WO2019210786A1 - 噁嗪类化合物及其应用 - Google Patents

噁嗪类化合物及其应用 Download PDF

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WO2019210786A1
WO2019210786A1 PCT/CN2019/083615 CN2019083615W WO2019210786A1 WO 2019210786 A1 WO2019210786 A1 WO 2019210786A1 CN 2019083615 W CN2019083615 W CN 2019083615W WO 2019210786 A1 WO2019210786 A1 WO 2019210786A1
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compound
group
formula
oxazine compound
light
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French (fr)
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樊江莉
姚起超
李海东
边雅娜
李明乐
王静云
彭孝军
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大连理工大学
大连科荣生物技术有限公司
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Priority to EP19796624.5A priority Critical patent/EP3792265A4/en
Priority to US17/053,042 priority patent/US11639360B2/en
Priority to JP2021510504A priority patent/JP7117453B2/ja
Publication of WO2019210786A1 publication Critical patent/WO2019210786A1/zh

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    • C07D513/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains three hetero rings
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

  • the invention belongs to the field of design, synthesis and application of anticancer drugs. Specifically, it relates to the synthesis of novel oxazine-based light/sound sensitizers and their specific recognition for tumors and new applications in diagnosis and treatment.
  • Photodynamic action refers to the function or morphological change of organism cells or biomolecules under the action of photosensitizers. In severe cases, it causes cell damage and necrosis. In the early stage, this kind of action must involve aerobic participation, so It is called photosensitization-oxidation, which is called photosensitization in chemistry. It is called photodynamic action in biology and medicine. Photodynamic therapy is called photodynamic therapy. PDT). Photodynamic therapy is a new disease treatment based on the interaction of light, photosensitizer and oxygen. The study of photosensitizers (photodynamic therapy drugs) is the key to the future of photodynamic therapy. Photosensitizers are special chemicals whose basic function is to transfer energy. It absorbs photons and is excited.
  • PDT has also been used for the treatment of non-tumor diseases such as condyloma acuminata, psoriasis, port wine stains, rheumatoid arthritis, fundus macular degeneration, restenosis after angioplasty.
  • the representative photosensitizers in China mainly include: Ela (5-ALA, external use of ketoacetate) for research and development produced by Shanghai Fudan Zhangjiang Bio-Pharmaceutical Co., Ltd.
  • dermatologists are convenient to use the abbreviation of Ella or Photodynamics.
  • Sonodynamic therapy uses ultrasound to have a strong penetration of biological tissue, especially focused ultrasound that non-invasively focuses acoustic energy on deep tissue and activates some sound-sensitive drugs (such as hematoporphyrin). Produces an anti-tumor effect.
  • some sound-sensitive drugs such as hematoporphyrin.
  • the light/sonic sensitizers used in clinical practice are mainly represented by porphyrins and phthalocyanines. Although these compounds have achieved great success in cancer treatment, there are still many defects, such as: proportion of therapeutic system composition Unstable, slow metabolism in the body, shortest excitation wavelength, prone to phototoxic side effects. These shortcomings have seriously affected the practical effects and clinical applications of photodynamic therapy.
  • photosensitizers there are certain guiding ideas in the world that can play a guiding role.
  • sound sensitive agents there is no relevant theoretical guidance, and it can be applied to clinical sound sensitivity. There are very few agents. Therefore, designing a suitable light/sound sensitive agent has a great advancing effect on tumor diagnosis and treatment.
  • One of the objects of the present invention is to develop a novel compound having an oxazine-based basic structure and having both photosensitivity and sound sensitivity.
  • the compound should be able to utilize the high molar extinction coefficient, long absorption emission wavelength, and oil/water amphiphilic properties originally possessed by the oxazine compound.
  • the new compound should also have a particularly prominent tumor cell targeting recognition. , marking and killing features.
  • the present invention first provides a class of oxazin compounds having the structure of formula F:
  • the A is selected from the group consisting of sulfur (S), selenium (Se), and tellurium (Te) elements;
  • R 1 , R 2 and R 3 are each independently selected from hydrogen and a substituted or unsubstituted alkyl group of C 1-20 ;
  • the substituted alkyl group is optionally substituted by a halogen, a hydroxyl group, an alkoxy group, an aldehyde group, a carbonyl group, an amino group, a carboxyl group, an ester group, an amide group, a nitro group or a sulfonic acid group;
  • the X is selected from the group consisting of phosphate, sulfate, hydrogen sulfate, nitrate, chloride anion, bromide, iodine or perchlorate.
  • the oxazin compound described in the present invention is a kind of photo/acoustic active organic molecule having near-infrared absorbing emission and having photosensitive and acoustic sensitivity characteristics.
  • the maximum absorption and emission wavelengths of these compounds are all greater than 660 nm, and the triplet conversion rate is high.
  • reactive oxygen species can be produced with higher efficiency, which has better killing effect on cancer cells and cancer tissues. It achieves no toxic side effects on normal tissues while achieving photo/acoustic power therapy.
  • another aspect of the invention discloses the use of the oxazide compounds described in the preparation of light/sonic sensitizers.
  • the photo/acoustic agent is a near-infrared long-wavelength fluorescent probe for labeling tumor cells.
  • the oxazine compound of the present invention can be self-assembled into nanoparticles or coated with other materials to form nano ions in addition to small molecule administration operations, and the nanometer scale effective range is 1-1000 nm.
  • Figure 1 is a structural formula of the compound F-1.
  • Figure 2 is a result of the spectral test of Compound F-1 (Example 2).
  • Fig. 3 is a graph showing the results of the singlet oxygen yield measurement experiment under the condition of the compound F-1 (Example 3).
  • Figure 4 is a result of an in vitro cell anticancer test under the condition of Compound F-1 under ultrasound conditions (Example 4).
  • Figure 5 is a result of an in vitro cell anticancer test under the light condition of Compound F-1 (Example 5).
  • Figure 6 is a result of the spectral test of Compound F-2 (Example 7).
  • Figure 7 is a graph showing the results of singlet oxygen production measurement under the illumination condition of Compound F-2 (Example 8).
  • Figure 8 is a graph showing the results of singlet oxygen production measurement under the ultrasonic condition of Compound F-2 (Example 9).
  • Figure 9 is a result of an in vitro cell anticancer test under the light condition of Compound F-2 (Example 10).
  • Figure 10 is a result of the spectral test of Compound F-3 (Example 12).
  • Figure 11 is a graph showing the results of the singlet oxygen production measurement experiment under the condition of the compound F-3 (Example 13).
  • Figure 12 is a graph showing the results of singlet oxygen production measurement under the ultrasonic condition of Compound F-3 (Example 14).
  • Figure 13 is a graph showing the in vitro cell anticancer test results of Compound F-3 under light conditions (Example 15).
  • Figure 14 is a scanning electron microscopy image of a compound F-1 self-assembled nano drug delivery system (Example 16).
  • the present invention provides a class of oxazide compounds having the structure of Formula F:
  • the A is selected from the group consisting of sulfur (S), selenium (Se), and tellurium (Te) elements;
  • R 1 , R 2 and R 3 are each independently selected from hydrogen and a substituted or unsubstituted alkyl group of C 1-20 ;
  • the substituted alkyl group is optionally substituted by a halogen, a hydroxyl group, an alkoxy group, an aldehyde group, a carbonyl group, an amino group, a carboxyl group, an ester group, an amide group, a nitro group or a sulfonic acid group;
  • the X is selected from the group consisting of phosphate, sulfate, hydrogen sulfate, nitrate, chloride anion, bromide, iodine or perchlorate.
  • R 1 , R 2 and R 3 in the formula F are each independently selected from hydrogen and a substituted or unsubstituted alkyl group of C 1-14 .
  • R 1 , R 2 and R 3 are each independently selected from hydrogen and a C 1-6 substituted or unsubstituted alkyl group.
  • one of R 1 and R 2 in the formula F is hydrogen.
  • R 3 is hydrogen, constituting a further preferred technical solution.
  • the following nine compounds are used in the preparation of a near-infrared fluorescent probe, and the compound is selected. From F-1, F-2, F-3, F-4, F-5, F-6, F-7, F-8, F-9, F-10 and F-11:
  • alkyl as used herein includes both straight chain alkyl and branched alkyl groups. When referring to a single alkyl group such as “propyl”, it is specifically referred to as a straight-chain alkyl group, and a single branched-chain alkyl group such as “isopropyl” is specifically referred to as a branched alkyl group.
  • C 1-6 alkyl includes C 1-4 alkyl, C 1-3 alkyl, methyl, ethyl, n-propyl, isopropyl and t-butyl. Similar rules apply to the other groups used in this specification.
  • halogen as used herein includes fluoro, chloro, bromo and iodo.
  • the oxazine compound described in the present invention is synthesized by the following method: an azo compound formed using an aromatic amine or a derivative thereof is condensed with 8-hydroxyjulolidine in an acid-containing DMF to prepare a target oxazine dye. .
  • the synthesis method has a simple process and a high conversion rate. More specifically, the synthetic route of the compound of the formula F of the present invention is:
  • the preparation method of the compound of the formula F represented by the above route includes the following steps:
  • a compound of the formula F is prepared by reacting a compound of the formula II with an 8-hydroxyzuridine (quinoline) in a molar ratio of 1:1 in acidic DMF at 135 to 145 ° C for 2 to 4 hours.
  • the light/sonic sensitizer with the oxazine as the precursor of the invention has the following characteristics:
  • the invention introduces oxygen heavy elements into the therapeutic molecule, so that the molecules have the advantages of photodynamic therapy, acoustic power therapy and chemical therapy at the same time, and the tumor can be diagnosed and treated more efficiently.
  • the compound provided by the invention has simple molecular structure, stableness, small toxic and side effects; easy preparation and purification, and easy availability of raw materials, and has great advantages for industrial production of drugs for photo/acoustic treatment of tumors.
  • the near-infrared light/acoustic agent of the present invention can be used for tumor and non-tumor cell and tissue markers.
  • compositions containing the near-infrared fluorescent probe compounds of the present invention can also be used for staining markers of tumor cells and tissues.
  • One of the two-photon fluorescent probe compounds provided by the present invention should be included in the composition in an effective amount.
  • other components required for dyeing biological samples such as solvents, pH adjusters, and the like, may also be included. These components are known in the industry.
  • the above composition may be present in the form of an aqueous solution or may be present in other suitable forms which are formulated as solutions in water.
  • the present invention also provides a method of labeling tumor cells and tissue biological samples using the near-infrared light/acoustic agent of the present invention described above, the method comprising the step of contacting the compound with a biological sample.
  • contacting as used herein may include contacting in a solution or a solid phase.
  • the compound of p-nitrodiazobenzene chloride and 1-I is reacted at a molar ratio of 1:1 at 25 to 35 ° C for 0.5 to 2 hours, and the reaction is completed, and the brick red is obtained after the suction filtration operation.
  • the solid powder crude product gave the compound of formula 1-II in a yield of 95%.
  • the F-1-DMSO solution was added to dichloromethane and mixed well.
  • the spectral properties were tested using an ultraviolet-visible spectrophotometer and a fluorescence spectrometer.
  • the F-1 molecule had a maximum absorption of 680 nm in dichloromethane and a maximum emission of 700 nm.
  • the F-1-DMSO solution was added to methanol and uniformly mixed, and then 1,3-diphenylisobenzofuran (DPBF) was added to adjust the DPBF concentration to make the absorbance value about
  • DPBF 1,3-diphenylisobenzofuran
  • the 650 nm wavelength xenon lamp source (adjusted by grating filtering) was used for illumination, and the UV-visible absorption curve of the system was measured at equal intervals. According to the absorbance change of DPBF at 411 nm wavelength, the correlation curve between absorbance and time was plotted.
  • the singlet oxygen quantum yield of compound F-1 was calculated. The results are shown in Fig. 3. The figure shows the change of the UV-visible absorption spectrum of the mixed system with the illumination time. According to the relevant formula, the singlet oxygen quantum yield of the compound F-1 is about 0.018.
  • the F-1-DMSO solution was added to ethylene glycol monomethyl ether and uniformly mixed, and then 1,3-diphenylisobenzofuran (DPBF) was added to adjust the DPBF concentration.
  • DPBF 1,3-diphenylisobenzofuran
  • the absorbance value was about 1.0, 1.5 W/cm 2
  • 50% cycle ultrasonic stimulation was used, and the UV-visible absorption curve of the system was measured at equal intervals.
  • the correlation curve between absorbance and time was plotted.
  • the result is shown in Fig. 4.
  • the figure shows the change of the UV-visible absorption spectrum of the mixed system with the extension of the ultrasonic time. It can be seen from the figure that the compound F-1 can generate singlet oxygen under the promotion of ultrasound.
  • Example 5 In vitro cell anticancer test under the condition of compound F-1 under illumination
  • MCF-7 human breast cancer cells
  • F-1, F-1-DMSO solution was added to 10% fetal bovine serum DMEM to prepare different concentration solutions, the prepared solution was added to a 96-well plate and placed in a cell culture incubator for 30 minutes, with a wavelength of 660 nm. The red light is illuminated for a certain period of time. After the irradiation, 96 empty plates were placed in a cell incubator and incubation was continued for 12 hours.
  • the compound F-1 has little killing effect on cells under the condition of no light, and has almost no toxicity; in the presence of light, compound F-1 can produce obvious killing effect on cells, and with illumination The energy density is enhanced and the phototoxicity of compound F-1 is significantly improved.
  • the compound of p-nitrodiazobenzene chloride and 2-I is reacted at a molar ratio of 1:1 at 25 to 35 ° C for 0.5 to 2 hours, and the reaction is completed, and after washing and washing, a brick is obtained.
  • the crude product of red solid powder gave the compound of formula 2-II in a yield of 95%.
  • the F-2-DMSO solution was added to dichloromethane and mixed well.
  • the spectral properties were tested using an ultraviolet-visible spectrophotometer and a fluorescence spectrometer.
  • the F-2 molecule had a maximum absorption of 686 nm in methylene chloride and a maximum emission of 712 nm.
  • the F-2-DMSO solution was added to methanol and mixed uniformly, and then 1,3-diphenylisobenzofuran (DPBF) was added to adjust the DPBF concentration so that the absorbance value was about
  • DPBF 1,3-diphenylisobenzofuran
  • the 650 nm wavelength xenon lamp source (adjusted by grating filtering) was used for illumination, and the UV-visible absorption curve of the system was measured at equal intervals. According to the absorbance change of DPBF at 411 nm, the correlation curve between absorbance and time was plotted.
  • the singlet oxygen quantum yield of compound F-2 was calculated. The results are shown in Fig. 7. The figure shows that the UV-visible absorption spectrum of the mixed system is prolonged with the illumination time. According to the relevant formula, the singlet oxygen quantum yield of the compound F-2 is about 0.47.
  • the F-2-DMSO solution was added to ethylene glycol monomethyl ether and uniformly mixed, and then 1,3-diphenylisobenzofuran (DPBF) was added to adjust the DPBF concentration.
  • DPBF 1,3-diphenylisobenzofuran
  • the absorbance value was about 1.0, 1.5 W/cm 2
  • 50% cycle ultrasonic stimulation was used, and the UV-visible absorption curve of the system was measured at equal intervals.
  • the absorbance change of DPBF at the wavelength of 411 nm the correlation curve between absorbance and time was plotted, and the result is shown in Fig. 8.
  • the figure shows the change of the UV-visible absorption spectrum of the mixed system with the ultrasonication time. It can be seen that the compound F-2 can generate singlet oxygen under the promotion of ultrasound.
  • Example 10 In vitro cell anticancer test under the condition of compound F-2 under illumination
  • MCF-7 human breast cancer cells
  • F-2, F-2-DMSO solution was added to 10% fetal bovine serum DMEM to prepare different concentration solutions, the prepared solution was added to a 96-well plate and placed in a cell culture incubator for 30 minutes, with a wavelength of 660 nm. The red light is illuminated for a certain period of time. After the irradiation, 96 empty plates were placed in a cell incubator and incubation was continued for 12 hours.
  • the p-nitrodiazobenzene chloride is reacted with the compound of 3-I at a molar ratio of 1:1 at 25 to 35 ° C for 0.5 to 2 hours, and the reaction is completed, and the brick is obtained by suction filtration.
  • the crude product of red solid powder gave the compound of formula 3-II in a yield of 94%.
  • the F-3-DMSO solution was added to dichloromethane and mixed well.
  • the spectral properties were tested using an ultraviolet-visible spectrophotometer and a fluorescence spectrometer.
  • the F-3 molecule had a maximum absorption of 697 nm in methylene chloride and a maximum emission of 740 nm.
  • the F-3-DMSO solution was added to methanol and uniformly mixed, and then 1,3-diphenylisobenzofuran (DPBF) was added to adjust the DPBF concentration so that the absorbance value was about
  • DPBF 1,3-diphenylisobenzofuran
  • the 650 nm wavelength xenon lamp source (adjusted by grating filtering) was used for illumination, and the UV-visible absorption curve of the system was measured at equal intervals.
  • the absorbance change of DPBF at 411 nm wavelength the correlation curve between absorbance and time was plotted, and the singlet oxygen quantum yield of compound F-3 was calculated by using methylene blue as a reference.
  • the results are shown in Fig. 11.
  • the figure shows the change of the ultraviolet visible absorption spectrum of the mixed system with the illumination time. According to the relevant formula, the singlet oxygen quantum yield of the compound F-3 is about 0.73.
  • the F-3-DMSO solution was added to ethylene glycol monomethyl ether and uniformly mixed, and then 1,3-diphenylisobenzofuran (DPBF) was added to adjust the DPBF concentration.
  • DPBF 1,3-diphenylisobenzofuran
  • the absorbance value was about 1.0, 1.5 W/cm 2
  • 50% cycle ultrasonic stimulation was used, and the UV-visible absorption curve of the system was measured at equal intervals.
  • the correlation curve between absorbance and time was plotted, and the results are shown in Fig. 12.
  • the figure shows the change of the UV-visible absorption spectrum of the mixed system with the ultrasonication time. It can be seen that the compound F-3 can generate singlet oxygen under the promotion of ultrasound.
  • Example 15 In vitro cell anticancer test under compound F-3 illumination conditions
  • MCF-7 human breast cancer cells
  • F-3, F-3-DMSO solution was added to 10% fetal bovine serum DMEM to prepare different concentrations of solution, the solution was added to a 96-well plate and placed in a cell culture incubator for 30 minutes, with a wavelength of 660 nm The red light is illuminated for a certain period of time. After the irradiation, 96 empty plates were placed in a cell incubator and incubation was continued for 12 hours.
  • compound F-3 has a certain killing effect on cells in the absence of light, but its chemical effect is weak; in the presence of light, compound F- 3 in the low light energy density can produce significant killing effect on the cells. It can be seen that compound F-3 has dual effects of chemotherapy and light/acoustic dynamic therapy for tumor cells.
  • phosphate buffer (PBS) or ultrapure water as needed, and the compound F-1 was PBS or ultrapure after shaking.
  • the nanoparticle system is formed by self-assembly process in water, and the scanning electron microscope of the system is shown in Fig. 14. It can be seen from Fig. 14 that the size of the compound F-1 formed by self-assembly in the nano-system is about 200 nm, the particle shape is regular, the size is uniform, and the dispersion property is excellent.

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Abstract

噁嗪类化合物及其应用。所述的噁嗪类化合物，具有通式F的结构。本发明中所述及的噁嗪类化合物是一类具有近红外吸收发射的同时具备光敏及声敏特性的光/声动力活性有机分子。该类化合物最大吸收和发射波长均大于660nm，三线态转化率高，在光照或超声条件下，可较高效率产生活性氧物种，对于癌细胞和癌组织具有较好的杀伤作用，在对肿瘤达到光/声动力治疗的同时几乎不对正常组织产生毒副作用。

Description

噁嗪类化合物及其应用 技术领域
本发明属于抗癌药物设计、合成及应用领域。具体涉及新型噁嗪类光/声敏剂的合成及其对肿瘤的专一性识别及在诊疗中的新应用。
背景技术
光动力作用是指在光敏剂参与下，在光的作用下，使有机体细胞或生物分子发生机能或形态变化，严重时导致细胞损伤和坏死作用，早期这种作用必须有氧的参与，所以又称光敏化-氧化作用，在化学上称这种作用为光敏化作用，在生物学及医学上称之为光动力作用，用光动力作用治病的方法，称为光动力疗法(photodynamic therapy,PDT)。光动力疗法是以光、光敏剂和氧的相互作用为基础的一种新的疾病治疗手段，光敏剂(光动力治疗药物)的研究是影响光动力治疗前景的关键所在。光敏剂是一些特殊的化学物质，其基本作用是传递能量，它能够吸收光子而被激发，又将吸收的光能迅速传递给另一组分的分子，使其被激发而光敏剂本身回到基态。随着第一个光敏剂Porfimer Sodium于1993--1997年在美国、加拿大、欧盟、日本及韩国陆续被批准上市，PDT领域的研究、开发和应用迅速活跃起来。随着新的光动力治疗药物的研发成功及激光设备技术的提高，PDT又迎来了前所未有的发展高峰。国际上，已批准上市或正在临床研究的新的光敏剂近十种。同时，PDT也被用于非肿瘤型疾病，如尖锐湿疣、牛皮癣、鲜红斑痣、类风湿关节炎、眼底黄斑病变、血管成型术后再狭窄等疾病的治疗。国内有代表性的光敏剂主要有：上海复旦张江生物医药股份有限公司研发生产的艾拉(5-ALA，外用盐酸氨酮戊酸散)。医学临床和实践中，皮肤科医生方便使用简称艾拉或光动力。
声动力疗法(Sonodynamic therapy，SDT)利用超声波对生物组织有较强的穿透能力，尤其是聚焦超声能无创伤地将声能聚焦于深部组织，并激活一些声敏药物(如血卟啉)产生抗肿瘤效应。1990年，Yumitai.q等报道了超声联合HP(血卟啉)对小鼠移植肿瘤生长抑制的协同效应，表现为单用HP无抑制作用，单用超声仅有轻微抑制作用.而两者合用则有明显抑制作用.并将之命名为声动力疗法。
目前，应用于临床的光/声敏剂主要以卟啉和酞菁类化合物为主要代表，尽管该类化合物在肿瘤治疗方面取得了较大成功，但是依然存在很多缺陷，例如： 治疗体系组成比例不稳定，在肌体内代谢缓慢，最大激发波长较短，易发生光毒副作用等。这些不足严重影响了光动力疗法的实际效果及临床应用。对于光敏剂的制备及应用研究，国际上已经有一定的指导思想能够起到指导作用，但对于声敏剂的制备及应用研究，并未有相关的理论指导，而能够应用于临床的声敏剂更是寥寥无几。因此设计合适的光/声敏剂，对于肿瘤诊疗具有很大推进作用。
发明内容
本发明目的之一在于开发一种具有噁嗪类基础结构，并同时具有光敏及声敏特性的新的化合物。该化合物应当能够利用噁嗪类化合物原本具有的高摩尔消光系数、长吸收发射波长、油/水两亲的特性，在此基础上，该新的化合物还应当具有特别突出的肿瘤细胞靶向识别、标记及杀伤的功能。
为此，本发明首先提供一类噁嗪类化合物，其具有通式F的结构：
Figure PCTCN2019083615-appb-000001
通式F中，
所述的A选自硫(S)，硒(Se)，碲(Te)元素；
所述的R 1、R 2和R 3各自独立地选自氢和C 1-20的取代或未取代烷基；
所述的取代烷基由下述基团任意取代：卤素、羟基、烷氧基、醛基、羰基、氨基、羧基、酯基、酰胺基、硝基或磺酸基；
所述的X选自磷酸根，硫酸根，硫酸氢根，硝酸根，氯负离子，溴负离子，碘负离子或高氯酸根。
本发明中所述及的噁嗪类化合物是一类具有近红外吸收发射的同时具备光敏及声敏特性的光/声动力活性有机分子。该类化合物最大吸收和发射波长均大于660nm，三线态转化率高，在光照或超声条件下，可较高效率产生活性氧物种，对于癌细胞和癌组织具有较好的杀伤作用，在对肿瘤达到光/声动力治疗的同时几乎不对正常组织增加毒副作用。因此，本发明另一方面公开所述的噁嗪类化合物在制备光/声敏剂中的应用。该所述的光/声敏剂是近红外长波长荧光探针，用于肿瘤细胞的标记。本发明所述的噁嗪类化合物除了可以进行小分子给药操作，亦可自组装成纳米粒子或被其它材料包被成纳米离子进行给药操作， 纳米尺度有效范围为1-1000nm。
附图说明
图1是化合物F-1的结构式。
图2是化合物F-1光谱测试实验结果(实施例2)。
图3是化合物F-1光照条件下单线态氧产量测定实验结果(实施例3)。
图4是化合物F-1超声条件下体外细胞抗癌测试实验结果(实施例4)。
图5是化合物F-1光照条件下体外细胞抗癌测试实验结果(实施例5)。
图6是化合物F-2光谱测试实验结果(实施例7)。
图7为化合物F-2光照条件下单线态氧产量测定实验结果(实施例8)。
图8为化合物F-2超声条件下单线态氧产量测定实验结果(实施例9)。
图9为化合物F-2光照条件下体外细胞抗癌测试实验结果(实施例10)。
图10为化合物F-3光谱测试实验结果(实施例12)。
图11为化合物F-3光照条件下单线态氧产量测定实验结果(实施例13)。
图12为化合物F-3超声条件下单线态氧产量测定实验结果(实施例14)。
图13是化合物F-3光照条件下体外细胞抗癌测试结果(实施例15)。
图14是化合物F-1自组装纳米给药体系的扫描电镜成像图(实施例16)。
具体实施方式
本发明提供一类噁嗪类化合物，具有通式F的结构：
Figure PCTCN2019083615-appb-000002
通式F中，
所述的A选自硫(S)，硒(Se)，碲(Te)元素；
所述的R 1、R 2和R 3各自独立地选自氢和C 1-20的取代或未取代烷基；
所述的取代烷基由下述基团任意取代：卤素、羟基、烷氧基、醛基、羰基、氨基、羧基、酯基、酰胺基、硝基或磺酸基；
所述的X选自磷酸根，硫酸根，硫酸氢根，硝酸根，氯负离子，溴负离子，碘负离子或高氯酸根。
具体的技术方案中，所述的通式F中的R 1、R 2和R 3各自独立地选自氢和 C 1-14的取代或未取代烷基。其中作为优选地，所述的R 1、R 2和R 3各自独立地选自氢和C 1-6的取代或未取代烷基。
更为具体的技术方案中，所述通式F中的R 1和R 2其中之一是氢。该特征可以结合R 3是氢的技术特征，构成进一步优选的技术方案。
进一步对各化合物的RNA特异性识别能力进行甄别和比较的基础上，本发明提供的最优选的实施方式中，下述9个化合物在制备近红外荧光探针中得以应用，所述的化合物选自F-1、F-2、F-3、F-4、F-5、F-6、F-7、F-8、F-9、F-10和F-11：
Figure PCTCN2019083615-appb-000003
Figure PCTCN2019083615-appb-000004
除另有说明外，本文中使用的术语具有以下含义。
本文中使用的术语“烷基”包括直链烷基和支链烷基。如提及单个烷基如“丙基”，则只特指直链烷基，如提及单个支链烷基如“异丙基”，则只特指支链烷基。例如，“C 1-6烷基”包括C 1-4烷基、C 1-3烷基、甲基、乙基、正丙基、异丙基和叔丁基。类似的规则也适用于本说明书中使用的其它基团。
本文中使用的术语“卤素”包括氟、氯、溴和碘。
上文中所述及的具有通式F的化合物，其制备方法并未被现有技术所公开，本领域技术人员应当能够结合本说明书中的合成技术信息及有机合成的基本理论和技术来完成本发明所述化合物的获得。本说明书中描述的下述噁嗪类化合物的制备方法提供该类化合物合成的一种具体方案，但不应当理解为对其的限定。
本发明中所述及的噁嗪类化合物通过下述方法合成：使用芳胺或其衍生物形成的偶氮化合物与8-羟基久洛尼定在含酸DMF中缩合，制备得到目标噁嗪染料。该合成方法工艺简洁，转化率高。更为具体的，本发明通式化合物F合成路线为：
Figure PCTCN2019083615-appb-000005
上述路线所表示的通式F的化合物的制备方法包括如下步骤：
(1)盐酸酸化体系中，氯化对硝基重氮苯与式I的化合物按照摩尔比1:1在 25～35℃条件下反应0.5～2小时，制备式II化合物；
Figure PCTCN2019083615-appb-000006
(2)式II化合物与8-羟基久洛里定(quinoline)按照摩尔比1:1在酸性DMF中于135～145℃条件下反应2～4小时制备通式F的化合物。
本发明所述的以噁嗪为母体的光/声敏剂具备以下特点：
(1)具备一定的水溶性，同时具有良好的细胞膜通透性；
(2)具有优异的近红外发射特性，应用于生物成像时具有底生物光漂白、光损伤和光毒性，并且产生的荧光信号可以穿透较深的生物组织；
(3)在700nm波长的光照或超声条件下，可以大量产生单线态氧；
(4)在测试条件下暗毒性低，生物相容性好，具备一定水溶性，光稳定性较好，可以作为优良的光敏剂应用于光动力及声动力肿瘤治疗领域；
本发明将氧族重元素引入治疗分子中，使得分子同时具有光动力治疗，声动力治疗以及化学治疗的多方面优点，可以更加高效地对肿瘤进行诊疗。并且，本发明所提供的化合物分子结构简单、稳定、毒副作用小；易于制备和纯化，原料易得，对于产业化生产制备光/声动力治疗肿瘤的药物具有极大优势。
基于上述特点，本发明所述的近红外光/声敏剂可用于肿瘤与非肿瘤细胞和组织标记。除了以本文中所述的形式直接用于肿瘤与非肿瘤细胞和组织的染色标记外，含有本发明的近红外荧光探针化合物的组合物也可以用于肿瘤细胞和组织的染色标记。所述组合物中应当包含有效量的本发明所提供的双光子荧光探针化合物之一。另外，还可以包含生物样品染色所需要的其它组分，例如溶剂、pH调节剂等。这些组分都是本行业内已知的。上述组合物可以以水溶液形式存在，或者可以在使用时用水配制为溶液的其它合适形式存在。
本发明还提供使用上述本发明的近红外光/声敏剂标记肿瘤细胞和组织生物样品的方法，该方法包括使所述化合物与生物样品接触的步骤。本文中使用的术语“接触”可包括在溶液或固相中接触。
下述非限制性实施例可以使本领域的普通技术人员更全面地理解本发明， 但不以任何方式限制本发明。
实施例1：制备化合物F-1
Figure PCTCN2019083615-appb-000007
(1)中间体1-II的合成
盐酸酸化体系中，氯化对硝基重氮苯与1-I的化合物按照摩尔比1:1在25～35℃条件下反应0.5～2小时，反应完毕，经过抽滤洗涤操作后得到砖红色固体粉末粗产品得式1-II的化合物，收率95％。
(2)化合物F-1的合成
将上述反应(1)制备得到的中间体1-II与硫代久洛里定(quinoline)加入到含有DMF的圆底烧瓶中，滴入1mL高氯酸溶液。滴加完毕，体系搅拌2.5h后停止反应，经硅胶柱色谱分离，二氯甲烷:甲醇(v:v)＝8:1洗脱提纯得具金属光泽的深蓝色针状晶体目标化合物F-1(结构式如图1)，收率83.2％。
1H NMR(400MHz,DMSO)δ9.13(s,1H),8.77(d,J＝8.2Hz,1H),8.34(d,J＝8.1Hz,1H),7.84(t,J＝7.4Hz,1H),7.77(t,J＝7.5Hz,1H),7.44(s,1H),7.22(s,1H),3.54(d,J＝17.3Hz,5H),2.96–2.71(m,3H),2.49(s,6H),2.05–1.99(m,2H),1.98–1.92(m,2H),1.35(t,J＝7.2Hz,3H).
实施例2：化合物F-1光谱测试实验
使用实施例1中合成的化合物F-1，将F-1-DMSO溶液加入到二氯甲烷中混合均匀。用紫外可见分光光度计和荧光光谱仪测试其光谱性质。结果如图2所示，F-1分子在二氯甲烷中最大吸收680nm，最大发射700nm。
实施例3：化合物F-1光照条件下单线态氧产量测定实验
使用实施例1中合成的化合物F-1，将F-1-DMSO溶液加入到甲醇中混合均 匀后加入1,3-二苯基异苯并呋喃(DPBF)，调节DPBF浓度使其吸光度值约为1.0后使用660nm波长的氙灯光源(通过光栅滤光调节)照射，等时间间隔测定体系紫外可见吸收曲线。根据DPBF在411nm波长处吸光度变化，绘制吸光度与时间的相关性曲线，采用亚甲基蓝作为参比，计算化合物F-1的单线态氧量子产率，结果如图3。该附图显示了随光照时间延长混合体系紫外可见吸光光谱变化情况，根据相关公式可得，化合物F-1的单线态氧量子产率约为0.018。
实施例4：化合物F-1超声条件下单线态氧产量测定实验
使用实施例1中合成的化合物F-1，将F-1-DMSO溶液加入到乙二醇单甲醚中混合均匀后加入1,3-二苯基异苯并呋喃(DPBF)，调节DPBF浓度使其吸光度值约为1.0后使用1.5W/cm 2,50％循环超声刺激，等时间间隔测定体系紫外可见吸收曲线。根据DPBF在411nm波长处吸光度变化，绘制吸光度与时间的相关性曲线，结果如图4。该附图显示了随超声时间延长混合体系紫外可见吸光光谱变化情况，由图可知，化合物F-1可在超声促进下产生单线态氧。
实施例5：化合物F-1光照条件下体外细胞抗癌测试
将MCF-7(人乳腺癌细胞)以每孔5000个细胞密度种植于96孔培养板中于细胞培养箱中培养24小时(37℃，5％CO 2)，使用实施例1中合成的化合物F-1，将F-1-DMSO溶液加入到含10％胎牛血清DMEM中配制成不同浓度溶液，将配好的溶液加入96孔板置于细胞培养箱中孵育30分钟后，用660nm波长的红光照射一定时间。照射完毕，96空板放置于细胞培养箱中继续孵育12小时。随后每孔加入含5mg/ml MTT的培养基100μl，并至于细胞培养箱中孵育4小时。除去板孔中的培养液，每孔加入100μl DMSO充分溶解MTT氧化产物后用酶标仪测定每孔于570nm和630nm处吸光度，计算细胞存活率，结果如图5所示。
从图5可以看出，在没有光照的条件下化合物F-1对细胞的杀伤效应非常小，几乎没有毒性；在有光照的条件下，化合物F-1可以细胞产生明显杀伤效果，并且随光照能量密度增强，化合物F-1光毒性呈现显著性提高。
实施例6：制备化合物F-2
Figure PCTCN2019083615-appb-000008
(1)中间体2-II的合成
盐酸酸化体系中，氯化对硝基重氮苯与2-I的化合物按照摩尔比1:1在25～35℃条件下反应0.5～2小时，反应完毕，经过抽滤洗涤操作后，得到砖红色固体粉末粗产品得式2-II的化合物，收率95％。
(2)化合物F-2的合成
将上述反应(1)制备得到的中间体2-II与硒代久洛里定(quinoline)加入到含有DMF的圆底烧瓶中，滴入1mL高氯酸溶液。滴加完毕，体系搅拌2.5h后停止反应，经硅胶柱色谱分离，二氯甲烷:甲醇(v:v)＝8:1洗脱提纯得具金属光泽的深蓝色针状晶体目标化合物F-2，收率54.4％。
1H NMR(400MHz,DMSO)δ9.26(s,1H),8.91(d,J＝7.7Hz,1H),8.40(d,J＝8.0Hz,1H),7.90–7.74(m,2H),7.67(d,J＝21.0Hz,2H),3.65–3.46(m,6H),3.30(s,3H),2.88(s,2H),2.02(d,J＝38.7Hz,4H),1.36(t,J＝6.8Hz,3H).
实施例7：化合物F-2光谱测试实验
使用实施例6中合成的化合物F-2，将F-2-DMSO溶液加入到二氯甲烷中混合均匀。用紫外可见分光光度计和荧光光谱仪测试其光谱性质。结果如图6所示，F-2分子在二氯甲烷中最大吸收686nm，最大发射712nm。
实施例8：化合物F-2光照条件下单线态氧产量测定实验
使用实施例6中合成的化合物F-2，将F-2-DMSO溶液加入到甲醇中混合均匀后加入1,3-二苯基异苯并呋喃(DPBF)，调节DPBF浓度使其吸光度值约为1.0后使用660nm波长的氙灯光源(通过光栅滤光调节)照射，等时间间隔测定体系紫外可见吸收曲线。根据DPBF在411nm波长处吸光度变化，绘制吸光 度与时间的相关性曲线，采用亚甲基蓝作为参比，计算化合物F-2的单线态氧量子产率，结果如图7。该附图显示随光照时间延长混合体系紫外可见吸光光谱变化情况，根据相关公式可得，化合物F-2的单线态氧量子产率约为0.47。
实施例9：化合物F-2超声条件下单线态氧产量测定实验
使用实施例6中合成的化合物F-2，将F-2-DMSO溶液加入到乙二醇单甲醚中混合均匀后加入1,3-二苯基异苯并呋喃(DPBF)，调节DPBF浓度使其吸光度值约为1.0后使用1.5W/cm 2,50％循环超声刺激，等时间间隔测定体系紫外可见吸收曲线。根据DPBF在411nm波长处吸光度变化，绘制吸光度与时间的相关性曲线，结果如图8。该附图显示随超声时间延长混合体系紫外可见吸光光谱变化情况，可见，化合物F-2可在超声促进下产生单线态氧。
实施例10：化合物F-2光照条件下体外细胞抗癌测试
将MCF-7(人乳腺癌细胞)以每孔5000个细胞密度种植于96孔培养板中于细胞培养箱中培养24小时(37℃，5％CO 2)，使用实施例6中合成的化合物F-2，将F-2-DMSO溶液加入到含10％胎牛血清DMEM中配制成不同浓度溶液，将配好的溶液加入96孔板置于细胞培养箱中孵育30分钟后，用660nm波长的红光照射一定时间。照射完毕，96空板放置于细胞培养箱中继续孵育12小时。随后每孔加入含5mg/ml MTT的培养基100μl，并至于细胞培养箱中孵育4小时。除去板孔中的培养液，每孔加入100μl DMSO充分溶解MTT氧化产物后用酶标仪测定每孔于570nm和630nm处吸光度，计算细胞存活率，如图9所示。
从图9可以看出，在没有光照的条件下化合物F-2对细胞的杀伤效应非常小，几乎没有毒性；在有光照的条件下，化合物F-1在低光照能量密度可以细胞产生明显杀伤效果。
实施例11：制备化合物F-3
Figure PCTCN2019083615-appb-000009
Figure PCTCN2019083615-appb-000010
(1)中间体3-II的合成
盐酸酸化体系中，氯化对硝基重氮苯，与3-I的化合物按照摩尔比1:1在25～35℃条件下反应0.5～2小时，反应完毕，经过抽滤洗涤操作后得到砖红色固体粉末粗产品得式3-II的化合物，收率94％。
(2)化合物F-3的合成
将上述反应(1)制备得到的中间体3-II与碲代久洛里定(quinoline)加入到含有DMF的圆底烧瓶中，滴入1mL高氯酸溶液。滴加完毕，体系搅拌2.5h后停止反应，经硅胶柱色谱分离，二氯甲烷:甲醇(v:v)＝8:1洗脱提纯得具金属光泽的深蓝色针状晶体目标化合物F-3，收率65.9％。
1H NMR(400MHz,DMSO)δ9.13(s,1H),8.90(d,J＝8.1Hz,1H),8.36(d,J＝8.1Hz,1H),8.07(s,1H),7.80(t,J＝7.5Hz,1H),7.74(t,J＝7.4Hz,1H),7.66(s,1H),3.54(dd,J＝13.7,6.7Hz,2H),3.43(d,J＝5.0Hz,4H),2.90–2.75(m,2H),2.35(t,J＝6.1Hz,2H),2.07–2.00(m,2H),1.98–1.86(m,3H),1.36(t,J＝7.2Hz,3H).
实施例12：化合物F-3光谱测试实验
使用实施例11中合成的化合物F-3，将F-3-DMSO溶液加入到二氯甲烷中混合均匀。用紫外可见分光光度计和荧光光谱仪测试其光谱性质。结果如图10所示，F-3分子在二氯甲烷中最大吸收697nm，最大发射740nm。
实施例13：化合物F-3光照条件下单线态氧产量测定实验
使用实施例11中合成的化合物F-3，将F-3-DMSO溶液加入到甲醇中混合均匀后加入1,3-二苯基异苯并呋喃(DPBF)，调节DPBF浓度使其吸光度值约为1.0后使用660nm波长的氙灯光源(通过光栅滤光调节)照射，等时间间隔测定体系紫外可见吸收曲线。根据DPBF在411nm波长处吸光度变化，绘制吸光度与时间的相关性曲线，采用亚甲基蓝作为参比，计算化合物F-3的单线态氧量子产率，结果如图11。该附图显示随光照时间混合体系紫外可见吸光光谱变化情况，根据相关公式可得，化合物F-3的单线态氧量子产率约为0.73。
实施例14：化合物F-3超声条件下单线态氧产量测定实验
使用实施例11中合成的化合物F-3，将F-3-DMSO溶液加入到乙二醇单甲醚中混合均匀后加入1,3-二苯基异苯并呋喃(DPBF)，调节DPBF浓度使其吸光度值约为1.0后使用1.5W/cm 2,50％循环超声刺激，等时间间隔测定体系紫外可见吸收曲线。根据DPBF在411nm波长处吸光度变化，绘制吸光度与时间的相关性曲线，结果如图12。该附图显示随超声时间延长混合体系紫外可见吸光光谱变化情况，可见，化合物F-3可在超声促进下产生单线态氧。
实施例15：化合物F-3光照条件下体外细胞抗癌测试
将MCF-7(人乳腺癌细胞)以每孔5000个细胞密度种植于96孔培养板中于细胞培养箱中培养24小时(37℃，5％CO 2)，使用实施例11中合成的化合物F-3，将F-3-DMSO溶液加入到含10％胎牛血清DMEM中配制成不同浓度溶液，将配好的溶液加入96孔板置于细胞培养箱中孵育30分钟后，用660nm波长的红光照射一定时间。照射完毕，96空板放置于细胞培养箱中继续孵育12小时。随后每孔加入含5mg/ml MTT的培养基100μl，并至于细胞培养箱中孵育4小时。除去板孔中的培养液，每孔加入100μl DMSO充分溶解MTT氧化产物后用酶标仪测定每孔于570nm和630nm处吸光度，计算细胞存活率，如图13所示。
从图13可以看出，由于具有重原子碲的存在，化合物F-3在没有光照的条件下对细胞的具有一定杀伤效果，但其化学疗效较弱；在有光照的条件下，化合物F-3在低光照能量密度可以细胞产生明显杀伤效果。可见化合物F-3具有对于肿瘤细胞具有化学疗法和光/声动力疗法双重功效。
实施例16：自组装纳米给药体系建立测试
使用实施例1中合成的化合物F-1，根据需要将适量F-1-DMSO溶液加入到磷酸缓冲液(PBS)或者超纯水中，摇匀后化合物F-1即可在PBS或超纯水中通过自组装过程，形成纳米颗粒体系，体系扫描电镜如图14所示。由图14可以看出，纳米体系中化合物F-1通过自组装形成的纳米颗粒尺寸约为200nm，颗粒形状规整、大小均一，具有优良的分散性质。

Claims (10)

  1. 噁嗪类化合物，具有通式F的结构：
    Figure PCTCN2019083615-appb-100001
    通式F中，
    所述的A选自硫，硒，碲元素；
    所述的R 1、R 2和R 3各自独立地选自氢和C 1-20的取代或未取代烷基；
    所述的取代烷基由下述基团任意取代：卤素、羟基、烷氧基、醛基、羰基、氨基、羧基、酯基、酰胺基、硝基或磺酸基；
    所述的X选自磷酸根，硫酸根，硫酸氢根，硝酸根，氯负离子，溴负离子，碘负离子或高氯酸根。
  2. 根据权利要求1所述的噁嗪类化合物，其特征在于，通式F中所述的R 1、R 2和R 3各自独立地选自氢和C 1-14的取代或未取代烷基。
  3. 根据权利要求2所述的噁嗪类化合物，其特征在于，通式F中所述的R 1、R 2和R 3各自独立地选自氢和C 1-6的取代或未取代烷基。
  4. 根据权利要求3所述的噁嗪类化合物，其特征在于，通式F中所述的R 1和R 2其中之一是氢。
  5. 根据权利要求3所述的噁嗪类化合物，其特征在于，通式F中所述的R 3是氢。
  6. 根据权利要求1所述的噁嗪类化合物，其特征在于，所述化合物选自F-1、F-2、F-3、F-4、F-5、F-6、F-7、F-8、F-9、F-10和F-11：
    Figure PCTCN2019083615-appb-100002
    Figure PCTCN2019083615-appb-100003
  7. 权利要求1所述的噁嗪类化合物在制备光/声敏剂中的应用。
  8. 根据权利要求7所述的应用，其特征在于，所述的光/声敏剂是近红外长波长荧光探针。
  9. 根据权利要求8所述的应用，其特征在于，所述的光/声敏剂用于肿瘤细胞的标记。
  10. 根据权利要求8所述的应用，其特征在于，光/声敏剂是自组装的纳米粒子或由其所制成的纳米粒子，纳米粒子粒径1-1000nm。
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