CN102603695B - Amino acid-fluorophore compound and application thereof - Google Patents
Amino acid-fluorophore compound and application thereof Download PDFInfo
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 27
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 title claims abstract description 7
- 102100022749 Aminopeptidase N Human genes 0.000 claims abstract description 46
- QZDDFQLIQRYMBV-UHFFFAOYSA-N 2-[3-nitro-2-(2-nitrophenyl)-4-oxochromen-8-yl]acetic acid Chemical compound OC(=O)CC1=CC=CC(C(C=2[N+]([O-])=O)=O)=C1OC=2C1=CC=CC=C1[N+]([O-])=O QZDDFQLIQRYMBV-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 13
- 108010049990 CD13 Antigens Proteins 0.000 claims abstract description 11
- -1 2-methylthioethyl Chemical group 0.000 claims abstract description 7
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims abstract description 4
- CJIJXIFQYOPWTF-UHFFFAOYSA-N 7-hydroxycoumarin Natural products O1C(=O)C=CC2=CC(O)=CC=C21 CJIJXIFQYOPWTF-UHFFFAOYSA-N 0.000 claims abstract description 3
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 claims abstract description 3
- ORHBXUUXSCNDEV-UHFFFAOYSA-N umbelliferone Chemical compound C1=CC(=O)OC2=CC(O)=CC=C21 ORHBXUUXSCNDEV-UHFFFAOYSA-N 0.000 claims abstract description 3
- 238000012216 screening Methods 0.000 claims description 12
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- 125000004861 4-isopropyl phenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 abstract 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明涉及一种氨基酸-荧光团类化合物及其应用。其结构通式为(I)、(II)、(III)、(IV)或者它们的游离形式，式中：R1为各种氨基酸侧链，优选甲基、丙基、2-甲硫基乙基、苯甲基、苯乙基、2-环己基乙基、4-异丙基苯基、4-二甲氨基苯基；R2为各种荧光团，优选7-羟基香豆素、萘二酰亚胺类荧光团、尼罗红系列和Cy系列荧光团；X为C或者N。该类荧光探针分子可用来检测氨肽酶N的活性(酶水平和细胞水平)，可作为探针工具来检测氨肽酶N的组织分布和进行肿瘤组织成像，可作为各类氨肽酶N异常表达疾病的诊断工具。此外，该类化合物制备方法反应条件温和，原料便宜易得，操作及后处理简单。 The invention relates to an amino acid-fluorophore compound and its application. Its structural formula is (I), (II), (III), (IV) or their free forms, in which: R1 is various amino acid side chains, preferably methyl, propyl, 2-methylthioethyl Base, benzyl, phenethyl, 2-cyclohexylethyl, 4-isopropylphenyl, 4-dimethylaminophenyl; R2 is various fluorophores, preferably 7-hydroxycoumarin, naphthalenedi Imide fluorophores, Nile Red series and Cy series fluorophores; X is C or N. This type of fluorescent probe molecule can be used to detect the activity of aminopeptidase N (enzyme level and cell level), and can be used as a probe tool to detect the tissue distribution of aminopeptidase N and perform tumor tissue imaging. A diagnostic tool for N abnormal expression diseases. In addition, the preparation method of this type of compound has mild reaction conditions, cheap and easy-to-obtain raw materials, and simple operation and post-treatment.
Description
Technical field
The present invention relates to amino acid-linker-fluorophore compounds and as the application in small molecules fluorescent probe and the screening aminopeptidase N inhibitor of Aminopeptidase N, belong to technical field of pharmaceuticals.
Background technology
Aminopeptidase N (A PN, CD13) wide expression, in kidney and IBB cell, marrow progenitor cell film, monocyte film, central nervous system cynapse cytolemma, inoblast, endothelial cell membrane, placenta cells film surface, can discharge amino acid from the N-terminal degraded of protein.With the moving phase ratio of normal cell, this enzyme is in tumor cell surface overexpression, and invasion and attack, vasculogenesis to tumour have vital role.In addition, this proteolytic enzyme participates in corresponding physiological response as the acceptor of much virus (as cause the dissemination marcy agent of neonatal pig acute gastroenteritis and cause people's upper respiratory tract infection coronavirus HCV229E).It can also be expressed in antigen presenting cell surface, and degraded panimmunity active substance, declines immunity of organisms, weakens scavenger cell and identification and kill capability [Zhang, the X.P. of NK cell to tumour cell; Xu, W.F.Aminopeptidase N (APN/CD13) as a target for anti-cancer agent design.Curr Med Chem2008,15,2850-2865].
Although the mankind have had a lot of understandings to the function of APN, detection method active for APN and tissue distribution does not have too many research.Existing method comprises immune labeled method, isotope-labelling method and fluorescent method.Immune labeled method can not realize intravital detection, detects although isotope-labelling method can be realized in vivo, has radiotoxicity.Comparatively speaking fluorescent method has advantages of that sensitivity, toxicity are little, can realize in vivo and detecting.Fluorophore is connected with APN targeting part NGR, and utilizing the targeting of NGR to measure the in vivo tissue distribution situation of APN is research method [(a) von Wallbrunn, the A. of recent years; Waldeck, J.; Holtke, C.; Zuehlsdorf, M.; Mesters, R.; Heindel, W.; Schafers, M.; Bremer, C.In vivo optical imaging of CD13/APN-expression in tumor xenografts.J Biomed opt2008,13,011007-1--011007-9. (b) Smith, R.A.; Giorgio, T.D.Quantitative measurement of multifunctional quantum dot binding to cellular targets using flow cytometry.Cytometry Part A2009,75A, 465-474]. but the use of these molecular probes is owing to being restricted compared with strong background signal.Therefore, design synthesis of selective APN fluorescent probe good, highly sensitive, that signal to noise ratio is large, toxicity is little has great importance.
Research shows, self does not have fluorescence the activated probe based on enzymic activity, when discharging fluorophore and produce fluorescence [Chen, X.Q. after itself and enzyme effect; Sun, M.; Ma, H.M.Progress in spectroscopic probes with cleavable active bonds.Curr org chem2006,10,477-489].Because APN can be from the aminoterminal of the peptide chain peptide chain of progressively degrading, if APN specific amino acid residue and fluorophore are connected and are syntheticly had APN and optionally can activate small molecules fluorescent probe by peptide bond, and then measure APN activity and the tissue distribution in various APN high expression level animal models, study itself and tumour, vasculogenesis and immune relation, certainly will be able to provide foundation for getting up early diagnosis and the prevention of tumour and disease of immune system.In addition, to the screening of aminopeptidase N inhibitor or take ultraviolet absorption method as main, the method has the shortcomings such as sensitivity is low, cost is high, the test duration is long.The experiment if the enzyme that we can find a desirable fluorescent probe to carry out Aminopeptidase N is lived, the screening that also can be aminopeptidase N inhibitor provides a platform.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, one seed amino acid-fluoresceins compound and preparation method thereof and the application at pharmacy field are provided.
For achieving the above object, the present invention adopts following technical proposals:
One seed amino acid-fluorophore compounds and application thereof.Its general structure is (I), (II), (III) or (IV), or their free form,
In formula: R1 is methyl, propyl group or 2-methylmercaptoethyl; R2 is umbelliferone; X is C or N.
Preferably, above-claimed cpd, has one of following structure:
Compound of the present invention is as the application of the small molecules fluorescent probe of Aminopeptidase N.
Compound of the present invention at screening aminopeptidase N inhibitor (comprising enzyme level and cell levels), detects the tissue distribution of Aminopeptidase N and the application of tumour cell and imaging of tissue as reference fluorescent probe.
Compound of the present invention is the application in screening aminopeptidase N inhibitor (comprising enzyme level and cell levels) as reference fluorescent probe.
Compound of the present invention is the application in the medical diagnosis on disease of all kinds of Aminopeptidase N unconventionality expression as reference fluorescent probe.
Accompanying drawing explanation
Fig. 1 has the probe L1 of reference wavelength character and the transmitted wave spectrogram of APN effect.
Embodiment
The following examples can make the present invention of those skilled in the art comprehend, but do not limit the present invention in any way.
Example one: methylthio group-N-(4-(2-oxo-2,2-amino-4 hydrogen-chromene-7-base oxygen base) phenyl) preparation of butanamide hydrochloride [L3]
Synthetic route 1
The preparation of intermediate 1:
14.9g methionine(Met) is dissolved in to 110mL1M sodium hydroxide solution, ice bath, slowly drip the anhydrous THF solution of 50mL containing 21.18g dimethyl dicarbonate butyl ester, use the sodium hydroxide solution control reaction solution pH of 1M in 9 left and right simultaneously, about 1h dropwises, under condition of ice bath, stir 1h, remove ice bath, stirring at room temperature 15h.Whole process keeps system pH in 9 left and right, steam after completion of the reaction except THF, with petroleum ether (100mL × 3), water regulates pH2～3 with saturated citric acid solution, with ethyl acetate extraction (100mL × 3), anhydrous magnesium sulfate drying spends the night, and concentrates to obtain faint yellow oily matter 20.5g, yield 87%.
The preparation of intermediate 2:
Under ice bath, in 100mL anhydrous methanol, drip 8.73mL Acetyl Chloride 98Min., ice bath stirs 15 minutes, adds 5.4g para-amino benzoic acid, and ice bath stirs after 1 hour and refluxes 2 hours.Concentrate to obtain white solid, obtain white powder 6.55g with methyl alcohol-ether recrystallization, yield 95%.
The preparation of intermediate 3:
2.7g intermediate 1 is dissolved in to 100mL methylene dichloride, adds 1.5g HOBT, 2.1g EDCI, stirring at room temperature 15min, adds 1.9g intermediate 2 and 1.4mL triethylamine, stirring at room temperature 6 hours.By saturated citric acid (50mL × 3) for reaction solution wash, saturated sodium bicarbonate (50mL × 3) washes, saturated aqueous common salt (50mL × 1) is washed.Anhydrous magnesium sulfate drying spends the night.Concentrated, obtain white solid 3.1g with ethyl acetate-normal hexane recrystallization, productive rate 80%, fusing point 100-102 ℃.
The preparation of intermediate 4:
0.76g intermediate 3 is dissolved in to 20mL anhydrous diethyl ether, and cryosel is bathed lower gradation and is added 0.23g Lithium Aluminium Hydride, room temperature reaction 1 hour.Under ice bath, in reaction solution, add 3mL ethyl acetate, regulate PH to 7 with 2M hydrochloric acid.Filter, organic phase is washed with saturated aqueous common salt (20mL × 3), and anhydrous magnesium sulfate drying spends the night.Concentrated, combiflash separates to obtain colorless oil 0.34g, yield 48%.
Prepared by intermediate 5:
0.26g intermediate 4 is dissolved in to the anhydrous THF of 40mL, under ice bath, adds 0.3mL triethylamine and 0.18mL methylsulfonyl chloride, 5 ℃ of following stirring reactions, after 4 hours, reaction finishes.Reaction solution with 0.5M hydrochloric acid (20mL × 3) wash, saturated common salt washing (20mL × 1), anhydrous magnesium sulfate drying.Concentrate to obtain yellow oil 0.28g, thick productive rate 80%, without purification, is directly used in next step reaction.
The preparation of intermediate 6:
0.12g7-Hydroxycoumarin is dissolved in to 10mL DMF, adds 0.2g Anhydrous potassium carbonate, stirring at room temperature, after 15 minutes, adds 0.23g intermediate 5, and reaction is spent the night.Reaction solution is poured in 100mL ethyl acetate, washed with unsaturated carbonate potassium solution (50mL × 5), saturated aqueous common salt (50mL × 1) is washed.Concentrated, obtain white solid 0.12g with ethyl acetate-normal hexane recrystallization, yield 60%.Fusing point 128-129 ℃.
The preparation of compound L 3
0.06g intermediate 6 is dissolved in the saturated hydrogenchloride-ethyl acetate solution of 3mL, and stirring at room temperature, after 2 hours, has a large amount of white precipitates to produce, and filters, and is dried to obtain white powder 0.06g, yield 92%.Fusing point 234.2-235.7 ℃.
1HNMR(300MHz,D
2O)δppm:7.75(d,1H,J=9.3Hz),7.37-7.45(m,5H),6.85(d,1H,J=9.3Hz),6.75(d,1H,J=14.1Hz),6.17(d,1H,J=0.9Hz),5.01(s,2H),4.19(t,1H,J=6.6Hz),2.58(t,2H,J=7.2Hz),2.17-2.41(m,2H),2.01(s,3H)。
Example two: 2-amino-N-(2-((2-oxo-2 hydrogen-chromene-7-base oxygen base)-methyl) phenyl) preparation of valeramide hydrochloride [L38]
Synthetic route 2
The preparation of intermediate 7:
1.2g Boc-L-norvaline is dissolved in to 50mL methylene dichloride, adds 0.75g HOBT, 1.05g EDCI, stirring at room temperature 15min, adds the adjacent aminobenzyl alcohol of 0.62g, stirring at room temperature 10 hours.By saturated citric acid (50mL × 3) for reaction solution wash, saturated sodium bicarbonate (50mL × 3) washes, saturated aqueous common salt (50mL × 1) is washed.Anhydrous magnesium sulfate drying spends the night.Concentrated, obtain white solid 0.97g with ethyl acetate-normal hexane recrystallization, productive rate 60%, fusing point 110.0-122.3 ℃.
The preparation of intermediate 8:
Take intermediate 7 as raw material, obtain intermediate 8 according to the preparation method of intermediate 5, brown oil, yield 80%.
Prepared by intermediate 9:
Take intermediate 8 as raw material, obtain intermediate 9 according to the preparation method of intermediate 6, white solid, yield 21%.Fusing point: 158.0-160.1 ℃.
The preparation of compound L 38
Take intermediate 8 as raw material, obtain L38 according to the preparation method of L3, white solid, yield 95%.Fusing point: 230.7-232.2 ℃.
1HNMR(300MHz,D
2O)δppm:7.91(d,1H,J=9.6Hz),7.56(d,2H,J=3.0Hz),7.31-7.50(m,3H),6.96(dd,2H,J=9.3Hz,3.0Hz),6.28(d,1H,J=9.6Hz),5.13(d,1H,J=11.1Hz),5.03(d,1H,J=11.1Hz),4.07(t,1H,J=6.3Hz),1.61-1.74(m,2H),1.18-1.31(m,2H),0.57(t,3H,J=7.2Hz)。
Example three: 2-amino-N-(2-((2-oxo-2 hydrogen-chromene-7-base oxygen base)-methyl) pyridin-3-yl) preparation of propionamide hydrochloride [L55]
Synthetic route 3
Synthetic reference literature [(a) Beyermann, the M. of intermediate 10 and 11; Bienert, M.; Niedrich, H; Carpino, L.A.; Sadat-Aalaee; D.Rapid continuous peptide synthesis via FMOC amino acid chloride coupling and4-(aminomethyl) piperidine deblocking.J Org Chem; 1990; 55; 721-728. (b) Carpino, L A.; Xia, J.S.; El-Faham, A.3-Hydroxy-4-oxo-3,4-dihydro-5-azabenzo-1,2,3-triazene.J Org Chem, 2004,69,54-61.].
The preparation of intermediate 12:
2.67g intermediate 11 and 3mL pyridine are dissolved in to 50mL dioxane, add 0.63g intermediate 10, reflux and be cooled to room temperature after 1 hour.Concentrated, use 200mL acetic acid ethyl dissolution, to wash with saturated sodium bicarbonate (50mL × 3), saturated aqueous common salt (50mL × 1) is washed.Anhydrous magnesium sulfate drying spends the night.Concentrated, obtain white solid 0.44g with re-crystallizing in ethyl acetate, productive rate 25.0%, fusing point: 136.6-138.6 ℃.
The preparation of intermediate 13:
Take intermediate 12 as raw material, obtain intermediate 13 according to the preparation method of intermediate 4, white solid, yield 17.9%, fusing point: 166.8-168.7 ℃.
The preparation of intermediate 14:
Take intermediate 13 as raw material, obtain intermediate 14 according to the preparation method of intermediate 5, white solid, yield 66.6%, fusing point: 139.3-141.7 ℃.
The preparation of intermediate 15:
0.02g7-Hydroxycoumarin is dissolved in to 10mL acetone, adds 0.02g Anhydrous potassium carbonate, stirring at room temperature, after 30 minutes, adds 0.04g intermediate 14, reacts 30 minutes.Reaction solution is poured in 100mL ethyl acetate, washed with unsaturated carbonate potassium solution (50mL × 5), saturated aqueous common salt (50mL × 1) is washed.Anhydrous magnesium sulfate drying spends the night.Concentrated, silicagel column purifying obtains white solid 0.03g, yield 66%.Fusing point 150.1-151.9 ℃.
The preparation of compound L 55
0.03g intermediate 15 is dissolved in to 8mL20% piperidines-DMF solution, and stirring at room temperature one hour, removes piperidines and DMF under reduced pressure.Residue 100mL acetic acid ethyl dissolution, washes with water (50mL × 3), and saturated aqueous common salt (50mL × 1) is washed.Anhydrous magnesium sulfate drying spends the night.Concentrated, silicagel column purifying obtains white solid 0.015g, yield 83%.Fusing point 59.0-61.3 ℃.
1H-NMR(DMSO,600MHz):δ8.39(d,1H,J=6Hz),8.34(d,1H,J=6Hz),7.99(d,1H,J=12Hz),7.64(d,1H,J=12Hz),7.44(dd,1H,J=12Hz,6Hz),7.13(d,1H,J=6Hz),7.07(dd,1H,J=12Hz,6Hz),6.31(d,1H,J=12Hz),5.38(s,2H),3.45(q,1H,J=6Hz),1.24(d,3H,J=6Hz).
Example four: the avidity of KINETIC METHOD detection probes compound to APN.
Reference literature [Huang, H.Z.; Tanaka, H.; Hammock, B.D.; Morisseau, c.Novel and highly sensitive fluorescent assay for leucine aminopeptidases.Anal biochem2009,391,11-16], measure L1, L2, the kinetic parameter of L3 to APN, result is as following table.
Note: the K of reference substrate leucyl p-Nitroaniline to APN
mbe respectively 423 μ M, 0.125U/mL, K with LOD
mbe worth that more the bright compound of novel is stronger to the avidity of APN.The consumption of APN when LOD refers to that the detection signal strength producing is more than or equal to three times of background signal.
Result shows that test-compound has good kinetic property and avidity to APN.
Example five: the reference wavelength character of detection probes compound
Add APN (0.01U) to probe compound (200 μ M), excite by λ=330nm excitation wavelength, per minute once.Judge whether to have reference wavelength character according to its collection of illustrative plates, Fig. 1 has the probe L1 of reference wavelength character and the transmitted wave spectrogram of APN effect.
Result shows that test-compound is the fluorescent probe that reference wavelength character could be identified and have by APN to a class.
Example six: the application (enzyme level) of target compound in APN inhibitor screening
Reference literature [Chen, L.Z.; Mou, J.J.; Xun, Y.Y.; Fan, H.; Xu, W.F.Design, synthesis and activity study of aminopeptidase N targeted3-amino-2-hydroxy-4-phenyl-butanoic acid derivatives.Drug Discov Ther.2011,5,61-65], using compound L 1 as APN substrate, using marketed drug bestatin as APN inhibitor, measure the IC of bestatin
50, the application of examination target compound in APN inhibitor screening.Result is as following table.
| Compound | [S]/μM | [E]/mIU | IC 50/μM |
| L1 | 22.5 | 1 | 0.35±0.05 |
| Leu-p- |
400 | 10 | 2.3±0.07 |
Result demonstration is used test-compound to test the IC obtaining
50close with traditional Leu-p-nitroanilide, illustrate that it can use in APN inhibitor screening.And it is less to use this substrate to test enzyme amount used, greatly reduces testing cost, for the high flux screening of APN inhibitor provides an effective means.
Example seven: the application (cell levels) of target compound in APN inhibitor screening
Reference fluorescent probe is affected by environment little, can PBS and substratum in measure the inhibition activity that listing APN inhibitor ubenimex is lived to ES-2 cell (APN high expression level) surface enzyme, testing method and example six are similar, result is as shown in the table.
| Compound | Test system | [S]/μM | Cell/10 4 | IC 50/μM |
| L1 | Medium | 25 | 5 | 105.4±10.8 |
| L1 | PBS | 25 | 5 | 39.1±1.5 |
| Leu-p-nitroanilide | PBS | 320 | 20 | 20±1.7 |
Result shows, tested probe can be measured exactly the IC of APN inhibitor in substratum
50.Because the survival time of cell in substratum is far longer than buffer salt solution, compared with traditional testing method (PBS system), this probe can be more exactly at cell-based screening APN inhibitor.
Claims (4)
1. one seed amino acid-fluorophore compounds, its general structure is (I) or (II), or their free form,
In formula: R
1for methyl, propyl group or 2-methylmercaptoethyl; R
2for umbelliferone; X is C or N.
2. compound according to claim 1, is characterized in that, has one of following structure:
3. the compound as described in claim 1-2 any one is as the application of the small molecules fluorescent probe of Aminopeptidase N.
4. the application in screening aminopeptidase N inhibitor as reference fluorescent probe of the compound as described in claim 1-2 any one.
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| CN1281346A (en) * | 1997-10-10 | 2001-01-24 | 西托维亚公司 | Novel fluorescent reporter molecules and their applications, including assays for caspases |
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| CN1281346A (en) * | 1997-10-10 | 2001-01-24 | 西托维亚公司 | Novel fluorescent reporter molecules and their applications, including assays for caspases |
| US20090155837A1 (en) * | 2007-12-18 | 2009-06-18 | Bong-Rae Cho | Two-photon fluorescent probes for acidic vesicles in live cells and tissue and method of imaging acidic vesicles in live cells and tissue using the same |
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