 μm) at 523 nm after irradiation of Compound I1 with 520 nm light Figure 3 B is the time-dependent change in the fluorescence intensity (I - I0) of DHR123 (5 μm) at 520 nm after irradiation of Compound I1 with 520 nm light Figure 3 C is the time-dependent change in the fluorescence intensity (I - I0) of HPF (5 μm) at 515 nm after irradiation of Compound I1 with 520 nm light Figure 3 D is the time-dependent change in the fluorescence intensity (I - I0) of SOSG (1 μm) at 520 nm after irradiation of Compound I1 with 520 nm light Figure 3 E is the time-dependent change in the fluorescence intensity (I - I0) of DCFH (5 μm) at 523 nm after irradiation of Compound I1 with 808 nm light Figure 3 F is the time-dependent change in the fluorescence intensity (I - I0) of DHR123 (5 μm) at 520 nm after irradiation of Compound I1 with 808 nm light Figure 3 G is the time-dependent change in the fluorescence intensity (I - I0) of HPF (5 μm) at 515 nm after irradiation of Compound I1 with 808 nm light Figure 3 H is the time-dependent change in the fluorescence intensity (I - I0) of SOSG (1 μm) at 520 nm after irradiation of Compound I1 with 808 nm light

[0057] Figure 4 For Compound I2, I3, I9, I 12 Production of superoxide anions and hydroxyl radicals after irradiation with 808 nm light

[0058] Among them, Figure 4 A is the time-dependent fluorescence spectrum of DHR123 (5 μm) after irradiation of Compound I2 with 808 nm light Figure 4 B is the time-dependent fluorescence spectrum of DHR123 (5 μm) after irradiation of Compound I3 with 808 nm light Figure 4 C is the time-dependent fluorescence spectrum of DHR123 (5 μm) after irradiation of Compound I9 with 808 nm light Figure 4 D is for Compound I 12 Time-dependent fluorescence spectrum of DHR123 (5 μm) after irradiation with 808 nm light Figure 4 E is the time-dependent fluorescence spectrum of HPF (5 μm) after irradiation of Compound I2 with 808 nm light Figure 4F is the time-dependent fluorescence spectrum of HPF (5 μm) after compound I3 is irradiated with 808 nm light; Figure 4 G is the time-dependent fluorescence spectrum of HPF (5 μm) after compound I9 is irradiated with 808 nm light; Figure 4 H is the time-dependent fluorescence spectrum of compound I 12 after irradiation with 808 nm light; the compound concentration is 10 μm.

[0059] Figure 5 is the result of evaluating the dark toxicity of compound I1 by the MTT method and the photodynamic therapy under normoxia and hypoxia;

[0060] Among them, Figure 5 A is the viability of LO2 cells incubated with different concentrations of I1 detected by MTT assay under light protection; Figure 5 B is the viability of 4T1 cells incubated with different concentrations of I1 under normoxia or hypoxia detected by MTT assay under laser irradiation at 808 nm (500 mW cm -2 , 5 min). Specific Embodiments

[0061] The technical solutions of the present invention will be described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the described embodiments.

[0062] For those without specific techniques or conditions indicated in the examples, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For those reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0063] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

[0064] Example 1 Preparation of (E)-4-(5-((2,2-difluoro-5-(4-fluorophenyl)-2,3-dihydro-1,3λ 4 ,4,2λ 4 -oxadiazol-1-ylidene)methyl)thiophen-2-yl)-N,N-diphenylaniline (I1)

[0065]

[0066] 5-(4-(Diphenylamino)phenyl)thiophene-2-carbaldehyde (355 mg, 1 mmol) and 4-fluorobenzohydrazide (154 mg, 1 mmol) were dissolved in anhydrous acetonitrile, refluxed overnight until the reaction was complete, cooled, and recrystallized with ethanol. After filtration, washing, and vacuum drying, it was redissolved in anhydrous toluene, 1 mL of boron trifluoride diethyl ether (47.0%) solution was added, and the mixture was refluxed overnight until the reaction ended. After cooling and recrystallization with ethanol, 368 mg of orange-red solid (I1) was obtained by purification, with a yield of 68.3%.

[0067] ESI-MS (m / z): 540 [M+H] + ;

[0068] 1 1H NMR (400 MHz, CDCl3) δ (ppm) 8.25 - 8.20 (m, 2H), 8.08 (s, 1H), 7.80 (d, J = 4.2 Hz, 1H), 7.67 - 7.60 (m, 2H), 7.42 - 7.32 (m, 5H), 7.26 - 7.12 (m, 10H).

[0069] Example 2 Preparation of (E)-4-(5-((5-(4-chlorophenyl)-2,2-difluoro-2,3-dihydro-1,3λ 4 ,4,2λ 4 -oxadiazol-1-ylidene)methyl)thiophen-2-yl)-N,N-diphenylaniline (I2)

[0070] Referring to the synthesis method of (I1) in Example 1, 4-chlorobenzohydrazide was used instead of 4-fluorobenzohydrazide in the method, and finally 321 mg of orange solid (I2) was obtained, with a yield of 57.8%.

[0071] ESI-MS (m / z): 556 [M+H] + ;

[0072] 1 1H NMR (400 MHz, CDCl3) δ (ppm) 8.28 - 8.21 (m, 2H), 8.09 (s, 1H), 7.81 (d, J = 4.2 Hz, 1H), 7.68 - 7.62 (m, 2H), 7.42 - 7.34 (m, 6H), 7.27 - 7.13 (m, 9H).

[0073] Example 3 Preparation of (E)-4-(5-((5-(4-bromophenyl)-2,2-difluoro-2,3-dihydro-1,3λ 4 ,4,2λ 4 -oxadiazol-1-ylidene)methyl)thiophen-2-yl)-N,N-diphenylaniline (I3)

[0074] Referring to the synthesis method of (I1) in Example 1, replace p-fluorobenzohydrazide in the method with p-bromobenzohydrazide, and finally obtain 410 mg of red solid (I3) with a yield of 68.3%.

[0075] ESI-MS (m / z): 601 [M+H] + ;

[0076] 1 H NMR (400 MHz, CDCl3) δ (ppm) 8.28 - 8.20 (m, 2H), 8.07 (s, 1H), 7.79 (d, J = 4.2 Hz, 1H), 7.66 - 7.61 (m, 2H), 7.42 - 7.33 (m, 5H), 7.26 - 7.11 (m, 10H).

[0077] Example 4 Preparation of (E)-4-(5-((2,2-difluoro-5-(4-iodophenyl)-2,3-dihydro-1,3λ 4 ,4,2λ 4 -dioxadiazol-1-ylidene)methyl)thiophen-2-yl)-N,N-diphenylaniline (I4)

[0078] Referring to the synthesis method of (I1) in Example 1, replace p-fluorobenzohydrazide in the method with p-iodobenzohydrazide, and finally obtain 420 mg of red solid (I4) with a yield of 64.9%.

[0079] ESI-MS (m / z): 648 [M+H] + ;

[0080] 1 H NMR (400 MHz, CDCl3) δ (ppm) 8.26 - 8.19 (m, 2H), 8.06 (s, 1H), 7.80 (d, J = 4.2 Hz, 1H), 7.67 - 7.62 (m, 2H), 7.40 - 7.32 (m, 5H), 7.25 - 7.10 (m, 10H).

[0081] Example 5 Preparation of (E)-4-(5-((2,2-difluoro-5-phenyl-2,3-dihydro-1,3λ 4 ,4,2λ 4 -dioxadiazol-1-ylidene)methyl)thiophen-2-yl)-N,N-dianiline (I5)

[0082] Referring to the synthesis method of (I1) in Example 1, replace p-fluorobenzohydrazide in the method with benzohydrazide, and finally obtain 370 mg of orange solid (I5) with a yield of 71.0%.

[0083] ESI-MS (m / z): 522 [M+H] + 。

[0084] 1 H NMR (400 MHz, CDCl3) δ (ppm) 8.24 - 8.16 (m, 2H), 8.02 - 7.84 (m, 3H), 7.66 - 7.60 (m, 2H), 7.38 - 7.09 (m, 15H).

[0085] Example 6 Preparation of (E)-4-(5-((2,2-difluoro-5-(p-tolyl)-2,3-dihydro-1,3λ 4 ,4,2λ 4 -oxadiazol-1-ylidene)methyl)thiophen-2-yl)-N,N-diphenylaniline (I6)

[0086] Referring to the synthesis method of (I1) in Example 1, p-methylbenzohydrazide was used instead of p-fluorobenzohydrazide in the method, and finally 440 mg of orange solid (I6) was obtained with a yield of 82.2%.

[0087] ESI-MS (m / z): 536 [M+H] + 。

[0088] 1 H NMR (400 MHz, CDCl3) δ (ppm) 8.26 - 8.20 (m, 2H), 8.08 (s, 1H), 7.80 - 7.76 (m, 1H), 7.68 - 7.62 (m, 2H), 7.40 - 7.32 (m, 6H), 7.25 - 7.10 (m, 9H), 2.40 (s, 3H).

[0089] Example 7 Preparation of (E)-4-(5-((5-(4-(tert-butyl)phenyl)-2,2-difluoro-2,3-dihydro-1,3λ 4 ,4,2λ 4 -oxadiazol-1-ylidene)methyl)thiophen-2-yl)-N,N-diphenylaniline (I7)

[0090] Referring to the synthesis method of (I1) in Example 1, p-tert-butylbenzohydrazide was used instead of p-fluorobenzohydrazide in the method, and finally 460 mg of orange solid (I7) was obtained with a yield of 79.7%.

[0091] ESI-MS (m / z): 578 [M+H] + 。

[0092] 11H NMR (400 MHz, CDCl3) δ (ppm) 8.24 - 8.18 (m, 2H), 8.05 (s, 1H), 7.80 - 7.75 (m, 1H), 7.65 - 7.60 (m, 2H), 7.38 - 7.31 (m, 5H), 7.24 - 7.08 (m, 10H), 1.35 (s, 9H).

[0093] Example 8 Preparation of (E)-4-(3-((5-(4-(diphenylamino)phenyl)thiophen-2-yl)methylene)-2,2-difluoro-2,3-dihydro-1,3λ 4 ,4,2λ 4 -oxadiazol-1-yl)phenol (I8)

[0094] Referring to the synthesis method of (I1) in Example 1, p-hydroxybenzohydrazide was used instead of p-fluorobenzohydrazide in the method, and finally 415 mg of orange solid (I8) was obtained with a yield of 77.1%.

[0095] ESI-MS (m / z): 538 [M + H] + .

[0096] 1 1H NMR (400 MHz, CDCl3) δ (ppm) 9.68 (s, 1H), 8.20 - 8.14 (m, 2H), 8.02 (s, 1H), 7.78 - 7.72 (m, 1H), 7.62 - 7.58 (m, 2H), 7.34 - 7.28 (m, 6H), 7.21 - 7.03 (m, 9H).

[0097] Example 9 Preparation of (E)-4-(5-((5-(3,4-dichlorophenyl)-2,2-difluoro-2,3-dihydro-1,3λ 4 ,4,2λ 4 -oxadiazol-1-ylidene)methyl)thiophen-2-yl)-N,N-diphenylaniline (I9)

[0098] Referring to the synthesis method of (I1) in Example 1, 3,4-dichlorobenzohydrazide was used instead of p-fluorobenzohydrazide in the method, and finally 450 mg of orange solid (I9) was obtained with a yield of 76.3%.

[0099] ESI-MS (m / z): 590 [M + H] + .

[0100] 11H NMR (400 MHz, CDCl3) δ (ppm) 8.26 - 8.20 (m, 2H), 8.08 (s, 1H), 7.78 - 7.58 (m, 4H), 7.36 - 7.28 (m, 5H), 7.20 - 7.06 (m, 9H).

[0101] Example 10 Preparation of (E)-4-(5-((2,2-difluoro-5-(4-methoxyphenyl)-2,3-dihydro-1,3λ 4 ,4,2λ 4 -oxadiazol-1-ylidene)methyl)thiophen-2-yl)-N,N-diphenylaniline (I 10 )

[0102] Referring to the synthesis method of (I1) in Example 1, 4-methoxybenzohydrazide was used instead of p-fluorobenzohydrazide in the method, and finally 445 mg of orange solid (I 10 ) was obtained with a yield of 80.6%.

[0103] ESI-MS (m / z): 552 [M + H] + .

[0104] 1 1H NMR (400 MHz, CDCl3) δ (ppm) 8.29 - 8.22 (m, 2H), 8.07 (s, 1H), 7.80 - 7.60 (m, 4H), 7.39 - 7.28 (m, 6H), 7.24 - 7.09 (m, 9H), 3.92 (s, 3H).

[0105] Example 11 Preparation of (E)-4-(5-((5-(3,5-dimethoxyphenyl)-2,2-difluoro-2,3-dihydro-1,3λ 4 ,4,2λ 4 -oxadiazol-1-ylidene)methyl)thiophen-2-yl)-N,N-diphenylaniline (I 11 )

[0106] Referring to the synthesis method of (I1) in Example 1, 3,5-dimethoxybenzohydrazide was used instead of p-fluorobenzohydrazide in the method, and finally 460 mg of orange solid (I 11 ) was obtained with a yield of 79.2%.

[0107] ESI-MS (m / z): 582 [M + H] + .

[0108] 11H NMR (400 MHz, CDCl3) δ (ppm) 8.22 - 8.16 (m, 2H), 8.05 (s, 1H), 7.82 - 7.64 (m, 3H), 7.38 - 7.28 (m, 5H), 7.26 - 7.08 (m, 9H), 6.62 (s, 1H), 3.82 (s, 6H).

[0109] Example 12 Preparation of (E)-4-(5-((5-(2,6-difluorophenyl)-2,2-difluoro-2,3-dihydro-1,3λ 4 ,4,2λ 4 -oxadiazol-1-yl)methyl)thiophen-2-yl)-N,N-diphenylaniline (I 12 )

[0110] Referring to the synthesis method of (I1) in Example 1, replace p-fluorobenzoyl hydrazide in the method with 2,6-difluorobenzoyl hydrazide, and finally obtain 311 mg of orange solid (I 12 ), with a yield of 55.8%.

[0111] ESI-MS (m / z): 558 [M + H] + .

[0112] 1 1H NMR (400 MHz, CDCl3) δ (ppm) 8.02 (s, 1H), 7.84 - 7.66 (m, 4H), 7.36 - 7.26 (m, 6H), 7.21 - 7.02 (m, 10H).

[0113] Example 13 Aggregation-Induced Emission Fluorescence Spectrum Detection of the Compounds of the Present Invention

[0114] Dissolve the compound I1 of the present invention in water / DMSO solutions with different ratios (0%, 99%) respectively, and the concentration of the detection solution is 10 μM. Use a fluorescence spectrometer to test its fluorescence emission spectrum data, with an excitation wavelength of 500 nm, and collect data from 550 - 800 nm.

[0115] Figure 1 The fluorescence spectrum of the compound (10 μM) of the present invention in water / DMSO solutions with different ratios (0%, 99%). Among them, Figure 1 A is the fluorescence spectrum of compound I1 in water / DMSO solutions with different ratios (0%, 99%); Figure 1 B is the fluorescence spectrum of compound I2 in water / DMSO solutions with different ratios (0%, 99%); Figure 1 C) is the fluorescence spectrum of compound I3 in water / DMSO solutions with different ratios (0%, 99%); Figure 1D is the fluorescence spectrum of Compound I9 in aqueous / DMSO solutions with different ratios (0%, 99%); Figure 1 E is Compound I 12 in aqueous / DMSO solutions with different ratios (0%, 99%). The concentration of the compound is 10 μM for all cases.

[0116] The results show that: for the compounds of the present invention in pure DMSO solution, the maximum emission peak is between 630 nm and 660 nm; in 99% aqueous / DMSO solution, the maximum emission peak exhibits a blue shift to between 610 nm and 630 nm; however, the fluorescence intensity is higher than that in pure DMSO solution. This proves that the triphenylamine group contained in the compounds of the present invention has an aggregation-induced emission fluorescence effect.

[0117] Example 14 Detection of two-photon fluorescence spectrum of the compounds of the present invention

[0118] The compound of the present invention (10 μM) was dissolved in THF solution, and the detection solution concentration was 10 μM. The two-photon optical properties were studied using a pulsed laser with a pulse width of 120 fs, a wavelength of 808 nm, and a repetition frequency of 1 kHz (Ti:sapphire laser), and the fluorescence emission spectral data were collected by a fluorescence spectrometer. The two-photon fluorescence emission spectra of the compound in THF solution at 200 mW and 500 mW powers of the 808 nm laser are shown in Figure 2 .

[0119] Among them, Figure 2 A is the two-photon fluorescence spectrum of Compound I1 in THF solution; Figure 2 B is the two-photon fluorescence spectrum of Compound I2 in THF solution; Figure 2 C is the two-photon fluorescence spectrum of Compound I3 in THF solution; Figure 2 D is the two-photon fluorescence spectrum of Compound I9 in THF solution; Figure 2 E is Compound I 12 in THF solution; the excitation wavelength is 808 nm (200 mW cm -2 / 500 mW cm -2 ).

[0120] The results show that: after excitation at 808 nm, the compounds of the present invention produce a maximum fluorescence emission peak between 610 nm and 660 nm, and the fluorescence intensity increases with the increase of laser power, proving that the compounds of the present invention can achieve two-photon excited fluorescence imaging.

[0121] Example 15 Test for the types of ROS generated by the compounds of the present invention

[0122] The ability of the compounds of the present invention to generate ROS was detected by fluorescence spectroscopy. The detection mechanisms of the total ROS indicator DCFH, the superoxide anion indicator DHR123, the hydroxyl radical indicator HPF, and the singlet oxygen indicator SOSG are as follows:

[0123]

[0124] When the total ROS content indicator DCFH (Bidepharm) generates ROS in the system, it will be oxidized to DCF, and the fluorescence intensity increases at 523 nm; DHR123 (MKBio), as the superoxide anion indicator, will be oxidized to R123 by superoxide anions, and the fluorescence intensity increases at 520 nm; SOSG (Meilunbio), as the singlet oxygen indicator, will be oxidized to fluorescein by singlet oxygen, and the fluorescence intensity increases at 520 nm; HPF (MKBio), as the hydroxyl radical indicator, will be oxidized to fluorescein by hydroxyl radicals, and the fluorescence intensity increases at 515 nm.

[0125] The compounds of the present invention were dissolved in an aqueous solution containing 5% DMSO, the concentration of the detection solution was 10 μM, and the corresponding indicator was added. After irradiation with a laser at 520 nm (50 mW cm -2 ) or 808 nm (800 mW cm -2 ), the fluorescence spectrum of the system from 500 nm to 600 nm was detected; the control was Ru(bpy)3 2+ (Bidepharm).

[0126] The results of the total amount and types of ROS generated after irradiating the compound I1 (10 μM) of the present invention with 520 nm and 808 nm are shown in Figure 3 .

[0127] Among them, Figure 3 A is the time-dependent change of the fluorescence intensity (I - I0) of DCFH (5 μm) at 523 nm after irradiating compound I1 with 520 nm; Figure 3 B is the time-dependent change of the fluorescence intensity (I - I0) of DHR123 (5 μm) at 520 nm after irradiating compound I1 with 520 nm; Figure 3 C is the time-dependent change of the fluorescence intensity (I - I0) of HPF (5 μm) at 515 nm after irradiating compound I1 with 520 nm; Figure 3 D is the time-dependent change of the fluorescence intensity (I - I0) of SOSG (1 μm) at 520 nm after irradiating compound I1 with 520 nm; Figure 3 E is the time-dependent change of the fluorescence intensity (I - I0) of DCFH (5 μm) at 523 nm after irradiating compound I1 with 808 nm; Figure 3F is the time-dependent change of the fluorescence intensity (I-I0) of DHR123 (5 μm) at 520 nm after compound I1 is irradiated with 808 nm light; Figure 3 G is the time-dependent change of the fluorescence intensity (I-I0) of HPF (5 μm) at 515 nm after compound I1 is irradiated with 808 nm light; Figure 3 H is the time-dependent change of the fluorescence intensity (I-I0) of SOSG (1 μm) at 520 nm after compound I1 is irradiated with 808 nm light.

[0128] The results show that, as Figure 3 shown in A-D, the singlet oxygen generation ability of compound I1 of the present invention is significantly weaker than that of Ru(bpy)3 after irradiation at 520 nm 2+ , the superoxide anion generation ability is comparable, and it can generate hydroxyl radicals, while Ru(bpy)3 2+ does not generate hydroxyl radicals, and the total ROS content is higher than that of the control Ru(bpy)3 2+ . As Figure 3 shown in E-H, when excited at 808 nm, compound I1 of the present invention does not generate singlet oxygen, can generate superoxide anions and hydroxyl radicals, while the control Ru(bpy)3 2+ does not generate any ROS. Therefore, compound I1 of the present invention has single-photon / two-photon triggered ROS generation, and the main products are superoxide anions and hydroxyl radicals, which can be applied to type I photodynamic therapy.

[0129] The present invention further detected the type I photodynamic ROS generation of compounds I2, I3, I9, I 12 using the above method. The generation of superoxide anions and hydroxyl radicals after compounds I2, I3, I9, I 12 are irradiated with 808 nm light can be seen in Figure 4 . Among them, Figure 4 A is the time-dependent fluorescence spectrum of DHR123 (5 μm) after compound I2 is irradiated with 808 nm light; Figure 4 B is the time-dependent fluorescence spectrum of DHR123 (5 μm) after compound I3 is irradiated with 808 nm light; Figure 4 C is the time-dependent fluorescence spectrum of DHR123 (5 μm) after compound I9 is irradiated with 808 nm light; Figure 4 D is the time-dependent fluorescence spectrum of DHR123 (5 μm) after compound I 12 is irradiated with 808 nm light; Figure 4 E is the time-dependent fluorescence spectrum of HPF (5 μm) after compound I2 is irradiated with 808 nm light; Figure 4 F is the time-dependent fluorescence spectrum of HPF (5 μm) after compound I3 is irradiated with 808 nm light;Figure 4 G is the time-dependent fluorescence spectrum of HPF (5 μm) of compound I9 after irradiation at 808 nm; Figure 4 H is compound I 12 Time-dependent fluorescence spectra of HPF (5 μM) after irradiation at 808 nm; compound concentration was 10 μM.

[0130] The results showed that under 808 nm excitation, the compounds all produced superoxide anions and hydroxyl radicals, thus proving that the compounds of the present invention having a triphenylamine group and a five-membered boron-containing heterocycle have a type I ROS generation effect.

[0131] Example 16 In vitro photodynamic therapy experiment of the compound of the present invention

[0132] The phototoxicity of the compound of the present invention is evaluated by an in vitro toxicity experiment using methylthiazolium blue (MTT) colorimetry to study the effect of in vitro photodynamic therapy.

[0133] First, the dark toxicity was verified by taking a bottle of normal LO2 cells (Shanghai Institute of Cell Biology, China) in a good exponential growth phase, digesting them and making 1×10 4 The cell suspension of cells / mL was spread on a 96-well plate and kept at 37°C in the dark for 24 hours. Compound I1 of the present invention (0, 1, 5, 10, 20, 50 and 100 μM) was then added and cultured for another 24 hours. MTT was added and reacted for 4 hours, the supernatant was discarded, 100 μL of DMSO was added, the absorbance at 570 nm was detected, and the cell survival rate was calculated.

[0134] Cell survival rate formula:

[0135] Survival rate (%) = (OD value of experimental group - OD value of blank control group) / (OD value of control group - OD value of blank control group) × 100%

[0136] Among them, OD value refers to the optical density value. The OD value of the experimental group is the absorbance value of the treated cells, the OD value of the control group is the absorbance value of the untreated cells, and the OD value of the blank control group is the absorbance value of the culture medium.

[0137] The photodynamic cell experiment method was basically the same as the above method. Tumor cells 4T1 cells (Shanghai Institute of Cell Biology, China) were cultured in normoxia or hypoxia, and then compound I1 of the present invention (0, 5, 10, 15, 20, 25 and 30 μM) was added and cultured for 2 hours. The photodynamic cell experiment was performed at 808 nm (500 mW / cm 2 ) After irradiation for 5 minutes, the cells were cultured for another 24 hours and the cell survival rate was calculated.

[0138] The dark toxicity of compound I1 was evaluated by MTT method, and the results of photodynamic therapy under normoxia and hypoxia are shown in Figure 5Among them, Figure 5 A is the viability of LO2 cells incubated with different concentrations of I1 under light avoidance detected by MTT assay. Figure 5 B is the viability of 4T1 cells incubated with different concentrations of I1 under normoxia or hypoxia under 808 nm (500 mW / cm 2 , 5 min) laser irradiation detected by MTT assay.

[0139] The experimental results show that: even when the concentration of the compound I1 of the present invention reaches 100 μM, the survival rate of LO2 cells still exceeds 80%, and the dark toxicity is relatively small ( Figure 5 A). After 808 nm laser irradiation, 4T1 tumor cells are effectively killed under both normoxia and hypoxia ( Figure 5 B), which proves that the compound of the present invention can be applied to type I photodynamic therapy, overcome the difficulty of treating tumor hypoxia, provide a new method for the treatment of deep hypoxic tumors in clinical practice, and have broad application prospects.

[0140] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as a limitation of the present invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A type I two-photon photosensitizer without heavy atoms and with aggregation-induced emission ability having the structure shown in general formula I: Wherein, Each R is independently selected from H, halogen, hydroxyl, nitro, alkyl, alkoxy, alkylthio, alkenyl, alkynyl, cycloalkyl, cycloalkyloxy, cycloalkylthio, acyl, ester, amide, aryl, heterocyclic group, heteroaryl, heterocycloalkyl, monoalkylamino or dialkylamino; n = 1 or 2.

2. The type I two-photon photosensitizer according to claim 1, wherein R is selected from H, -CH3, -F, -Cl, -Br, -I, -OH, tert-butyl or methoxy.

3. The type I two-photon photosensitizer according to claim 1, wherein Selected from:

4. A method for preparing a type I two-photon photosensitizer represented by the general formula I of claim 1, characterized in that, Comprising the following steps: (1) Condensing 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde with benzohydrazide containing different substituents R under heating conditions; (2) The condensation product is then complexed with boron trifluoride diethyl ether solution to obtain the type I two-photon photosensitizer shown in general formula I; Wherein, R is as defined in general formula I.

5. The preparation method according to claim 4, characterized in that, In step (1), 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde and benzohydrazide containing different substituents R are refluxed overnight in anhydrous acetonitrile or ethanol until the reaction is complete, and the solid is obtained by filtration, which is the condensation product; wherein, R is as defined in general formula I.

6. The preparation method according to claim 4, characterized in that, In step (2), the condensation product and boron trifluoride diethyl ether solution are refluxed overnight in toluene until the reaction ends, and the type I two-photon photosensitizer shown in general formula I is obtained by recrystallization.

7. A pharmaceutical composition, characterized in that: Comprising the type I two-photon photosensitizer according to any one of claims 1-3 and a pharmaceutically acceptable carrier or excipient.

8. Use of the type I two-photon photosensitizer according to any one of claims 1-3 in the preparation of a tumor imaging agent, characterized in that, The type I two-photon photosensitizer realizes single-photon or two-photon fluorescence imaging in vivo and in vitro.

9. Use of the type I two-photon photosensitizer according to any one of claims 1-3 in the preparation of anti-tumor drugs.

10. The application according to claim 9, characterized in that The type I two-photon photosensitizer can effectively ablate tumor cells after being irradiated with single-photon or two-photon laser under normoxic or hypoxic conditions.

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