CN102268191B - Heptamethine indocyanine dye, synthetic method thereof and applications thereof - Google Patents
Heptamethine indocyanine dye, synthetic method thereof and applications thereof Download PDFInfo
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- CN102268191B CN102268191B CN 201010192236 CN201010192236A CN102268191B CN 102268191 B CN102268191 B CN 102268191B CN 201010192236 CN201010192236 CN 201010192236 CN 201010192236 A CN201010192236 A CN 201010192236A CN 102268191 B CN102268191 B CN 102268191B
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Abstract
The invention relates to a heptamethine indocyanine dye containing N-fatty esters or N-fatty amide side chains, a synthetic method thereof and applications thereof in tumor targeting imaging and treatment. The heptamethine indocyanine dye which possesses or individually possesses near infrared absorption, fluorography and antineoplastic activity allows present research and development ideas and treatment levels of tumor targeting treatment medicaments to be greatly improved, and has great significances to aspects of early discovery, control and the like of various tumors.
Description
Technical field
The present invention relates to biological field, particularly a kind of seven methine indoles cyanine dyes and synthetic method and application.
Background technology
Malignant tumour is the major disease of serious harm human health and life, become first cause of death of China's population, at present malignant tumour is still lacked special method of early diagnosis and desirable treatment measure, improving the tumor diagnosis and treatment level, is the great demand of China's economy, society and people ' s health.The proposition of the molecular targeted treatment concept of tumour and the successful Application of target therapeutic agent are the inexorable trends of major progress and the future development in oncotherapy field.At present the method for targeted therapy mainly be utilize tumor tissues or cell to have specificity (or special relatively) biomolecules as target spot, by chemical process the antibody of medicine and these targeted molecular specific combination, part etc. are coupled to prepare targeted drug.This method spininess is to single target and specific tumors type, and lower to tumour cell target usefulness.Report is arranged, through vein administered antibodies molecule, have only 100,000/can arrive targeting moiety (Wang X, Yang L, Chen ZG, Shin DM.Applicationof nanotechnology in cancer therapy and imaging.CA Cancer J Clin.2008; 58 (2): 97-110).And chemical coupled reaction also can influence the activity of medicine, and increased the difficulty of preparation.Therefore, press for the novel tumor targeted system that develops and set up efficient more and wide spectrum.
Because the tumor targeted molecular imaging technique can be the dynamic real-time monitoring of the early diagnosis and therapy effect of tumour important means is provided, therefore, the target of following tumour individualized treatment is the multi-functional antitumor drug of development cancer target imaging simultaneously and lethal effect, namely have good tumor-targeting and cytotoxicity simultaneously, and can carry out the target real-time visualization of tumour by corresponding imaging device, the tumor treatment process is realized visual dynamic monitoring (Nie S, Xing Y, Kim GJ, Simons JW.Nanotechnology applicationsin cancer.Annu Rev Biomed Eng.2007; 9:257-88).
Indoles seven methine cyanine dyes belong to a class nir dye, at wavelength 700-1000nm place strong absorption are arranged, and biological sample is very little in this area light absorption or fluorescence intensity.Utilize this property difference, this type of dyestuff is combined with biological sample, such as nucleic acid, protein bound, carry out nucleic acid marking, bioanalysis etc., because background interference is little, the sensitivity and the accuracy that have improved biological sample analysis and mark greatly.We have at first found one group of indoles, seven methine cyanine dye compounds early stage in the world, indoles seven methine cyanine dyes classes chemistry small molecules (the Shi C that has cancer target near-infrared fluorescent imaging characteristic simultaneously, Zhu Y, Cheng T, Su Y.Effectivetargeting, imaging and photodynamic killing of cancer cells with a new class of heptamethine3H-indocyanine dyes.Experimental Hematology.2008; 36 (Suppl): S50).This compounds has the characteristic of good near-infrared fluorescent imaging characteristic and lipophilic cation compound, can utilize the higher mitochondrial membrane potential of tumour cell and optionally be accumulated in the tumour plastosome, and realization near-infrared fluorescent imaging, transplanted tumor and spontaneous tumor to human tumor cells, animal tumor cell and the animal of nearly all test all have good target near-infrared fluorescent imaging characteristic, greatly reduce background interference.And be chemical small molecules, can not produce the immunogenicity of biomacromolecule carriers such as similar monoclonal antibody, and propose the innovative approach based on the micromolecular cancer target imaging research of chemiluminescence.
But this type of chemical small molecules itself does not have the activity of direct killing tumour cell, has limited its application aspect antineoplaston, and the ultimate aim of tumour early discovery is to realize the early treatment of tumour.Therefore, this area presses for the target therapeutic agent that development has tumor imaging and lethal effect simultaneously concurrently, so that both realize can the specific killing tumour cell, simultaneously again can be to tumor tissues target real-time visualization, thus the residual focus in the tumor treatment process is realized visual dynamic monitoring.
Summary of the invention
The purpose of this invention is to provide seven methine indoles cyanine dyes and synthetic method and its purposes in cancer target imaging and treatment that an a kind of class contains N-fatty ester or N-fatty amide side chain.Described seven methine indoles cyanine dyes have or have separately near infrared light absorption, fluorography and anti-tumor activity.The present invention improves greatly research and development theory and the treatment level of existing neoplasm targeted therapy medicine, and aspects such as the early discovery of various tumours and control all are significant.
Realize that technical scheme of the present invention is:
One class thing of seven methine indoles cyanine dyes of a kind of N-of containing fatty ester or N-fatty amide side chain has following universal architecture:
Formula I
Wherein, R
1, R
2, R
3, R
4Be in hydrogen, methyl, methoxyl group, hydroxyl, carboxyl, carboxylate salt, sulfonic group, the sulfonate etc. any one independently of one another; Z, Y are Sauerstoffatom (O) or nitrogen-atoms (N) independently of one another; X can be among iodide ion, chlorion, bromide anion, alkyl azochlorosulfonate, tetrafluoroborate, the perchlorate a kind of arbitrarily; N1, n2, n3, n4 are 0～14.
An above-mentioned class thing that contains seven methine indoles cyanine dyes of N-fatty ester or N-fatty amide side chain:
(1) works as R
1, R
2, R
3, R
4Be hydrogen, n1, n2, n3, n4 are 0～14 integer independently of one another, Z, Y are Sauerstoffatom (O) or nitrogen-atoms (N), when X is a kind of arbitrarily among iodide ion, chlorion, bromide anion, alkyl azochlorosulfonate, tetrafluoroborate, the perchlorate, its structure is the seven methine indoles cyanine dyes that contain the symmetry of N-fatty ester side chain, and structural formula is compound 1, compound 2; Or structure is the seven methine indoles cyanine dyes that contain the symmetry of N-fatty amide side chain, and structural formula is compound 3, compound 4;
(2) work as R
1, R
2, R
3, R
4Be hydrogen, n1, n2, n3, n4 are 0～14, Z, Y are respectively Sauerstoffatom (O) or nitrogen-atoms (N) separately, X can be among iodide ion, chlorion, bromide anion, alkyl azochlorosulfonate, tetrafluoroborate, the perchlorate etc. when a kind of arbitrarily, its structure is for containing the asymmetric seven methine indoles cyanine dyes of N-fatty ester, N-fatty amide side chain individually or simultaneously, and structural formula is compound 5, compound 6;
(3) work as R
1, R
2Be in hydroxyl, carboxyl, sulfonic group, carboxylate salt, the sulfonate etc. any one independently of one another, R
3, R
4Be hydrogen, Z, Y are Sauerstoffatom (O) or nitrogen-atoms (N), X can be among iodide ion, chlorion, bromide anion, alkyl azochlorosulfonate, tetrafluoroborate, the perchlorate etc. a kind of arbitrarily, n1, n2, n3, n4 are 0～14 o'clock, its structure is the seven methine indoles cyanine dyes that contain the symmetry of N-fatty ester side chain, and its structural formula is compound 7, compound 8; Or be the seven methine indoles cyanine dyes that contain the symmetry of N-fatty amide side chain, structure is compound 9, compound 10;
(4) work as R
1, R
2Be in hydroxyl, carboxyl, sulfonic group, carboxylate salt, the sulfonate etc. any one independently of one another, R
3, R
4Be hydrogen, Z, Y are respectively Sauerstoffatom (O) or nitrogen-atoms (N) separately, X can be among iodide ion, chlorion, bromide anion, alkyl azochlorosulfonate, tetrafluoroborate, the perchlorate etc. a kind of arbitrarily, n1, n2, n3, n4 be 0～14 the time, its structure is for containing N-fatty ester, N-fatty amide side chain individually or simultaneously) asymmetric seven methine indoles cyanine dyes, structural formula is compound 11, compound 12;
(5) work as R
1, R
2, R
3, R
4Be in methyl, methoxyl group, hydroxyl, carboxyl, carboxylate salt, sulfonic group, the sulfonate etc. any one independently of one another, Z, Y are Sauerstoffatom (O) or nitrogen-atoms (N), X can be among iodide ion, chlorion, bromide anion, alkyl azochlorosulfonate, tetrafluoroborate, the perchlorate etc. a kind of arbitrarily, n1, n2, n3, n4 are 0～14 o'clock, its structure is the seven methine indoles cyanine dyes that contain the symmetry of N-fatty ester side chain, and structural formula is compound 13, compound 14; Or structure is the seven methine indoles cyanine dyes that contain the symmetry of N-fatty amide side chain, and structural formula is compound 15, compound 16;
(6) work as R
1, R
2, R
3, R
4Be in methyl, methoxyl group, hydroxyl, carboxyl, carboxylate salt, sulfonic group, the sulfonate etc. any one independently of one another, Z, Y are respectively Sauerstoffatom (O) and nitrogen-atoms (N) separately, X can be among iodide ion, chlorion, bromide anion, alkyl azochlorosulfonate, tetrafluoroborate, the perchlorate etc. a kind of arbitrarily, n1, n2, n3, n4 are 0～14 o'clock, its structure is for containing the asymmetric seven methine indoles cyanine dyes of N-fatty ester, N-fatty amide side chain individually or simultaneously, and structural formula is compound 17, compound 18
The seven methine indoles cyanine dye part-structure examples (but being not limited to above structure) that the present invention relates to
Contain the synthetic method of seven methine indoles cyanine dyes of N-fatty ester or N-fatty amide side chain, following steps arranged:
The universal synthesis method that contains seven methine indoles cyanine dyes of N-fatty ester or N-fatty amide side chain
(1) in the organic solvent, below 0 ℃, pimelinketone and DMF/POCl
3The Vilsmeimer-Haack formylation reaction takes place; reacted 1-6 hour; (A refers to compd A to synthetic dialdehyde chloro tetrahydrobenzene intermediate A; its structure is seen among the universal synthesis method figure of seven methine indoles cyanine dyes of the above-mentioned N-of containing fatty ester or N-fatty amide side chain; down together), pimelinketone and DMF, POCl
3Mol ratio be 1: 1～10: 1～15, compd A gets pure product through the organic solvent recrystallization;
(2) in organic solvent, under 90-180 ℃, contain R
32 of replacement, 3,3-trimethylammonium 3H indoles and the various halohydrocarbons reaction that contains fatty ester or fatty amide, react to become in 10-20 hour and contain 2,3 of N-fatty ester or N-fatty amide side chain, 3-trimethylammonium 3H indoles quaternary ammonium salt B, 2, the mol ratio of 3,3-trimethylammonium 3H indoles and N-alkylating reagent is 1: 1～2, and compd B gets pure product through the organic solvent recrystallization;
(3) in organic solvent, under 90-180 ℃, contain R
42 of replacement, 3,3-trimethylammonium 3H indoles and the reaction of N-alkylating reagent, react to be transformed in 10-20 hour and contain 2,3 of N-fatty ester or N-fatty amide side chain, 3-trimethylammonium 3H indoles quaternary ammonium salt C, 2, the mol ratio of 3,3-trimethylammonium 3H indoles and N-alkylating reagent is 1: 1～2, and Compound C gets pure product through the organic solvent recrystallization;
(4) in organic solvent, under 60-90 ℃, in the presence of schiff bases, dialdehyde chloro tetrahydrobenzene intermediate A and 1～2 times of equivalent contain 2 of fatty acid ester or fatty amide side chain, 3,3-trimethylammonium 3H indoles quaternary ammonium salt B, C compound generation condensation reaction must contain in the seven methine indoles cyanine dye D.D structures of N-fatty ester or N-fatty amide side chain;
(5) under refluxad, make solvent separately or mix with toluene or benzene or hexanaphthene to make solvent with alcohol, dialdehyde chloro tetrahydrobenzene intermediate A and 1～2 times of equivalent contain 2 of fatty acid ester or fatty amide side chain, 3,3-trimethylammonium 3H indoles quaternary ammonium salt B, C compound generation condensation reaction must contain seven methine indoles cyanine dye D of N-fatty ester or N-fatty amide side chain.
Organic solvent is methylene dichloride or N described in the above-mentioned steps (1), dinethylformamide, the organic solvent that described recrystallization organic solvent is used recrystallization in the above-mentioned steps is the mixed solvent of any one or a few combination of methyl alcohol, ethanol, propyl alcohol, acetone, ethyl acetate, acetic acid.
Organic solvent is adjacent chlorodiphenyl described in above-mentioned steps (2), (3), DMF, DMSO, any one of toluene, glycol dimethyl ether.
Organic solvent described in the above-mentioned steps (4) is any one of diacetyl oxide, ethanol, benzene, toluene; Described schiff bases comprises any one in sodium-acetate, pyridine, the triethylamine; Dialdehyde chloro tetrahydrobenzene intermediate A and 1～2 times of equivalent contain 2 of fatty acid ester or fatty amide side chain, 3, dialdehyde chloro tetrahydrobenzene intermediate A and B or C press 1: 2 molar equivalent reaction in 3-trimethylammonium 3H indoles quaternary ammonium salt B, the C compound generation condensation reaction, and A and B, C obtain symmetry or asymmetric seven methine indole cyanine dyes respectively by reaction in 1: 1: 1.
Alcohol described in the above-mentioned steps (5) is any one in methyl alcohol, ethanol, propyl alcohol, Virahol, propyl carbinol, isopropylcarbinol, amylalcohol, hexanol, enanthol, the octanol etc., dialdehyde chloro tetrahydrobenzene intermediate A and 1～2 times of equivalent contain 2 of fatty ester or fatty amide side chain, 3, dialdehyde chloro tetrahydrobenzene intermediate A and B or C press 1: 2 molar equivalent reaction in 3-trimethylammonium 3H indoles quaternary ammonium salt B, the C compound generation condensation reaction, and A and B, C obtain symmetry or asymmetric seven methine indole cyanine dyes respectively by reaction in 1: 1: 1.
Seven methine indoles cyanine dyes are for detection of the application in the tumour.
Making the emission wavelength of tumor tissues fluorescence imaging in the above-mentioned application when detecting tumour is 600-1600nm.
The preferred 630-950nm of emission wavelength in the above-mentioned application.
The application of seven methine indoles cyanine dyes in preparation medicine for treating tumor thing.
The present patent application people further carries out the research of novel near-infrared fluorescent targeting anti-tumor compound on the basis of the micromolecular cancer target imaging research based on chemiluminescence.By designing the analog that synthetic a series of applicants of being different from have found compound early stage, carry out external and the interior structure activity study to tumour cell near-infrared fluorescent imaging characteristic and killing activity of body, successfully obtained to have simultaneously the Multifunction compound of cancer target, near infrared imaging, antitumor action, both having realized can the specific killing tumour cell, also can realize the target real-time visualization to tumor tissues simultaneously, the residual focus in the tumor treatment process is realized visual dynamic monitoring.
Seven methine indoles cyanine dye synthetic methods of the present invention have characteristics such as convenient post-treatment, yield height, can replace propyl carbinol with other alcohol in the building-up process, obtain seven methine indole cyanine dyes of multiple ester (namely containing the fatty ester side chain).Adopt the method for the invention, can obtain following 10 compounds:
The design that contains the seven methine indole cyanine dyes (compound 26-35) of fatty ester side chain reaches synthetic
Synthetic route of the present invention is that the 6-bromocaproic acid is raw material with 2,3,3-dimethyl-3H indoles (24), and 1,2-orthodichlorobenzene is solvent, and reaction obtains N-caproic acid indoles quaternary ammonium bromine salt (25); With N-caproic acid indoles quaternary ammonium bromine salt (25) with 2-chloro-3-hydroxyl methene-1-formyl radical tetrahydrobenzene (20) is raw material; the toluene less with toxicity replaces benzene; with various alcohol (methyl alcohol, n-propyl alcohol, Virahol, propyl carbinol, n-hexyl alcohol etc.) back flow reaction 5-15 hour; obtain various diester compound 26-30 and monoester compound 31-36 respectively, these compounds there is no synthetic report.
The seven methine indoles cyanine dyes that a class of the present invention contains N-fatty ester or N-fatty amide side chain have the tumour cell targeting, it can selectivity be accumulated in tumor tissues, and various tumour cells are had lethal effect, all can significantly suppress the growth of tumour cell in vitro and in vivo; Described dyestuff has the characteristic of fluorescence imaging in addition near infrared wavelength region simultaneously, can realize the target real-time visualization to tumor tissues, and can realize visual dynamic monitoring to the tumor treatment process.
The present invention also provides suc as formula seven methine indoles cyanine dye near infrared fluorescent compound pharmaceutically acceptable derivates shown in the I, comprises suc as formula the near infrared fluorescent compound of seven methine indoles cyanine dyes shown in the I and the conjugate of bioprotein molecule.
Provided by the invention have compound shown by formula I or its pharmaceutically acceptable derivates demonstrates the powerful ability of being absorbed by the tumour cell selectivity efficient, to the mankind's various different tissues organ primary or metastatic tumo(u)r, all demonstrate the effect of near infrared light imaging and killing tumor cells, have advantages such as efficient, safety, can realize the two approach of cancer target imaging and treatment simultaneously.
The composition of seven methine indoles cyanine dyes of the present invention and pharmaceutically acceptable derivates and pharmaceutically acceptable carrier is near infrared fluorescent compound, described composition can be the dosage form of this area routine, comprises peroral administration preparation and para-oral preparation.Peroral administration solids composition of the present invention can adopt forms such as tablet, pill, capsule, powder, granule, drops.Mixed one or more active substance and at least a inert diluent in these solids compositions, also can make the additive that contains in the composition except inert agents according to common method, in this way tablet or pill.Peroral administration liquid composition comprises the opacifying agent that allows on the medicament, solution, suspension agent, syrup etc., can also comprise wetting agent, suspension agent, etc. auxiliary agent, sweeting agent, correctives, perfume compound and sanitas.
The preferred para-oral injection of the present invention.Para-oral injection of the present invention comprises sterile aqueous or non-aqueous solution agent, suspension agent and opacifying agent.Also can comprise isotonic agent, sanitas, wetting agent, emulsifying agent, dispersion agent, stablizer, solubility promoter in these compositions.Composition provided by the invention can be used separately, also comprise with the means of other existing oncotherapies and unite use (for example: as radiotherapy, chemotherapy and biotherapy etc.); Application approach comprises systemic vein administration and tissue local application.
Composition provided by the invention can be applied to Mammals, preferred people, and suitable disease comprises the diseases such as various tissue abnormalities hyperplasia that various malignant tumours (as cancer and sarcoma), innocent tumour, precancerous lesion and a variety of causes cause.
Above-mentioned composition has the effect of the effect of tumour photoimaging and targeted therapy simultaneously.
In the present invention, the dosage to providing compound does not have special restriction, available any proper dosage.The type of carrier and quantity also can be very inequality, and this depends on degree of damage of mammiferous kind, body weight and tumor tissues to be treated etc.Usually, during as imaging contrast or as medicine, the effective dosage ranges of compound be generally the 0.01-100 mg/kg/time or higher, 0.1-50 mg/kg that preferable is/time, 0.2-20 mg/kg that better is/time.
The present composition for preparing can carry out administration by conventional route, comprising: intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous, oral or tumor by local administration.In addition, composition of the present invention can also use with the agent of other treatment tumor treatment.
Seven methine indoles cyanine dyes of the present invention have tumour cell, and (described tumour comprises animal body histoorgan primary or the various malignant tumours of transitivity (comprising cancer and sarcoma), innocent tumour, precancerous lesion.So specifically comprise the tumour of histoorgans such as digestive system tumor, tumor in respiratory system, hematological system tumor, urogenital neoplasm, skin and soft tissue neoplasm) targeting, selectivity is accumulated in tumor tissues, and tumour cell had lethal effect, all can significantly suppress the growth of tumour cell in vitro and in vivo; This compounds also has the characteristic of fluorescence imaging simultaneously, can realize the target real-time visualization to tumor tissues, and can realize visual dynamic monitoring to the tumor treatment process.
Major advantage of the present invention is:
1, the invention provides compound and have good light stability and tissue penetration performance, in 600-1600nm wavelength region good imaging quality, is the good material of near-infrared fluorescent imaging;
2, the invention provides in the chemical combination object through behind vein or the topical administration, can efficiently be absorbed by the various tumor tissue cells of human and animal, have the tumour cell highly selective;
3, the invention provides compound and absorbed by tumor tissue cell after, near infrared range, can make histocyte in vivo desirable video picture of organism;
4, the invention provides compound and give after, has good metabolic characteristic in vivo, after this compounds enters in the organism, only absorbed by tumor tissues, and not by any healthy tissues specificity picked-up, tumor tissues and on every side such compound concentration ratio of normal control tissue can reach more than 10 times, the compound that is absorbed by tumor tissues can not got rid of external by body fully;
5, the invention provides compound and have the effect of killing tumor cell significantly, after tumour cell absorbs this compounds, can bring out cellular form and change, apoptosis takes place; After giving compound in the body, at the tumor tissues selective enrichment, and inducing death of neoplastic cells, gross tumor volume is dwindled even completely dissolve;
6, it is good to the invention provides the compound biological safety, under the test conditions, awards animal and is higher than the dosage of effective dose more than 100 times, and animal can be well tolerable, and tangible acute toxic reaction does not take place;
7, the invention provides compound and can realize visual video picture and targeted therapy to unknown tumor tissues simultaneously.
8, the invention provides compound and can realize the target real-time visualization to tumor tissues, thereby the tumor treatment process is realized visual dynamic monitoring.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is usually according to normal condition or the condition of advising according to manufacturer.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.
Description of drawings
Fig. 1 has shown the tumor locus fluorescent imaging situation after preferred compound 29 that the embodiment of the invention 1 provides gives lotus people lung cancer A549 tumour nude mice;
Fig. 2 has shown the tumor locus fluorescent imaging situation after preferred compound 34 that the embodiment of the invention 1 provides gives lotus people lung cancer A549 tumour nude mice;
Fig. 3 has shown the tumor locus fluorescent imaging situation after preferred compound 29 that the embodiment of the invention 1 provides gives lotus vicious transformation corium multipotential stem cell tumour rat;
Fig. 4 has shown the tumor locus fluorescent imaging situation after preferred compound 34 that the embodiment of the invention 1 provides gives lotus vicious transformation corium multipotential stem cell tumour rat;
Fig. 5 has shown that preferred compound 29 that embodiment 1 provides and 34 compounds can be by the tumour cell huge uptakes, and is positioned cell mitochondrial, and the normal cell intake is starkly lower than tumour cell.
Fig. 6 has shown compound 29 provided by the invention whole body near infrared imaging in vein gives mouse 48 hours.
Fig. 7 has shown the tissue distribution content of compound 34 provided by the invention each organ of whole body in vein gives mouse 48 hours.
Fig. 8 has shown compound 34 provided by the invention tumor growth curve after vein gives tumor-bearing rat.
Fig. 9 shown compound 34 provided by the invention when 5mg/kg dosage after vein gives tumor-bearing rat tumor histology's (HE dyeing) situation, wherein the left side be control tumor, the right side is treatment group tumour.
Figure 10 has shown compound 34 provided by the invention tumor growth curve after vein gives lotus people lung cancer A549 tumour nude mice.
Figure 11 shown compound 34 provided by the invention when 5mg/kg dosage after vein gives mouse each histoorgan histology (HE dyeing) situation, wherein go up row and be the administration group, following row is control group.
Specific embodiment
The instrument and equipment that adopts among the embodiment:
Institute responds and all follows the tracks of with thin-layer chromatography, the efficient tlc silica gel plate (model C F-254) that uses the yellow affair silica gel of Yantai City's Zhifu development experiments factory to produce, phospho-molybdic acid, triketohydrindene hydrate, tetrabromo-mcresolsulfonphthalein or iodine colour developing.
The chromatographic silica gel (10-40 μ) that column chromatography is produced with the yellow affair silica gel of Yantai City's Zhifu development experiments factory, chromatography is analytical pure with organic solvent, and through the dry processing of overweight evaporate to dryness.Reagent D MF, POCl
3All pass through distillation purifying and drying treatment;
Fusing point is measured with the WRR fusing point instrument that Shanghai Precision Scientific Apparatus Co., Ltd produces.
All compounds
1H NMR and
13C NMR is measured by the Mercury Plus-400 nuclear magnetic resonance spectrometer that U.S. Varian company produces, and mark in TMS does without specified otherwise, is all used CDCl
3Make solvent, δ value unit is ppm.
Mass spectrum is measured by HP5989A type mass spectrograph, and IR is measured by Testscan Schimadzu FTIR 8000series.
The preparation of embodiment 1 dialdehyde chloro tetrahydrobenzene (2-chloro-3-hydroxyl methylene-1-formyl radical tetrahydrobenzene)
Interior warm 0-5 ℃ of control will be stirred down slowly adding 20ml (new evaporate to dryness is dry) POCl in the reactor that fills 60ml (new evaporate to dryness is dry) DMF
3, drip and finish, keep 0-5 ℃ of restir after 30 minutes, slowly rise to room temperature.Add the 5ml pimelinketone, be warming up to 50 ℃ of reaction 6h.Stopped reaction, reaction solution incline to water, behind the stirring 3h. and a large amount of yellow solids are separated out, suction filtration, filter cake is extremely neutral with cold wash.Crude product with ethyl acetate refining yellow granular crystal 5.2g, yield 65.3%.Lucifuge low temperature (4 ℃) is preserved.Fusing point: 130-131 ℃ of (document fusing point: 130-131 ℃) thin-layer chromatography detects: hexanaphthene: ethyl acetate (3: 1), the Rf value is about 0.4.UV (methyl alcohol): λ max=408nm[OHC-C=C];
1H NMR:
1H NMR (400MHz, DMSO-d): 1.573 (m, 2H, 5-CH2), 2.351 (t, 4H, 4,6-CH2), 3.347 (s, 2H, 3-CH2OH), 10.851 (s, 1H, 1-CHO).
The preparation of embodiment 2N-caproic acid indoles quaternary ammonium bromine salt (2,3,3-trimethylammonium-1-caproic acid-3H-indoles bromine salt)
Add 2,2,3-trimethylammonium-3H indoles in the reactor, 6-bromocaproic acid and 1,2-orthodichlorobenzene (or DMF, or DMSO or toluene or glycol dimethyl ether), react 12h. down in 110 ℃.Check reaction solution with fluorescence or bromine cresols chlorine, the 6-bromocaproic acid participates in reaction fully.Stopped reaction is chilled to room temperature, leaves standstill 10 hours.System is separated out a large amount of yellowish brown solids, and suction filtration uses acetone repetitive scrubbing filter cake to pale solid.Crude product need not continue purifying, can directly carry out the next step.Melting range: 90-92 ℃; Thin-layer chromatography detects: methyl alcohol: chloroform (1: 5), Rf value are about under 0.3, the 254nm ultraviolet to be inspected.UV (methyl alcohol) λ max=230nm[indole];
1H NMR (400MHz, DMSO-d):: 1.423 (m, 2H, γ-CH2), 1.532 (s, 6H, 3-CH 3), 1.576 (m, 2H, β-CH2), 1.840 (m, 2H, δ-CH2), 2.228 (t, 2H, α-CH2), 2.834 (s, 3H, 2-CH3), 4.449 (t, 2H, N-CH2), 7.613-7.981 (m, 4H, Ar-H), 12.057 (s, 1H, COOH).
Synthetic (29,34) of seven methine indole cyanine dyes of 3 pairs of positive butyl esters of embodiment or mono side chain
Add 2.780g indoles quaternary ammonium salt 25 (7.809mmol) in the 500ml single port reaction flask; 0.645g condensing agent 29 (3.728mmol); use the 250ml propyl carbinol separately; or make solvent with benzene (or toluene or hexanaphthene) mixed solution (7: 3); water trap and prolong are installed; under the nitrogen protection, in 120 ℃ of back flow reaction 5h of outer temperature, system transfers green to by redness.Stopped reaction, underpressure distillation (0.098Mp, 60 ℃) eliminates solvent benzol and propyl carbinol, gets 3.395g glassy yellow solid (blanking bar green), get 3.395g glassy yellow solid (blanking bar green), get compound 29 (yield 60%) and compound 34 (yield 30%) respectively through silica gel column chromatography.
29 structural analysis data:
Mp：198-200℃；
1H-NMR(DMSO，400MHz)：8.275(2H，d，J＝13.6Hz，βCH＝C)，7.650-7.247(8H，m，Ar-H)，6.326(2H，d，J＝13.6Hz，αCH＝C)，4.224(4H，t，J＝6.8Hz，N-CH
2)，3.972(4H，t，J＝9.6Hz，OCH
2)，2.673(4H，t，J＝7.2Hz，0＝C-CH
2)，2.289(4H，t，J＝7.2Hz，Cyclohexene-CH
2)，1.865-1.235(22H，m，other-CH
2)，1.687(12H，s，indol-CH
3)，0.865(6H，t，J＝7.2Hz，n-Bu-CH
3).
13C-NMR(DMSO，200MHz)：173.334(C＝O)，172.148(C＝O)，144.152，142.026，140.879，128.711，127.345，125.215，122.154，110.815，101.258，64.102，49.197，44.592，33.765，30.476，27.993，27.054，26.480，26.330，24.454，20.585，18.968，13.577(Bu-CH
3)；
UV/Vis(MeOH)ε
max＝ε(781nm)256000(L.mol
-1.cm
-1)；
IR (KBr): 1732 (C=O); 30012866 (ring C-H ,-CH2O); 1060-1019 (=C-Cl)
HRMS(ESI+)：795.4874[M-Br]，796.4936[M-Br+1]，797.4918[M-Br+2]；
calcd for C
50H
68ClN
2O
4：795.49[M-Br]，796.49[M-Br+1]，797.48[M-Br+2].
34 related data:
Mp：158-160℃；
1H NMR(CDCl
3，400MHz)：8.329(1H，t，J＝15.2Hz，1’-H)，8.291(1H，t，J＝15.2Hz，7’-H)，7.447-7.049(8H，m，Ar-H)，6.287(1H，dd，J 1＝14.0Hz，J 2＝14.0Hz，2”-CH＝C)，6.068(1H，dd，J1＝14.0Hz ，J2＝14.0Hz，6”-CH＝C)，4.254-4.004(6H，m，6’”，1””，6’””-CH2)，2.756-2.662(4H，m，2’”，2’””-CH2-C＝O)，2.543-2.313(4H，m，8”，10”-CH2)，1.712-1.702(12H，d，3，3’-CH3)，1.989-1.210(18H，m，other-CH
2)，0.906(3H，t，J＝7.2Hz，4””-CH3).
UV/Vis(MeOH)：λmax/nm＝781；εmax＝28000(L.mol-1.cm-1)；IR(KBr)：3400.5(COOH)；1727.4(C＝O)；1520(O＝CO-)，1136.8(C-N)，918.9(OH)。
MS (ESI+): 739.63 (M-Br), 741.45 (M-Br+2H), 742.57 (M-Br+3H), 785.41 (M-Br+2Na); Theoretical value C
46H
60ClN
2O
4: 739.42.
Embodiment 4
Interior tumor cell picked-up and fluorescence imaging test
Material and method
Embodiment 4
The storage liquid that the preferred compound 29 that embodiment 1 is provided and 34 usefulness methyl-sulphoxides (DMSO) are mixed with 10mM concentration places-20 ℃ to keep in Dark Place.
Select rat vicious transformation corium multipotential stem cell (the Shi C of this research department's separation and Culture for use, Mai Y, ZhuY, Cheng T, Su Y.Spontaneous transformation of a clonal population of dermi s-derived multipotentcells in culture.In Vitro Cellular and Developmental Biology-Animal, 2007; 43:290-296), cultivate based on 37 ° with the IMDM that contains 10% foetal calf serum, cultivate under the 5%CO2 condition; Kodak In-vivoFx multifunctional active animal fluorescence imaging system is available from Kodak.
Prepare nude mice lotus people lung cancer A549 model and rat lotus vicious transformation corium multipotential stem cell model respectively, by tumour cell 2 * 106 every to be inoculated in the nude mice mouse subcutaneous, by 5 * 106 every be inoculated in subcutaneous rat, preparation animal lotus knurl model, when treating that tumor growth is big or small to diameter 0.5-1 centimetre, the preferred compound 29 and 34 that provides with embodiment 1 is provided by the tail vein, dosage is the 0.5mg/kg body weight, detect the fluorescent imaging situation of tumor locus by Kodak In-vivo Fx multifunctional active animal fluorescence imaging system respectively at different time after the administration, in Kodak's living imaging instrument, observe, excitation wavelength 770nm, emission wavelength 830nm, the time shutter is 20 seconds.
The preferred compound 29 and 34 that embodiment 1 provides is after giving tumor animal, with the blood circulation whole body that distributes rapidly, the integral animal fluorescent signal obviously strengthens, after the administration 30 minutes, the near-infrared fluorescent signal that is higher than other healthy tissuess can be seen in the tumor tissues position, the tumor locus fluorescent signal strengthens gradually subsequently, can reach the contrast gradient of highly significant in 6 hours, after the administration 48 hours, tumor locus then enrichment a large amount of fluorescent signals, the compound that is not ingested then is excluded external, healthy tissues fluorescence significantly reduces, tumor tissues has obtained best contrast images, the ratio of tumor tissues and other healthy tissues fluorescence signal intensities can reach 5-15 doubly, the results are shown in Figure 1 (nude mice lotus knurl model injection compound compound 29), Fig. 2 (nude mice lotus knurl model injection chemical combination materialization, Fig. 3 (rat lotus knurl model injection compound compound 29) and Fig. 4 (rat lotus knurl model injection compound 34) (arrow is depicted as tumor locus).
The result shows, after giving in the chemical combination object provided by the invention, have by the great ability of the efficient picked-up of tumor tissues, healthy tissues is not then absorbed this compounds, demonstrate at tumor tissues and healthy tissues and have the extremely distribution characteristics of significant difference, simultaneously can carry out tumor imaging at biological tissue, this has great importance to specific diagnosis and the targeted therapy of carrying out tumour pointedly.
Embodiment 5
External test of being absorbed by tumour cell
Material and method
The preferred compound 29 and 34 that embodiment 1 provides;
Tumor cell line is selected HeLa Cells for use, available from U.S. ATCC company, cultivates based on 37 ° with the RPMI 1640 that contains 10% foetal calf serum, cultivates under the 5%CO2 condition, changes nutrient solution every other day one time.Normal cell separates preservation for separating from healthy human bone marrow substrate cell by this research department.
Get well-grown, reach 70% cell that merges, digest being inoculated in the observation of 35mm Laser Scanning Confocal Microscope culture dish, overnight incubation after centrifugal, discard substratum, in culturing bottle, add the serum free medium 1ml that contains compound 29 or 34 respectively, in 37 °, hatched under the 5%CO2 condition 15 minutes, discard substratum, give a baby a bath on the third day after its birth time with the PBS damping fluid, with rhodamine 123 labeled mitochondrias, under Laser Scanning Confocal Microscope, observe in the fluorescence intensity of compound 29 and 34 and the cell and locate.
The result shows that compound 29 and 34 can efficiently be absorbed by tumour cell under condition of in vitro culture, demonstrate very strong fluorescent signal (red positive signal), and the normal bone marrow stroma cell then absorbs seldom; The red fluorescence signal can with overlap from mitochondrial green, confirm that compound enters cell after, mainly be distributed in the cell mitochondrial (Fig. 5).
Embodiment 6
Antitumor activity in vitro
Material and method
The preferred compound 29 and 34 that embodiment 1 provides;
Tumour cell is selected the strain of people's lung cancer A549 cell, human breast carcinoma MCF-7 cell strain for use, and human cervical carcinoma HeLa is all available from U.S. ATCC company.
Above-mentioned cell is placed the RPIM1640 substratum that contains 10% foetal calf serum and 1% penicillin, Streptomycin sulphate, at 37 ℃, the conventional cultivation in the 5%CO2 incubator.
The density of cell by 5 * 104/ml is inoculated in 96 well culture plates every hole 200ul.Test is divided into blank group and compound group, 6 every group multiple holes.Test the preferred compound 29 and 34 that used compound provides for embodiment 1, the test final concentration is followed successively by 0.2,1,5,10 respectively, 20 and 40uM.Cultivated 48 hours, the CCK-8 method detects cell viability, adds aseptic CCK-8 liquid 10ul to each test holes, continues behind the mixing to cultivate 2 hours, detects the absorbance value at 490nm place at enzyme connection instrument.The half-inhibition concentration value (IC50) of computerized compound.
The result: two kinds of compounds all have lethal effect to above tumour cell, and its IC50 is respectively compound 29 (4.42,5.89,0.26), compound 34 (3.78,6.33,0.47).This result shows that the preferred compound 29 that embodiment 1 provides all has good lethal effect with 34 pairs of different tumour cells, and this all has specificity picked-up effect with different tumour cells to this compounds is consistent.
Embodiment 7
Bio distribution and fluorescence imaging research in the body
Material and method
The preferred compound 34 of selecting for use embodiment 1 to provide;
Choose the normal adult kunming mice and carry out the pharmacokinetics test, the preferred compound 34 that provides by 1mg/kg (0.3ml PBS solution) tail vein injection embodiment 1, by Kodak's animalcule living imaging systematic observation execute preferred compound 34 that example 1 provides different time points (before the injection, injected back 1 minute, 30 minutes, 1 hour, and 48 hours) detect whole body fluorescent signal (Fig. 6); After different time points is put to death mouse (n=3), get main organs and comprise that heart, liver, spleen, lung, kidney, small intestine sample carry out tissue homogenate, getting whole blood puts in the 0.5ml heparinization plastic centrifuge tube, centrifugal back separated plasma, the preferred compound 34 concentration-absorbancy curve that provides according to embodiment 1 carries out content analysis, observes preferred compound 34 each histoorgan distribution situation (Fig. 7) in the different time points body that embodiment 1 provides.The result shows, in the normal mouse body, preferred compound 34 content that embodiment 1 provides in the blood plasma are the highest during with 30 minutes, after reduce gradually, eliminated substantially in vivo to 48 hours.The preferred compound 34 that embodiment 1 provides after intravenous injection is very fast to be absorbed by liver, and fluorescent signal increases gradually to 1 hour peaking, and signal is reduced to lower level gradually afterwards.
The result shows, after giving in the chemical combination object provided by the invention, have good internal metabolism and get rid of feature, healthy tissues is not absorbed this compounds, can be got rid of by body fully, thereby can demonstrate at tumor tissues and healthy tissues and have the extremely distribution characteristics of significant difference.
Embodiment 8
Anti-tumor activity test after whole body is used
Material and method
The preferred compound 34 of selecting embodiment 1 to provide;
Set up rat lotus knurl model by subcutaneous transplantation rat vicious transformation corium multipotential stem cell, inject the preferred compound 34 (5 that embodiment 1 provides through the abdominal cavity, 10, the 20mg/kg body weight), observe preferred compound 34 anti-tumour effects that embodiment 1 provides, with clinical the positive medication therapy groups of anticancer drugs, doxorubicin (2mg/kg) is often arranged, and set up the solvent control group, every group of 6 rats.Treated that the knurl piece began the administration of dividing into groups in the time of can touching on the 4th day after the lotus knurl, medication every other day once, totally five times.Record rat tumor major diameter and transverse diameter calculate gross tumor volume, draw tumor growth curve.Put to death experimental animal on the 3rd day after the drug withdrawal, carry out that tumor tissues is weighed and the related pathologies credit is analysed.
Measure the long and short footpath of tumour respectively, and calculate gross tumor volume by following formula:
Select the strain of people's lung cancer A549 cell, press tumour cell 2 * 10
6Every to be inoculated in the nude mice mouse subcutaneous, inject the preferred compound 34 (5mg/kg) that embodiment 1 provides through the abdominal cavity, observe preferred compound 34 anti-tumour effects that embodiment 1 provides, with clinical (20mg/kg) positive medication therapy groups of cancer therapy drug endoxan (CTX) often arranged, and set up solvent control group, every group of 6 rats.Treated that the knurl piece began the administration of dividing into groups in the time of can touching on the 4th day after the lotus knurl, medication every other day once totally five times, was put to death experimental animal on the 3rd day after the drug withdrawal.Record rat tumor major diameter and transverse diameter calculate gross tumor volume, draw tumor growth curve.
The result shows: the preferred compound 34 that embodiment 1 provides all has the effect of obvious suppression tumor growth when three kinds of different concns, gross tumor volume significantly dwindles, suitable with simple positive drug control group, and the normal control group is to tumor growth unrestraint effect (Fig. 8).The visible control group tumor growth of histological examination is vigorous, no necrosis region, and tumor group is woven with extensive necrosis (Fig. 9).Tumor bearing nude mice antineoplaston result of study shows: in people's lung cancer A549 cell tumor model, treatment group gross tumor volume poor growth even reduce is significantly less than control group (P＜0.01).Preferred compound 34 (5mg/kg) the treatment group that endoxan 20mg/kg treatment group and embodiment 1 provide is obvious to the restraining effect of tumor growth.There were significant differences between two treatment groups, illustrates that preferred compound 34 that embodiment 1 provides presses down knurl effect (Figure 10) what 5mg/kg treatment group was better than endoxan 20mg/kg treatment group.
Embodiment 9
The studies on acute toxicity of the preferred compound 34 that embodiment 1 provides
The preferred compound 34 that Application Example 1 provides carries out the medicine studies on acute toxicity, select the healthy adult kunming mice for use, inject the preferred compound 34 that 10 days embodiment 1 provide every other day through the abdominal cavity, dosage is 5mg/kg, the result do not find mouse occur obviously losing weight, One's spirits are drooping and stool and urine is unusual.Each main histoorgan of mouse comprises heart, liver, spleen, lung, kidney, brain, testis pathological examination after putting to death, and relatively shows no obvious abnormalities (Figure 11) with the normal control group.Through single-dose 200mg/kg (n=6), observed continuously 30 days, do not see dead mouse.
The result shows that under this experiment condition, The compounds of this invention 34 tumor group to tumor-bearing rat after giving through tumor by local is woven with remarkable lethal effect, tumor growth is slowed down, the medicine of effect and clinical use is suitable, and is harmless to other healthy tissues organs simultaneously, is safe.
The above only is preferred embodiment of the present invention, be not in order to limit essence technology contents scope of the present invention, essence technology contents of the present invention is broadly to be defined in the claim scope of application, any technology entity or method that other people finish, if it is defined identical with the claim scope of application, also or a kind of change of equivalence, all will be regarded as being covered by among this claim scope.
Claims (3)
1. the application of seven methine indoles cyanine dye compounds 29 of two positive butyl esters or mono side chain and 34 in preparation treatment people lung-cancer medicament, the chemical structural formula of seven methine indoles cyanine dye compounds 29 of two positive butyl ester side chains is as follows:
The chemical structural formula of seven methine indoles cyanine dye compounds 34 of mono side chain is as follows:
。
2. the application of seven methine indoles cyanine dye compounds 29 of two positive butyl esters or mono side chain and 34 in preparation treatment human breast carcinoma medicine, the chemical structural formula of seven methine indoles cyanine dye compounds 29 of two positive butyl ester side chains is as follows:
The chemical structural formula of seven methine indoles cyanine dye compounds 34 of mono side chain is as follows:
。
3. the application of seven methine indoles cyanine dye compounds 29 of two positive butyl esters or mono side chain and 34 in preparation treatment human cervical carcinoma medicine, the chemical structural formula of seven methine indoles cyanine dye compounds 29 of two positive butyl ester side chains is as follows:
The chemical structural formula of seven methine indoles cyanine dye compounds 34 of mono side chain is as follows:
。
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| CN102703569B (en) * | 2012-05-24 | 2014-04-09 | 中国科学院化学研究所 | New use of cyanine dyes |
| WO2013174262A1 (en) * | 2012-05-24 | 2013-11-28 | 中国科学院化学研究所 | Cancer diagnosing method, reagent kit, and system |
| CN103911017B (en) | 2012-12-28 | 2017-09-15 | 浙江海正药业股份有限公司 | Cyanine dye compound and preparation method thereof, for dual-use function agent of photodynamic therapy and preparation method thereof |
| CN103834382B (en) * | 2014-01-02 | 2016-03-02 | 东南大学 | A kind of near-infrared fluorescent molecular probe and preparation method thereof and purposes |
| KR101590527B1 (en) * | 2014-03-12 | 2016-02-02 | 한국외국어대학교 연구산학협력단 | New indocyanine derivatives, composition comprising same, and detection probes for thiol-containing compounds |
| CN104448891B (en) * | 2014-10-31 | 2016-11-09 | 山东师范大学 | There is the near infrared fluorescent dye of big Stokes shift and synthetic method and application |
| CN104804463B (en) * | 2015-03-10 | 2016-08-24 | 西安交通大学第一附属医院 | Near-infrared fluorescent stain and preparation method and application for target tumor tissue |
| CN105566938B (en) * | 2016-02-02 | 2017-12-22 | 史春梦 | A kind of seven Mitochondrially targeted methine indoles cyanine dyes and preparation method and application |
| CN109796779B (en) * | 2017-11-17 | 2024-05-07 | 中国科学院宁波材料技术与工程研究所 | Preparation method of heptamethine benzindole cyanine dye |
| CN109796780A (en) * | 2017-11-17 | 2019-05-24 | 中国科学院宁波材料技术与工程研究所 | Seven methine benzindole cyanine dyes of one kind and its preparation method and application |
| CN108624080B (en) * | 2018-07-20 | 2020-03-10 | 青岛大学 | Near-infrared dye |
| CN111662567A (en) * | 2019-03-08 | 2020-09-15 | 中国科学院宁波材料技术与工程研究所 | Heptamethine carboxyl indole cyanine dye and preparation method and application thereof |
| CN111662566A (en) * | 2019-03-08 | 2020-09-15 | 中国科学院宁波材料技术与工程研究所 | Heptamethine hydroxyindole cyanine dye, and synthesis method and application thereof |
| CN110064064B (en) * | 2019-05-14 | 2020-10-23 | 四川大学 | Contrast agent and application thereof in photoacoustic imaging of brown fat |
| CN111592482B (en) * | 2020-05-15 | 2021-06-25 | 江南大学 | PH reversible activation type photo-thermal/photodynamic/fluorescent integrated probe molecule |
| CN111925311B (en) * | 2020-08-28 | 2021-11-02 | 上海库擘生物技术有限公司 | Tumor contrast compound, preparation method thereof and application thereof in tumor diagnosis imaging |
| CN113214672B (en) * | 2021-04-13 | 2022-11-04 | 大连理工大学 | Amide substituted azaindole-pentamethine cyanine dye, its synthesis method and application |
| CN113350336B (en) * | 2021-07-01 | 2022-06-10 | 中国人民解放军陆军军医大学 | Application of heptamethine indocyanine or derivative thereof |
| CN113683602B (en) * | 2021-09-08 | 2022-06-10 | 中国人民解放军陆军军医大学 | Heptamethine cyanine micromolecule for multi-modal treatment of hypoxic tumors, and preparation method and application thereof |
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