Preparation method and application of near-infrared two-region chemiluminescence afterglow probe based on xanthene dye skeleton

Abstract

The invention relates to the technical field of afterglow imaging, and discloses a preparation method and application of a near-infrared two-region chemiluminescent material based on a xanthene dye skeleton, and the molecular structure of an afterglow probe is shown as the following formula 8-F. According to the invention, a fluorescent skeleton is constructed to solve many challenges of current cancer diagnosis and treatment, including early diagnosis sensitivity, treatment tolerance, relapse, metastasis and the like. In cancer therapy, traditional chemotherapeutic drugs may be distributed in various tissues and organs in the body, resulting in serious toxic and side effects. After the afterglow probe is excited, the organic molecules transfer energy to the luminous group through the conjugated structure, so that the luminous group can emit light in NIR-II, and specifically react with a tumor microenvironment (TME) to generate ROS so as to carry out PDT treatment, and the fluorescence is kept in a closed state in normal tissues.

Description

Technical Field

[0001] This invention relates to the field of afterglow imaging technology, specifically to a method for preparing and applying near-infrared II chemiluminescent materials based on a dye skeleton. Background Technology

[0002] Cancer is the leading cause of cancer worldwide, affecting nearly one in six people. Unfortunately, there is often no noticeable pain or sensation before the onset of obvious clinical symptoms. Therefore, once diagnosed using conventional medical instruments such as magnetic resonance imaging (MRI), computed tomography (CT), ultrasound (US), and photoacoustic imaging (PA), real-time dynamic and visual monitoring of tumor development, progression, and malignancy plays a crucial role in personalized treatment. Cancer is often already in its middle or late stages. Even after a tumor is detected in the body, surgery remains one of the primary treatment options at this stage. During surgery, a key goal is to remove the tumor as completely as possible to prevent recurrence while preserving healthy tissue to minimize impact on the patient. However, successful tumor resection largely depends on the surgeon's experience and ability to observe anatomical features in the operating room. During cancer surgery, it is often difficult to accurately identify some non-palpable and invisible lesions. Furthermore, while pathological staining (such as hematoxylin and eosin staining) can provide pathological information, the use of fixed samples is not suitable for dynamic observation of tumor progression in vivo. Therefore, visual tracking of early tumor lesions and metastatic microlesions can assist surgeons in the diagnosis and resection of tumors.

[0003] Chemiluminescence (CL) is a photoluminescence phenomenon that accompanies the chemical reaction of certain substances. CL bioassays, especially CL immunoassays, have advantages such as low background, simple instruments, easy operation, and low cost, making them one of the most advanced and widely used detection methods in clinical diagnosis and biomedical research. In recent years, various nanomaterials have seen significant development. Nanomaterials, polymers, small molecules, and other functional systems (such as liposomes and extracellular vesicles) exhibit remarkable tumor accumulation, EPR effects, penetration, and drug delivery capabilities. They can load chemotherapeutic drugs and safely and accurately transport them to the lesion site, then release the drugs through some means, reducing chemotherapeutic drug loss and protecting normal tissue. Besides chemotherapy, radiotherapy is also used to treat tumors. Radiotherapy, or radiation therapy, uses rays of various energies (X-rays, gamma rays, electron beams) to irradiate tumors, thereby inhibiting and killing cancer cells. It is suitable for local treatment and can assist surgical treatment to improve efficacy. Like chemotherapy, it suffers from the problem of indiscriminate killing of both cancer cells and normal cells, and also has drawbacks such as the effective dose and toxic dose being very close, resulting in significant toxic side effects.

[0004] To date, surgery remains the most common and effective treatment for both primary and metastatic tumors. Highly sensitive and precise visualization of tumors, metastases, and tumor-positive sentinel lymph nodes is crucial for prognosis but remains challenging. For decades, surgeons have relied primarily on low-accuracy palpation and gross examination. High-sensitivity intraoperative visualization of small tumors is essential for complete resection of lesions but remains a challenge for conventional imaging modalities. In short, computed tomography (CT) and magnetic resonance imaging (MRI) require long acquisition and reconstruction times, and ultrasound imaging has low spatial resolution. In contrast, one of the significant advantages of optical imaging is its ability to visualize tissue in real-time and dynamically with high spatial resolution, serving as a powerful tool for image-guided surgery. Therefore, developing a technique utilizing chemiluminescent afterglow probes for tumor-specific imaging and treatment is of significant practical importance. Summary of the Invention

[0005] The purpose of this invention is to design an organic small molecule framework that emits long afterglow after X-ray irradiation. This framework can penetrate approximately 15 cm of muscle tissue to activate the material and emit long afterglow imaging, with an imaging time lasting up to 6 minutes. This allows for clear visualization of tumor regions within the organism, resulting in better tumor localization. Furthermore, after X-ray irradiation, it can generate a large amount of [unclear - possibly related to radiation] within the tumor tissue. 1 O2 is used to achieve the therapeutic effect.

[0006] To achieve the above objectives, on the one hand, the present invention provides a near-infrared II chemiluminescence afterglow probe based on a dye backbone, the molecular structure of which is shown in Formula 8-F below:

[0007] .

[0008] According to a second aspect of the present invention, the present invention also provides a method for preparing a near-infrared II chemiluminescence afterglow probe based on a tannin dye backbone, comprising the following steps:

[0009] S1. Compound A of formula A, malononitrile and acetic anhydride are mixed and reacted at 130~150 °C to obtain compound B of formula B.

[0010] , ;

[0011] S2. Compound B, hexamethylenetetramine and acetic acid are mixed and reacted at 60-70 °C. Then ice water is added to continue the reaction to obtain compound C with the following formula C.

[0012] ;

[0013] S3. Compound C, ethylene glycol, methylbenzenesulfonic acid monohydrate and toluene are reacted at 140~150 °C to obtain compound D with the following formula D;

[0014] ;

[0015] S4. Compound D, compound 8 of formula 8, acetonitrile, and piperidine are mixed and reacted at 80~90 °C to obtain compound 8-D of formula 8-D;

[0016] , ;

[0017] S5. Compound 8-D, tetrahydrofuran, and hydrochloric acid are mixed and reacted at room temperature to obtain compound 8-E of formula 8-E;

[0018] ;

[0019] S6. Compound a, compound 8-E, and acetic acid are mixed and refluxed at 80-95°C to obtain the target product shown in formula 8-F;

[0020] , .

[0021] As a further preferred technical solution of the present invention, 4-diethylaminoketo acid, cyclohexanone, perchloric acid and sulfuric acid are mixed and reacted at -5~0℃ to obtain compound a shown in formula a.

[0022] As a further preferred technical solution of the present invention, after the reaction in step S1 is completed, the compound B is separated and purified by silica gel column chromatography with a volume ratio of PE:EA:DCM=30:1~2:1~2, and the obtained compound B is a yellow solid; and / or, the molar ratio of compound A to malononitrile in step S1 is 1:5~10.

[0023] As a further preferred technical solution of the present invention, after the reaction in step S2 is completed, the compound C is obtained by filtration through a Buchner funnel and is a yellow solid; and / or, the molar ratio of compound B to hexamethylenetetramine in step S2 is 1:8~12.

[0024] As a further preferred technical solution of the present invention, after the reaction in step S3 is completed, the mixture is extracted with hydrochloric acid and ethyl acetate, dried with anhydrous Na2SO4, and evaporated under reduced pressure to obtain compound D as a yellow solid; and / or, the molar ratio of compound C, ethylene glycol and methylbenzenesulfonic acid monohydrate in step S3 is 1:5~10:0.05~0.2.

[0025] As a further preferred technical solution of the present invention, after the reaction in step S4 is completed, the mixture is extracted with 0.3~0.5M hydrochloric acid and ethyl acetate, dried with anhydrous Na2SO4, and rotary evaporated under reduced pressure to obtain compound 8-D as an orange-yellow solid; and / or, the molar ratio of compound D, compound 8, acetonitrile and piperidine in step S4 is 1:1~1.2:0.5~2:0.5~2.

[0026] As a further preferred technical solution of the present invention, after the reaction in step S5 is completed, the mixture is extracted with EA and saturated brine, dried with anhydrous Na2SO4, and evaporated under reduced pressure to obtain compound 8-E as a red solid; and / or, the concentration of hydrochloric acid in step S5 is 1~3 mol / L.

[0027] As a further preferred technical solution of the present invention, after the reaction in step S6 is completed, the product is extracted with ethyl acetate and saturated NH4Cl, dried with anhydrous Na2SO4, and evaporated under reduced pressure to obtain a red solid; and / or, the molar ratio of compound 8-E to compound a in step S6 is 1~1.5:1.

[0028] As a further preferred embodiment of the present invention, compound A is prepared by reacting 2,6-dimethyl-4H-1-benzothiaran-4-one with N-bromosuccinimide (NBS), benzoyl peroxide (BPO), and carbon tetrachloride (CCL4) at 80-90°C.

[0029] According to a third aspect of the invention, the invention also provides the application of a chemiluminescent afterglow probe of formula 8-F in the preparation of drugs for the prevention and treatment of cancer. The chemiluminescent afterglow probe can be used for tumor-specific imaging and treatment. The afterglow probe material is injected into the body via intraperitoneal or tail vein injection. Under irradiation, the aldehyde portion of the afterglow material generates singlet oxygen at the tumor site for phototherapy (PDT). Because the adamantane portion can transfer stored energy to the aldehyde portion, it can interact with water molecules in the environment to continuously generate singlet oxygen, enhancing the therapeutic effect of PDT. The adamantane moiety, upon entering the tumor site, can perform fluorescence imaging in the near-infrared II region, ultimately achieving fluorescence imaging-guided PDT.

[0030] Compared with the prior art, the present invention can achieve the following beneficial effects:

[0031] (1) A novel near-infrared II chemiluminescent framework was constructed, which is an organic small molecule framework that can emit long afterglow after X-ray irradiation. It can penetrate 15 cm of muscle tissue to activate the material and emit long afterglow imaging. It has a long imaging time and a sufficient time window, which is convenient for clear tumor areas to be seen in the body in clinical practice and for better tumor localization. In addition, after X-ray irradiation, a large amount of tumor tissue is generated in the tumor tissue. 1 O2 is used to achieve the therapeutic effect.

[0032] (2) The afterglow wavelength can reach the near-infrared II region, which can be used for tumor-specific imaging and treatment. Specifically, the afterglow material can be injected into the body through the peritoneal cavity or tail vein. Under irradiation, the aldehyde portion of the afterglow material generates singlet oxygen at the tumor site for PDT. Since the adamantane portion can transfer the stored energy to the aldehyde portion, it can react with water molecules in the environment to continuously generate singlet oxygen, thereby enhancing the therapeutic effect of PDT. The afterglow material can perform fluorescence imaging in the near-infrared II region after entering the tumor site, ultimately realizing fluorescence imaging-guided PDT.

[0033] (3) Radiation dynamics therapy can be performed under low-dose radiation.

[0034] (4) It has a good enrichment effect in mouse tumors and can observe clear tumor boundaries. Its high sensitivity and accurate visualization of tumors, metastases and tumor-positive sentinel lymph nodes are of great significance for prognosis. Attached Figure Description

[0035] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0036] Figure 1 This is a schematic diagram of the synthesis circuit of the afterglow probe in Example 1.

[0037] Figure 2 This is the NMR spectrum of compound A.

[0038] Figure 3 This is the NMR spectrum of compound B.

[0039] Figure 4 This is the NMR spectrum of compound C.

[0040] Figure 5 This is the NMR spectrum of compound 8-D.

[0041] Figure 6 This is the NMR spectrum of compound 8-E.

[0042] Figure 7 This is the NMR spectrum of compound 8-F.

[0043] Figure 8 In the figure, a represents the change in chemiluminescence intensity before and after X-ray irradiation; b represents the change in chemiluminescence intensity after the addition of NaN3.

[0044] Figure 9 In the diagram, a represents the change in cell activity with 8-F concentration; b represents the change in cell activity with radiation dose.

[0045] Figure 10 This is a chemical imaging result of tumors after X-ray excitation of 8-F in mice.

[0046] The objectives, features, and advantages of this invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0047] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0048] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which this invention pertains. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0049] Example 1

[0050] See Figure 1 The synthetic route provided in this embodiment is as follows: A method for preparing a near-infrared II chemiluminescence afterglow probe based on a dye backbone.

[0051] Step S1, Compound B:

[0052] First, 2,6-dimethyl-4H-1-benzothiaran-4-one (1 eq) was mixed with N-bromosuccinimide (NBS) (1 eq), benzoyl peroxide (BPO) (1 eq), and carbon tetrachloride (CCL4) and reacted at 85 °C to prepare compound A, the NMR spectrum of which is shown below. Figure 2 As shown;

[0053] Then, malononitrile (8 eq) and acetic anhydride were added to a single-necked round-bottom flask containing compound A (1 eq), and the mixture was stirred in an oil bath at 140 °C for 5 h. After the reaction was complete, the product was purified by silica gel column chromatography (PE:EA:DCM = 30:1:1, volume ratio) to obtain compound B. The product was a yellow solid, and its NMR spectrum is shown below. Figure 3 As shown.

[0054] Step S2, Compound C:

[0055] Add 10 eq of hexamethylenetetramine and acetic acid to a single-necked round-bottom flask containing compound B (1 eq), and stir in an oil bath at 70°C for 12 h. Add ice water to the flask and react for 10 min. Finally, filter through a Buchner funnel; the product is a yellow solid, and its NMR spectrum is shown below. Figure 4 As shown.

[0056] Step S3, Compound D:

[0057] Ethylene glycol (10 eq) and toluenesulfonic acid monohydrate (0.1 eq) were added to a single-necked round-bottom flask containing compound C (1 eq). Toluene was then added, and the mixture was stirred in an oil bath at 150 °C for 12 h. After the reaction was complete, the mixture was extracted with ethyl acetate and brine, dried over anhydrous Na₂SO₄, and rotary evaporated under reduced pressure to obtain a yellow solid. Its NMR spectrum is shown below. Figure 4 As shown.

[0058] Step S4, Compound 8-D:

[0059] Acetonitrile (1 eq) and piperidine (1 eq) were added to a two-necked round-bottom flask containing compound D (1 eq) and compound 8 (1.2 eq), and the mixture was stirred in an oil bath at 85°C for 1.5 h–12 h. After the reaction was complete, the mixture was extracted with 0.5 M hydrochloric acid and ethyl acetate, dried over anhydrous Na₂SO₄, and rotary evaporated under reduced pressure to obtain an orange-yellow solid. Its NMR spectrum is shown in the figure below. Figure 5 As shown.

[0060] Step S5, Compound 8-E:

[0061] THF and 2 mol / L hydrochloric acid were added to a single-necked flask containing compound 8-D, and the mixture was stirred at room temperature for 1 h. After the reaction was complete, the mixture was extracted with EA and saturated brine, dried over anhydrous Na₂SO₄, and rotary evaporated under reduced pressure to obtain a red solid. Its NMR spectrum is shown below. Figure 6 As shown.

[0062] Step S6, Compound 8-F:

[0063] First, 4-diethylaminoketo acid (1 eq), cyclohexanone (1 eq), perchloric acid (1 eq), and sulfuric acid (1 eq) were mixed and reacted at 0 °C to obtain compound a.

[0064] Then, acetic acid was added to a two-necked flask containing compound 8-E (1.2 eq) and compound a (CAS: 1816997-05, 1 eq), and the mixture was refluxed at 90 °C and stirred for 12 h. After the reaction was complete, the sample was extracted with ethyl acetate and saturated NH4Cl, dried over anhydrous Na2SO4, and rotary evaporated under reduced pressure to obtain a red solid afterglow probe, the NMR spectrum of which is shown below. Figure 7 As shown.

[0065] The prepared afterglow probe was tested as follows:

[0066] Under radiation ( Figure 8 After (a), its chemiluminescence intensity was significantly enhanced, and by (a) Figure 8 (b) The addition of an active oxygen quencher resulted in a decrease in chemiluminescence, demonstrating its predictive ability against active oxygen.

[0067] In vitro cell experiments showed that, Figure 9 In Figure a, cell activity decreases with increasing 8-F concentration under a certain dose of radiation. Furthermore, at a certain concentration, approximately 20% killing effect can be achieved at 1 Gy with increasing radiation dose, demonstrating its good killing and therapeutic effect. Figure b shows the change in cell activity with radiation dose.

[0068] like Figure 10 In mice, 8 h after injection of 8-F, good enrichment was observed within the tumor, with clear tumor boundaries, demonstrating its imaging effectiveness. In this experiment, the afterglow probe was activated to emit long-persistence imaging by penetrating approximately 15 cm of mouse muscle tissue.

[0069] This invention constructs a fluorescent backbone to address numerous challenges in current cancer diagnosis and treatment, including sensitivity for early diagnosis, treatment tolerance, recurrence, and metastasis. In cancer therapy, traditional chemotherapy drugs can distribute to various tissues and organs throughout the body, leading to severe toxic side effects. Upon excitation, the organic molecule in this invention transfers energy to the luminescent group through a conjugated structure, enabling it to emit light in NIR-II and specifically interact with the tumor microenvironment (TME) to generate ROS for PDT treatment, while maintaining fluorescence in a closed state in normal tissues.

[0070] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. The scope of protection of the present invention is defined only by the appended claims.

Claims

1. A near-infrared II chemiluminescence afterglow probe based on a dye backbone, characterized in that, Its molecular structure is shown in Equation 8-F below: 。 2. A method for preparing the afterglow probe according to claim 1, characterized in that, Includes the following steps: S1. Mix compound A (shown in formula A), malononitrile, and acetic anhydride, and react at 130~150 °C to obtain compound B (shown in formula B). ， ； S2. Mix compound B, hexamethylenetetramine and acetic acid, react at 60-70 °C, then add ice water to continue the reaction, to obtain compound C as shown in formula C; ； S3. Compound C, ethylene glycol, methylbenzenesulfonic acid monohydrate and toluene were reacted at 140~150 °C to obtain compound D as shown in the following formula D; ； S4. Compound D, compound 8 of formula 8, acetonitrile, and piperidine are mixed and reacted at 80-90 °C to obtain compound 8-D as shown in formula 8-D; ， ； S5. Compound 8-D, tetrahydrofuran, and hydrochloric acid are mixed and reacted at room temperature to obtain compound 8-E as shown in formula 8-E; ； S6. Mix compound a, compound 8-E, and acetic acid as shown in formula a, and reflux at 80~95℃ to obtain the target product shown in formula 8-F; 。 3. The method for preparing the afterglow probe according to claim 2, characterized in that, After the reaction in step S1 is completed, the compound B is separated and purified by silica gel column chromatography with a volume ratio of PE:EA:DCM = 30:1~2:1~2, and the obtained compound B is a yellow solid; and / or, the molar ratio of compound A to malononitrile in step S1 is 1:5~10.

4. The method for preparing the afterglow probe according to claim 2, characterized in that, After the reaction in step S2 is completed, the compound C is obtained by filtration through a Buchner funnel as a yellow solid; and / or, the molar ratio of compound B to hexamethylenetetramine in step S2 is 1:8~12.

5. The method for preparing the afterglow probe according to claim 2, characterized in that, After the reaction in step S3 is completed, the mixture is extracted with hydrochloric acid and ethyl acetate, dried over anhydrous Na2SO4, and evaporated under reduced pressure to obtain compound D as a yellow solid; and / or, the molar ratio of compound C, ethylene glycol and methylbenzenesulfonic acid monohydrate in step S3 is 1:5~10:0.05~0.

2.

6. The method for preparing the afterglow probe according to claim 2, characterized in that, After the reaction in step S4 is completed, the mixture is extracted with 0.3-0.5M hydrochloric acid and ethyl acetate, dried over anhydrous Na2SO4, and rotary evaporated under reduced pressure to obtain compound 8-D as an orange-yellow solid; and / or, the molar ratio of compound D, compound 8, acetonitrile and piperidine in step S4 is 1:1-1.2:0.5-2:0.5-2.

7. The method for preparing the afterglow probe according to claim 2, characterized in that, After the reaction in step S5 is completed, the mixture is extracted with EA and saturated brine, dried over anhydrous Na2SO4, and evaporated under reduced pressure to obtain compound 8-E as a red solid; and / or, the concentration of hydrochloric acid in step S5 is 1~3 mol / L.

8. The method for preparing the afterglow probe according to claim 2, characterized in that, After the reaction in step S6 is completed, the product is extracted with ethyl acetate and saturated NH4Cl, dried over anhydrous Na2SO4, and evaporated under reduced pressure to obtain a red solid; and / or, the molar ratio of compound 8-E to compound a in step S6 is 1~1.5:

1.

9. The method for preparing the afterglow probe according to claim 2, characterized in that, Compound A was prepared by reacting 2,6-dimethyl-4H-1-benzothiaran-4-one with N-bromosuccinimide, benzoyl peroxide, and carbon tetrachloride at 80-90 °C.

10. The use of the afterglow probe according to claim 1 in the preparation of drugs for the prevention and treatment of cancer.

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