A dual-activated photodynamic immune prodrug based on semicyanine and IDO inhibitor and its preparation method and application

Abstract

The present invention provides a dual-activated photodynamic immune prodrug based on semi-cyanine and IDO inhibitor, and a preparation method and application thereof. The first purpose is to provide a dual-activated photodynamic immune prodrug based on semi-cyanine and IDO inhibitor, the product has the effect of inhibiting the activity of photosensitizer and immune drug at the same time, performing efficient synergistic immunotherapy, obtaining excellent tumor treatment efficiency, and avoiding the effect of serious side effects during tumor treatment. The second purpose is to provide a method for preparing a dual-activated photodynamic immune prodrug, and the synthesis method can be used for the covalent connection synthesis of the same type of photosensitizer and immune checkpoint inhibitor. The third purpose is to provide a dual-activated photodynamic immune prodrug that can be used for the selective treatment and diagnosis of tumors and non-tumor cells, can activate photodynamic therapy, and can effectively improve the specificity of drug molecules to tumor cells, thereby further realizing effective photodynamic immune combined therapy.

Description

Technical Field

[0001] The present invention relates to the field of biochemical technology, and in particular to a dual-activated photodynamic immune prodrug based on semi-cyanine and IDO inhibitor, and a preparation method and application thereof. Background Art

[0002] Immunotherapy is an effective cancer treatment strategy that has developed rapidly in recent years. It mainly activates the immune system to clear tumor tissue and prevent tumor recurrence and metastasis. However, clinical cancer immunotherapy still faces the problems of low response rate and easy immune-related side effects. Combining photodynamic therapy with immune checkpoint blockade is one of the effective methods to maximize the effect of immunotherapy by enhancing tumor immunogenicity and regulating the immunosuppressive tumor microenvironment. Tumor photodynamic therapy has been widely studied due to its outstanding characteristics such as non-invasiveness, spatial precision and synergistic effects, and has important application prospects. However, in the actual treatment process, the biological activity of photosensitizers or immune checkpoint inhibitors is always in an activated state, which may lead to uncontrollable side effects and limit their in vivo therapeutic effects.

[0003] Therefore, the design of activatable prodrug molecules that specifically respond to tumor cells is of great significance for improving the selectivity of tumor treatment. Summary of the invention

[0004] In response to the problem that current drug molecules have insufficient specific selectivity for cancer cells, the first purpose of the present invention is to provide a dual-activated photodynamic immunotherapy prodrug based on semi-cyanine and IDO inhibitor. The product has the effect of simultaneously inhibiting the activity of photosensitizer and immune drug, performing efficient synergistic immunotherapy, achieving excellent tumor treatment efficiency, and avoiding serious side effects during tumor treatment.

[0005] The second object of the present invention is to provide a method for preparing a dual-activated photodynamic immune prodrug based on semi-cyanine and IDO inhibitor, and the synthesis method can be used for the covalent connection synthesis of the same type of photosensitizers and immune checkpoint inhibitors.

[0006] The third object of the present invention is to provide a dual-activated photodynamic immunotherapy prodrug based on semi-cyanine and IDO inhibitor that can be used for the selective treatment and diagnosis of tumor and non-tumor cells, can activate photodynamic therapy, and can effectively improve the specificity of drug molecules for tumor cells, thereby further realizing effective photodynamic immunotherapy.

[0007] To achieve the above first objective, the present invention provides the following technical solution: a dual-activated photodynamic immune prodrug based on hemicyanine and IDO inhibitor, wherein the dual-activated photodynamic immune prodrug has the following general formula (I):

[0008]

[0009] wherein a and b are each independently selected from an integer of 1-9;

[0010] X1 is a halogen ion;

[0011] X2 is O, S or Se; preferably, X2 is O;

[0012] X3, X4 and X5 are each independently selected from -O- or -NH-; preferably, when X3 is -NH-, X4 is selected from -O-, and X5 is selected from -O-;

[0013] R1 is selected from one of the following groups:

[0014]

[0015] R2 is selected from one of the following groups:

[0016]

[0017] R3 is an IDO inhibitor or 1-methyl-tryptophan.

[0018] Further, X1 is Cl, Br or I; further, X1 is I.

[0019] To achieve the above second purpose, the present invention provides the following technical solution: a method for preparing a dual-activated photodynamic immunoprodrug based on hemicyanine and IDO inhibitor, wherein the compound of formula I is prepared by the following method:

[0020]

[0021] S1: A halogenated 2,3,3-trimethylindole compound A reacts with a compound B containing an R1 group in the presence of a first organic solvent and a first weak base to obtain a first intermediate C;

[0022] S2: The first intermediate C reacts with a condensing agent 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde in the presence of a second organic solvent and a second weak base to obtain an intermediate, and the intermediate reacts with a compound D in the presence of a third organic solvent under the protection of an inert gas to obtain a second intermediate E;

[0023] S3: The second intermediate and the first catalyst are dissolved in a fourth organic solvent to obtain a mixture, and the mixture is reacted with a compound F containing an R2 group in a fifth organic solvent and a third weak base under the protection of an inert gas to obtain a third intermediate G;

[0024] S4: Compound H containing an immune checkpoint inhibitor and a second catalyst are dissolved in a sixth organic solvent to obtain a mixture. Under the protection of an inert gas, the mixture reacts with a third intermediate G in a seventh organic solvent and a fourth weak base to obtain a dual-activated photodynamic immune prodrug based on semicyanine and an IDO inhibitor of structure I.

[0025] Further, in step S1, the reaction time is 24-48 hours, and the first organic solvent used is selected from at least one of toluene, benzene, xylene, and acetonitrile;

[0026] The first weak base used is selected from at least one of triethylamine, sodium acetate, ammonium acetate, sodium carbonate and sodium bicarbonate.

[0027] Further, in step S2, the second organic solvent is selected from at least one of acetic acid, ethanol, acetonitrile, DMF, tetrahydrofuran, and acetone;

[0028] The second weak base is selected from at least one of sodium acetate, ammonium acetate, potassium acetate, sodium carbonate, potassium carbonate, and sodium bicarbonate;

[0029] The third organic solvent is selected from at least one of acetonitrile, DMF, DMSO, dichloromethane and chloroform.

[0030] Further, in step S3, the reaction time is 12-24 hours, and the first catalyst used is selected from at least one of carbonyl imidazole, triphosgene, and p-nitrophenyl chloroformate;

[0031] The fourth organic solvent used is selected from at least one of dichloromethane, DMF, acetonitrile, dichloroethane, chloroform and tetrahydrofuran;

[0032] The third weak base used is selected from at least one of DMAP, DIPEA, DBU and triethylamine;

[0033] The fifth organic solvent used is at least one selected from dichloromethane, tetrahydrofuran and DMF.

[0034] Further, in step S4, the second catalyst is selected from at least one of carbonyl imidazole, triphosgene, and p-nitrophenyl chloroformate;

[0035] The sixth organic solvent is selected from at least one of dichloromethane, DMF, acetonitrile, dichloroethane, chloroform, and tetrahydrofuran;

[0036] The fourth weak base is selected from at least one of DMAP, DIPEA, DBU and triethylamine;

[0037] The seventh organic solvent is selected from at least one of dichloromethane, tetrahydrofuran and DMF.

[0038] Furthermore, in steps S2, S3 and S4, the inert gas is independently selected from at least one of helium, nitrogen and argon.

[0039] To achieve the third objective, the present invention provides the following technical solution: A dual-activated photodynamic immunoprodrug based on hemicyanine and IDO inhibitor has at least one of the following effects:

[0040] The dual-activated photodynamic immunoprodrug based on hemicyanine and IDO inhibitor is used for activatable fluorescence imaging tumor diagnosis based on hemicyanine dye;

[0041] The dual-activated photodynamic immune prodrug based on hemicyanine and IDO inhibitor is used for activatable photodynamic therapy based on hemicyanine photosensitizing dye;

[0042] The dual-activated photodynamic immunoprodrug based on hemicyanine and IDO inhibitor is used for selective treatment of tumor and non-tumor cells;

[0043] The dual-activated photodynamic immune prodrug based on hemicyanine and IDO inhibitor is used for immune checkpoint inhibitor release;

[0044] The dual-activated photodynamic immunotherapy prodrug based on hemicyanine and IDO inhibitor is used for photodynamic immunotherapy combined with enhanced immune response.

[0045] In summary, the present invention has the following beneficial effects:

[0046] First, the present invention obtains a photodynamic immunotherapy prodrug by connecting the semi-cyanine dye and the IDO inhibitor through a covalent bond responsive to a tumor marker, thereby achieving dual inhibition of the activity of the photosensitizer and the immune drug. The fluorescence of the obtained photodynamic immunotherapy molecular prodrug is quenched, the ability to produce active oxygen is greatly reduced (reduced to 40% of the semi-cyanine dye parent), and the IDO inhibition ability is significantly weakened (EC 50 =5.9 μM, while the EC of free NLG919 50 =1 μM);

[0047] Second, after the drug molecules respond in tumor cells, the drug activity is restored, the fluorescence of the semi-cyanine photosensitizer is restored, and the ability to produce reactive oxygen species is significantly enhanced. 2 , 5min) can effectively kill tumor cells under light (IC 50=2μM), achieving photodynamic therapy while causing immunogenic cell death to enhance the immunogenicity of tumor cells. Further in vivo experiments, tumor-associated antigens released after photodynamic killing and activated immune checkpoint inhibitors jointly induced synergistic anti-tumor immune responses, thereby inhibiting the growth of in situ / distant tumors and preventing tumor lung metastasis; the activatable photodynamic immune prodrug molecules not only achieved synergistic therapy and obtained excellent tumor treatment efficiency, but also avoided serious side effects during tumor treatment;

[0048] Third, activatable fluorescence imaging based on semi-cyanine dyes is used for tumor diagnosis, which can achieve a fluorescence enhancement effect of about 8-10 times before and after activation, and can be used for tumor identification; it is used for activatable photodynamic therapy based on semi-cyanine photosensitizer dyes, and the photodynamic effect can be increased by 2-3 times after activation; it is used for selective treatment of tumor and non-tumor cells, and the therapeutic effect differs by 5-6 times; it is used for the release of immune checkpoint inhibitors, and the drug activity before and after release differs by 5-6 times; it is used for photodynamic immunotherapy to enhance immune response, and the infiltration of cytotoxic T lymphocytes in the in situ tumor is 4-5 times that of the control group, and 12-15 times that of the distal tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0050] Figure 1 is a high-resolution mass spectrum of the second intermediate disclosed in the embodiments of the present invention;

[0051] Figure 2 is a nuclear magnetic hydrogen spectrum of the second intermediate disclosed in the embodiments of the present invention;

[0052] Figure 3 is a high-resolution mass spectrum of the third intermediate disclosed in the embodiments of the present invention;

[0053] Figure 4 is a nuclear magnetic hydrogen spectrum of the third intermediate disclosed in the embodiments of the present invention;

[0054] Figure 5 is the NMR carbon spectrum of the third intermediate disclosed in the embodiments of the present invention;

[0055] Figure 6 A high-resolution mass spectrum of the target activatable prodrug molecule disclosed in the embodiments of the present invention;

[0056] Figure 7is a hydrogen NMR spectrum of the target activatable prodrug molecule disclosed in the embodiments of the present invention;

[0057] Figure 8 The NMR carbon spectrum of the target activatable prodrug molecule disclosed in the embodiments of the present invention;

[0058] Fig. 9 A high-resolution mass spectrum of the target activatable prodrug molecule disclosed in the comparative example of the present invention;

[0059] Fig.10 The H NMR spectrum of the target activatable prodrug molecule disclosed in the comparative example of the present invention;

[0060] Fig.11 The NMR carbon spectrum of the target activatable prodrug molecule disclosed in the comparative example of the present invention;

[0061] Fig.12 : are absorption and fluorescence spectra of the second intermediate, the target activatable prodrug molecule disclosed in the embodiments of the present invention and the target activatable prodrug molecule disclosed in the comparative example; A is an absorption spectrum; B is a fluorescence spectrum;

[0062] Fig.13 Comparison of singlet oxygen generation capabilities of the second intermediate and target activatable prodrug molecule disclosed in the embodiments of the present invention and the target activatable prodrug molecule disclosed in the comparative examples;

[0063] Fig.14 Drug activity testing of the target activatable prodrug molecules disclosed in the examples of the present invention and the target activatable prodrug molecules disclosed in the comparative examples and NLG919;

[0064] Fig.15 The present invention discloses in the embodiments a fluorescence recovery process of a target activatable prodrug molecule;

[0065] Fig.16 The disclosed target in the embodiments of the present invention can activate the fluorescence expression of prodrug molecules in different cells;

[0066] Fig.17 The present invention discloses a cell therapy experiment in which the target can activate the prodrug molecule, including normal cells and tumor cells;

[0067] Fig.18 Cell phototoxicity experiments of the target activatable prodrug molecules and the third intermediate disclosed in the examples of the present invention and the target activatable prodrug molecules disclosed in the comparative examples;

[0068] Fig.19 Mouse in situ tumor inhibition test of the third intermediate, the target activatable prodrug molecule disclosed in the examples of the present invention and the target activatable prodrug molecule disclosed in the comparative examples;

[0069] Fig. 20 Mouse distal tumor inhibition assay of target-activatable prodrug molecules disclosed in the examples of the present invention;

[0070] Fig.21 The target disclosed in the examples of the present invention can activate the prodrug molecules in the lung metastasis experiment of mouse transplanted tumors;

[0071] Fig. 22 Analysis of in vivo immune responses in mice after treatment with target-activatable prodrug molecules disclosed in the examples of the present invention. DETAILED DESCRIPTION

[0072] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the following will be combined with the appended drawings of the embodiments of the present invention. Figure 1-22 , the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0073] The instruments or chemical and biological reagents involved in this application that are not otherwise specified are conventional products that can be purchased from the market.

[0074] Sources of raw materials for this application: Chemical drugs are from Anage or Aladdin Chemical Reagent Company; Immunofluorescence labeling antibodies are from Biolegend Biological Company; biological consumables fetal bovine serum, trypsin, and DMEM are purchased from Gibco; and the cells used are from ATCC cell bank.

[0075] Example

[0076] The reaction mechanism involved in this application is:

[0077]

[0078] Example

[0079] In this embodiment, R1 is The preparation method of a dual-activated photodynamic immunoprodrug based on hemicyanine and IDO inhibitor, wherein R2 is -SS-; R3 is an IDO inhibitor (NLG919); X1 is I; X2 is O; X3 is -NH-, X4 is -O-, and X5 is -O-, is as follows:

[0080] S1: Preparation of the first intermediate:

[0081]

[0082] 5-iodo-2,3,3-trimethylindole (10 mmol) and iodoethane (50 mmol) were dissolved in 20 mL of anhydrous toluene, and 3 drops of triethylamine were added thereto. The reaction was refluxed for 12 h until the reaction was completed. The product was a light pink precipitate. The light pink precipitate was filtered and washed with ether to obtain the first intermediate, which was a light pink powder solid. It can be directly used for the next step without further purification;

[0083] S2: Preparation of the second intermediate (E)-2-(2-(6-amino-2,3-dihydro-1H-oxanthene)vinyl)-1-ethyl-5-iodo-3,3-dimethyl-3H-indole salt:

[0084]

[0085] The first intermediate (2 mmol) was reacted with a condensing agent (1mmol) was dissolved in 5mL acetic acid, sodium acetate (2mmol) was added under stirring, heated to 110℃ and refluxed for 2h, and the organic solvent was removed by vacuum rotary evaporation to obtain an intermediate. The intermediate was dissolved in 10mL acetonitrile, and then m-hydroxyaniline (3mmol) and potassium carbonate (3mmol) were added. Under nitrogen protection, the reaction was stirred for 12h at a reaction temperature of 20℃. After 20min of reaction, stannous chloride (4mmol) was added and refluxed at 80℃ for 8h. The mixed solvent was removed under reduced pressure distillation to obtain a crude product, which was purified by silica gel chromatography column with an eluent of dichloromethane: methanol = 30:1 (v / v) to obtain a second intermediate, namely (E)-2-(2-(6-amino-2,3-dihydro-1H-xanthene)vinyl)-1-ethyl-5-iodo-3,3-dimethyl-3H-indole salt (0.6mmol), blue, with a yield of 60%;

[0086] HRMS(ESI):m / zC 27 H 28 IN2O + Calculated value [M] + 523.1241, test value 523.1249, its high-resolution mass spectrum is as follows Figure 1 shown.

[0087] 1H NMR (500MHz, DMSO-d6) δ8.39(d,J=10.3Hz,1H),8.03(d,J=1.4Hz,1H),7.78(s,2H),7.76(d,J=1.5Hz,1H),7.46(d,J=8.6Hz,1H),7.26(d,J =8.4Hz,1H),7.04(s,1H),6.79(dd,J=8.6,1.8Hz,1H),6.69(d,J=1.3Hz,1H) ,4.19(q,J=7.1Hz,2H),2.73(t,J=2.0Hz,2H),2.67(t,J=5.9Hz,2H),1.88(m, J=5.0Hz,2H),1.69(s,6H),1.28(t,J=7.1Hz,3H). Its H NMR spectrum is as follows Figure 2 As shown;

[0088] S3: Preparation of the third intermediate 5-iodo-2,3,3-trimethylindole:

[0089]

[0090] The second intermediate (0.2 mmol) and p-nitrophenyl chloroformate (0.6 mmol) were dissolved in 10 mL of dichloromethane and stirred for 1 h to obtain a mixed solution. Then, 2 mL of dichloromethane was added to the mixed solution. A mixed solution of (0.2mmol) and DIPEA (0.2mmol) was stirred at room temperature for 12h to terminate the reaction. The mixed solvent was removed under reduced pressure distillation to obtain a crude product, which was purified using a silica gel chromatography column with an eluent of dichloromethane:methanol = 20:1 (v / v) to obtain a blue third intermediate (0.16mmol) with a yield of 80%;

[0091] HRMS(ESI):m / zC 32 H 36 N2O4S2 + Calculated value [M] + 703.1156, test value 703.1135. Its high-resolution mass spectrum is as follows Figure 3 shown.

[0092] 1H NMR(500MHz,CD3OD)δ8.78(d,J=14.7Hz,1H),8.03(d,J=1.5Hz, 1H),7.94(s,1H),7.87(dd,J=8.4,1.6Hz,1H),7.50–7.44(m,2H),7.33(d,J =8.4Hz,1H),7.25(dd,J=8.5,2.0Hz,1H),6.49(d,J=14.6Hz,1H),4.47(t,J= 6.4Hz,2H),4.35(q,J=7.2Hz,2H),3.82(t,J=6.4Hz,2H),3.06(t,J=6.4Hz,2H),2.90(t,J=6.4Hz,2H),2.81(t,J=4.4Hz,2H),2.74(t,J=6.0Hz,2H), 1.99–1.91(m,2H),1.82(s,6H),1.47(t,J=7.3Hz,3H). Its H NMR spectrum is shown in Figure 4 shown.

[0093] 13 C NMR (400MHz, MeOD) δ177.80,163.80,155.38,155.11,147.36, 145.62,144.71,142.49,139.39,135.78,133.20,129.62,129.50,118.68,117.46,116.36,115.31,105.46,104.15,92.01,64.44,61.25,51.82,42.31,41.46,38.36, 35.42,30.07,28.18,25.06,21.62,12.74. Its NMR carbon spectrum is as follows Figure 5 shown.

[0094] S4: Preparation method of target product (4) dual-activated photodynamic immunoprodrug based on hemicyanine and IDO inhibitor:

[0095]

[0096] The prodrug molecule NLG919 (0.1mmol) and carbonyldiimidazole (0.5mmol) were dissolved in 5mL tetrahydrofuran and heated under reflux at 60°C for 10h. Then the organic solvent was removed, and the residue and the third intermediate (0.1mmol) were dissolved in 5mL chloroform, DBU (0.2mmol) was added thereto, and the reaction was stirred for 12h until the reaction was completed. Under reduced pressure distillation, the mixed solvent was removed to obtain a crude product, which was purified using a silica gel chromatography column with an eluent of dichloromethane: methanol = 15:1 (v / v) to obtain the target activatable prodrug molecule with a yield of 65%;

[0097] HRMS(ESI):m / z C 51 H 56 IN4O6S2 + Calculated value [M] + 1011.2680, test value 1011.2671; [M+H] 2+ 506.1276, test value 506.1373. Its high-resolution mass spectrum is as follows Figure 6 shown.

[0098] 1 H NMR (400MHz, CD3OD) δ8.73(d,J=14.8Hz,1H),8.04(s,1H), 7.97(d,J=1.5Hz,1H),7.93(s,1H),7.86(dd,J=8.4,1.5Hz,1H),7.55(dd, J=15.1,7.6Hz,2H),7.49–7.45(m,1H),7.44(d,J=4.5Hz,1H),7.37– 7.29(m,3H),7.19–7.16(m,1H),6.47(d,J=14.7Hz,1H),5.41(t,J=4.7Hz,1H),4.45(dd,J=12.6,6.3Hz,2H),4.33(q,J=7.2Hz,2H),4.20–4.13 (m,2H),3.52–3.45(m,1H),3.06(t,J=6.4Hz,2H),2.93(t,J=6.3Hz,2H),2.80(t,2H),2.72(t,J=5.8Hz,2H),2.56–2.40(m,2H),2.19(t,J=7.2Hz, 1H), 2.05–1.99 (m, 1H), 1.94 (t, J = 4.8 Hz, 2H), 1.78 (s, 6H), 1.46 (t, J = 6.2 Hz, 3H), 1.32–1.28 (m, 8H), 1.18 (t, J = 7.0 Hz, 2H). Its H NMR spectrum is shown in Figure 7 shown.

[0099] 13 C NMR(500MHz,MeOD)δ177.76,163.80,155.84,155.37,155.06, 147.35,145.58,144.70,142.50,139.39,135.81,133.14,131.10,129.66,129 .49,128.68,128.25,126.29,125.52,121.34,118.67,117.50,116.35,115.32, 105.46,104.13,91.99,79.12,66.81,64.29,60.21,51.78,43.53,42.60,41.45, 38.46,38.33,37.19,35.69,32.76,30.76,29.57,28.20,27.99,27.02,23.70,22.99,21.62,14.43,12.74,11.76. Its NMR carbon spectrum is as follows Figure 8 shown.

[0100] Comparative Example

[0101] This comparative example is a method for preparing a conjugate of a drug and a semi-cyanine molecule that is not activatable by connecting the drug with an unbreakable carbon chain. The difference from the embodiment is that in step S3, replace The mixed solvent was removed under reduced pressure distillation to obtain a crude product, which was purified using a silica gel chromatography column with an eluent of dichloromethane:methanol=22:1 (v / v) to obtain a blue third intermediate (50 mg) with a yield of 75%; the target activatable prodrug molecule was finally obtained with a yield of 60%;

[0102] HRMS(MALDI):m / z C 53 H 60 IN4O6 + Calculated value [M] + 975.3553, test value 975.3508. Its high-resolution mass spectrum is as follows Fig. 9 shown.

[0103] 1H NMR (500MHz, MeOD) δ8.74(d,J=14.8Hz,1H),8.16(dd,J=11.6,1.4Hz,1H),7.99(dd,J=8.4,1.5Hz,1H),7.86(dd,J=8.4,1.5Hz, 1H),7.60–7.54(m,2H),7.50–7.44(m,2H),7.42(s,1H),7.39–7.29(m,5H),6.48(d,J=14.8Hz,1H),5.39(dd,J=5.0,3.0Hz,1H),4.60–4.56 (m, 1H), 4.34 (q, J = 7.2 Hz, 2H), 3.62 (dd, J = 4.4, 3.4 Hz, 2H), 3.40 (d, J = 5.6 Hz, 1H), 2.81–2.78 (t, J = 5.8 Hz, 2H), 2.72 (t, J = 6.0 Hz, 2H), 2.59–2.52 (m, 2H), 2.43 (t, J = 7.3 Hz, 3H), 1.96–1.92 (t, J = 5.9 Hz, 2H), 1.81–1.78 (m, 8H), 1.73–1.66 (m, 6H), 1.62–1.54 (m, 4H), 1.46 (t, J = 7.2 Hz, 3H), 1.28 (s, 6H). Its H NMR spectrum is shown in Fig.10 shown.

[0104] 13 C NMR(126MHz,MeOD)δ177.97,174.98,174.51,163.61, 155.12,147.49,145.65,144.12,142.45,139.40,135.42,133.16,129.90, 129.61,129.48,128.05,121.38,119.31,118.22,116.38,115.41,106.92, 104.39,92.16,74.40,73.72,71.56,69.24,68.61,54.82,51.85,43.52,41.53,37.99,36.36,34.54,33.08,30.76,30.48,30.10,29.82,28.89,28.19,27.44, 27.13,26.39,25.53,25.04,23.75,21.61,19.55,14.46,12.79. Its NMR carbon spectrum is as follows Fig.11 shown.

[0105] Performance testing

[0106] 1. Test of the dual quenching effect of photodynamic therapy and IDO inhibitor;

[0107] (1) In the preparation process of the above-mentioned dual-activated photodynamic immune prodrug embodiment, the absorption spectra of the second intermediate prepared in step S2 and the target activatable prodrug molecule prepared in step S4 are tested by a UV-visible spectrophotometer, specifically as follows: Fig.12 As shown in (A), it can be seen that compared with the second intermediate at the amino end, the absorption of the target unactivated molecule in the solvent is blue-shifted, and the absorption is greatly reduced, which is about 1 / 2 of the activated structure. The absorption spectrum proves the inhibitory effect of this structure on the photosensitizer molecule.

[0108] The fluorescence spectra of the second intermediate prepared in step S2 and the target activatable prodrug molecule prepared in step S4 are tested using a fluorescence spectrometer. Fig.12 As shown in (B), it can be seen that the second intermediate with an activated amino terminal structure has a strong fluorescence emission at 730nm, while the target molecule in the inactivated state exhibits extremely low fluorescence due to the quenching effect.

[0109] Combining the embodiments and Fig.12 (A) and Fig.12 (B) It can be seen that compared with the second intermediate prepared in step S2 of the embodiment, the target activatable prodrug molecule exhibits obvious absorption attenuation at long wavelengths, the absorption peak is blue-shifted, and the fluorescence of the target activatable prodrug molecule is quenched, indicating that the ICT effect of its semi-cyanine photosensitizing dye molecule is significantly inhibited, thereby affecting the photodynamic therapy ability of the molecule.

[0110] (2) During the preparation of the above-mentioned dual-activated photodynamic immune prodrug embodiment, the singlet oxygen generating ability of the second intermediate prepared in step S2 and the target activatable prodrug molecule prepared in step S4 was tested:

[0111] Add 3 mM of the test sample into a test quartz dish containing 3 mL of dichloromethane solution, adjust the absorbance at 415 nm to about 1 with DPBF solution, and place the quartz dish at 700 nm, 10 mW / cm 2 The solution was irradiated under a light source and the absorption spectrum of the solution was recorded every 120 seconds. The test results are shown in Figure 13.

[0112] Combining the embodiments and Fig.13 It can be seen that compared with the second intermediate molecule with a complete photosensitizer form, the target activatable prodrug molecule prepared in the embodiment has a significantly lower degradation rate of DPBF under light than the second intermediate (only 40% of the second intermediate) when not activated, representing its low singlet oxygen production ability.

[0113] (3) The above examples and comparative examples provide dual-activated photodynamic immune prodrugs to test the inhibitory activity of immune drugs on IDO in tumor cells:

[0114] The test sample of 3mM was prepared into medium solutions of different concentrations. After the cells were stimulated with interferon-γ for 12 hours, IDO was produced. Different concentrations of the example and comparative example molecules were added and incubated for 12 hours. The inhibitory effect of the molecules on IDO was evaluated. Fig.14 .

[0115] Combining the embodiments and comparative examples and Fig.14 It can be seen that the drug activity of the non-cuttable comparative example is greatly reduced due to the inability to release free inhibitors (EC 50 =5.9 μM, EC of NLG919 50 =1 μM), proving that covalent modification of drug active sites can inhibit drug activity for prodrug design. In this example, after tumor cells respond, the inhibitor can be released to restore its drug activity (EC 50 =1.6μM). The verification results show that if the embodiment is in normal cells, since the drug cannot be released and activated, it can neither produce effective photodynamic killing effect nor release immune drugs. Further, such a dual-activation prodrug strategy avoids the toxic side effects caused by drug molecules entering normal cells or tissues.

[0116] 2. Imaging and testing of photodynamic therapy activation effects

[0117] (1) At the solution level, under physiological conditions simulated by a phosphate buffer solution, 10 μM of the example solution was reacted with 5 mM of GSH (glutathione) on a shaker at 37° C. for 1 h, and then the fluorescence spectrum was measured using a fluorescence spectrometer, as shown in FIG. Fig.15 shown.

[0118] from Fig.15 It can be seen that the fluorescence enhancement effect increases with time, indicating that the imaging ability of the embodiment under GSH is activated.

[0119] (2) The activation effect of the above fluorescence imaging was verified in different cells (cancer cells and normal cells):

[0120] Different cells were cultured in a 37°C, 5% CO2 constant temperature cell culture incubator for adherent culture. The culture medium was 10% serum and 1% double antibody-containing DMEM high glucose. The cells were digested and cultured in a 35mm culture dish. After 12 hours of growth, the cells adhered to the wall. 1μM dye molecules were added and cultured for 30 minutes. After washing, fluorescence imaging of different cells at different times was performed under a confocal microscope. The detection results are shown in Fig.16In tumor cells (4T1 and Hela), the molecules of the embodiment can achieve fluorescence activation over time until saturation, while in normal cells (COS-7 and NIH-3T3), they show low fluorescence response.

[0121] The cells were cultured in a 96-well plate and grown for 12 hours until the cells adhered to the wall and the cell density reached 60-70%. The target of the embodiment with different concentrations was added to the wells containing cells in the 96-well plate. After incubation for 2 hours, the cells were treated with light for 5 minutes (50 mW / cm 2 ), then add MTT solution, incubate for 4 hours, add DMSO, and use a microplate reader to detect the absorbance at 490nm. The difference in the therapeutic ability of the target molecule under light in different cells is calculated. The test results are shown in Fig.17 .from Fig.17 It can be seen that the killing effect of the prodrug molecules provided in the examples on tumor cells is 1-5 times that on normal cells.

[0122] Similar to the above cell survival rate experiment, the target molecule, the third intermediate and the control sample were tested for their different cell killing effects, with the same illumination conditions. Fig.18 .from Fig.18 It can be seen that in cells, the third intermediate provided in the embodiment and step S3 exhibited similar photodynamic effects, while the non-activatable control sample exhibited a lower therapeutic effect (0-0.5 times), indicating that whether the molecule can be activated is the key to exerting a therapeutic effect.

[0123] 3. Mouse tumor inhibition and lung metastasis inhibition experiments were performed on the third intermediate prepared in step S3 of the above embodiment, the target activatable prodrug molecule prepared in step S4, and the molecule provided in the comparative example.

[0124] (1) Tumor inhibition in mice: A bilateral subcutaneous breast cancer (4T1) tumor model was established in 5-6 week-old Balb / C female mice. A distal tumor model was established 3 days after orthotopic tumor establishment. When the orthotopic tumor size reached 100 mm, the distal tumor model was established. 3 The drug was administered by paratumoral injection and then incubated for 2 hours before 10 minutes of phototherapy (700 nm, 100 mW / cm 2 ), record the volume changes of the in situ tumor and distal tumor, and the test results are shown in Fig.19 .

[0125] Combining the embodiments and comparative examples and Fig.19 It can be seen that the target activatable prodrug molecule and the single activated third intermediate prepared in step S4 both showed inhibitory effects on the photoirradiated in situ tumor, while the non-activatable control group and the blank control group did not show obvious therapeutic effects.

[0126] For distal tumors, the test results are shown in Fig. 20 , only the example showed an effective inhibitory effect, while the third intermediate prepared by single activation of S3 for only single photodynamic therapy failed to inhibit it.

[0127] (2) Lung metastasis inhibition:

[0128] The breast-lung metastasis model was established in 8-9 week old Balb / C female mice. 3 The mice in different groups were irradiated with light for 10 min (700 nm, 100 mW / cm 2 ), and the mice were euthanized 30 days later, and their lung tissues were sampled and fixed with formalin, and the number of lung metastatic tumor nodules was analyzed and counted. Fig.21 Compared with the blank control, the third intermediate and the comparative example, the embodiment showed the best lung metastasis inhibition effect.

[0129] 4. Experiments were conducted on the immune effects of the third intermediate obtained in step S3 of the preparation process of the above embodiment, the target activatable prodrug molecule obtained in step S4, and the molecule provided in the comparative example.

[0130] To investigate the immune response in mice, we first tested the maturation of dendritic cells in vivo. Tumor-draining lymph nodes from mice in different treatment groups were collected and ground and filtered to obtain single-cell suspensions. Cells were stained with fluorescent antibodies and analyzed by flow cytometry. Fig. 22 It was observed that the target activatable prodrug molecule prepared in step S4 and the third intermediate prepared in step S3 can promote the activation of dendritic cells (CD11 + CD80 + CD86 + )’s maturity.

[0131] To further analyze the infiltration of cytotoxic T lymphocytes in the tumor, the tumor was cut into small pieces and digested with collagenase for 2 h, then ground and filtered to obtain a single cell suspension, and then stained with fluorescent antibody (CD45 + CD3 + CD8 + ) and then used for flow cytometry analysis. The analysis results showed that the target activatable prodrug molecule prepared in step S4 significantly enhanced the infiltration of cytotoxic T lymphocytes (0-4.2 times for in situ tumors and 0-14 times for distal tumors), while the third intermediate prepared in step S3 had no significant effect. At the same time, for the immunosuppressive regulatory T cells (CD3 + CD4 + Foxp3 +) was tested and the target activated prodrug molecule regulated a significant decrease in the proportion of T cells, proving that the introduction of immune checkpoints in the embodiment can reverse the immunosuppressive microenvironment. The second intermediate could not reverse the immunosuppressive microenvironment without the introduction of immune drugs, indicating that single photodynamic therapy could not induce a sufficiently strong immune response, thereby reflecting the importance of combined therapy. Although the control example introduced immune checkpoint inhibitors, the drug activity could not be restored due to the non-severable covalent connection, and no obvious therapeutic effect was shown.

[0132] In summary, compared with the prior art, the dual-activated photodynamic immunoprodrug based on hemicyanine and IDO inhibitor provided by the present invention has the following inventiveness:

[0133] Drug molecules have the problem of insufficient specific selectivity for cancer cells when treating tumors. The present invention realizes covalent connection between photodynamic molecules and immune drug IDO inhibitors through a linker that responds to tumor cell overexpression markers. The covalent connection simultaneously achieves dual inhibition of the drug activity of photosensitizer and IDO inhibitor, effectively improving the selectivity of molecules for tumor cells, reducing damage to normal cells during treatment, and avoiding serious side effects during tumor treatment.

[0134] Due to the specific selective response of this molecule to tumor cells, this drug molecule can also be used for fluorescence imaging and treatment of malignant tumors such as breast cancer, endometrial cancer, ovarian cancer, lung adenocarcinoma, brain cancer, colorectal cancer, and testicular cancer;

[0135] This drug molecule achieves dual activation of photosensitizer and IDO inhibitor after tumor cell response, and performs efficient synergistic immunotherapy after killing the tumor after light irradiation, achieving excellent tumor treatment effects and avoiding serious side effects during tumor treatment, providing a strategy for the covalent binding pathway of photosensitizers and immune drugs.

[0136] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A dual-activated photodynamic immunoprodrug based on hemicyanine and IDO inhibitor, It is characterized in that The dual-activated photodynamic immune prodrug has the following general formula (I): （I） wherein a and b are each independently selected from an integer of 1-9; X 1 is a halogen; X 2 is O; X 3 is -NH-; X 4 and X 5 for -O-; R 1 Selected from or ; R 2 Select from one of the following groups: , , ; R 3 for .

2. A dual-activated photodynamic immunoprodrug based on hemicyanine and IDO inhibitor according to claim 1, It is characterized in that X 1 is Cl, Br or I.

3. A method for preparing a dual-activated photodynamic immune prodrug based on hemicyanine and IDO inhibitor according to any one of claims 1-2, It is characterized in that The compound of general formula (I) is prepared by the following method: ； S1: Halogenated 2,3,3-trimethylindole compound A and containing R 1 The compound B of the group reacts under the action of a first organic solvent and a first weak base to obtain a first intermediate C; S2: The first intermediate C reacts with a condensing agent 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde in the presence of a second organic solvent and a second weak base to obtain an intermediate, and the intermediate reacts with a compound D in the presence of a third organic solvent under the protection of an inert gas to obtain a second intermediate E; S3: The second intermediate E and the first catalyst are dissolved in a fourth organic solvent to obtain a mixed solution. Under the protection of an inert gas, the mixed solution is mixed with a 2 The compound F of the group reacts with a fifth organic solvent and a third weak base to obtain a third intermediate G; S4: Compound H and the second catalyst are dissolved in a sixth organic solvent to obtain a mixture, and the mixture is reacted with the third intermediate G in a seventh organic solvent and a fourth weak base under the protection of an inert gas to obtain a dual-activated photodynamic immune prodrug based on a semicyanine and an IDO inhibitor having a structure of the general formula (I); Wherein, the first catalyst is selected from at least one of carbonyl imidazole, triphosgene, and p-nitrophenyl chloroformate; The second catalyst is selected from at least one of carbonyl imidazole, triphosgene and p-nitrophenyl chloroformate.

4. A method for preparing a dual-activated photodynamic immunoprodrug based on hemicyanine and IDO inhibitor according to claim 3, It is characterized in that In step S1, the reaction time is 24-48 hours, and the first organic solvent used is selected from at least one of toluene, benzene, xylene, and acetonitrile; The first weak base used is selected from at least one of triethylamine, sodium acetate, ammonium acetate, sodium carbonate and sodium bicarbonate.

5. A method for preparing a dual-activated photodynamic immunoprodrug based on hemicyanine and IDO inhibitor according to claim 3, It is characterized in that In step S2, the second organic solvent is selected from at least one of acetic acid, ethanol, acetonitrile, DMF, tetrahydrofuran, and acetone; The second weak base is selected from at least one of sodium acetate, ammonium acetate, potassium acetate, sodium carbonate, potassium carbonate, and sodium bicarbonate; The third organic solvent is selected from at least one of acetonitrile, DMF, DMSO, dichloromethane and chloroform.

6. A method for preparing a dual-activated photodynamic immune prodrug based on hemicyanine and IDO inhibitor according to claim 3, It is characterized in that In step S3, the reaction time is 12-24h; The fourth organic solvent used is selected from at least one of dichloromethane, DMF, acetonitrile, dichloroethane, chloroform and tetrahydrofuran; The third weak base used is selected from at least one of DMAP, DIPEA, DBU and triethylamine; The fifth organic solvent used is at least one selected from dichloromethane, tetrahydrofuran and DMF.

7. A method for preparing a dual-activated photodynamic immune prodrug based on hemicyanine and IDO inhibitor according to claim 3, Features The sixth organic solvent is selected from at least one of dichloromethane, DMF, acetonitrile, dichloroethane, chloroform, and tetrahydrofuran; The fourth weak base is selected from at least one of DMAP, DIPEA, DBU and triethylamine; The seventh organic solvent is selected from at least one of dichloromethane, tetrahydrofuran and DMF.

8. A method for preparing a dual-activated photodynamic immune prodrug based on hemicyanine and IDO inhibitor according to claim 3, It is characterized in that In steps S2, S3 and S4, the inert gas is independently selected from at least one of helium, nitrogen and argon.

9. The use of a dual-activated photodynamic immunoprodrug based on hemicyanine and IDO inhibitor according to any one of claims 1-2, It is characterized in that At least one of the following uses: The use of the dual-activated photodynamic immunoprodrug based on hemicyanine and IDO inhibitor for the preparation of activatable fluorescent imaging tumor diagnosis and treatment drugs based on hemicyanine dyes; The use of the dual-activated photodynamic immune prodrug based on hemicyanine and IDO inhibitor in the preparation of an activatable photodynamic tumor therapy drug based on hemicyanine photosensitizer dye; The use of the dual-activated photodynamic immunoprodrug based on hemicyanine and IDO inhibitor for selective treatment of tumors; The tumor is any one of breast cancer, endometrial cancer, ovarian cancer, lung adenocarcinoma, brain cancer, colorectal cancer, and testicular cancer.

Images