Near-infrared photosensitizer that can be activated in slightly acidic tumor environment and preparation method therefor and use thereof

Abstract

The present invention relates to the technical field of biomedical synthesis. Specifically disclosed are a near-infrared photosensitizer capable of being activated in a slightly acidic tumor environment and a preparation method therefor and the use thereof. The near-infrared photosensitizer capable of being activated in a slightly acidic tumor environment has a chemical structural formula as shown in any one of formula (I) to formula (VI). According to the near-infrared photosensitizer capable of being activated in a slightly acidic tumor environment, a cyanine or hemicyanine derivative with good biocompatibility and a high near-infrared light absorption capacity is used as a basic skeleton, an ether oxygen bond is introduced at the ortho position of an indole nitrogen atom as an acid-responsive group, and the ability of the photosensitizer molecules to produce active oxygen is adjusted by means of substituting different halogen atoms with R1, wherein an R2 group is hydroxyl, amino or a biomarker-responsive group. The photosensitizer of the present invention is specifically activated in tumors, has strong near-infrared absorption and fluorescence emission capabilities, has a photodynamic therapeutic effect, can realize accurate treatment of tumor sites, reduces systemic toxicity, and provides a new strategy for the accurate treatment of tumors.

Description

Near-infrared photosensitizer activated by tumor microacidic environment and its preparation method and application Technical Field

[0001] The present invention relates to the field of biomedical synthesis technology, and in particular to a near-infrared photosensitizer that can be activated by a slightly acidic environment of a tumor, and a preparation method and application thereof. Background Art

[0002] Cancer has a high incidence and mortality rate, seriously endangering people's life and health. Photodynamic therapy (PDT) is an emerging tumor treatment option that uses photosensitizers to transfer laser energy to dissolved oxygen in tumors, generating cytotoxic singlet oxygen, thereby achieving the purpose of killing tumor cells. This light-controlled therapy has the following advantages: (1) Minimal tissue trauma: Through light treatment, it is non-invasive; with the help of optical fiber, endoscope and other interventional technologies, the laser can also be guided deep into the body for treatment, avoiding the trauma and pain caused by thoracotomy, laparotomy and other surgeries. (2) High spatiotemporal selectivity and low systemic toxicity: Only when the photosensitizer enters the tissue and reaches a certain concentration and is exposed to sufficient light will it trigger a photodynamic reaction and kill the target cells. The part that is not exposed to light does not produce this killing reaction, causing little damage to organs and tissues in other parts, and does not affect hematopoietic function. Therefore, this treatment method that selectively and only locally irradiates the diseased tissue has low systemic toxic side effects, which is difficult to achieve with many other treatment methods. (3) Good applicability: Different types of tumor cells have different sensitivities to radiotherapy and chemotherapy, so its application is limited. However, photodynamic therapy is effective for lesions of different cell types and has a wide range of applications. (4) Repeatable treatment: Tumor cells have no resistance to photosensitizing drugs, and patients will not experience increased toxic reactions due to multiple treatments, so repeated treatment is possible.

[0003] At present, the photosensitizers used in clinical practice are mainly porphyrin derivatives. These photosensitizers have the following main problems: (1) The diffuse distribution of photosensitizer drugs throughout the body causes excessive accumulation of drugs in normal tissues, causing clinical side effects such as skin phototoxicity that affect the quality of life of patients. For example, It is one of the most widely used photosensitizer drugs in clinical practice in recent years. Its main ingredient is a derivative of hematoporphyrin. The drug lacks tumor selectivity and is widely distributed in the skin all over the body after administration. It causes severe skin inflammation under sunlight. The FDA requires that patients must not expose their skin and eyes to sunlight for up to 1 month after taking the drug, which seriously affects their lifestyle. (2) The laser penetration depth of photosensitizers is relatively shallow, and the therapeutic effect on deep tumors is poor. (3) The active oxygen production rate of photosensitizers is generally low. In addition, the inherent hypoxic characteristics of the tumor microenvironment and the continuous consumption of oxygen during treatment greatly weaken the effect of photodynamic therapy. Therefore, higher doses of photosensitizer drugs have to be used to meet treatment needs, thereby increasing the long-term safety risks of medication. Therefore, overcoming these problems and developing new photosensitizer molecules are of great significance to the clinical application of photodynamic therapy.

[0004] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a near-infrared photosensitizer that can be activated by the slightly acidic environment of tumors, as well as a preparation method and application thereof, aiming to solve the problems of high skin phototoxicity, shallow treatment depth and low reactive oxygen production of existing photosensitizers.

[0006] The technical solutions of the present invention are as follows:

[0007] In a first aspect, the present invention provides a series of near-infrared photosensitizers that can be activated by a slightly acidic environment of a tumor, having a chemical structure shown in any one of Formula (I) to Formula (VI):

[0008] Wherein, R1 is H, F, Cl, Br or I atom, R2 is -OH, -NH2, A is a self-eliminating group, and B is a biomarker-responsive group.

[0009] The photosensitizer provided by the present invention uses cyanine / hemicyanine as the parent structure, and introduces an ether oxygen bond as an acid-responsive group at the ortho position of the indole nitrogen atom. The ability of the photosensitizer molecule to generate reactive oxygen is adjusted by replacing different halogen (fluorine, chlorine, bromine and iodine) atoms by R1. With respect to the semi-cyanine skeleton, in addition to the acid-responsive group, an R2 group (hydroxyl, amino or a group with biomarker responsiveness) is connected to one end of the photosensitizer molecule. Among them, the cyanine molecular skeleton of formula (I) to formula (III) has a high molar absorption coefficient and near-infrared photosensitivity, and the positive charge of the indole structure makes it easy to target mitochondria. The semi-cyanine molecular skeleton of formula (IV) to formula (VI) is a variant of the cyanine molecule, and has photophysical and photochemical properties very similar to those of the cyanine molecule, but has higher photostability and structural modifiability. The -OH group of the acid-responsive group forms a closed ring structure with an ether oxygen bond adjacent to the indole nitrogen atom. In its inactive state, the photosensitizer exhibits no absorption or fluorescence in the near-infrared region, and thus no near-infrared photodynamic therapy effect. However, upon activation in the slightly acidic environment of the tumor, the ether oxygen bond formed by the acid-responsive group breaks, forming an open ring structure. The molecule then reverts to the classic cyanine / hemicyanine structure, resulting in strong near-infrared absorption and fluorescence, and exhibiting near-infrared photodynamic therapy effects. Different halogen atoms substituted by R1 in the photosensitizer have different atomic effects during the molecular photosensitization process, resulting in a higher reactive oxygen species yield. The R2 biomarker-responsive group introduced into the hemicyanine skeleton, in combination with the acid-responsive group, yields a dual-responsive photosensitizer molecule with enhanced tumor selectivity.

[0010] It should be noted that the chemical structure of the photosensitizer All of them are indole structures, and the three can be equivalently replaced with each other in the present invention. However, as the degree of conjugation of the molecular skeleton increases successively, the absorption and emission wavelengths thereof also increase successively.

[0011] Optionally, the self-eliminating group A is

[0012] Optionally, the biomarker responsive group B is:

[0013] Optionally, after being activated in a slightly acidic environment, the photosensitizers represented by formula (I) to formula (VI) have the structures represented by corresponding formula (I') to formula (VI'):

[0014] Optionally, the near-infrared photosensitizer activated by the slightly acidic environment of the tumor has the following structural formula:

[0015] The use of the near-infrared photosensitizer molecule activated by the micro-acidic environment of the tumor or its pharmaceutically acceptable enantiomers, diastereomers, and tautomers in the preparation of photosensitizer molecules is also within the scope of protection of the present invention.

[0016] The second aspect of the present invention provides a method for preparing the near-infrared photosensitizer that can be activated by the slightly acidic environment of tumors, comprising the steps of:

[0017] Step S1: Compound 1 represented by Formula 1 and Compound 4 represented by Formula 4 undergo condensation reaction to obtain Compound 6 represented by Formula 6;

[0018] Or compound 2 represented by formula 2 and compound 4 represented by formula 4 undergo condensation reaction to obtain compound 7 represented by formula 7;

[0019] Or compound 3 represented by formula 3 and compound 4 represented by formula 4 undergo condensation reaction to obtain compound 8 represented by formula 8;

[0020] or compound 1 represented by formula 1 and compound 5 represented by formula 5 undergo condensation reaction to obtain compound 9 represented by formula 9;

[0021] or the compound 2 represented by formula 2 and the compound 5 represented by formula 5 undergo a condensation reaction to obtain the compound 10 represented by formula 10;

[0022] or the compound 3 represented by formula 3 and the compound 5 represented by formula 5 undergo a condensation reaction to obtain the compound 11 represented by formula 11;

[0023] In step S2, compound 6 undergoes ester hydrolysis reaction and ring closure reaction in sequence under alkaline conditions to obtain a near-infrared photosensitizer activated by the slightly acidic environment of tumors as shown in formula (I);

[0024] Alternatively, compound 7 undergoes ester hydrolysis and ring closure reactions in sequence under alkaline conditions to obtain a near-infrared photosensitizer activated by the slightly acidic environment of the tumor as shown in formula (II);

[0025] Or compound 8 undergoes a ring-closing reaction under alkaline conditions to obtain a near-infrared photosensitizer that can be activated by the slightly acidic environment of the tumor as shown in formula (III);

[0026] Alternatively, compound 9 undergoes ester hydrolysis and ring closure reactions in sequence under alkaline conditions to obtain a near-infrared photosensitizer activated by the slightly acidic environment of the tumor as shown in formula (IV);

[0027] Alternatively, compound 10 undergoes ester hydrolysis and ring closure reactions in sequence under alkaline conditions to obtain a near-infrared photosensitizer activated by the slightly acidic environment of the tumor as shown in formula (V);

[0028] Alternatively, compound 11 undergoes a ring-closing reaction under alkaline conditions to obtain a near-infrared photosensitizer that can be activated by the slightly acidic environment of the tumor as shown in formula (VI).

[0029] The structural formulas of compounds 1 to 11 are shown in the following formulas 1 to 11:

[0030] Optionally, the temperature of the condensation reaction in step S1 is 50-100° C., and the time of the condensation reaction is 2-12 hours.

[0031] Optionally, the solvent used in the condensation reaction in step S1 is one or more of acetic anhydride, anhydrous methanol, anhydrous ethanol and anhydrous N,N-dimethylformamide, and the optional activating agent is one or more of sodium acetate, potassium acetate, potassium carbonate and cesium carbonate.

[0032] Optionally, the alkaline conditions required for the ring-closure reaction in step S2 include one or more of potassium carbonate, cesium carbonate, potassium hydroxide, sodium hydroxide, sodium hydride, ammonia water or triethylamine.

[0033] Optionally, the solvent used in the ring-closing reaction in step S2 is one or more of dichloromethane, methanol, ethanol, chloroform and N,N-dimethylformamide.

[0034] Optionally, the temperature of the ring-closing reaction is 0 to 50° C., and the time of the ring-closing reaction is 2 to 12 hours.

[0035] Optionally, the compound 1 in step S1 is prepared by a substitution reaction between compound 12 and compound 13. The temperature of the substitution reaction is 50 to 150° C., the time of the substitution reaction is 24 to 72 hours, and the solvent used in the substitution reaction is one or more of acetonitrile, N,N-dimethylformamide, dichloromethane, chloroform, toluene, and o-dichlorobenzene.

[0036] The structural formulas of compound 12 and compound 13 are shown below:

[0037] Optionally, the compound 3 in step S1 is prepared by the following steps:

[0038] Compound 14 undergoes sulfurization reaction to obtain compound 15;

[0039] The compound 15 undergoes iodination reaction to obtain compound 16;

[0040] The compound 16 undergoes a substitution reaction with the compound 17 to obtain the compound 18;

[0041] The compound 18 undergoes a substitution reaction with the compound 13 to obtain the compound 19;

[0042] The compound 19 undergoes a hydrolysis reaction to obtain the compound 3.

[0043] The structural formulas of compounds 13 to 19 are as follows:

[0044] Optionally, the reaction temperature involved in the preparation step of compound 3 is 50-150° C., the reaction time is 24-72 h, and the solvent used in the reaction can be one or more of acetonitrile, N,N-dimethylformamide, dichloromethane, chloroform, toluene and o-dichlorobenzene.

[0045] The third aspect of the present invention provides a use of the near-infrared photosensitizer that can be activated by the slightly acidic environment of a tumor, for preparing a tumor diagnostic agent and / or a tumor therapeutic agent.

[0046] Optionally, the tumor diagnostic agent comprises a fluorescent imaging agent or a photoacoustic imaging agent.

[0047] Optionally, the tumor treatment agent includes a photodynamic therapy agent or an agent for combining photodynamic therapy with other treatment options.

[0048] Optionally, the dosage form of the medicament is capsule, tablet, oral preparation, injection, suppository, spray or ointment.

[0049] The tumor microacid environment that the present invention provides can be activated by near-infrared dye molecules and can be used as photosensitizers for photodynamic therapy, and can be prepared into preparations for various modes of administration (intravenous injection, microneedle patch, intraperitoneal injection or spraying). After the tumor microacid environment is activated by intravenous injection, due to the normal tissue microenvironment pH being 7.4, the acid response group of the photosensitizer molecule exists in a closed-loop form, and the molecule is not activated, so obvious near-infrared light absorption and emission will not be detected in normal tissue, and under near-infrared light irradiation, the photosensitizer will not produce reactive oxygen species, and therefore will not cause damage to normal tissue. When the photosensitizer molecule is in the tumor microacid environment (pH < 7), the acid response group exists in an open-loop form, and the photosensitizer molecules shown in formula (I) to formula (III) are activated, producing strong near-infrared absorption and emission, so that fluorescence or photoacoustic imaging can be performed, and reactive oxygen species can be produced under near-infrared light irradiation, thereby killing tumor tissue. After being further activated by specific biomarkers expressed in large quantities in tumors, the photosensitizer molecules represented by formulas (IV) to (VI) can produce near-infrared light absorption and emission, guiding photodynamic therapy through fluorescence or photoacoustic imaging, achieving precise visualization of photodynamic therapy, improving treatment effects, and providing new ideas for the development of photodynamic therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG1 is a schematic diagram showing the activation principle of the near-infrared photosensitizer that can be activated by the slightly acidic environment of a tumor provided by the present invention.

[0051] FIG2 is a synthetic route of the activatable near-infrared photosensitizers LET-H, LET-Cl, LET-Br, and LET-I (collectively referred to as LET-R) of Examples 1 to 4 of the present invention.

[0052] FIG3 is a synthetic route of the activatable near-infrared photosensitizer LET-BCy of Example 5 of the present invention.

[0053] FIG4 is a synthetic route of the activatable near-infrared photosensitizer LET-Hcy-N of Example 6 of the present invention.

[0054] FIG5 is a synthetic route of the activatable near-infrared photosensitizers LET-BHcy-ROS and LET-BHcy-GSH of Examples 7 and 8 of the present invention.

[0055] FIG6 is a pH-responsive absorption spectrum of the activatable near-infrared photosensitizers of Examples 1 to 8 of the present invention.

[0056] Figure 7 is a fluorescence emission spectrum of the present invention, where a and b are pH-responsive fluorescence spectra of LET-H, with excitation wavelengths of 420 and 760 nm, respectively; c and d are pH-responsive fluorescence spectra of LET-I, with excitation wavelengths of 420 and 760 nm, respectively; and e is a pH-responsive fluorescence spectrum of LET-Hcy-N, with an excitation wavelength of 660 nm.

[0057] In Figure 8, LET-R' (R = H, Cl, Br, I) represents the molecules after activation of the photosensitizer LET-R, and a represents the molecular structure of LET-R and LET-R' under 808 nm laser (power 0.2 W cm -2 ) is a comparison of the degradation rates of DPBF under irradiation, b is a comparison of the singlet oxygen quantum yield of LET-R', and c is the electron paramagnetic resonance spectrum detection of the production of singlet oxygen by LET-H' or LET-I' with and without light irradiation.

[0058] FIG9 is a graph showing the test results of the cell killing ability of the activated near-infrared photosensitizers LET-H and LET-I under different illumination times.

[0059] In Figure 10, a is the fluorescence imaging of folic acid-modified nanoengineered LET-I (denoted as LET-I-FA) in mouse tumors and legs, b is the corresponding change in fluorescence intensity over time, c is the photoacoustic imaging of LET-I-FA in mouse tumors and legs, and d is the corresponding change in photoacoustic signal intensity over time.

[0060] Figure 11, a, is a graph evaluating the therapeutic effect of the photosensitizer LET-I-FA on the 4T1 tumor-bearing mouse model; b is a statistical graph of the tumor weight of mice after the end of treatment; c is a graph showing the weight changes of mice in each treatment group during the treatment period, where LET-H-FA represents the folic acid-modified nanoengineered photosensitizer LET-H.

[0061] FIG12 a is a diagram showing tissue sections of major organs in each group after treatment, and b is a diagram showing the blood biochemical evaluation results in each group. DETAILED DESCRIPTION

[0062] The present invention provides near-infrared photosensitizers activated by a slightly acidic tumor environment, as well as methods for their preparation and use. To clarify the objectives, technical solutions, and effects of the present invention, the present invention is described in further detail below. It should be understood that the specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention.

[0063] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0064] The following describes it in detail through specific examples.

[0065] Example 1

[0066] The synthetic route for preparing the tumor slightly acidic environment-activated near-infrared photosensitizer LET-H in this embodiment is shown in FIG2 . The specific synthetic steps are as follows:

[0067] Under a nitrogen atmosphere, phosphorus oxychloride (8.7 mL, 58 mmol) was slowly added to a solution of 10 mL of dichloromethane and 10 mL of N,N-dimethylformamide. After stirring in an ice bath for 30 minutes, compound A1 (2.5 g, 25 mmol) was added. Stir at 80°C for 6 hours, then cool to room temperature and add ice water and stir overnight. The mixture was filtered, and the precipitate was washed with a small amount of ice water, then with ice ethanol, and finally oven-dried to obtain compound B1 (4.1 g, 96% yield) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ10.85(s,1H),2.36(m,4H),1.59(m,2H).

[0068] Compound D1 (1.07 g, 4.71 mmol) was added dropwise to a solution of compound C1 (500 mg, 3.14 mmol) in acetonitrile and refluxed for 1 d. After cooling, the mixture was poured into a mixture of 22.5 mL of dichloromethane and 2.5 mL of methanol. 10 mL of cold ether was added to the mixture, and the resulting precipitated crystals were collected, washed with 3 × 3 mL of diethyl ether, and dried under vacuum to afford Compound E1 (1.055 g, 87% yield) as a light brown solid. 1 H NMR (400MHz, CDCl3) δ7.73 (m, 1H), 7.59 (m, 3H), 4.89 (t, J = 8Hz, 2H), 4.23 (t, J = 4Hz, 2H), 3.17 (s, 3H), 2.38 (m, 2H), 2.00 (s, 3H), 1.67 (s, 6H). HRMS(ESI)calcd for C16 H 22 NO2 + 260.1645,found 260.1636.

[0069] Under nitrogen, compound B1 (107 mg, 0.65 mmol), compound E1 (500 mg, 1.3 mmol), and sodium acetate (106 mg, 1.3 mmol) were added to 5 mL of acetic anhydride. The solution was heated to 70°C for 2 h. The mixture was poured into cold ether, and the precipitate was collected to obtain compound F1 (491 mg, 96% yield) as a solid with a metallic green sheen. 1 H NMR (500MHz, DMSO-d6) δ8.29(d,J=10Hz,2H),7.66(d,J=10Hz,2H),7.45(m,4H),7.30(m,2H),6.36(d,J=15Hz,2H),4.30(t, J=7Hz,4H),4.09(t,J=6Hz,4H),2.72(t,J=5Hz,4H),2.08(m,4H),1.94(s,6H),1.86(m,2H),1.69(s,12H).HRMS(ESI)calcd for C 40 H 48 ClN2O4 + 655.3297, found 655.3275.

[0070] Under nitrogen protection, compound F1 (464 mg, 0.59 mmol) was dissolved in 30 mL of methanol. Potassium carbonate solid (164 mg, 1.19 mmol) was then added. The mixture was stirred at room temperature for 3 h. After ring closure of intermediate compound G1, the final product was synthesized. The solvent was removed by rotary evaporation, and 10 mL of dichloromethane was added. The resulting mixture was washed twice with 10 mL of saturated sodium bicarbonate aqueous solution, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude product was purified by aluminum oxide column (dichloromethane as eluent) to obtain a light yellow solid compound (208 mg, 62% yield), which is a near-infrared photosensitizer that can be activated by the slightly acidic environment of tumors and is designated as LET-H. 1 H NMR (400MHz, DMSO-d6) δ8.27(d,J=8Hz,2H),7.64(d,J=4Hz,2H),7.45(m,4H),7.29(td,J1=4.6Hz,J2=1.2Hz,2H),6. 44(d,J=8Hz,2H),4.87(bro,2H),4.26(t,J=4Hz,4H),3.50(bro,4H),2.70(t,J=4Hz,4H),1.90(m,4H),1.68(m,14H). 13C NMR (100MHz, CDCl3) δ172.6,144.7,142.1,139.2,128.9,127.2,125.3,122.1,119.2,110.8,108 .7,68.2,65.7,62.3,58.0,41.2,40.0,29.9,29.6,28.1,26.2,22.0,20.6,18.3.HRMS(ESI)calcd for C 36 H 43 ClN2O2 + 570.3013,found[M+H] + 571.3078.

[0071] Example 2

[0072] The synthetic route for preparing the tumor slightly acidic environment-activated near-infrared photosensitizer LET-Cl in this embodiment is shown in FIG2 . The specific synthetic steps are as follows:

[0073] Compound C2 and D1 were synthesized by the same synthesis steps as in Example 1 to synthesize brown solid compound E2 with a yield of 78%. 1 H NMR (500MHz, CDCl3) δ7.88 (d, J = 5Hz, 1H), 7.52 (s, 1H), 7.51 (d, J = 2Hz, 1H), 4.81 (t, J=5Hz,2H),4.20(t,J=5Hz,2H),3.11(s,3H),2.35(m,2H),1.97(s,3H),1.65(s,6H).

[0074] Compound E2 was used to obtain green solid compound F2 through the same synthesis steps as in Example 1 with a yield of 92%. 1 HNMR (500MHz, CDCl3) δ8.33(d,J=10Hz,2H),7.37(dd,J1=7Hz,J2=2Hz,2H),7.33(d,J=2Hz,2H),7.12(d,J=7Hz,2H),6.34(d,J =10Hz, 2H), 4.38 (t, J = 6Hz, 4H), 4.18 (t, J = 5Hz, 4H), 2.79 (t, J = 5Hz, 4H), 2.21 (m, 4H), 2.04 (s, 6H), 1.98 (m, 2H), 1.73 (s, 12H).

[0075] Compound F2 was synthesized in the same manner as in Example 1, and the intermediate compound G2 was subjected to a ring-closure reaction to obtain a yellow solid compound with a yield of 58%. This compound was a near-infrared photosensitizer activated by the slightly acidic environment of tumors and was designated as LET-Cl. 1H NMR (500MHz, CD3OD) δ8.45(d,J=15Hz,2H),7.59(d,J=5Hz,2H),7.44(dd,J1=10Hz,J2=5Hz,2H),7.36(d,J=10Hz,2H),6.4 5(d,J=15Hz,2H),4.25(t,J=10Hz,4H),3.65(t,J=5Hz,4H),2.74(t,J=5Hz,4H),2.03(m,4H),1.95(m,2H),1.74(s,12H). 13 C NMR(100MHz,CD3OD)δ174.1,145.6,144.5,142.5,132.1,129.9,128.9,124.1,113.5,59.3,50.7,42.5,31.0,28.2,27.3,22.1.HRMS(ESI)calcd for C 36 H 41 Cl3N2O2 + 640.2204,found[M+H] + 641.2263.

[0076] Example 3

[0077] The synthetic route for preparing the tumor-activated near-infrared photosensitizer LET-Br in this embodiment is shown in FIG2 . The specific synthetic steps are as follows:

[0078] Compound C3 and D1 were used to synthesize light yellow solid compound E3 by the same steps as those for preparing E1 in Example 1, with a yield of 82%. 1 H NMR(500MHz, CDCl3)δ7.80(d,J=5Hz,1H),7.71(d,J=5Hz,1H),7.68(s,1H),4 .85(t,2H),4.22(t,2H),3.12(s,3H),2.36(t,2H),2.00(s,3H),1.67(s,6H).

[0079] Compound E3 was used to obtain green compound F3 by the same steps as in Example 1 with a yield of 95%. 1H NMR (500MHz, CDCl3) δ8.34(d,J=15Hz,2H),7.52(dd,J1=7Hz,J2=1.5Hz,2H),7.47(d,J=1.5Hz,2H),7.07(d,J=5Hz,2H),6.34(d,J= 10Hz, 2H), 4.37 (t, J = 5.5Hz, 4H), 4.18 (t, J = 4.5Hz, 4H), 2.79 (t, J = 5Hz, 4H), 2.22 (m, 4H), 2.04 (s, 6H), 1.97 (m, 2H), 1.73 (s, 12H).

[0080] Compound F3 was synthesized in the same manner as in Example 1, and intermediate compound G3 was ring-closed to obtain a yellow solid compound with a yield of 67%. This compound is a near-infrared photosensitizer activated by the slightly acidic environment of tumors and is designated as LET-Br. 1 H NMR (500MHz, CD3OD) δ8.45(d,J=15Hz,2H),7.72(d,J=2.5Hz,2H),7.58(dd,J1=11Hz,J2=2.5Hz,2H),7.31(d,J=10.5Hz,2H), 6.46(d,J=8Hz,2H),4.25(t,J=9Hz,4H),3.65(t,J=6.5Hz,4H),2.74(t,J=7Hz,4H),2.02(m,4H),1.95(m,2H),1.74(s,12H). 13 C NMR(100MHz,CD3OD)δ172.6,143.4,141.5,131.5,127.5,125.6,118.0,112.5,101.6,57.8,49.3,41.0,29.6,26.8,25.9,20.7.HRMS(ESI)calcd for C 36 H 41 Br2ClN2O2 + 728.1203,found[M+H] + 729.1257.

[0081] Example 4

[0082] The synthetic route for preparing the tumor slightly acidic environment-activated near-infrared photosensitizer LET-I in this embodiment is shown in FIG2 . The specific synthetic steps are as follows:

[0083] Compounds C4 and D1 were used to synthesize brown solid compound E4 by similar steps with a yield of 60%. 1H NMR (400MHz, DMSO-d6) δ8.32 (s, 1H), 8.03 (d, J = 8Hz, 1H), 7.78 (d, J = 8Hz, 1H), 4.51 (t, J = 8Hz, 2H),4.13(t,J=4Hz,2H),2.81(s,3H),2.18(m,2H),1.93(s,3H),1.54(s,6H).HRMS(ESI)calcd for C 16 H 22 INO2 + 386.0611,found 386.0608.

[0084] The green compound F4 was obtained by similar synthetic steps using compound E4 with a yield of 41%. 1 H NMR (400MHz, CDCl3) δ8.34(d,J=11.2Hz,2H),7.71(dd,J1=6.4Hz,J2=0.8Hz,2H),7.65(d,J=0.8Hz,2H),6.97(d,J=6.8Hz,2H),6.36(d,J =11.2Hz,2H),4.37(t,J=5.6Hz,4H),4.18(t,J=4.8Hz,4H),2.79(t,J=4Hz,4H),2.21(m,4H),2.04(s,6H),1.72(s,14H).HRMS(ESI)calcd for C 40 H 46 ClI2N2O4 + 907.1230, found 907.1226.

[0085] Compound F4 was used through similar synthetic steps to obtain a yellow solid compound through ring closure of intermediate compound G4 with a yield of 50%. This compound is a near-infrared photosensitizer that can be activated by the slightly acidic environment of tumors and is recorded as LET-I. 1H NMR (500MHz, CD3OD) δ8.52(d,J=20Hz,1H),8.40(d,J=15Hz,1H),7.93(d,J=1.5Hz,1H),7.86(d,J =2Hz,1H),7.79(dd,J1=10.5Hz,J2=2Hz,1H),7.74(dd,J1=10.5Hz,J2=2Hz,1H),7.25(d,J=10Hz, 1H),7.11(d,J=10Hz,1H),6.55(d,J=20Hz,1H),6.26(d,J=15Hz,1H),4.31(m,2H),4.24(m,2H),4 .17(t,J=5Hz,2H),3.66(m,4H),2.77(m,4H),2.21(m,2H),1.97(m,2H),1.75(s,6H),1.73(s,6H). 13 C NMR (100MHz, CDCl3) δ173.9,169.9,167.1,149.9,146.7,142.8,141.6,141.4,140.5,140.1,137.2,136.5,130.4,128.7,126.8 ,113.3,110.2,104.7,97.7,89.9,85.9,60.3,56.9,47.3,41.6,39.8,27.3,27.1,26.9,25.5,25.1,19.9,19.7.HRMS(ESI)calcd for C 36 H 41 I2ClN2O2 + 822.0946,found[M+H] + 823.1015.

[0086] The structural formulas of the tumor slightly acidic environment-activated near-infrared photosensitizers LET-H, LET-Cl, LET-Br, and LET-I prepared in Examples 1 to 4 are shown below:

[0087] Example 5

[0088] The synthesis route for preparing the tumor-activated near-infrared photosensitizer LET-BCy in this embodiment is shown in FIG3 . The specific synthesis steps are as follows:

[0089] This embodiment provides a near-infrared photosensitizer that can be activated by the slightly acidic environment of tumors. The synthesis route is shown in FIG3 . The specific synthesis method comprises the following steps:

[0090] Compound A5 (2 g, 11.8 mmol) and phosphorus pentasulfide (2.25 g, 11.8 mmol) were dissolved in 30 mL of pyridine and heated to 115°C under reflux overnight under nitrogen. After completion of the reaction, the mixture was cooled and added to 400 mL of water. The mixture was then allowed to cool at 4°C for 3-4 hours. The solid was isolated by filtration and washed three times with water to obtain the crude yellow solid, Compound B5. After vacuum drying, the product was directly used in the next reaction (1.79 g, 82.1% yield).

[0091] Compound B5 (12.4 g, 66.9 mmol) and iodomethane (5.0 mL, 80 mmol) were dissolved in 20 mL of acetone and heated under reflux at 45°C for 30 min. The precipitated product was crystallized from methanol to obtain compound C5 as a golden solid. The product was unstable and was used directly in the next reaction without purification (crude yield 76%).

[0092] A mixture of the crude product, compound C5 (16.4 g, 0.05 mol), compound D5 (14.4 g, 0.1 mol), and sodium acetate (8.2 g, 0.1 mol) in anhydrous ethanol was reacted at 50°C for 1 h. After completion of the reaction, the residue was cooled, isolated by filtration, and washed with water and ethanol (volume ratio 1:1) to obtain compound E5 (13.8 g, 94% yield). 1 H NMR (500MHz, DMSO-d6) δ12.78(s,1H),9.41(d,J=8.0Hz,1H),8.40(d,J=8.0Hz,1H),7.95(t ,J=8Hz,1H),7.89(d,J=8.5Hz,1H),7.79(d,J=7.0Hz,1H),7.69(t,J=8Hz,1H),1.73(s,6H).

[0093] Compound E5 (443 mg, 1.5 mmol) and potassium carbonate (622 mg, 4.5 mmol) were mixed in 10 mL of N,N-dimethylformamide and stirred at 25°C for 15 min, followed by the dropwise addition of compound D1 (1.02 g, 4.5 mmol). The mixture was then heated to 100°C and allowed to react for 12 h, quenched with 250 mL of ice water, and extracted with dichloromethane. The crude product was purified by silica gel column chromatography (dichloromethane / methanol as eluent) to afford compound F5 (367 mg, 62% yield) as a solid. 1H NMR (500MHz, CDCl3) δ8.72(d,J=7.0Hz,1H),8.58(d,J=7.5Hz,1H),8.48(d,J=7.0Hz,1H),8.27(d,J=8.0Hz,1H ), 8.08 (t, J = 7.5Hz, 1H), 7.95 (t, J = 8Hz, 1H), 5.06 (s, 2H), 3.84 (s, 2H), 3.53 (s, 3H), 2.44 (s, 2H), 1.26 (s, 6H).

[0094] Compound F5 (1.14 g, 2.9 mmol) was dissolved in 4 mL of acetic acid, and the mixture was refluxed at 90°C for 1 h. 4 mL of concentrated hydrochloric acid was added dropwise to the refluxing mixture until the color changed from red to green. The mixture was cooled to room temperature, and then saturated potassium iodide solution was added until the product began to precipitate. The product was filtered, washed with ether, and dried under vacuum to obtain solid Compound G5 (314.7 mg, 48% yield). 1 H NMR(500MHz,DMSO-d6)δ9.0(d,J=7.5Hz,1H),8.81(d,J=8.0Hz,1H),8.52(d,J=7.0Hz,1H),8.46(d,J=8.0Hz,1H),8 .150(t,J=7.5Hz,1H),8.02(t,J=8Hz,1H),4.75(t,J=7.0Hz,2H),3.55(t,J=5.5Hz,2H),3.26(s,3H),2.13(m,2H).

[0095] Compound G5 (762.64 mg, 2.19 mmol), compound B1 (173 mg, 1 mmol), and sodium acetate (160 mg, 2 mmol) were dissolved in 5 mL of acetic anhydride and stirred at 40°C for 2 h to synthesize the condensation intermediate compound H5. Potassium carbonate solid (164 mg, 1.19 mmol) was then added and stirred for 2 h. The crude product was then precipitated with 60 mL of diethyl ether to obtain a crude product. The product was finally purified by column chromatography to obtain a solid compound, a near-infrared photosensitizer activated by the slightly acidic environment of tumors, designated LET-BCy. HRMS (ESI) calculated for C 38 H 36 ClN2O2 + 587.2460,found[M+H] + 587.2459.

[0096] The structural formula of the near-infrared photosensitizer LET-BCy, which can be activated by the slightly acidic environment of tumors, prepared in Example 5 is as follows:

[0097] Example 6

[0098] The synthetic route for preparing the tumor microacid environment-activatable near-infrared photosensitizer LET-Hcy-N in this embodiment is shown in FIG4 . The specific synthetic steps are as follows:

[0099] Under nitrogen protection and an ice bath, phosphorus oxychloride (7.7 mL, 82 mmol) was slowly added dropwise to anhydrous N,N-dimethylformamide (7.9 mL, 64 mmol) and stirred for 15 minutes. Compound A1 (5 g, 50.9 mmol) was then added dropwise to the reaction mixture and stirred at room temperature for 2 hours. After the reaction, the reaction mixture was poured into 200 g of ice water and sodium bicarbonate was slowly added to adjust the pH to approximately 7. The mixture was then extracted multiple times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain Compound A6 (6.8 g, 93% yield) as an orange oil. This was used directly in the next reaction without further purification.

[0100] Compound A6 (0.97 g, 4 mmol), compound B6 (0.39 g, 2 mmol), and cesium carbonate (1.95 g, 6 mmol) were dissolved in 20 mL of anhydrous N,N-dimethylformamide and stirred at room temperature for 3 days. After completion of the reaction, the mixture was washed with dichloromethane and filtered. The filtrate was collected and washed several times with saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. This was then separated and purified by silica gel chromatography (dichloromethane) to obtain Compound C6 (0.36 g, 64% yield) as a yellow solid. 1 H NMR (400MHz, CDCl3) δ10.27(s,1H),7.08(m,2H),6.65(s,1H),6.44(s,1H),3.40 (d, J=6.7Hz, 4H), 2.59 (m, 2H), 2.45 (t, J=6.0Hz, 2H), 1.42 (s, 2H), 1.21 (s, 6H).

[0101] Under nitrogen protection, compound C6 (0.28 g, 1.0 mmol), compound E1 (0.32 g, 1.2 mmol), potassium carbonate (0.28 g, 2.0 mmol) and 5 mL of anhydrous acetic anhydride were added to a reaction flask and stirred at room temperature for 1 d. After the reaction, dichloromethane was added to the reaction solution, and the mixture was washed with water three times. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product, which was then separated and purified by silica gel chromatography (dichloromethane / methanol, volume ratio 20:1) to obtain a dark green solid compound D6 (0.35 g, yield 67%). 1H NMR(500MHz, CDCl3)δ8.50(d,J=14.1Hz,1H),7.56(s,1H),7.44(dd,J=26.1,9.9Hz,3 H),7.20(d,J=8.0Hz,1H),6.82(d,J=9.0Hz,1H),6.52(s,1H),6.18(d,J=14.1Hz,1H) ,4.35(t,J=6.9Hz,2H),4.21(t,J=5.9Hz,2H),3.56(q,J=7.1Hz,4H),3.49(s,1H),2. 83(m,4H),2.29(m,2H),1.95(s,3H),1.79(s,6H),1.72(s,2H),1.31(t,J=7.1Hz,6H).

[0102] Potassium carbonate (56 mg, 0.4 mmol) was added to a solution of compound D6 (105 mg, 0.2 mmol) in methanol (2 mL) and stirred at room temperature overnight. D6 further underwent a ring-closure reaction via intermediate compound E6. After completion of the reaction, the mixture was extracted with dichloromethane / saturated sodium bicarbonate solution. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. This was then separated and purified by basic alumina chromatography (petroleum ether / dichloromethane, volume ratio 50:1) to obtain a yellow solid, a near-infrared photosensitizer activated by the slightly acidic environment of tumors, designated LET-Hcy-N (86 mg, 89% yield). 1 H NMR (500MHz, MeOD) δ8.65(d,J=12.6Hz,1H),7.55(d,J=8.0Hz,2H),7.48(m,2H),7.39(d,J=7. 9Hz,1H),7.31(td,J=7.4,1.0Hz,1H),6.93(dd,J=9.0,2.4Hz,1H),6.73(d,J=2.5Hz,1H),6.38 (d,J=12.7Hz,1H),4.29(t,J=7.2Hz,2H),3.69(t,J=5.7Hz,2H),3.60(q,J=7.1Hz,4H),2.76(d d,J=26.1,6.3Hz,4H),2.08(m,2H),1.94(t,J=5.2Hz,2H),1.80(s,6H),1.28(d,J=7.1Hz,6H). HRMS(ESI)calcd for C 32 H 38 N2O2 + 483.293, found [M+H] + 483.765.

[0103] The structural formula of the near-infrared photosensitizer LET-Hcy-N, which can be activated by the slightly acidic environment of tumors, prepared in this example is as follows:

[0104] Example 7

[0105] The synthesis route for preparing the tumor slightly acidic environment-activated near-infrared photosensitizer LET-BHcy-ROS in this embodiment is shown in FIG5 . The specific synthesis steps are as follows:

[0106] Compound A6 (1 g, 6.94 mmol) and compound A7 (0.88 g, 5.78 mmol) were dissolved in 40 mL of N,N-dimethylformamide and stirred at room temperature for 3 days. After the reaction, the mixture was filtered and the filtrate was concentrated under vacuum. The residue was dissolved in 50 mL of dichloromethane and washed with distilled water (25 × 3 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under vacuum. Purification by chromatography (n-hexane / ethyl acetate, volume ratio 1:3) afforded compound B7 (1.09 g, 78%) as a yellow solid. This was carried directly to the next step.

[0107] Compound B7 (1 g, 2.19 mmol) was dissolved in anhydrous dichloromethane. Boron tribromide (2.2 mL, 22.6 mmol) was added dropwise in an ice bath at 0°C under nitrogen. The reaction was stirred overnight at room temperature, then quenched with water and extracted with dichloromethane. The organic phase was rinsed three times with water, dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified by silica gel chromatography (dichloromethane / methanol, volume ratio 20:1) to obtain compound C7 (0.34 g, 34% yield) as a brown solid. 1 H NMR (500MHz, DMSO-d6): δ10.20(s,2H),7.19(d,J=8.5Hz,1H),6.93(s,1H),6.63(d,J= 2.0Hz, 1H), 6.60 (dd, J = 8.5, 2.5Hz, 1H), 2.55 (m, 2H), 2.29 (t, J = 6Hz, 2H), 1.64 (m, 2H).

[0108] Compound D7 (297 mg, 1.0 mmol) and potassium carbonate (138.21 mg, 1.0 mmol) were added to a dichloromethane solution of compound C7 (114.1 mg, 0.5 mmol), and the reaction mixture was stirred at room temperature overnight. After the reaction, the excess potassium carbonate was filtered off, the filtrate was dried over anhydrous sodium sulfate, and then concentrated. The crude product was collected by silica gel column chromatography (dichloromethane / methanol, volume ratio 30:1), and the solvent was removed to obtain a yellow solid compound E7 (193 mg, yield 53%). HRMS (ESI) calculated for C27 H 30 Bo5 + 445.2108,found[M+H] + 445.217. Under nitrogen protection, compound G5 (68.9 mg, 0.2 mmol) and compound E7 (50 mg, 0.1 mmol) were dissolved in 10 mL of anhydrous ethanol, heated to 80°C, and stirred for 12 h. The condensation intermediate compound F7 was synthesized. Potassium carbonate solid (16.5 mg, 0.12 mmol) was then added and stirred for 2 h. The crude product was then precipitated with 30 mL of diethyl ether. After the reaction, the solvent was removed by spin drying. The crude product was separated and purified by column chromatography (dichloromethane / methanol, volume ratio 20:1). The resulting solid compound (45 mg, 55.4% yield) was a near-infrared photosensitizer activated by the slightly acidic environment of tumors and designated LET-BHcy-ROS. 1 H NMR(500MHz,MeOD)δ8.89(d,J=14.5Hz,1H),8.52(d,J=7.5Hz,1H),8.15(d,J=4.0Hz,1H) ,7.96(t,J=7.5Hz,1H),7.73(dd,J=8.0,4.5Hz,3H),7.59(m,2H),7.41(m,4H),7.16(d,J =4.0Hz,1H),7.02(dd,J=8.5,2.0Hz,1H),6.76(d,J=14.5Hz,1H),5.24(s,2H),4.42(t,J =7.0Hz,2H),3.65(t,J=6.0Hz,2H),2.70(m,4H),2.04(m,2H),1.88(m,2H),1.33(s,12H).

[0109] Example 8

[0110] The synthesis route for preparing the tumor microacid environment-activatable near-infrared photosensitizer LET-BHcy-GSH in this embodiment is shown in FIG5 . The specific synthesis steps are as follows:

[0111] Under nitrogen protection and a 0°C ice bath, compound D8 (0.66 g, 2.5 mmol) and triethylamine (0.26 mL, 3 mmol) were added sequentially to a solution of compound C7 (114.1 mg, 0.5 mmol) in anhydrous dichloromethane. The reaction solution was stirred at room temperature overnight. After the reaction, the reaction solution was washed with saturated sodium chloride and distilled water, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain compound E8 (106 mg, 48% yield) as a yellow solid. HRMS (ESI) calculated for C 20 H 15 N2SO9 +459.0420,found[M+H] + 459.067.

[0112] Under nitrogen protection, compound G5 (66.8 mg, 0.2 mmol) and compound E8 (50 mg, 0.1 mmol) were dissolved in 10 mL of anhydrous ethanol. The reaction solution was heated to 80°C and stirred overnight to synthesize the condensation intermediate compound F8. Subsequently, solid potassium carbonate (16.5 mg, 0.12 mmol) was added and stirred for 2 h. The crude product was then precipitated with 30 mL of ether to obtain a crude product. After the reaction, the solvent was removed by spin-drying. The crude product was separated and purified by column chromatography (dichloromethane / methanol, volume ratio 20:1), collected and spin-dried to obtain a solid compound (34 mg, yield 45.9%), which was a near-infrared photosensitizer activated by the slightly acidic environment of the tumor and was designated as LET-BHcy-GSH. 1 H NMR(500MHz,MeOD)δ9.15(d,J=15Hz,1H),8.96(d,J=5Hz,1H),8.74(d,J=10Hz,1H),8. 52(dd,J=10,5Hz,1H),8.37(d,J=5Hz,1H),8.00(m,2H),7.84(d,J=10Hz,1H),7.60(d, J=5Hz,1H),7.43(d,J=5Hz,1H),7.37(s,1H),7.14(dd,J=10,5Hz,1H),7.03(d,J=15Hz ,1H),4.60(t,J=5Hz,2H),3.67(t,J=5Hz,2H),2.82(m,4H),2.14(m,2H),1.97(m,2H).

[0113] The structural formulas of the tumor slightly acidic environment-activated near-infrared photosensitizers LET-BHcy-ROS and LET-BHcy-GSH prepared in Examples 7 and 8 are shown below:

[0114] Performance Testing

[0115] (1) Spectral test

[0116] Each photosensitizer prepared in Examples 1 to 8 was dissolved in dimethyl sulfoxide to prepare a photosensitizer stock solution with a concentration of 10 mM and stored at 4°C for subsequent use. All tests were performed in a PBS buffer (1×) system containing 1% dimethyl sulfoxide (DMSO). 2 μL of the photosensitizer stock solution was added to 2 mL of PBS buffer at different pH values ​​and tested using a UV-visible spectrophotometer and a fluorescence spectrophotometer to obtain absorption and fluorescence spectra of each photosensitizer under different pH conditions.

[0117] The absorption test results of each photosensitizer are shown in Figure 6. Since the activation mechanism of each photosensitizer molecule is the same (as shown in Figure 1), which is the cleavage of the ortho-ether oxygen bond of the indole, the trend of change in their UV-visible absorption spectra with changes in pH is similar. That is, as the pH of the solution decreases, the absorbance of the photosensitizer molecule in the near-infrared region gradually increases, while the absorbance in the visible region gradually decreases. The fluorescence emission of the photosensitizers is shown as LET-H, LET-I, and LET-Hcy-N. Their fluorescence spectra are shown in Figures a, b, c, d, and e in Figure 7. In the visible light region, the fluorescence emission intensity of the photosensitizer molecules LET-H and LET-I decreases with increasing pH, while in the near-infrared region, the fluorescence emission intensity of LET-H, LET-I, or LET-Hcy-N increases with increasing pH, indicating that the photosensitizers prepared by the present invention can be activated by a slightly acidic environment, thereby producing near-infrared absorption and fluorescence emission.

[0118] (2) Singlet oxygen generation test

[0119] The photosensitizers LET-R (R = H, Cl, Br, I) prepared in Examples 1 to 4 were tested. 1,3-Diphenylisobenzofuran (DPBF) was selected as a singlet oxygen scavenger, and the ability of each photosensitizer molecule to generate singlet oxygen was determined.

[0120] First, prepare a 1mM DPBF stock solution for later use. Then, add an appropriate volume of the freshly prepared DPBF solution to DMSO, adjusting the DPBF absorbance at 415nm to approximately 1.0. Then, add the appropriate concentration of the photosensitizer solution. Measure the UV-visible absorption spectrum of the solution under near-infrared laser irradiation for varying durations. By monitoring changes in DPBF absorbance at 415nm, the ability of each photosensitizer to generate singlet oxygen is evaluated.

[0121] The test results, shown in Figure 8a, show that compared to the unactivated photosensitizer molecule LET-R (R = H, Cl, Br, I), the activated photosensitizer LET-R' (R = H, Cl, Br, I) has the ability to generate singlet oxygen, indicating that the photosensitizers prepared in Examples 1 to 4 of the present invention can produce near-infrared photodynamic therapy effects after activation. In addition, LET-I' has a more efficient singlet oxygen generation ability, indicating that the halogen atom modification of the photosensitizer molecular backbone can enhance the photosensitizer molecule's ability to generate singlet oxygen.

[0122] The singlet oxygen yield of the photosensitizer LET-R (R = H, Cl, Br, I) was tested using indocyanine green (ICG) as a reference. First, an appropriate volume of freshly prepared DPBF solution was added to dichloromethane, and the absorbance of DPBF at 415 nm was adjusted to approximately 1.0. Then, an appropriate concentration of the test solution (photosensitizer molecule or ICG) was added. Subsequently, a near-infrared laser was used to irradiate the solution, and the changes in the UV spectrum at different time points were measured. The singlet oxygen quantum yield of the molecule was calculated using the following formula (1): Φ Δ =Φ MB *(k ps *F MB ) / (k MB *F ps ) Formula (1)

[0123] Φ Δ is the singlet oxygen quantum yield of the molecule, Φ ICG is the singlet oxygen quantum yield of ICG. ps represents the molecule to be measured, and k represents the slope of the absorbance of DPBF at 415 nm over time. The correction factor F is calculated by the following formula (2): F = 1-10 OD Formula (2)

[0124] In formula (2), OD represents the absorbance of the solution at the laser wavelength.

[0125] The test results are shown in FIG8 b, which further quantitatively confirm that the photosensitizer LET-I′ has a higher singlet oxygen generation ability.

[0126] Electron paramagnetic resonance (ESR) was used to further examine the generation and type of singlet oxygen. Using the photosensitizers LET-H' and LET-I' as examples, 2,2,6,6-tetramethyl-4-piperidinone hydrochloride (TEMP) was used as a singlet oxygen scavenger. First, two photosensitizer stock solutions were diluted and then TEMP was added. One group remained untreated, while the other was irradiated with a near-infrared laser. A blank control was also obtained.

[0127] The test results are shown in Figure 8(c). Near-infrared light irradiation of samples containing activated photosensitizers LET-H' or LET-I' can produce three paramagnetic peaks of equal height (1:1:1), indicating that both photosensitizers LET-H' and LET-I' produce singlet oxygen.

[0128] (3) Evaluation of therapeutic effects at the cellular level

[0129] The standard MTT method was used to evaluate the effect of photodynamic therapy of photosensitizer molecules on the survival rate of mouse breast cancer cells (4T1 cells).

[0130] Cell culture was performed in DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% double-antibody (penicillin-streptomycin mixture) and incubated at 37°C in a 5% CO2 incubator. When the cell density reached 80%, the relevant experiments were performed. Taking photosensitizers LET-H and LET-I as an example, 4T1 cells were seeded in 96-well plates (100 μL, 5×10 3 cells / well) and cultured for 24 h. The original culture medium was then discarded and a culture medium with a concentration of 20 μM photosensitizer molecules was added. After incubation for 6 h, the culture medium in each well was replaced with fresh culture medium and the cells were illuminated with 808 nm (0.2 W / cm 2 ) laser irradiation for different times (0, 5, 10, and 20 minutes) and continued incubation in a cell culture incubator. After 12 hours of incubation, 10 μL of CCK-8 solution was added to each well and incubated in the cell culture incubator for another hour. The absorbance at 450 nm was then measured using a microplate reader.

[0131] The test results are shown in Figure 9. After 20 minutes of laser irradiation, the cell survival rate of the LET-I group was less than 30%, and was significantly lower than the cell survival rate of the LET-H group after 20 minutes of light irradiation, indicating that the photosensitizer LET-I exhibits stronger phototoxicity.

[0132] (4) Visual evaluation of photodynamic therapy effects at the in vivo level

[0133] Establish a mouse tumor model for visualized photodynamic therapy.

[0134] Female BALB / c nude mice (6 weeks old) were purchased to establish a mouse tumor model. 4T1 cells in the logarithmic growth phase were digested and resuspended in serum-free medium. Before inoculation, the cells were kept on ice. All mice were randomly divided into groups of 5. 4T1 cells were inoculated into the right hind limb of the mouse under the armpit, with 100 μL injected subcutaneously at each inoculation site. When the tumor volume reached 80 mm 3 During the experiment, the photosensitizer molecules were injected into the mice via intratumoral injection or tail vein injection, and the changes in the fluorescence and photoacoustic signals in the tumor area over time were detected using the small animal fluorescence imaging system (IVIS Spectrum) and the small animal photoacoustic imaging system (VisualSonics Vevo LAZR system). During the imaging experiment, all mice were anesthetized with isoflurane (2% in oxygen). Taking LET-H and LET-I as examples, in order to enhance the biocompatibility of the photosensitizer molecules, the photosensitizer molecules were nano-engineered using amphiphilic polymers modified with folic acid, which were recorded as LET-H-FA and LET-I-FA, respectively.

[0135] As shown in a and b in Figure 10, after intratumoral injection of LET-I-FA, the fluorescence signal of the tumor gradually increased, while its fluorescence intensity remained basically unchanged after subcutaneous injection into normal tissue (leg muscle). 20 minutes after injection, the fluorescence intensity of the mouse tumor was significantly higher than that of normal tissue. At the same time, the PBS control group showed a low and unchanged fluorescence signal in both tumor and normal tissue, indicating that LET-I-FA can be specifically activated in the tumor and produce a strong fluorescence signal. At the same time, as shown in c and d in Figure 10, after LET-I-FA was injected intratumorally or subcutaneously into the tumor or normal tissue, the photoacoustic signal intensity in the tumor reached its maximum after 20 minutes, while the photoacoustic signal of normal tissue remained basically unchanged and was lower than the signal intensity of the tumor. This further shows that LET-I-FA can be specifically activated in the tumor and produce a strong photoacoustic signal.

[0136] Tumor-bearing mice were randomly divided into 7 groups: (1) PBS group; (2) simple light group; (3) LET-I-FA group; (4) LET-H-FA group; (5) LET-I light group; (6) LET-H-FA light group; (7) LET-I-FA light group. The photosensitizer solution of each group was 10 μmol / kg, and PBS was 100 μL in dose. The mice were injected into the tail vein. Twelve hours after the injection, the group that needed laser irradiation was irradiated with 808 laser at 0.2 W / cm 2 The mice were irradiated with a power of 100 nm for 30 minutes each. Starting from the time of dosing, the weight of each group of mice was observed every other day. As shown in Figure 11(a), the results show no significant changes in mouse weight during treatment, demonstrating the good biosafety of the photosensitizer molecule used.

[0137] After 14 days of treatment, mice in each group were euthanized, and the ex vivo tumors were obtained, weighed, and photographed. As shown in Figure 11 (b) and (c), the tumors in the LET-I-FA group were significantly inhibited, and the treatment effect in this group was significantly better than that in the LET-H-FA group.

[0138] In addition, after the mice were dissected, major organs (heart, liver, spleen, lungs, and kidneys) were removed and fixed with paraformaldehyde. Hematoxylin-eosin (H&E) staining was performed to obtain images of tissue sections. The results, shown in Figure 12(a), indicate that no significant changes were observed in these major organs after photosensitizer treatment.

[0139] Three groups of mice, each containing three mice, were injected via tail vein with a 10 μmol / kg solution of LET-H-FA or LET-I-FA photosensitizer in 100 μL of PBS. Fourteen days later, blood was collected from each group via orbital bleeding, centrifuged, and the supernatant serum was collected. Biochemical parameters were analyzed to assess the effects on liver and kidney function in each group. As shown in Figure 12(b), no significant abnormalities were observed in the blood biochemical profiles of the mice in each group, further demonstrating the good biosafety of the photosensitizer molecules used.

[0140] In summary, the near-infrared photosensitizer activated by the micro-acidic environment of the tumor provided by the present invention can realize efficient and precise tumor photodynamic therapy guided by fluorescence imaging / photoacoustic imaging. By introducing pH-responsive groups, the near-infrared photosensitizer has better tumor selectivity, thereby effectively reducing the phototoxicity of the photosensitizer to normal tissues. At the same time, this tumor-specific activation property gives the photosensitizer molecule a higher imaging signal-to-noise ratio, which is conducive to precise visualization of photodynamic therapy. Moreover, by simply regulating the type of halogen atom substituted in the molecular skeleton, the ability of the photosensitizer molecule to produce singlet oxygen can be improved, effectively inhibiting tumor growth, and providing new ideas for the development of more novel near-infrared photosensitizers.

[0141] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A near-infrared photosensitizer that can be activated by the slightly acidic environment of a tumor, characterized in that: It has a chemical structure shown in any one of formula (I) to formula (VI): Wherein, R1 is H, F, Cl, Br or I atom, R2 is -OH, -NH2, A is a self-eliminating group, and B is a biomarker-responsive group.

2. The near-infrared photosensitizer activated by the slightly acidic environment of tumors according to claim 1, characterized in that The self-eliminating group A is 3. The near-infrared photosensitizer activated by the slightly acidic environment of tumors according to claim 1, characterized in that The biomarker responsive group B is:

4. The near-infrared photosensitizer activated by the slightly acidic environment of tumors according to claim 1, characterized in that After being activated in a slightly acidic environment, the photosensitizers represented by formula (I) to formula (VI) have the structures represented by formula (I') to formula (VI') in sequence:

5. The near-infrared photosensitizer activated by the slightly acidic environment of tumors according to claim 1, characterized in that It has the following structural formula:

6. A method for preparing the near-infrared photosensitizer activated by a slightly acidic environment of a tumor according to any one of claims 1 to 5, characterized in that: Including steps: S1, compound 1 represented by formula 1 and compound 4 represented by formula 4 undergo condensation reaction to obtain compound 6 represented by formula 6; Or compound 2 represented by formula 2 and compound 4 represented by formula 4 undergo condensation reaction to obtain compound 7 represented by formula 7; Or compound 3 represented by formula 3 and compound 4 represented by formula 4 undergo condensation reaction to obtain compound 8 represented by formula 8; or compound 1 represented by formula 1 and compound 5 represented by formula 5 undergo condensation reaction to obtain compound 9 represented by formula 9; or the compound 2 represented by formula 2 and the compound 5 represented by formula 5 undergo a condensation reaction to obtain the compound 10 represented by formula 10; or the compound 3 shown in formula 3 and the compound 5 shown in formula 5 undergo a condensation reaction to obtain the compound 11 shown in formula 11; S2 and compound 6 undergo ester hydrolysis and ring closure reactions in sequence under alkaline conditions to obtain a near-infrared photosensitizer that can be activated by the slightly acidic environment of tumors as shown in formula (I); Alternatively, compound 7 undergoes ester hydrolysis and ring closure reactions in sequence under alkaline conditions to obtain a near-infrared photosensitizer activated by the slightly acidic environment of the tumor as shown in formula (II); Or compound 8 undergoes a ring-closing reaction under alkaline conditions to obtain a near-infrared photosensitizer that can be activated by the slightly acidic environment of the tumor as shown in formula (III); Alternatively, compound 9 undergoes ester hydrolysis and ring closure reactions in sequence under alkaline conditions to obtain a near-infrared photosensitizer activated by the slightly acidic environment of the tumor as shown in formula (IV); Alternatively, compound 10 undergoes ester hydrolysis and ring closure reactions in sequence under alkaline conditions to obtain a near-infrared photosensitizer activated by the slightly acidic environment of the tumor as shown in formula (V); Alternatively, compound 11 undergoes a ring-closing reaction under alkaline conditions to obtain a near-infrared photosensitizer that can be activated by the slightly acidic environment of the tumor as shown in formula (VI); The structural formulas of compounds 1 to 11 are shown in the following formulas 1 to 11: The temperature of the condensation reaction in step S1 is 50-100° C., and the time of the condensation reaction is 2-12 hours; The alkaline conditions required for the ring-closure reaction in step S2 are one or more of potassium carbonate, cesium carbonate, potassium hydroxide, sodium hydroxide, sodium hydride, ammonia water and triethylamine; and / or the temperature of the ring-closure reaction is 0 to 50° C., and the time of the ring-closure reaction is 2 to 12 hours.

7. The method for preparing a near-infrared photosensitizer activated by a slightly acidic tumor environment according to claim 6, characterized in that: In step S1, compound 1 is prepared by a substitution reaction between compound 12 and compound 13; wherein the temperature of the substitution reaction is 50 to 150° C., and the time of the substitution reaction is 24 to 72 hours; and / or the solvent used in the substitution reaction is one or more of acetonitrile, N,N-dimethylformamide, dichloromethane, chloroform, toluene, and o-dichlorobenzene; The structural formulas of compound 12 and compound 13 are shown below:

8. The method for preparing a near-infrared photosensitizer activated by a slightly acidic environment of a tumor according to claim 6, characterized in that: The compound 3 in step S1 is prepared by the following steps: Compound 14 undergoes sulfurization reaction to obtain compound 15; The compound 15 undergoes iodination reaction to obtain compound 16; The compound 16 undergoes a substitution reaction with the compound 17 to obtain the compound 18; The compound 18 undergoes a substitution reaction with the compound 13 to obtain the compound 19; The compound 19 undergoes a hydrolysis reaction to obtain the compound 3; The structural formulas of compounds 13 to 19 are shown below:

9. Use of a near-infrared photosensitizer activated by a slightly acidic environment of a tumor according to any one of claims 1 to 5, characterized in that: Used for preparing tumor diagnosis agents and / or tumor treatment agents.

10. The use of the near-infrared photosensitizer activated by a slightly acidic environment of a tumor according to claim 9, characterized in that: The tumor diagnostic agent includes a fluorescent imaging agent or a photoacoustic imaging agent; and / or the tumor therapeutic agent includes a photodynamic therapy agent or an agent for combining photodynamic therapy with other treatment regimens.

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