CN111925369B - Beta-carboline cyano furan derivatives, preparation method and application thereof - Google Patents
Beta-carboline cyano furan derivatives, preparation method and application thereof Download PDFInfo
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- CN111925369B CN111925369B CN202010828568.8A CN202010828568A CN111925369B CN 111925369 B CN111925369 B CN 111925369B CN 202010828568 A CN202010828568 A CN 202010828568A CN 111925369 B CN111925369 B CN 111925369B
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- carboline
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- malononitrile
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- MAIOGFNXVJDUQM-UHFFFAOYSA-N C(#N)C=1OC=CC1.C1=NC=CC=2C3=CC=CC=C3NC12 Chemical class C(#N)C=1OC=CC1.C1=NC=CC=2C3=CC=CC=C3NC12 MAIOGFNXVJDUQM-UHFFFAOYSA-N 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 150000001875 compounds Chemical class 0.000 claims abstract description 43
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 23
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 claims abstract description 10
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims abstract description 8
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- 229920002554 vinyl polymer Polymers 0.000 claims description 20
- 125000000814 indol-3-yl group Chemical group [H]C1=C([H])C([H])=C2N([H])C([H])=C([*])C2=C1[H] 0.000 claims description 18
- -1 amino, methylamino Chemical group 0.000 claims description 14
- QXAUTQFAWKKNLM-UHFFFAOYSA-N methyl indole-3-carboxylate Chemical compound C1=CC=C2C(C(=O)OC)=CNC2=C1 QXAUTQFAWKKNLM-UHFFFAOYSA-N 0.000 claims description 12
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 claims description 11
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- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 3
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- 230000000694 effects Effects 0.000 abstract description 10
- AIFRHYZBTHREPW-UHFFFAOYSA-N β-carboline Chemical class N1=CC=C2C3=CC=CC=C3NC2=C1 AIFRHYZBTHREPW-UHFFFAOYSA-N 0.000 abstract description 9
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- WYTGDNHDOZPMIW-RCBQFDQVSA-N alstonine Chemical compound C1=CC2=C3C=CC=CC3=NC2=C2N1C[C@H]1[C@H](C)OC=C(C(=O)OC)[C@H]1C2 WYTGDNHDOZPMIW-RCBQFDQVSA-N 0.000 abstract 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
- C07D471/04—Ortho-condensed systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- 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
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/09—Geometrical isomers
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
- C09K2211/1033—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with oxygen
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
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- C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
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- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
Abstract
The invention relates to the technical field of biological medicines, in particular to a beta-carboline cyano furan derivative, a preparation method and application thereof, and the beta-carboline cyano furan derivative has a structure shown in a general formula I or II:
the structure of the beta-carboline derivative combined with the naturally-occurring indole alkaloid has pyridine [3,4-b ]]And a planar tricyclic framework of indole; five-membered ring cyano furan segments with strong electron withdrawing property are introduced into 1 or 3 positions of the beta-carboline mother ring through Knoevenagel condensation reaction, the conjugated system of the beta-carboline is further prolonged, and two types of beta-carboline cyano furan derivatives with photodynamic effect and pH sensitive fluorescence are designed and obtained by utilizing ICT effect. The compound has the characteristics of high stability, low dark toxicity and the like, can generate high-activity singlet oxygen under laser irradiation, can effectively eliminate tumors, provides an effective tool for monitoring and treating tumors in medicine, and has good application prospects in the aspects of tumor targeted imaging, treatment, monitoring and the like.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to beta-carboline cyano furan derivatives, and a preparation method and application thereof.
Background
Currently, photodynamic therapy (PDT) is being used in clinical trials for the treatment of tumors of the head and neck, brain, lung, pancreas, abdominal cavity, breast, prostate and skin. PDT involves two separate non-toxic components (a photosensitizer and a light source of appropriate wavelength) that combine to induce cellular and tissue effects in an oxygen-dependent manner. After absorption of a photon, the photosensitizer changes its state from its ground state (singlet) to a short excited singlet state and to a longer-lived excited electronic state (triplet). The triplet state can transfer its energy directly to oxygen, thereby forming highly reactive singlet oxygen. Due to the high reactivity and short half-life of singlet oxygen, the biological response to the photosensitizer occurs only in specific tissue regions where the photosensitizer is exposed to light are activated. Typically, the wavelength range for therapeutically activating photosensitizers is 600 to 800nm to avoid interference of endogenous chromophores in the body, while maintaining the energy necessary for singlet oxygen production. The half-life of singlet oxygen in biological systems is less than 0.04. mu.s, and therefore the radius of action of singlet oxygen is less than 0.02. mu.m. One of the advantages of PDT is that photosensitizers can be administered by a variety of means, such as by intravenous injection or topical application to the skin, but these can affect their biodistribution. Since the biodistribution varies with time, adjusting the exposure time also adjusts the PDT effect. Researchers are investigating the ability to increase tumor specificity by binding photosensitizers to tumor-associated antibodies. This approach has been successfully used in preclinical models of cancer therapy, as well as in the treatment of ocular angiogenesis-related diseases. However, there are some problems with the use of macromolecules (monoclonal antibodies) in PDT, such as complexity of synthesis, transport difficulties and potential toxicity. Thus, PDT based on small molecule photosensitizers may continue to be used as a stand-alone treatment modality or in combination with chemotherapy, surgery, radiation therapy or other new strategies (e.g., anti-angiogenic therapy).
Notably, excited singlet photosensitizers, which also can cause fluorescence emission due to relaxation to the ground state, can also be used as fluorescent imaging agents, typically using photoactivation in the 400nm range, and are very useful in diagnostic imaging applications. Thus, in addition to being a therapeutic agent for PDT, photosensitizers may also act as imaging agents, fluorescing in the visible region upon excitation at appropriate wavelengths. Photosensitizer fluorescence detection can play a role in disease diagnosis, but the technology is more of a tool to optimize surgical resection. Due to the tendency of photosensitizers to preferentially accumulate in tumor tissue, this method, often referred to as photodynamic diagnosis, is intrinsically well suited for selective visualization of tumors by fluorescence contrast to distinguish the boundaries of cancerous and healthy tissue. Although conventional fluorescent dyes are not therapeutic agents, the fluorescence quantum yield of most photosensitizers is also much lower than that of conventional fluorescent dyes. While the short wavelength excitation of the photosensitizer significantly limits the depth of tissue penetration, the volume probed under these conditions is relatively shallow. It is well known that the pH of the tumor microenvironment is primarily between 6.5 and 7, the pH of the endosomes of tumor cells is approximately 5.0, and the pH of lysosomes is approximately 4.5. Therefore, the photosensitizer with pH sensitive fluorescence is designed to hopefully improve the fluorescence intensity of the photosensitizer in the acidic region of the tumor, and the photosensitizer and the associated optical imaging have the advantages of large tissue penetration depth, low self-fluorescence background and the like, are more suitable for selectively detecting diseases and strengthening the inherent capability of the photosensitizer as a tool for optimizing treatment, and have more important application value.
Disclosure of Invention
Aiming at the problems, the invention provides a beta-carboline cyano furan derivative, a preparation method and application thereof, integrates pH sensitive fluorescence and photodynamic treatment effects, and uses thereof in tumor monitoring and treatment.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the beta-carboline cyano furan derivative has a structure shown in a general formula I or II:
wherein R is1Represents one of H, C1-C6 alkyl, alkynyl-substituted C1-C6 alkyl, halogenated C1-C6 alkyl, methoxy-substituted C1-C6 alkyl and morpholine-substituted C1-C6 alkyl; r2Representative H, COOCH3、COOH、COONH2、CH2OH；R3Represents one of H, C1-C6 alkyl and methoxy substituted phenyl; r4Represents one of H, nitryl, amino, methylamino and dimethylamino.
Preferably, said R is1Representative H, CH34-ethylmorpholine; r2Representative H, COOCH3、CH2OH；R3Represents CH3、C(CH3)3、3,4,5-trimethoxyphenyl；R4Representative H, NO2、NH2、N(CH3)2。
Preferably, the partial compound symbols of the general formula I or II and the corresponding structures are as follows:
table 1 partial compound symbols of general formula i or ii and corresponding structures
I1: (E) -methyl 1- (2- (4-cyano-5- (dicyanomethylene) -2, 2-dimethyl-2, 5-dihydrofuran-3-yl) vinyl) -9H-pyridine [3,4-b]Indole-3-carboxylic acid methyl ester;
I2: (E) -2- (3-cyano-4- (2- (3- (hydroxymethyl) -9-methyl-9H-pyrido [3, 4-b)]Indol-1-yl) vinyl) -5, 5-dimethylfuran-2 (5H) -methylene) malononitrile;
Ⅱ1: (E) -2- (3-cyano-4- (2- (1, 9-dimethyl-9H-pyridine [3,4-b ]]Indol-3-yl) vinyl) -5, 5-dimethylfuran-2 (5H) -methylene) malononitrile;
Ⅱ2: (E) -2- (3-cyano-5, 5-dimethyl-4- (2- (1-methyl-6-nitro-9H-pyrido [3, 4-b)]Indol-3-yl) vinyl) furan-2 (5H) -methylene) malononitrile;
Ⅱ3: (E) -2- (3-cyano-4- (2- (6- (dimethylamino) -1, 9-dimethyl-9H-pyridinyl [3,4-b ]]Indol-3-yl) ethenes-5, 5-dimethylfuran-2 (5H) -methylene) malononitrile;
Ⅱ4: (E) -2- (3-cyano-5, 5-dimethyl-4- (2- (1- (3,4, 5-trimethoxyphenyl) -9H-pyridinyl [3, 4-b)]Indol-3-yl) vinyl) furan-2 (5H) -methylene) malononitrile;
Ⅱ5: (E) -2- (4- (2- (1- (1- (tert-butyl) -9-methyl-9H-pyrido [3, 4-b)]Indol-3-yl) vinyl) -3-cyano-5, 5-dimethylfuran-2 (5H) -methylene) malononitrile;
Ⅱ6: (E) -2- (3-cyano-5, 5-dimethyl-4- (2- (1-methyl-9- (2-morpholinoethyl)) -9H-pyridinyl [3,4-b ]]Indol-3-yl) vinyl) furan-2 (5H) -methylene) malononitrile.
The invention also aims to provide a preparation method of the beta-carboline cyano furan derivative, which is to prepare 9-R1-3-R2-9H-pyrido [3,4-b]Indole-1-formyl or 9-R1-6-R4-1-R3-9H-pyrido [3,4-b]Dissolving indole-3-formyl and 2- (3-cyano-4, 5, 5-trimethyl furan-2 (5H) -methylene) malononitrile in absolute ethyl alcohol, adding a catalytic amount of ammonium acetate, heating and refluxing for 12-24 hours, performing Knoevenagel condensation reaction, and performing recrystallization or chromatographic column purification to obtain the beta-carboline cyano furan derivative.
Wherein, R is1Representative H, CH3N-ethylmorpholine; r2Represents COOCH3、CH2OH；R3Represents CH3、C(CH3)3、3,4,5-trimethoxyphenyl；R4Representative H, NO2、NH2、N(CH3)2。
The synthetic route of the beta-carboline cyano furan derivative is as follows:
the invention further aims to provide application of the beta-carboline cyano furan derivatives in preparing pH sensitive fluorescent compounds and realizing in-vivo and in-vitro tumor tissue or tumor cell fluorescence imaging, wherein the tumor cells are breast cancer cells, cervical cancer cells, colon cancer cells, liver cancer cells or stomach cancer cells.
The invention also aims to provide application of the beta-carboline cyano furan derivatives in preparing a medicament for photodynamic tumor therapy, wherein the beta-carboline cyano furan derivatives generate singlet oxygen to kill tumor cells after laser irradiation.
The beta-carboline cyano furan derivative has pH sensitivity fluorescence, greatly enhances the fluorescence under an acidic condition, and has the characteristics of low dark toxicity, high stability and tumor cell selective fluorescence imaging. Meanwhile, the compound can selectively target tumor tissues, and high-activity singlet oxygen is generated after the compound is excited by specific light so as to kill tumors.
The invention has the beneficial effects that:
1. the invention combines naturally-existing indole beta-carboline alkaloid, utilizes stronger rigidity and a conjugated system thereof as an electron donor, introduces a five-membered ring cyano segment with strong electron withdrawing property at 1 site or 3 site of a beta-carboline mother ring through Knoevenagel condensation reaction, and the strategy design achieves the purpose of prolonging the beta-carboline conjugated system; and simultaneously, by utilizing ICT effect, two types of beta-carboline cyano furan derivatives with pH sensitive fluorescence and photodynamic therapy effects are obtained.
2. According to the invention, through detection means such as an ultraviolet spectrophotometer, a fluorescence spectrometer, a cytotoxicity test and the like, the compounds are found to have the advantages of high stability, low dark toxicity, tumor cell selective fluorescence imaging and the like, and the compounds integrate diagnosis and treatment, are beneficial to tumor treatment and intraoperative imaging and have wide application prospects.
Drawings
FIG. 1 is a graph of the pH sensitive UV absorption spectrum of a portion of the compounds of the present invention in 5% DMSO in water (wavelength on the abscissa and absorbance on the ordinate);
FIG. 2 is a graph showing the pH-sensitive fluorescence emission spectrum (wavelength on abscissa and fluorescence intensity on ordinate) of a portion of the compounds of the present invention in a 5% DMSO aqueous solution;
FIG. 3 is an ultraviolet absorption spectrum (the abscissa is the wavelength and the ordinate is the absorbance value) of the compound of the present invention and a singlet oxygen scavenger DPBF mixed and irradiated;
FIG. 4 is a schematic diagram of the cytotoxicity test of the compound of the present invention in the presence and absence of light (the abscissa is the code of the compound, and the ordinate is the cell viability);
FIG. 5 is a confocal fluorescence imaging of a fluorescent compound of the present invention partially mixed with HepG2 cells at 1-25 μ M;
FIG. 6 is a confocal fluorescence imaging diagram of a partial compound of the fluorescent compound of the present invention and Hela cells under a condition of 1-25 μ M;
FIG. 7 is a confocal fluorescence image of partial compounds of the fluorescent compounds of the present invention and LO2 cells at 1-25 μ M.
Detailed Description
To further illustrate the present invention, a series of examples are given below, which are purely illustrative and are intended to be a detailed description of the invention only and should not be understood as limiting the invention.
The synthetic route of the beta-carboline cyano furan derivative is shown as follows:
example 1: (E) -methyl 1- (2- (4-cyano-5- (dicyanomethylene) -2, 2-dimethyl-2, 5-dihydrofuran-3-yl) vinyl) -9H-pyridine [3,4-b]Indole-3-carboxylic acid methyl ester (I)1) Preparing;
adding 1-formyl-9H-pyrido [3,4-b ] into an eggplant-shaped single-neck bottle]Methyl indole-3-carboxylate (254mg,1.0mmol) and 5ml of absolute ethanol, followed by addition of 2- (3-cyano-4, 5, 5-trimethylfuran-2 (5H) -methylene) malononitrile (219mg,1.1mmol), addition of a catalytic amount of ammonium acetate (7.7mg,0.1mmol), and stirring under reflux overnight. After TLC monitoring reaction, suction filtration, washing filter cake with cold ethanol for many times, vacuum drying filter cake, re-crystallizing to obtain yellow solid product 318 mg. The yield was 73.1%. (I)1) The spectrogram data is as follows: ESI-MS (M/z):436[ M + H]+；1H NMR(d6-DMSO,400MHz):δ12.69(s,1H,NH),9.03(s,1H,Ar-H),8.49(d,J＝7.8Hz,1H,Ar-H),8.27(d,J＝15.8Hz,1H,CH),8.04–7.94(m,1H,Ar-H),7.77–7.69(m,2H,Ar-H,CH),7.41–7.38(m,1H,Ar-H),3.96(s,3H,CH3),2.00(s,6H,CH3)。
Example 2: (E) -2- (3-cyano-4- (2- (3- (hydroxymethyl) -9-methyl-9H-pyrido [3, 4-b)]Indol-1-yl) vinyl) -5, 5-dimethylfuran-2 (5H) -methylene) malononitrile (I2) Preparing;
reference example 1 (I)1) The synthesis method of (1) from 3- (hydroxymethyl) -9-methyl-9H-pyrido [3,4-b]indole-1-formyl-9H-pyrido [3,4-b ] in place of method]Indole-3-carboxylic acid methyl ester to give (I) a yellow solid2)289mg, yield 68.6%. (I)2) The spectrogram data is as follows: ESI-MS (M/z) 422[ M + H]+；1H NMR(d6-DMSO,400MHz):δ9.01(s,1H,Ar-H),8.43(d,J＝7.2Hz,1H,Ar-H),8.22(d,J＝15.8Hz,1H,CH),8.00–7.92(m,1H,Ar-H),7.71–7.63(m,2H,Ar-H,CH),7.35–7.29(m,1H,Ar-H),4.98–4.94(m,1H,OH),4.21–4.17(m,2H,CH2),3.82(s,3H,CH3)，2.02(s,6H,CH3)。
Example 3: (E) -2- (3-cyano-4- (2- (1, 9-dimethyl-9H-pyridine [3,4-b ]]Indol-3-yl) vinyl) -5, 5-dimethylfuran-2 (5H) -methylene malononitrile (II)1) Preparing;
reference example 1 (I)1) The synthesis method of (1), 9-dimethyl-9H-pyrido [3, 4-b)]Indole-3-formyl substituting 1-formyl-9H-pyrido [3,4-b ] in process]Indole-3-carboxylic acid methyl ester to give a red solid (II)1)290mg, yield 71.6%. (II)1) The spectrogram data is as follows: ESI-MS (M/z):406[ M + H]+；1HNMR(d6-DMSO,400MHz):δ8.19(d,J＝7.2Hz,1H,Ar-H),7.83(d,J＝15.8Hz,1H,CH),7.59–7.55(m,2H,Ar-H,CH),7.46–7.42(m,2H,Ar-H),7.37–7.33(m,1H,Ar-H),3.91(s,3H,CH3),3.10(s,3H,CH3),1.93(s,6H,CH3)。
Example 4: (E) -2- (3-cyano-5, 5-dimethyl-4- (2- (1-methyl-6-nitro-9H-pyrido [3, 4-b)]Indol-3-yl) vinyl) furan-2 (5H) -methylene) malononitrile (II)2) Preparing;
reference example 1 (I)1) The synthesis method of (1-methyl-6-nitro-9H-pyrido [3, 4-b)]Indole-3-formyl substituting 1-formyl-9H-pyrido [3,4-b ] in process]Indole-3-carboxylic acid methyl ester to give a red solid (II)2)320mg, 73.6% yield. (II)2) The spectrogram data is as follows: ESI-MS (M/z) 437[ M + H]+；1H NMR(d6-DMSO,400MHz):δ12.55(s,1H,NH)，9.03(s,1H,Ar-H),8.82(s,1H,Ar-H),8.38(d,J＝8.2Hz,1H,Ar-H),7.94–7.88(m,2H,Ar-H,CH),7.58(d,J＝15.8Hz,1H,CH),3.07(s,3H,CH3),2.04(s,6H,CH3)。
Example 5: (E) -2- (3-cyano-4- (2- (6- (dimethylamino) -1, 9-dimethyl-9H-pyridinyl [3,4-b ]]Indol-3-yl) vinyl) -5, 5-dimethylfuran-2 (5H) -methylene malononitrile (II)3) Preparing;
reference example 1 (I)1) The synthesis method of (1) is characterized in that the compound is prepared from 6- (dimethylamino) -1, 9-dimethyl-9H-pyrido [3,4-b]Indole-3-formyl substituting 1-formyl-9H-pyrido [3,4-b ] in process]Indole-3-carboxylic acid methyl ester to give a deep red solid (II)3)341mg, 76.2% yield. (II)3) The spectrogram data is as follows: ESI-MS (M/z):449[ M + H ]]+；1H NMR(d6-DMSO,400MHz):δ9.43(s,1H,Ar-H),8.98(s,1H,Ar-H),8.45(d,J＝9.2Hz,1H,Ar-H),7.96–7.92(m,2H,Ar-H,CH),7.62(d,J＝15.8Hz,1H,CH),3.93(s,3H,CH3),3.34(s,6H,CH3),3.08(s,3H,CH3),1.92(s,6H,CH3)。
Example 6: (E) -2- (3-cyano-5, 5-dimethyl-4- (2- (1- (3,4, 5-trimethoxyphenyl) -9H-pyridinyl [3, 4-b)]Indol-3-yl) vinyl) furan-2 (5H) -methylene) malononitrile (II)4) Preparing;
reference example 1 (I)1) The synthesis method of (1) is carried out by 1- (3,4, 5-trimethoxyphenyl) -9H-pyrido [3,4-b]Indole-3-formyl substituting 1-formyl-9H-pyrido [3,4-b ] in process]Indole-3-carboxylic acid methyl ester to give a pale red solid (II)4)436mg, yield 80.3%. (II)4) The spectrogram data is as follows: ESI-MS (M/z) 544[ M + H]+；1H NMR(d6-DMSO,400MHz):δ12.12(s,1H,NH),8.65(s,1H,Ar-H),8.26(d,J＝7.9Hz,1H,Ar-H),8.17(d,J＝15.7Hz,1H,CH),7.98–7.85(m,2H,,Ar-H),7.72(d,J＝8.2Hz,1H,Ar-H),7.63(t,J＝7.6Hz,1H,Ar-H),7.42–7.36(m,2H,Ar-H,CH),3.95(s,6H,CH3),3.80(s,3H,CH3),1.90(s,6H,CH3)。
Example 7: (E) -2- (4- (2- (1- (1- (tert-butyl) -9-methyl-9H-pyrido [3, 4-b)]Indol-3-yl) vinyl) -3-cyano-5, 5-dimethylfuran-2 (5H) -methylene malononitrile (II)5) Preparing;
reference example 1 (I)1) The synthesis method of (1) from 1- (tert-butyl) -9-methyl-9H-pyrido [3,4-b]Indole-3-formyl substituting 1-formyl-9H-pyrido [3,4-b ] in process]Indole-3-carboxylic acid methyl ester to give a red solid (II)5)289mg, yield 64.5%. (II)5) The spectrogram data is as follows: ESI-MS (M/z):448[ M + H]+；1H NMR(d6-DMSO,400MHz):δ8.15(d,J＝6.8Hz,1H,Ar-H),7.91(d,J＝15.7Hz,1H,CH),7.66–7.60(m,3H,Ar-H,CH),7.55–7.51(m,1H,Ar-H),7.40–7.36(m,1H,Ar-H),3.12(s,3H,CH3),2.63(s,9H,CH3),1.82(s,6H,CH3)。
Example 8: (E) -2- (3-cyano-5, 5-dimethyl-4- (2- (1-methyl-9- (2-morpholinoethyl)) -9H-pyridinyl [3,4-b ]]Indol-3-yl) vinyl) furan-2 (5H) -methylene) malononitrile (II)6) Preparing;
reference example 1 (I)1) The synthesis method of (1-methyl-9- (2-morpholinoethyl) -9H-pyrido [3, 4-b)]Indole-3-formyl substituting 1-formyl-9H-pyrido [3,4-b ] in process]Indole-3-carboxylic acid methyl ester to give a deep red solid (II)6)433mg, yield 85.7%. (II)6) The spectrogram data is as follows: ESI-MS (M/z) 505[ M + H]+；1HNMR(CDCl3,400MHz):δ8.17(d,J＝5.9Hz,2H,Ar-H),7.93(d,J＝15.7Hz,1H,CH),7.68–7.62(m,2H,Ar-H,CH),7.57(d,J＝8.3Hz,1H,Ar-H),7.42–7.38(m,1H,Ar-H),4.79–4.75(m,2H,CH2),3.73–3.64(m,4H,CH2),3.15(s,3H,CH3),2.84–2.80(m,2H,CH2),2.58–2.46(m,4H,CH2),1.85(s,6H,CH3)。
Example 9: pH sensitive ultraviolet absorption Spectroscopy testing of fluorescent Compounds of the invention
Referring to fig. 1, the fluorescent compound of the present invention is dissolved in an aqueous solution containing 5% DMSO to prepare a detection solution of 1 to 25 μ M. The ultraviolet absorption spectrum data of the fluorescent compound is tested by adopting an ultraviolet-visible spectrophotometer, and the result shows that the maximum ultraviolet absorption wavelength of the fluorescent compound is in the range of 460-550nm and is reduced along with the reduction of pH. Wherein the compound II3The maximum absorption wavelength of ultraviolet is about 475nm, the peak value of the ultraviolet is reduced along with the reduction of pH, and the ultraviolet blue shifts to about 465nm (figure 1 a); compound II4The maximum absorption wavelength of ultraviolet is around 490nm, the peak value decreases with the decrease of pH, and blue shifts to around 482nm (FIG. 1 b); compound II6The maximum absorption wavelength of the UV is around 480nm, the peak decreases with decreasing pH and shifts blue to around 472nm (FIG. 1 c).
Example 10: pH sensitive fluorescence Spectroscopy testing of fluorescent Compounds of the invention
Referring to fig. 2, the fluorescent compound of the present invention is dissolved in an aqueous solution containing 5% DMSO to prepare a detection solution of 1 to 25 μ M, and the pH values are adjusted to 7.4, 6.5, 6.0, 5.5, and 4.5, respectively. The fluorescence emission spectrum data of the fluorescent compound is tested by adopting a fluorescence spectrometer, and the result shows that the maximum emission wavelength of the fluorescent compound is within the range of 600-680nm, and the fluorescence is obviously increased along with the reduction of the pH value. Wherein the compound I1The fluorescence peak around 635nm increases with decreasing pH, λ ex ═ 472nm (fig. 2 a); wherein the compound II1The peak at around 645nm blue shifts to 630nm and the fluorescence intensity increases with decreasing pH, λ ex 490nm (fig. 2 b); wherein the compound II3The fluorescence peak around 615nm increases with decreasing pH, λ ex 490nm (fig. 2 c); wherein the compound II4The fluorescence peak around 665nm increases with decreasing pH, λ ex 490nm (fig. 2 d); wherein the compound II6The peak at around 650nm shifts blue to 640nm and the fluorescence intensity increases with decreasing pH, with λ ex being 488nm (fig. 2 e).
Example 11: dark toxicity test of fluorescent Compounds of the invention on cells
Referring to fig. 4, toxicity of the compound of the present invention to human colon cancer cell HT29 cell line under the condition of illumination and without illumination was respectively detected by using tetramethylazoles blue colorimetry (MTT). HT29 cells are collected at logarithmic phase, digested by adding 0.25% trypsin to exfoliate adherent cells to 2 × 10/ml4～4×104A suspension of individual cells. Inoculating the cell suspension on a 96-well plate with 200 μ L per well, and placing in constant temperature CO2The culture was carried out in an incubator for 24 hours. Changing the solution, adding a test compound (the compound is dissolved in DMSO and then diluted by PBS, the concentration of the test compound is 1-25 mu M, and each hole is 20 mu L, and the dark toxicity test needs to be carried out at 37 ℃ and contains 5% CO2Culturing in incubator, continuously culturing in dark for 48 hr, replacing fresh complete culture medium, continuously culturing at 37 deg.C with 5% CO2After 24 hours of incubation in an incubator, MTT was added to a 96-well plate at 20. mu.L per well and the reaction was carried out in the incubator for 4 hours. The supernatant was aspirated, 150. mu.L DMSO was added to each well, and shaken on a shaker for 5 minutes. The absorbance of each well was measured at a wavelength of 570nm using an enzyme linked immunosorbent assay to calculate the cell viability. The experimental result shows that the activity of the cell of the compound is basically unchanged and is maintained at about 95% under the condition of no light source irradiation (figure 4), and the compound has little influence on the survival rate of the cell and low dark toxicity in the dark.
Example 12: phototoxicity assay of fluorescent Compounds of the invention on cells
Phototoxicity assay referring to FIG. 4, a 650nm laser (15 mW/cm) was used after the dark toxicity assay described above was completed with the test compound2) Irradiating for 15 min, replacing fresh complete culture medium after irradiation, and continuing to make culture at 37 deg.C with 5% CO2After 24 hours of incubation in an incubator, MTT was added to a 96-well plate at 20. mu.L per well and the reaction was carried out in the incubator for 4 hours. The supernatant was aspirated, 150. mu.L DMSO was added to each well, and shaken on a shaker for 5 minutes. The absorbance of each well was measured at a wavelength of 570nm using an enzyme linked immunosorbent assay to calculate the cell viability. Experiments prove that the survival rate of cells is sharply reduced and combined after the laser light source is irradiated under the condition without illuminationThe decrease is particularly obvious when the concentration of the compound is 10 mu M (figure 4), which proves that the compound has stronger phototoxicity and low dark toxicity, and can be used for photodynamic ablation of cancer cells.
Example 13: cell imaging using confocal microscopy
Referring to FIGS. 5-7, confocal microscopy was used to image cells, one day before imaging HepG2, HeLa or LO2 cells were cultured in DEME or 1640 medium, placed in a confocal laser dish, 1-25 μ M of the test compound was added to the cells, placed at 37 deg.C and 5% CO2Is incubated in the cell culture chamber for half an hour. After washing 3 times with phosphate buffer solution with pH 7.4, the incubated cells were placed on the stage of a confocal microscope for confocal fluorescence imaging, and the excitation wavelength of the test compound was set: λ em is 460-. The result shows that the beta-carboline cyano furan derivative can be effectively absorbed by tumor cells, and the fluorescent compound can selectively perform fluorescence imaging on a plurality of tumor cells (figure 5, figure 6 and figure 7), has weak fluorescence imaging on normal liver cells, provides a feasible means for in vivo and in vitro tumor tissue or cell imaging research, and has wide application prospect.
The embodiments of the present invention have been described in detail, but the description is only for the preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.
Claims (8)
1. The beta-carboline cyano furan derivative is characterized by having a structure shown in a general formula I or II:
wherein R is1Represents H, C1-C6 alkyl, alkynyl-substituted C1-C6 alkyl, halogenated C1-C6 alkyl, methoxy-substituted C1-C6 alkyl and morpholine-substituted C1-one of C6 alkyl groups; r2Representative H, COOCH3、COOH、COONH2、CH2OH；R3Represents one of H, C1-C6 alkyl and methoxy substituted phenyl; r4Represents one of H, nitryl, amino, methylamino and dimethylamino.
2. The class of β -carboline cyanofuran derivatives of claim 1, wherein: the R is1Representative H, CH32- (N-morpholinyl) ethyl; r2Representative H, COOCH3、CH2OH；R3Represents CH3、C(CH3)33,4, 5-trimethoxyphenyl; r4Representative H, NO2、NH2、N(CH3)2。
3. The class of β -carboline cyanofuran derivatives of claim 2, wherein: the code numbers of the partial compounds of the general formula I or II and the corresponding structures are as follows:
I1: (E) -1- (2- (4-cyano-5- (dicyanomethylene) -2, 2-dimethyl-2, 5-dihydrofuran-3-yl) vinyl) -9H-pyridine [3,4-b]Indole-3-carboxylic acid methyl ester;
I2: (E) -2- (3-cyano-4- (2- (3- (hydroxymethyl) -9-methyl-9H-pyrido [3, 4-b)]Indol-1-yl) vinyl) -5, 5-dimethylfuran-2 (5H) -methylene) malononitrile;
Ⅱ1: (E) -2- (3-cyano-4- (2- (1, 9-dimethyl-9H-pyridine [3,4-b ]]Indol-3-yl) vinyl) -5, 5-dimethylfuran-2 (5H) -methylene) malononitrile;
Ⅱ2: (E) -2- (3-cyano-5, 5-dimethyl-4- (2- (1-methyl-6-nitro-9H-pyrido [3, 4-b)]Indol-3-yl) vinyl) furan-2 (5H) -methylene) malononitrile;
Ⅱ3: (E) -2- (3-cyano-4- (2- (6- (dimethylamino) -1, 9-dimethyl-9H-pyridinyl [3,4-b ]]Indol-3-yl) vinyl) -5, 5-dimethylfuran-2 (5H) -methylene) malononitrile;
Ⅱ4: (E) -2- (3-cyano-5, 5-dimethyl-4- (2- (1- (3,4, 5-trimethoxyphenyl)-9H-pyridinyl [3,4-b ]]Indol-3-yl) vinyl) furan-2 (5H) -methylene) malononitrile;
Ⅱ5: (E) -2- (4- (2- (1- (1- (tert-butyl) -9-methyl-9H-pyrido [3, 4-b)]Indol-3-yl) vinyl) -3-cyano-5, 5-dimethylfuran-2 (5H) -methylene) malononitrile;
Ⅱ6: (E) -2- (3-cyano-5, 5-dimethyl-4- (2- (1-methyl-9- (2-morpholinoethyl)) -9H-pyridinyl [3,4-b ]]Indol-3-yl) vinyl) furan-2 (5H) -methylene) malononitrile.
4. A preparation method of beta-carboline cyano furan derivatives is characterized by comprising the following steps: reacting 9-R1-3-R2-9H-pyrido [3,4-b]Indole-1-formyl or 9-R1-6-R4-1-R3-9H-pyrido [3,4-b]Dissolving indole-3-formyl and 2- (3-cyano-4, 5, 5-trimethyl furan-2 (5H) -methylene) malononitrile in absolute ethyl alcohol, adding a catalytic amount of ammonium acetate, heating and refluxing for 12-24 hours, performing Knoevenagel condensation reaction, and performing recrystallization or chromatographic column purification to obtain a beta-carboline cyano furan derivative;
the R is1Representative H, CH32- (N-morpholinyl) ethyl; r2Represents COOCH3、CH2OH；R3Represents CH3、C(CH3)33,4, 5-trimethoxyphenyl; r4Representative H, NO2、NH2、N(CH3)2。
5. The use of a class of beta-carboline cyanofuran derivatives according to any one of claims 1-3 in the preparation of pH-sensitive fluorescent compounds, and reagents for achieving in vivo and in vitro tumor tissue or tumor cell fluorescence imaging.
6. The application of the beta-carboline cyano furan derivatives of any one of claims 1-3 in preparing pH-sensitive fluorescent compounds and reagents for realizing in vivo and in vitro tumor tissue or tumor cell fluorescence imaging, which is characterized in that: the tumor is breast cancer, cervical cancer, colon cancer, liver cancer or gastric cancer.
7. The use of a class of β -carboline cyanofuran derivatives according to any one of claims 1-3 for the preparation of a medicament for photodynamic tumour therapy.
8. The use of a class of β -carboline cyanofuran derivatives of claim 7 in the preparation of a medicament for photodynamic tumor therapy, wherein: the beta-carboline cyano furan derivative generates singlet oxygen to kill tumor cells after laser irradiation.
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