A near-infrared photoactivated nitrogen mustard drug compound, its preparation method and application

Abstract

This invention discloses a near-infrared photoactivated nitrogen mustard drug compound, its preparation method, and its applications. The general structural formula of the near-infrared photoactivated nitrogen mustard drug compound is shown below: where R1 and R2 are both optionally selected from methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl. This compound reduces the toxic side effects of nitrogen mustard through a prodrug strategy. This compound utilizes the synergistic effect of nitrogen mustard chemotherapy and photodynamic therapy with a photosensitizer to effectively inhibit tumor growth. Simultaneously, the introduction of the photosensitizer imparts fluorescence changes before and after nitrogen mustard drug release, enabling visualization of drug release. This compound has broad application prospects in reducing drug toxicity and anti-tumor activity. The near-infrared photoactivated nitrogen mustard prodrug / drug preparation method provided by this invention is simple, uses inexpensive and readily available raw materials, has a simple synthesis process, and is easy to separate and purify, making it suitable for large-scale production and widespread application.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis and biomedicine technology, specifically relating to a near-infrared light-activated nitrogen mustard drug compound, its preparation method, and its application. Background Technology

[0002] Chemotherapy plays a crucial role in cancer treatment and is one of the most effective treatment methods currently available. Nitrogen mustard, as a widely used anticancer drug, has been used clinically for over 70 years. Its main anticancer mechanism involves the formation of a highly reactive nitrogen heterocyclic intermediate, which reacts with the N7 position of a deoxyguanosine residue, thereby initiating DNA monoalkylation or DNA interstrand crosslinking. However, the high toxicity and limited selectivity of nitrogen mustard often lead to severe damage to normal tissues and organs, causing most patients to experience significant systemic toxicity. Therefore, developing novel nitrogen mustard drugs to reduce toxicity and improve anticancer selectivity is an urgent research challenge.

[0003] Prodrugs are compounds that are metabolized into active drugs in vivo and are designed to improve drug targeting, pharmacokinetic properties, and safety. Prodrug strategies can optimize drug solubility, stability, and metabolic efficiency, reduce systemic toxicity, and enhance therapeutic efficacy. Typically, prodrugs are activated in vivo by specific enzymes, pH changes, or other physiological stimuli, releasing the active ingredient with anticancer activity. For example, patent CN 110305036B reports a hydrogen peroxide-responsive nitrogen mustard antitumor prodrug with rapid response, strong cell selectivity, and low toxicity; patent CN107266483A introduces a photosensitive targeted antitumor prodrug based on a hydrogen peroxide-responsive mechanism, which can improve drug targeting and achieve visualized release; patent CN 101747300A reports a paclitaxel-nitrogen mustard synergistic prodrug that helps reduce drug toxicity. However, these near-infrared light-activated nitrogen mustard prodrugs still have certain limitations in visualizing drug release.

[0004] Furthermore, photodynamic therapy (PDT), as an emerging non-invasive cancer treatment strategy, has broad application prospects. Its basic principle is to utilize photosensitizers to generate reactive oxygen species (ROS) under specific wavelengths of light irradiation, thereby inducing oxidative damage to key intracellular biomolecules, ultimately leading to apoptosis or necrosis and achieving an anti-tumor effect. Simultaneously, many photosensitizers also possess fluorescence imaging capabilities, providing possibilities for integrated tumor diagnosis and treatment. Methylene blue (MB) is currently the only photosensitizer approved for cancer PDT, exhibiting significant advantages in fluorescence imaging, ROS generation capacity, and biosafety. However, the dark toxicity of methylene blue remains a concern, potentially causing adverse reactions or local tissue damage under high concentrations or overactivation conditions. Therefore, optimizing the use of methylene blue to reduce its dark toxicity while enhancing its anti-cancer effects is a direction worthy of in-depth research in the field of PDT.

[0005] Therefore, there is an urgent need for a method that can combine photodynamic therapy and prodrug design strategies to achieve low-toxicity and highly visualized anti-tumor therapy. Summary of the Invention

[0006] One of the objectives of this invention is to provide a near-infrared photoactivated nitrogen mustard drug compound with low toxicity and side effects, high anti-tumor activity, and the ability to visualize the drug release process.

[0007] A second objective of this invention is to provide a method for preparing the near-infrared light-activated nitrogen mustard drug compound.

[0008] A third objective of this invention is to provide the application of the near-infrared light-activated nitrogen mustard drug compound.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] This invention provides a near-infrared photoactivated nitrogen mustard drug compound (NBMB), the general structural formula of which is shown below:

[0011]

[0012] R1 and R2 are one or two of methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl.

[0013] Preferably, the structural formula of the near-infrared photoactivated nitrogen mustard drug compound includes any one of the following compounds 1 to 7:

[0014]

[0015]

[0016] This invention also provides a method for preparing the near-infrared photoactivated nitrogen mustard drug compound, comprising the following steps:

[0017] S1. Synthesis of compound NB: A mixture of phenothiazine, iodine, and disubstituted amine was stirred. After the reaction was complete, the mixture was cooled to room temperature, evaporated under reduced pressure, dissolved in dichloromethane, and added dropwise to diethyl ether. The mixture was filtered, and the resulting residue was washed with chloroform and dried to obtain compound NB. The synthetic route is as follows:

[0018]

[0019] S2. Synthesis of compound LNBCl: A mixture of compound NB, base 1, reducing agent, and solvent 1 was heated under nitrogen protection. Then, a mixture of triphosgene and solvent was added dropwise under an ice-water bath. After the addition was complete, the mixture was moved to room temperature. After the reaction was completed, the reaction solution was poured into water, and compound LNBCl was obtained by separation and purification. The synthetic route is as follows:

[0020]

[0021] S3. Synthesis of compound NMNO2: A mixture of N-(4-nitrophenyl)diethanolamine, N,N-dimethylformamide (DMF), and thionyl chloride was stirred and reacted at room temperature. After the reaction was completed, the reaction solution was poured into water, and the compound NMNO2 was obtained by separation and purification. The synthetic route is as follows:

[0022]

[0023] S4. Synthesis of compound NM: A mixture of compound NMNO2, stannous chloride, and concentrated hydrochloric acid was stirred and reacted at room temperature. After the reaction was complete, the solvent was evaporated, and hydrochloric acid ethanol solution was added and stirred. During stirring, diethyl ether was added, and then the reaction solution was filtered. The resulting residue was NM. The synthetic route is as follows:

[0024]

[0025] S5. Synthesis of compound NBNM: A mixture of compound LNBC1, NM, base 2, and solvent 2 was reacted under nitrogen protection. After the reaction was completed, the reaction solution was poured into water, and compound NBNM was obtained by separation and purification. The synthetic route is as follows:

[0026]

[0027] Furthermore, in step S1, the molar ratio of phenothiazine to iodine is 1:3-4; the molar volume ratio of phenothiazine to disubstituted amine is 1:4-5 mmol / mL; the reaction temperature is 40-50℃, and the reaction time is 20-24 h; the molar volume ratio of phenthiazine to dichloromethane is 1:8-10 mmol / mL, and the volume ratio of dichloromethane to diethyl ether is 1:12-15.

[0028] Preferably, the disubstituted amine includes dimethylamine, methyl ethylamine, diethylamine, di-n-propylamine, di-n-butylamine, di-n-pentylamine, and di-n-hexylamine.

[0029] Further, in step S2, solvent 1 is a mixture comprising dichloromethane and water, with a volume ratio of dichloromethane to water of 1:1 to 2; alkali 1 comprises sodium carbonate and potassium carbonate; the reducing agent comprises sodium dithionite; the molar volume ratio of compound NB to solvent 1 is 1:(2.5 to 4) mmol / mL; the molar ratio of compound NB to sodium dithionite is 1:4 to 5; the molar ratio of compound NB to alkali 1 is 1:4 to 5; the molar ratio of compound NB to triphosgene is 1:0.6 to 0.8; the reaction temperature before the ice-water bath is 35-40℃, and the reaction time is 30-45 min; the time for adding the mixture dropwise in the ice-water bath is 15-20 min; the reaction temperature after the ice-water bath is room temperature, and the reaction time is 6-8 h; the volume ratio of the reaction solution to water is 1:10 to 12.

[0030] Further, in step S2, the separation and purification specifically involves: first, extracting with CH2Cl2 to obtain an organic layer, then drying the organic layer with anhydrous Na2SO4 and rotating it dry on a rotary evaporator, and then separating and purifying it using silica gel column chromatography; the eluent used in the silica gel column chromatography separation and purification is n-hexane:ethyl acetate = 10:1, v / v.

[0031] Further, in step S3, the molar volume ratio of N-(4-nitrophenyl)diethanolamine to thionyl chloride is 1:(3-4) mmol / mL; the molar volume ratio of N-(4-nitrophenyl)diethanolamine to N,N-dimethylformamide is 1:(0.4-0.6) mmol / mL; the reaction temperature is 25-30℃, the reaction time is 4-6 h; the volume ratio of reaction solution to water is 1:10-12; and the eluent used in the silica gel column chromatography separation and purification is dichloromethane.

[0032] Further, in step S4, the molar volume ratio of compound NMNO2 to concentrated hydrochloric acid is 1:(3-4) mmol / mL; the molar ratio of compound NMNO2 to stannous chloride is 1:(4-5); the reaction time is 6-8 h; the volume ratio of concentrated mixture to hydrochloric acid ethanol solution is 1:5-8; the volume ratio of hydrochloric acid ethanol to diethyl ether is 1:10-15; and the stirring time is 30-45 min.

[0033] Further, in step S5, solvent 2 is an organic solvent, including acetonitrile and chloroform; base 2 includes potassium carbonate and triethylamine; the molar volume ratio of compound LNBC1 to solvent is 1:(4-6) mmol / mL; the molar ratio of compound LNBC1 to NM is 1:1-1.5; the molar ratio of compound LNBC1 to base 2 is 1:(1-1.5); the reaction temperature is 35-40℃, and the reaction time is 4-6 h; the eluent used in the silica gel column chromatography separation and purification is n-hexane:ethyl acetate = 10:1, v / v.

[0034] The present invention also provides an application of the near-infrared light-activated nitrogen mustard drug compound in drug release visualization, reducing nitrogen mustard toxicity, and photodynamic therapy combined with chemotherapy for synergistic anti-tumor effects.

[0035] Furthermore, the near-infrared light-activated nitrogen mustard drug compound, under preset conditions, can be used for photodynamic therapy combined with chemotherapy to enhance its ability to kill tumor cells.

[0036] The preset condition is a 660nm illumination condition.

[0037] The beneficial effects of this invention are:

[0038] (1) The near-infrared light-activated nitrogen mustard drug compound provided by the present invention links nitrogen mustard to methylene blue derivative through urea bond, cleverly combining the photodynamic antitumor effect of photosensitive group with the antitumor mechanism of chemotherapy, enhancing the killing effect on tumor cells, and has the advantages of high antitumor cell activity and good biocompatibility.

[0039] (2) The near-infrared light-activated nitrogen mustard drug compound provided by the present invention destroys the conjugated structure of methylene blue derivative through urea bond, quenching its fluorescence properties. After the urea bond is broken and nitrogen mustard is released, the fluorescence is turned on, realizing the visualization of nitrogen mustard release.

[0040] (3) The near-infrared light-activated nitrogen mustard drug compound provided by the present invention reduces the electron cloud density of nitrogen mustard through the electron-withdrawing effect of urea bonds, inhibits the formation of its active intermediate, and reduces the toxic side effects of nitrogen mustard;

[0041] (4) The near-infrared light-activated nitrogen mustard drug compound provided by the present invention can be rapidly released under near-infrared light irradiation and has high spatiotemporal selectivity;

[0042] (5) The method for preparing near-infrared light-activated nitrogen mustard drug compounds provided by the present invention is simple, the raw materials are cheap and readily available, the synthesis process is simple, the separation and purification are easy, the yield is high, the dark stability is strong, and it is suitable for large-scale production and promotion. Attached Figure Description

[0043] Figure 1 The chemical synthesis route of compound 1 in Example 1 is shown below;

[0044] Figure 2 Figure A shows the response release spectrum of compound 1. Figure B shows the change of the UV-Vis absorption spectrum of MBNM with increasing illumination time and the change of the fluorescence spectrum of MBNM with increasing illumination time.

[0045] Figure 3 HRMS validation of the response release of compound 1;

[0046] Figure 4 The response of compound 1 at different pH values;

[0047] Figure 5 Evaluation of the dark stability of compound 1;

[0048] Figure 6 To evaluate the reactive oxygen species (ROS) generation capacity of compound 1 before and after release; Figure A is a schematic diagram of DCFH detection of ROS, Figure B is the fluorescence spectrum change of DCFH with added pre-irradiated MBNM as the irradiation time increases, and Figure C is the fluorescence change of DCFH, DCFH with added un-irradiated MBNM, and DCFH with added pre-irradiated MBNM at 378 nm as the irradiation time increases.

[0049] Figure 7 The cytotoxicity of compound 1;

[0050] Figure 8 To evaluate the in vitro killing ability of compound 1 against B16F10 cells using the live-death staining method; Figure A shows the live-death staining of B16F10 cells treated with 10 μM MBNM at different irradiation times, and Figure B shows the live-death staining of B16F10 cells treated with different concentrations of MBNM after irradiation for 15 min.

[0051] Figure 9 Imaging of nuclear damage caused by compound 1;

[0052] Figure 10 To evaluate the apoptosis-inducing ability of compound 1 using cell flow cytometry;

[0053] Figure 11Evaluation of the hemolytic activity of compound 1;

[0054] Figure 12 Figure 1 shows the changes in mouse weight and tumor volume during treatment with compound 1, and the tumor mass after treatment. Figure A shows the changes in mouse weight during treatment, Figure B shows the changes in tumor volume during treatment, and Figure C shows the tumor mass after treatment. Detailed Implementation

[0055] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. Unless otherwise specified, the methods described are conventional methods. Unless otherwise specified, the raw materials are all available from publicly available commercial sources.

[0056] In the following examples, the reagent DCM is dichloromethane; TEA is triethylamine; DMF is N,N-dimethylformamide; and DCFH is 2',7'-dichlorofluorescein diacetic acid.

[0057] Example 1: Synthesis of Compound 1

[0058] The synthetic route for compound 1 is shown below. Figure 1 As shown, the specific steps include:

[0059] Step 1: Synthesis of compound NB(1)

[0060] A mixture of phenothiazine (4000 mg, 20.10 mmol), iodine (1525 mg, 60.20 mmol), and dimethylamine (65 mL) was stirred at 50 °C for 21 h, then cooled to room temperature, evaporated under reduced pressure, dissolved in DCM, added dropwise to diethyl ether, filtered, and washed to give compound NB(1) (3517 mg, yield 45%). 1 H NMR(500MHz,DMSO-d6)δppm 7.88(d,J=9.7Hz,2H),7.47(d,J=2.7Hz,2H),7.25(dd,J=9.7,2.7Hz,2H),3.36(q,J=7.2Hz,8H).HRMS(m / z):calculated for C 16 H 18 N3S + [M] + :284.1216; found:284.1219.

[0061] Step 2: Synthesis of compound LNBCl(1)

[0062] Compound NB(1) (1000 mg, 3.12 mmol), Na₂CO₃ (1320 mg, 12.60 mmol), and Na₂S₂O₄ (2200 mg, 12.60 mmol) were dissolved in water (10 mL) and DCM (5 mL), and stirred at 40 °C for 30 min under nitrogen protection. Triphosgene (560 mg, 1.88 mmol) dissolved in DCM was slowly added dropwise to the reaction system in an ice-water bath. The mixture was stirred at room temperature for 6 h. The solution was poured into water and then extracted with CH₂Cl₂. The organic layer was dried over anhydrous Na₂SO₄ and evaporated under reduced pressure. The mixture was purified by silica gel column chromatography, eluting with n-hexane and ethyl acetate (10 / 1, v / v), to give compound LNBCl(1) (705 mg, 65% yield). 1 H NMR (500MHz, Chloroform-d) δppm 7.38 (d, J = 8.3 Hz, 2H), 6.69 (d, J = 2.8 Hz, 2H), 6.61 (dd, J = 9.0, 2.8 Hz, 2H), 2.95 (s, 12H). 13 C NMR(126MHz, CDCl3)δppm149.85,149.33,133.97,127.86,126.75,110.68,110.19,40.52.HRMS(m / z):calculated for C 17 H 18 ClN3OS + [M] + :348.0937; found:348.0920.

[0063] Step 3: Synthesis of compound NMNO2(1)

[0064] N-(4-nitrophenyl)diethanolamine (226 mg, 1.00 mmol) was dissolved in SOCl2 (5 mL), and DMF (0.5 mL) was added. The mixture was stirred at room temperature for 6 h. After cooling to room temperature, the solution was poured into water and then extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure. The mixture was purified by silica gel column chromatography, eluted with dichloromethane, to give compound NMNO2(1) (216 mg, 82% yield). 1 H NMR (500MHz, Chloroform-d) δppm 8.07-8.01 (m, 2H), 7.04-6.98 (m, 2H), 3.70 (t, J = 3.3Hz, 4H), 3.62 (t, J = 3.3Hz, 4H). 13C NMR (125MHz, Chloroform-d) δ152.20,137.72,125.97,112.26,52.15,41.55.

[0065] Step 4: Synthesis of compound NM(1)

[0066] Compound NMNO2(1) (131 mg, 0.50 mmol) and SnCl2 (474 ​​μL, 2.50 mmol) were added to 2 mL of concentrated HCl and stirred at room temperature for 6 h. The mixture was poured into water, the pH was adjusted to neutral, and then extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residual oil was dissolved in a saturated hydrochloric acid-ethanol solution and stirred for 30 min. An appropriate amount of diethyl ether was added, stirred, filtered, and washed to give compound NM(1) (74 mg, yield 57%). 1 H NMR (500MHz, DMSO-d6) δppm 10.10 (s, 2H), 7.21 (d, J = 8.7Hz, 2H), 6.82 (d, J = 8.6Hz, 2H), 3.73 (s, 8H). 13 C NMR(126MHz,DMSO-d6)δ46.64,124.67,120.77,112.89,52.42,41.47.HRMS(m / z):calculated for C 10 H 15 Cl2N2 + [M] + :233.0607; found:233.0614.

[0067] Step 5: Synthesize Compound 1

[0068] LNBCl(1) (282 mg, 0.81 mmol), NM(1) (217 mg, 0.81 mmol), and triethylamine (0.45 mL, 3.24 mmol) were dissolved in 5 mL of CH3CN and stirred at 40 °C for 4 h. After the reaction was complete, the system was evaporated to dryness, purified by silica gel column chromatography, and eluted with n-hexane:ethyl acetate (10:1, v / v) to give compound 1 (203 mg, 46% yield). 1 H NMR (500MHz, DMSO-d6) δppm 10.10 (s, 2H), 7.21 (d, J = 8.7Hz, 2H), 6.82 (d, J = 8.6Hz, 2H), 3.73 (s, 8H). 13C NMR(126MHz,DMSO-d6)δ153.62,149.00,142.73,133.57,130.11,128.85,127.5 1,122.97,112.57,111.70,110.88,52.88,41.74,40.71.HRMS(m / z):calculated forC 27 H 31 Cl2N5S + [M] + :544.1700; found:544.1709.

[0069] Example 2 describes the synthesis of compound 2, which specifically includes the following steps:

[0070] Step 1: Synthesis of compound NB(2)

[0071] A mixture of phenothiazine (4000 mg, 20.10 mmol), iodine (1525 mg, 60.20 mmol), and diethylamine (65 mL) was stirred at 50 °C for 23 h, then cooled to room temperature, evaporated under reduced pressure, dissolved in DCM, added dropwise to diethyl ether, filtered, and washed to give compound NB(2) (4210 mg, yield 45%). 1 H NMR(500MHz,DMSO-d6)δppm 7.83(d,J=9.7Hz,1H),7.46(d,J=2.7Hz,1H),7.22(dd,J=9.7,2.7Hz,1H),3.72(q,J=7.2Hz,4H),1.34(t,J=7.2Hz,6H).HRMS(m / z):calculated for C 20 H 26 N3S + [M] + :340.1842; found:340.1848.

[0072] Step 2: Synthesis of compound LNBCl(2)

[0073] Compound NB(2) (1457 mg, 3.12 mmol), Na₂CO₃ (1320 mg, 12.60 mmol), and Na₂S₂O₄ (2200 mg, 12.60 mmol) were dissolved in water (10 mL) and DCM (5 mL), and stirred at 40 °C for 30 min under nitrogen protection. Triphosgene (560 mg, 1.88 mmol) dissolved in DCM was slowly added dropwise to the reaction system in an ice-water bath. The mixture was stirred at room temperature for 6 h. The solution was poured into water and then extracted with CH₂Cl₂. The organic layer was dried over anhydrous Na₂SO₄ and evaporated under reduced pressure. The mixture was purified by silica gel column chromatography, eluting with n-hexane and ethyl acetate (10 / 1, v / v), to give compound LNBCl(2) (587 mg, 62% yield). HRMS (m / z): calculated for C 21 H 26 ClN3OS + [M] + :403.1485; found:403.1482.

[0074] Step 3: Synthesis of compound NMNO2(2)

[0075] N-(4-nitrophenyl)diethanolamine (226 mg, 1.00 mmol) was dissolved in SOCl2 (5 mL), and DMF (0.5 mL) was added. The mixture was stirred at room temperature for 6 h. After cooling to room temperature, the solution was poured into water and then extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure. The solution was purified by silica gel column chromatography, eluted with dichloromethane, to give compound NMNO2(2) (216 mg, 82% yield). 1 H NMR (500MHz, Chloroform-d) δppm 8.07-8.01 (m, 2H), 7.04-6.98 (m, 2H), 3.70 (t, J = 3.3Hz, 4H), 3.62 (t, J = 3.3Hz, 4H). 13 C NMR (125MHz, Chloroform-d) δ152.20,137.72,125.97,112.26,52.15,41.55.

[0076] Step 4: Synthesis of compound NM(2)

[0077] Compound NMNO2(2) (131 mg, 0.50 mmol) and SnCl2 (474 ​​μL, 2.50 mmol) were added to 2 mL of concentrated HCl and stirred at room temperature for 6 h. The mixture was poured into water, the pH was adjusted to neutral, and then extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residual oil was dissolved in a saturated hydrochloric acid-ethanol solution and stirred for 30 min. An appropriate amount of diethyl ether was added, stirred, filtered, and washed to give compound NM(2) (74 mg, yield 57%). 1 H NMR (500MHz, DMSO-d6) δppm 10.10 (s, 2H), 7.21 (d, J = 8.7Hz, 2H), 6.82 (d, J = 8.6Hz, 2H), 3.73 (s, 8H). 13 C NMR(126MHz,DMSO-d6)δ46.64,124.67,120.77,112.89,52.42,41.47.HRMS(m / z):calculated for C 10 H 15 Cl2N2 + [M] + :233.0607; found:233.0614.

[0078] Step 5: Synthesize compound 2

[0079] LNBCl(2) (327 mg, 0.81 mmol), NM(2) (217 mg, 0.81 mmol), and triethylamine (0.45 mL, 3.24 mmol) were dissolved in 5 mL of CH3CN and stirred at 40 °C for 4 h. After the reaction was complete, the system was evaporated to dryness and purified by silica gel column chromatography, eluting with n-hexane:ethyl acetate (10:1, v / v) to give compound 2 (233 mg, 48% yield). HRMS (m / z): calculated for C 31 H 39 Cl2N5OS + [M] + :599.2252; found:599.2256.

[0080] Example 3 describes the synthesis of compound 3, which specifically includes the following steps:

[0081] Step 1: Synthesis of compound NB(3)

[0082] A mixture of phenothiazine (4000 mg, 20.10 mmol), iodine (1525 mg, 60.20 mmol), and methylethylamine (65 mL) was stirred at 50 °C for 22 h, then cooled to room temperature, evaporated to dryness under reduced pressure, dissolved in DCM, added dropwise to diethyl ether, filtered, and washed to give compound NB(3) (4035 mg, yield 47%). HRMS (m / z): calculated for C 18 H 22 N3S + [M] + :312.1529; found:312.1528.

[0083] Step 2: Synthesis of compound LNBCl(3)

[0084] Compound NB(3) (1120 mg, 3.12 mmol), Na2CO3 (1320 mg, 12.60 mmol), and Na2S2O4 (2200 mg, 12.60 mmol) were dissolved in water (10 mL) and DCM (5 mL), and stirred at 40 °C for 30 min under nitrogen protection. Triphosgene (560 mg, 1.88 mmol) dissolved in DCM was slowly added dropwise to the reaction system in an ice-water bath. The mixture was stirred at room temperature for 5 h. The solution was poured into water and then extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure. The mixture was purified by silica gel column chromatography, eluting with n-hexane and ethyl acetate (10 / 1, v / v), to give compound LNBCl(3) (520 mg, 59% yield). HRMS (m / z): calculated for C 19 H 22 ClN3OS + [M] + :375.1172; found:375.1178.

[0085] Step 3: Synthesis of compound NMNO2(3)

[0086] N-(4-nitrophenyl)diethanolamine (226 mg, 1.00 mmol) was dissolved in SOCl2 (5 mL), and DMF (0.5 mL) was added. The mixture was stirred at room temperature for 6 h. After cooling to room temperature, the solution was poured into water and then extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure. The mixture was purified by silica gel column chromatography, eluted with dichloromethane, to give compound NMNO2(3) (216 mg, 82% yield). 1H NMR (500MHz, Chloroform-d) δppm 8.07-8.01 (m, 2H), 7.04-6.98 (m, 2H), 3.70 (t, J = 3.3Hz, 4H), 3.62 (t, J = 3.3Hz, 4H). 13 C NMR (125MHz, Chloroform-d) δ152.20,137.72,125.97,112.26,52.15,41.55.

[0087] Step 4: Synthesis of compound NM(3)

[0088] Compound NMNO2(3) (131 mg, 0.50 mmol) and SnCl2 (474 ​​μL, 2.50 mmol) were added to 2 mL of concentrated HCl and stirred at room temperature for 6 h. The mixture was poured into water, the pH was adjusted to neutral, and then extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residual oil was dissolved in a saturated hydrochloric acid-ethanol solution and stirred for 30 min. An appropriate amount of diethyl ether was added, stirred, filtered, and washed to give compound NM(3) (74 mg, yield 57%). 1 H NMR (500MHz, DMSO-d6) δppm 10.10 (s, 2H), 7.21 (d, J = 8.7Hz, 2H), 6.82 (d, J = 8.6Hz, 2H), 3.73 (s, 8H). 13 C NMR(126MHz,DMSO-d6)δ46.64,124.67,120.77,112.89,52.42,41.47.HRMS(m / z):calculated for C 10 H 15 Cl2N2 + [M] + :233.0607; found:233.0614.

[0089] Step 5: Synthesize compound 3

[0090] LNBCl(4) (303 mg, 0.81 mmol), NM(4) (217 mg, 0.81 mmol), and triethylamine (0.45 mL, 3.24 mmol) were dissolved in 5 mL of CH3CN and stirred at 37 °C for 5 h. After the reaction was complete, the system was evaporated to dryness and purified by silica gel column chromatography, eluting with n-hexane:ethyl acetate (10:1, v / v) to give compound 3 (199 mg, 43% yield). HRMS (m / z): calculated for C 29 H 35 Cl2N5OS + [M]+ :571.1939; found:571.1944.

[0091] Example 4 describes the synthesis of compound 4, which specifically includes the following steps:

[0092] Step 1: Synthesis of compound NB(4)

[0093] A mixture of phenothiazine (4000 mg, 20.10 mmol), iodine (1525 mg, 60.20 mmol), and di-n-propylamine (65 mL) was stirred at 45 °C for 23 h, then cooled to room temperature, evaporated to dryness under reduced pressure, dissolved in DCM, added dropwise to diethyl ether, filtered, and washed to give compound NB(4) (5231 mg, yield 48%). HRMS (m / z): calculated for C 24 H 34 N3S + [M] + :396.2468; found:396.2473.

[0094] Step 2: Synthesis of compound LNBCl(4)

[0095] Compound NB(4) (1236 mg, 3.12 mmol), Na2CO3 (1320 mg, 12.60 mmol), and Na2S2O4 (2200 mg, 12.60 mmol) were dissolved in water (10 mL) and DCM (5 mL), and stirred at 40 °C for 30 min under nitrogen protection. Triphosgene (560 mg, 1.88 mmol) dissolved in DCM was slowly added dropwise to the reaction system in an ice-water bath. The mixture was stirred at room temperature for 6 h. The solution was poured into water and then extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure. The mixture was purified by silica gel column chromatography, eluting with n-hexane and ethyl acetate (10 / 1, v / v), to give compound LNBCl(4) (528 mg, 49% yield). HRMS (m / z): calculated for C 25 H 34 ClN3OS + [M] + :459.2111; found:459.2114.

[0096] Step 3: Synthesis of compound NMNO2(4)

[0097] N-(4-nitrophenyl)diethanolamine (226 mg, 1.00 mmol) was dissolved in SOCl2 (5 mL), and DMF (0.5 mL) was added. The mixture was stirred at room temperature for 6 h. After cooling to room temperature, the solution was poured into water and then extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure. The mixture was purified by silica gel column chromatography, eluted with dichloromethane, to give compound NMNO2(4) (216 mg, 82% yield). 1 H NMR (500MHz, Chloroform-d) δppm 8.07-8.01 (m, 2H), 7.04-6.98 (m, 2H), 3.70 (t, J = 3.3Hz, 4H), 3.62 (t, J = 3.3Hz, 4H). 13 C NMR (125MHz, Chloroform-d) δ152.20,137.72,125.97,112.26,52.15,41.55.

[0098] Step 4: Synthesis of compound NM(4)

[0099] Compound NMNO2(4) (131 mg, 0.50 mmol) and SnCl2 (474 ​​μL, 2.50 mmol) were added to 2 mL of concentrated HCl and stirred at room temperature for 6 h. The mixture was poured into water, the pH was adjusted to neutral, and then extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residual oil was dissolved in a saturated hydrochloric acid-ethanol solution and stirred for 30 min. An appropriate amount of diethyl ether was added, stirred, filtered, and washed to give compound NM(4) (74 mg, yield 57%). 1 H NMR (500MHz, DMSO-d6) δppm 10.10 (s, 2H), 7.21 (d, J = 8.7Hz, 2H), 6.82 (d, J = 8.6Hz, 2H), 3.73 (s, 8H). 13 C NMR(126MHz,DMSO-d6)δ46.64,124.67,120.77,112.89,52.42,41.47.HRMS(m / z):calculated for C 10 H 15 Cl2N2 + [M] + :233.0607; found:233.0614.

[0100] Step 5: Synthesize compound 4

[0101] LNBCl(4) (371 mg, 0.81 mmol), NM(4) (217 mg, 0.81 mmol), and triethylamine (0.45 mL, 3.24 mmol) were dissolved in 5 mL of CH3CN and stirred at 35 °C for 5 h. After the reaction was complete, the system was evaporated to dryness and purified by silica gel column chromatography, eluting with n-hexane:ethyl acetate (10:1, v / v) to give compound 4 (207 mg, 39% yield). HRMS (m / z): calculated for C 35 H 47 Cl2N5OS + [M] + :655.2878; found:655.2881.

[0102] Example 5: Synthesis of Compound 5

[0103] The synthesis of compound 5 was performed according to Example 1, except that the dimethylamine in step 3 was replaced with an equimolar amount of di-n-butylamine, and the remaining reaction conditions and methods were the same as in Example 1. HRMS (m / z): calculated for C 39 H 55 Cl2N5OS + [M] + :711.3504; found:711.3502.

[0104] Example 6: Synthesis of Compound 6

[0105] The synthesis of compound 6 was performed according to Example 2, except that the diethylamine in step 3 was replaced with an equimolar amount of di-n-pentaneamine, and the remaining reaction conditions and methods were the same as in Example 2. HRMS (m / z): calculated for C 43 H 63 Cl2N5OS + [M] + :767.4130; found:767.4133.

[0106] Example 7: Synthesis of Compound 7

[0107] The synthesis of compound 7 was performed according to Example 3, except that the methylethylamine in step 3 was replaced with an equimolar amount of di-n-hexylamine, and the remaining reaction conditions and methods were the same as in Example 3. HRMS (m / z): calculated for C 47 H 71 Cl2N5OS + [M] + :823.4756; found:767.4133.

[0108] Example 8: Response release experiment of compound 1

[0109] Compound 1 was dissolved in DMSO to prepare a 1 mM stock solution for subsequent use. 10 μL of the stock solution was added to 190 μL of DMSO and diluted to 1 mL with PBS to obtain a final concentration of 10 μM for Compound 1. The solution was then exposed to 660 nm light (100 mW / cm²). 2 The absorption spectrum was measured every 3 minutes using a UV-Vis spectrophotometer for 21 minutes. 10 μL of the stock solution was added to 190 μL of DMSO and diluted to 1 mL with PBS to obtain a 10 μM solution of compound 1. The solution was then exposed to 660 nm light (100 mW / cm²). 2 The fluorescence spectrum of the sample was measured every 3 minutes using a fluorescence spectrophotometer for 21 minutes. Figure 2 As shown, the intensity of ultraviolet absorption and fluorescence emission characteristic of methylene blue increases with increasing illumination time. The intensity reaches its maximum when the illumination time reaches 15 minutes.

[0110] Example 9: HRMS validation of the response release of compound 1

[0111] ESI-MS analysis was performed on methylene blue, nitrogen mustard, compound 1, and compound 1 after 15 minutes of 660 nm light irradiation. Figure 3 As shown, the HRMS results of compound 1 after illumination show molecular ion peaks for methylene blue and nitrogen mustard, indicating that 660 nm light irradiation can effectively release methylene blue and nitrogen mustard from compound 1.

[0112] Example 10: Response of Compound 1 at different pH values

[0113] Take 10 μL of the compound 1 stock solution and dilute it to 1 mL with PBS solutions of different pH values ​​to achieve a final concentration of 10 μM for compound 1. Then, expose the solutions at different pH values ​​to 660 nm light (100 mW / cm²). 2 ) 21 minutes, and its ultraviolet absorption was measured. For example Figure 4 As shown, the absorbance values ​​of solutions with different pH values ​​did not change significantly after light exposure, indicating that compound 1 has good release capacity at different pH values ​​and the ability to be released in the acidic microenvironment of tumors.

[0114] Example 11 Dark stability evaluation of compound 1

[0115] Add 10 μL of the compound 1 stock solution to 190 mL of DMSO, and dilute to 1 mL with PBS to achieve a final concentration of 10 μM for compound 1. Store the compound 1 solution at 4 °C protected from light, and measure its UV absorbance for seven consecutive days. The results are as follows: Figure 5 As shown, the ultraviolet absorption of compound 1 did not change significantly under light-shielded conditions, indicating good dark stability.

[0116] Example 12 Evaluation of the reactive oxygen species generation capacity of compound 1 before and after release

[0117] DCFH was used as an indicator of total reactive oxygen species (ROS) production. A mixed solution of DCFH and pre-irradiated compound 1 (containing DCFH: 10 μM, compound 1: 10 μM) was irradiated with 660 nm light (100 mW / cm²). 2 After different time intervals (0, 1, 2, 3, 4, 5, 6, 7, 8, and 9 seconds), the fluorescence emission spectra (500–600 nm) of each sample were measured. The generation capacity of reactive oxygen species was estimated by comparing the degree of decrease in absorbance at 522 nm. Figure 6 As shown, in the mixed solution of DCFH and pre-irradiated compound 1, the fluorescence intensity of DCFH at 522 nm gradually increases with the extension of illumination time.

[0118] Example 13 In vitro cytotoxicity test of compound 1

[0119] B16F10 cells were cultured in DMEM medium containing 10% fetal bovine serum, and the flasks were placed in an incubator at 37°C containing 5% carbon dioxide. When the cells nearly covered the bottom of the flask, they were separated with 0.25% trypsin and seeded into 96-well plates (approximately 1 × 10⁶ cells per well). 4 Cells were incubated in the above-mentioned incubator for 24 hours. After removing the culture medium, 100 μL of the compound dissolved in the culture medium at different concentrations was added to each well. After incubation for 4 hours, a 660 nm LED lamp (100 mW / cm²) was used. 2 Cells were irradiated with light for 15 minutes. Cell viability was determined using the MTT assay. 100 μL of fresh culture medium containing 10 μL of undiluted MTT (5 mg / mL) was added to each well, and the cells were incubated at 37°C for 48 h. After removing the culture medium from each well, 150 μL of DMSO was added to dissolve crystal violet, and the absorbance at 490 nm was measured. Cell viability in each group was compared with that in the untreated control group. Figure 7 As shown, when the concentration of compound 1 reached 10 μM, the viability of B16F10 cells in the irradiated group dropped to below 10%, while the viability of cells in the unirradiated group remained above 80%.

[0120] Example 14: Live / dead staining experiment of compound 1

[0121] First, B16F10 cells were seeded onto cell culture plates and cultured for 24 hours, followed by the following different treatments. In the concentration-dependent group, different concentrations of compound 1 were co-incubated with the cells for 4 hours at 37°C, and then irradiated with 660nm light (power density 100mW / cm²). 215 minutes. In the time-dependent group, 10 μM compound 1 was added to cell culture plates, incubated for 4 hours, and then irradiated with 660 nm light (power density 100 mW / cm²). 2 Different time intervals were observed. After light exposure and incubation for 30 minutes, Calein-AM and PI were added, and incubation was continued for 10 minutes. Observation was then performed using an inverted fluorescence microscope. Figure 8 As shown, when the concentration of compound 1 reached 10 μM, irradiation for 15 minutes was able to kill almost all B16F10 cells.

[0122] Example 15: Imaging Experiment of Cell Nuclear Damage

[0123] B16F10 cells were seeded in 6-well plates and cultured for 24 hours. The culture medium was then replaced with fresh medium containing PBS, nitrogen mustard (10 μM), methylene blue (10 μM), and compound 1 (10 μM), and incubated for another 8 hours. Subsequently, the light-treated group was exposed to a 660 nm LED lamp (power density 100 mW / cm²). 2 Cells were incubated for 10 minutes at 37°C for 1 hour. After washing with PBS, the cells were processed according to the instructions of the DNA damage detection kit. Finally, images were observed and collected using a fluorescence inverted microscope. Figure 9 As shown, the cell nuclei of the non-illuminated group of compound 1 showed almost no damage, while the cell nuclei of the illuminated group of compound 1 showed more significant damage compared to the nitrogen mustard group and the methylene blue illuminated group.

[0124] Example 16: Cell flow cytometry experiment of compound 1

[0125] B16F10 cells were seeded in 6-well plates and cultured for 24 hours. Then, the culture medium was replaced with fresh medium containing PBS, NM (10 μM), MB (10 μM), and Compound 1 (10 μM), and incubated for another 8 hours. Subsequently, the light-treated group was exposed to light (power density 100 mW / cm²). 2 Cells were incubated for 15 minutes and then at 37°C for 2 hours. Apoptosis was detected using the Annexin V-FITC apoptosis detection kit; cells were suspended in 195 μL Annexin V-FITC buffer and incubated with 5 μL Annexin V-FITC and 10 μL PI at room temperature in the dark for 20 minutes. Finally, the samples were analyzed by flow cytometry. Figure 10 As shown, the cells in the compound 1 light-illuminated group showed a higher proportion of early and late apoptosis compared to the nitrogen mustard group and the methylene blue light-illuminated group, while the proportion of apoptotic cells in the compound 1 non-light-illuminated group was consistent with that in the control group.

[0126] Example 17 In vitro hemolysis experiment of compound 1

[0127] Arterial blood (cardiac blood) was collected from mice and anticoagulated with heparin. The anticoagulated blood was centrifuged at 1500 rpm for 5 min, and red blood cells were collected. The obtained red blood cells were further washed twice with PBS and diluted. 500 μL of compound 1 at different concentrations (256, 128, 64, 32, 16, 8, 4, 2, 1 μM) was prepared, using physiological saline as a negative control and deionized water as a positive control, and mixed with 500 μL of red blood cell suspension. The mixture was incubated at 37℃ for 2 h, centrifuged, and the absorbance of the supernatant at 540 nm was measured. The hemolysis rate was calculated using the following formula:

[0128]

[0129] like Figure 11 As shown, the hemolysis assay results indicate that it is safe for in vivo treatment. Compound 1 has almost no hemolytic activity at concentrations below 256 μM.

[0130] Example 18: In vivo antitumor activity experiment of compound 1

[0131] All animal experimental procedures were approved by the Animal Protection and Use Committee of Central South University and complied with relevant ethical guidelines. Female BALB / c mice (6 weeks old) were purchased from the Experimental Animal Center of Central South University. The tumor volume in the tumor-bearing mice was 150 mm. 3 Intratumoral injection was administered. Mice were randomly divided into 5 groups: (1) PBS group (control) without any treatment; (2) Compound 1 group; (3) nitrogen mustard group; (4) methylene blue light irradiation group; (5) Compound 1 light irradiation group; (n=3 per group). For the light irradiation group, the tumor site was irradiated for 10 minutes with a 660nm laser (1W, 10cm away from the tumor) 2 hours after administration. Treatment was performed on days 1, 3, and 5, and tumor volume and mouse weight were measured and recorded for 7 consecutive days. On day 7, the tumor was dissected, and tumor mass was measured and recorded. The results are as follows: Figure 12 As shown, tumor growth in mice irradiated with laser was significantly inhibited, while tumor growth in the unirradiated group was not inhibited, and the mice's size did not change significantly during treatment. These results indicate that compound 1 has good tumor-killing ability after light irradiation and can significantly reduce the toxic side effects of nitrogen mustard.

Claims

1. A near-infrared photoactivated nitrogen mustard drug compound, characterized in that, The structural formula of the near-infrared photoactivated nitrogen mustard drug compound is any one of the following compounds 1 to 7: 。 2. A method for preparing the near-infrared light-activated nitrogen mustard drug compound according to claim 1, characterized in that, Includes the following steps: S1. Synthesis of compound NB: A mixture of phenothiazine, iodine, and disubstituted amine was stirred. After the reaction was complete, the mixture was cooled to room temperature, evaporated under reduced pressure, dissolved in dichloromethane, and added dropwise to diethyl ether. The mixture was filtered, and the resulting residue was washed with chloroform and dried to obtain compound NB. The synthetic route is as follows: ； S2. Synthesis of compound LNBCl: A mixture of compound NB, base 1, reducing agent, and solvent 1 was heated under nitrogen protection. Then, a mixture of triphosgene and solvent was added dropwise under an ice-water bath. After the addition was complete, the mixture was moved to room temperature. After the reaction was completed, the reaction solution was poured into water, and compound LNBCl was obtained by separation and purification. The synthetic route is as follows: ； S3. Synthesis of compound NMNO2: A mixture of N-(4-nitrophenyl)diethanolamine, N,N-dimethylformamide, and thionyl chloride was stirred and reacted at room temperature. After the reaction was completed, the reaction solution was poured into water, and the compound NMNO2 was obtained by separation and purification. The synthetic route is as follows: ； S4. Synthesis of compound NM: A mixture of compound NMNO2, stannous chloride, and concentrated hydrochloric acid was stirred and reacted at room temperature. After the reaction was complete, the solvent was evaporated, and hydrochloric acid ethanol solution was added and stirred. During stirring, diethyl ether was added, and then the reaction solution was filtered. The resulting residue was NM. The synthetic route is as follows: ； S5. Synthesis of compound NBNM: A mixture of compound LNBC1, NM, base 2, and solvent 2 was reacted under nitrogen protection. After the reaction was completed, the reaction solution was poured into water, and compound NBNM was obtained by separation and purification. The synthetic route is as follows: ； In step S1, the molar ratio of phenothiazine to iodine is 1:3-4; the molar volume ratio of phenothiazine to disubstituted amine is 1:4-5 mmol / mL; the reaction temperature is 40-50℃, and the reaction time is 20-24 h; the molar volume ratio of phenothiazine to dichloromethane is 1:8-10 mmol / mL, and the volume ratio of dichloromethane to diethyl ether is 1:12-15; the disubstituted amine is dimethylamine, methylethylamine, diethylamine, di-n-propylamine, di-n-butylamine, di-n-pentylamine, or di-n-hexylamine. In step S2, solvent 1 is a mixture of dichloromethane and water, with a volume ratio of dichloromethane to water of 1:1~2; alkali 1 is sodium carbonate or potassium carbonate; the reducing agent is sodium dithionite; the molar volume ratio of compound NB to solvent 1 is 1:(2.5~4) mmol / mL; the molar ratio of compound NB to sodium dithionite is 1:4~5; the molar ratio of compound NB to alkali 1 is 1:4~5; the molar ratio of compound NB to triphosgene is 1:0.6~0.8; the reaction temperature before the ice-water bath is 35-40℃, and the reaction time is 30~45 min; the time for adding the mixture dropwise in the ice-water bath is 15~20 min; the reaction temperature after the ice-water bath is room temperature, and the reaction time is 6~8 minutes. h; the volume ratio of the reaction solution to water is 1:10~12; the separation and purification are specifically as follows: first, extract with CH2Cl2 to obtain an organic layer, then dry the organic layer with anhydrous Na2SO4 and evaporate it on a rotary evaporator, and then separate and purify it using silica gel column chromatography; the eluent used in the silica gel column chromatography separation and purification is n-hexane:ethyl acetate = 10:1, v / v; Wherein, groups R1 and R2 are identical to the groups corresponding to the structural formula in claim 1.

3. The method for preparing near-infrared photoactivated nitrogen mustard drug compounds according to claim 2, characterized in that, In step S3, the molar volume ratio of N-(4-nitrophenyl)diethanolamine to thionyl chloride is 1:(3~4) mmol / mL; the molar volume ratio of N-(4-nitrophenyl)diethanolamine to N,N-dimethylformamide is 1:(0.4~0.6) mmol / mL; the reaction temperature is 25-30℃, the reaction time is 4~6 h; the volume ratio of reaction solution to water is 1:10~12; and the eluent used in the silica gel column chromatography separation and purification is dichloromethane.

4. The method for preparing near-infrared photoactivated nitrogen mustard drug compounds according to claim 2, characterized in that, In step S4, the molar volume ratio of compound NMNO2 to concentrated hydrochloric acid is 1:(3~4) mmol / mL; the molar ratio of compound NMNO2 to stannous chloride is 1:(4~5); the reaction time is 6~8 h; the volume ratio of concentrated mixture to hydrochloric acid ethanol solution is 1:5~8; the volume ratio of hydrochloric acid ethanol to diethyl ether is 1:10~15; and the stirring time is 30~45 min.

5. The method for preparing near-infrared photoactivated nitrogen mustard drug compounds according to claim 2, characterized in that, In step S5, solvent 2 is an organic solvent, namely acetonitrile or chloroform; base 2 is potassium carbonate or triethylamine; the molar volume ratio of compound LNBC1 to solvent is 1:(4~6) mmol / mL; the molar ratio of compound LNBC1 to NM is 1:1~1.5; the molar ratio of compound LNBC1 to base 2 is 1:(1~1.5); the reaction temperature is 35-40℃, and the reaction time is 4~6h; the eluent used in the silica gel column chromatography separation and purification is n-hexane:ethyl acetate = 10:1, v / v.

6. The application of a near-infrared photoactivated nitrogen mustard drug compound as described in claim 1 or a near-infrared photoactivated nitrogen mustard drug compound prepared by any one of claims 2 to 5 in the preparation of drugs for visualizing drug release, reducing the toxic side effects of nitrogen mustard, and synergistic anti-tumor effects of photodynamic therapy combined with chemotherapy.

7. The application according to claim 6, characterized in that, The near-infrared light-activated nitrogen mustard drug compound, when used in combination with chemotherapy under 660nm light irradiation, enhances the killing ability against tumor cells.

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