CN111187278A - Glaucocalyxin A small molecular probe and preparation method and application thereof - Google Patents
Glaucocalyxin A small molecular probe and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a glaucocalyxin A (GLA) small molecular probe and a preparation method and application thereof, belonging to the field of pharmaceutical chemistry. The glaucocalyxin small molecule probe structurally comprises three parts of Glaucocalyxin (GLA), a connecting group (Linker) and a reporting group (biotin), and has two isomer structures of a general formula I and a general formula I'. The result of the in vitro antitumor activity test of the glaucocalyxin A molecular probe designed by the invention shows that: the fluorescent probe has a good inhibition effect on tumor cells, and can be used as a small molecular probe for researching the action mechanism of glaucocalyxin A.
General formula I
Description
Technical Field
The invention relates to a glaucocalyxin A small molecular probe, a preparation method thereof and an anti-tumor effect, and belongs to the field of pharmaceutical chemistry.
Background
The discovery and structural modification of active natural products play an important role in the development of innovative drugs. According to statistics, about 50% of innovative drugs approved to be on the market in the world in 1980 are direct application of natural drugs or structural modifications of active natural products. However, most of the drugs are found through screening based on cell effect, the action target and action mechanism of the drugs are still unknown after years of clinical application, and the toxic and side effects of some drugs cause the drug development to be stopped in the clinical test stage and recalled even after entering the market, thereby causing huge economic loss and even threatening life safety. Therefore, the determination of the intracellular action target of the active small molecule compound is one of the key problems in medicinal chemistry, chemical biology, and particularly in the process of drug development.
Glaucocalyxin A (GLA) is an enantiomer-kaurane tetracyclic diterpenoid compound separated from a medicinal plant rabdosia glaucocalyx of Rabdosia of Labiatae, and is firstly separated from the plant rabdosia glaucocalyx in 1981, and the structure of the compound is identified by a spectrum method, wherein the content of the compound in dry leaves of the rabdosia glaucocalyx is up to 1.03% (Zhang Yuan Tung, Sadong Xuan, Shaming, et al. Chinese traditional medicine journal, 1991,16(11): 679.).
The research shows that the active center group of the glaucocalyxin A is α -unsaturated ketone unit, the in vitro anti-tumor experiment shows that the glaucocalyxin A has good effect of inhibiting the proliferation of tumor cell strains such as prostate cancer DU-145, rectal cancer Lovo, human promyelocytic leukemia cell HL-60 and the like, particularly is most sensitive to colorectal cancer (Lovo) and non-hormone-dependent prostate cancer (DU-145) cells, the morphological observation shows that the glaucocalyxin A with the concentration of 50 and 25 mu mol/L can induce the apoptosis of the cells of the Lovo tumor cell, the Chinese patent 201210015481.4 'a glaucocalyxin A derivative and a preparation method and application thereof' report that the glaucocalyxin A acetal derivative has obvious proliferation inhibition effect on liver cancer, lung cancer, breast cancer, cervical cancer, esophageal cancer, choriocarcinoma, prostate cancer, rectal cancer, acute myelocytic leukemia or chronic myelocytic leukemia, and is a promising anti-tumor activity lead compound, and the prospect of the glaucocalyxin A prodrug II is identified by the Adenoxin II and the Adenoxin B reductase target of the tetracyclic leukemia.
In conclusion, although glaucocalyxin A has a good anti-tumor effect in vitro, the research on the structure-activity relationship is less, the design and synthesis of an active compound are difficult points in the research center, and the action target and the action mechanism are not clear. Therefore, a small molecular probe with biological activity needs to be designed and prepared, and the method has important significance for researching the action target of the glaucocalyxin A medicament and an anti-tumor mechanism. As a commonly used labeling group, no experiment report that biotin is connected with glaucocalyxin A to form a biotin-glaucocalyxin A biotin labeled probe so as to adjust an action target point is found at present.
Disclosure of Invention
In order to overcome the defects of the existing action target and antitumor mechanism research of the glaucocalyxin A, the invention aims to design and synthesize a small molecular probe of the glaucocalyxin A, and the glaucocalyxin A and a biotin report group are connected to form a novel small molecular probe by introducing a connecting group consisting of amphiphilic polyethylene glycol (PEG) and dicarboxylic acid, wherein the active center, hydroxyl group and other active groups of α -unsaturated ketone of the glaucocalyxin A are reserved, and polyethylene glycol and diacid groups which are low in toxicity and easy to metabolize are introduced to obtain the molecular probe with good water solubility and good antitumor activity, so that a template molecule is provided for further researching the action mechanism of the glaucocalyxin A.
The technical scheme of the invention is as follows:
1. the glaucocalyxin A small molecule probe has a structure of a general formula I, I':
n is 1, 2 or 3; m is 2 or 3.
2. The glaucocalyxin A small molecular probe is preferably a compound shown by 1-6 and an isomer thereof:
3. the invention provides a synthesis method of a glaucocalyxin A molecular probe, which comprises the following reaction processes:
1)biotin-PEG-NH2the preparation of (1):
(1) dissolving polyethylene glycol (PEG) and p-toluenesulfonyl chloride or methanesulfonyl chloride in an organic solvent, performing substitution reaction under an alkaline condition in an ice bath, extracting with the organic solvent after the reaction is finished, drying, filtering and concentrating to obtain a midbody substituted by sulfonyl groups at two ends. The acyl chloride is preferably p-toluenesulfonyl chloride, and the molar ratio of the acyl chloride to the polyethylene glycol is 1: 2-1: 4, preferably 1: 2.2. The alkali is one or more of potassium hydroxide, sodium hydroxide, potassium carbonate, sodium carbonate, pyridine, triethylamine, DMAP (dimethyl acetamide), diisopropylethylamine and N-methylmorpholine;
(2) dissolving the p-toluenesulfonate derivative and sodium azide in an organic solvent, heating to perform substitution reaction, extracting with the organic solvent after the reaction is finished, drying, filtering and concentrating to obtain azide derivatives at two ends;
the organic solvent in the steps (1) and (2) is one or more selected from N, N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, dichloromethane, methanol, acetonitrile, ethyl acetate, petroleum ether, dioxane and ethanol.
(3) The azide derivatives at two ends and a reducing agent are dissolved in an organic solvent and undergo reduction reaction under the acidic condition and the inert gas atmosphere. After the reaction is finished, extracting with an organic solvent, drying, filtering and concentrating to obtain the amino azide derivative; the inert gas is selected from nitrogen or argon; the reducing agent is one or more selected from triphenylphosphine, hydrogen/palladium carbon, sodium borohydride, tetrabutylammonium borohydride, lithium aluminum hydride and dimethylamino borane.
(4) Dissolving the amino azide derivative and biotin in an organic solvent, reacting at room temperature overnight, spin-drying the solvent, and carrying out column chromatography to obtain biotin-PEG-N of the biotin azide derivative3(ii) a The organic solvent is one or two of N, N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, dichloromethane, methanol, acetonitrile, ethyl acetate, petroleum ether, dioxane, diethyl ether and ethanol;
(5)biotin-PEG-N3reducing azide into amino under the reducing condition, and obtaining biotin-PEG-NH after the solvent is dried by spinning after the reaction is finished2. The reducing agent is one or more selected from triphenylphosphine, hydrogen/palladium carbon, sodium borohydride, tetrabutylammonium borohydride, lithium aluminum hydride and dimethylamino borane.
2) The preparation of the compounds of the general formulae I and I' comprises the following steps:
(1) dissolving glaucocalyxin A and acid anhydride in an organic solvent, heating and refluxing under an alkaline condition to perform an esterification reaction to obtain a derivative of terminal carboxyl of 7 α and 14 β.
(2): dissolving the obtained glaucocalyxin A terminal carboxyl derivative and HOBT (1-hydroxybenzotriazole) in an organic solvent, and adding a reporter group biotin-PEG-NH2The obtained molecular probe containing the amide group is a molecular probe with two isomers at positions 7 α and 14 β of the glaucocalyxin A, because the glaucocalyxin A7 α and 14 β carboxyl derivatives can be subjected to tautomerization at positions 7 α and 14 β at normal temperature, and the catalyst is EDCI (1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride);
in the step (1), the organic solvent is THF or CH2Cl2DMF, DMSO; the acid anhydride is preferably succinic anhydride or glutaric anhydride, and the molar ratio of the glaucocalyxin A to the acid anhydride is 1:2. The heating reflux time of the reaction is preferably 3-4 h.
In the step (2), the biotin report group (RG-PEG-NH)2) The preferable molar ratio of the derivative of the terminal carboxyl group of the glaucocalyxin A7 α and 14 β to the EDCI and the HOBT is 1:1:1.5:1.2, and the organic solvent is DMF, THF and CH2Cl2One or more of the above; the reaction time is 6-12 h at room temperature.
In the steps (1) and (2), the alkali is selected from one of triethylamine, pyridine, DMAP, N-methylmorpholine and tetramethylethylenediamine.
4. The raw material is preferably one or more of the following compounds:
screening out a compound with good activity as a small molecular probe to research the antitumor mechanism of the glaucocalyxin A.
It can also be used in tumor inhibiting medicine. In particular to the preparation of the drugs for liver cancer, lung cancer, breast cancer, cervical cancer and esophageal cancer.
The invention has the following advantages:
1. a series of novel series of glaucocalyxin A small molecular probes taking PEG chains as connecting groups and biotin as a reporting group are synthesized.
2. The glaucocalyxin A small molecular probe has good water solubility and anti-tumor activity, provides a basis and a template molecule for 'fishing' the glaucocalyxin A target spot and researching an action mechanism, and has good application prospect.
3. The synthetic method is simpler and more convenient, the reaction condition is mild, and a better method is provided for the design of a natural product complex long-chain molecular probe.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The following examples describe the preparation process, all chemical reagents used were analytically pure, unless otherwise noted.
Example 1: biotin-PEG-NH2Preparation of
1. Adding 9.4mmol of diethylene glycol into a round-bottom flask, adding 20mL of anhydrous dichloromethane, stirring and dissolving in ice bath, adding 3.6g (18.8mmol) of p-toluenesulfonyl chloride, adding ground potassium hydroxide in batches, and reacting in ice bath for 3 h. After the reaction is stopped, water and dichloromethane are added for extraction, an organic layer is reserved, the organic layer is washed by saturated saline solution in sequence, dried for 3 hours by anhydrous sodium sulfate, filtered and concentrated to obtain white solid polyethylene glycol derivatives with two end-to-tosyl (Ts) groups substituted.
2. 1g (2.4mmol) of the Ts-substituted polyethylene glycol compound obtained in the above step was charged into a 50mL round-bottomed flask, and DMF15mL was added, followed by addition of 0.5g (7.2mmol) of sodium azide and reaction at 90 ℃ for 10 hours. And after the reaction is finished, adding ethyl acetate for extraction, washing with distilled water and saturated sodium chloride solution, reserving an organic layer, drying for 3 hours by using anhydrous sodium sulfate powder, filtering, and concentrating under reduced pressure by using a rotary evaporator to obtain a faint yellow oily polyethylene glycol derivative with azide substitution at two ends.
3. 1.3g (8mmol) of the azide compound in the previous step is taken and added into a round-bottom flask, 15mL of 0.65M phosphoric acid is added, the mixture is stirred uniformly, 1.8g (6.7mmol) of triphenylphosphine is dissolved in 10mL of diethyl ether, the solution is slowly dripped into the reaction solution by a speed-regulating dropping funnel, and the reaction is carried out for 24 hours at room temperature. The whole operation and reaction are carried out under the protection of nitrogen. And after the reaction is finished, extracting with a small amount of diethyl ether for three times, reserving a water layer, steaming until no diethyl ether smell exists, adding 1.2g of potassium hydroxide, refrigerating for 16h at 4 ℃, and filtering out the precipitated triphenylphosphine oxide. Adding potassium hydroxide 4.8g into the water layer, extracting with dichloromethane for more than 10 times, combining the organic layers, washing with equal amount of distilled water and saturated sodium chloride solution, retaining the organic layer, using anhydrous sodium sulfate powder, filtering, and concentrating under reduced pressure with a rotary evaporator to obtain light yellow oily amino azide derivative.
4. 150mg (1.2mmol) of aminoazide derivative was charged in a 25mL round-bottomed flask, 3mL of DMF was added, and 307mg (0.9mmol) of biotin succinimide was added thereto, followed by reaction at room temperature for 12 hours. After the reaction is finished, DMF is evaporated to dryness, the mixture is purified by silica gel column chromatography,performing column chromatography separation on dichloromethane/methanol/acetic acid at a ratio of 200:10:0.1 to obtain a white solid: biotin-PEG-N3。
5、biotin-PEG-N3200mg (0.6mmol) and 318mg Pd/C (3.0mmol) in a round bottom flask, 5mL MeOH, H2Reacting at room temperature for 12h under the atmosphere. After the reaction is finished, Pd/C is removed by filtration, and oily biotin-PEG-NH is obtained by vacuum concentration2。
Example 2: glaucocalyxin A carboxyl derivative
Adding 2mL of DMF (dimethyl formamide) into a dry round-bottom flask for dissolving, then adding 1mL of pyridine into a reaction system for reacting for 3h, cooling to room temperature, adding 30mL of HCl with the mass percentage of 10%, extracting 20mL of DMF with ethyl acetate, combining extract liquor, washing with saturated saline solution, drying with anhydrous sodium sulfate, concentrating under reduced pressure, separating by silica gel column chromatography, and eluting with petroleum ether/ethyl acetate/acetic acid (100:100:1) to obtain the glaucocalyxin carboxyl derivative as a white solid.
Example 3: preparation of series glaucocalyxin A small molecular probes
Dissolving the carboxyl derivative at the tail end of the glaucocalyxin A and HOBT in DMF, reacting for a certain time under the condition of ice-water bath, and then adding EDCI and biotin reporter group (biotin-PEG-NH)2) And triethylamine is added, stirring reaction is carried out at room temperature, a certain amount of distilled water is added after the reaction is stopped, dichloromethane is added for extraction, saturated NaCl solution is washed, anhydrous sodium sulfate is added for drying, filtration, reduced pressure concentration and silica gel column chromatography separation are carried out, and the eluent is dichloromethane/methanol according to the volume ratio of 15/1, so that the glaucocalyxin A small molecule probe target substance is obtained.
The glaucocalyxin A small molecular probe isomer compounds 1-6 are obtained through the method.
Compound 1 and 1' isomers: yield 65%, Isomer A + B1H NMR(400MHz,CDCl3)δ7.02–6.90(m,2H),6.80–6.76(m,2H),6.48(s,2H),6.11(s,2H),5.76(s,2H),5.40(s,2H),5.34(dd,J＝11.9,3.6Hz,2H),4.79(s,1H),4.46(dd,J＝6.9,5.2Hz,2H),4.27(dd,J＝7.4,4.6Hz,2H),3.49–3.47(m,8H),3.38–3.33(m,6H),3.13–3.08(m,4H),3.04(s,2H),2.86(dd,J＝13.4,5.3Hz,4H),2.68(d,J＝12.8Hz,4H),2.59–2.52(m,4H),2.43–2.33(m,4H),2.32–2.23(m,2H),2.23–2.10(m,10H),1.99–1.79(m,12H),1.74–1.66(m,6H),1.63–1.53(m,6H),1.49–1.36(m,10H),1.19(d,J＝15.4Hz,4H),1.09–1.07(m,6H),1.05–1.03(m,6H),1.02(s,6H).13C NMR(101MHz,CDCl3)δ216.95,216.53,206.93,205.70,173.90,173.84,173.30,173.08,172.79,171.16,146.99,146.41,118.78,118.11,77.43,75.75,75.53,74.19,72.58,69.91,69.85,69.79,69.72,62.31,62.02,61.51,60.41,55.87,54.43,53.64,53.41,52.11,51.30,46.99,46.76,45.82,44.43,40.74,39.30,39.27,39.20,39.17,39.13,39.06,38.27,38.19,35.92,35.38,35.16,33.82,33.72,33.64,33.28,32.23,31.00,29.87,28.65,28.37,28.36,28.18,28.06,27.77,26.57,25.80,25.77,21.24,21.19,20.96,20.80,18.70,18.60,18.23,18.11.HR-ESI-MS(m/z)calcd for C39H58N4O9S,[M+Na]+781.3817,found 781.2826.
Compound 2 and 2' isomers: yield 61%, Isomer A + B1H NMR(400MHz,CDCl3)δ7.04–7.01(m,2H),6.98–6.91(m,2H),6.51(s,2H),6.10(d,J＝4.5Hz,2H),5.92(s,1H),5.77(s,2H),5.39(d,J＝5.6Hz,2H),4.78(s,1H),4.46(dd,J＝7.4,4.9Hz,2H),4.27(dd,J＝7.6,4.7Hz,2H),4.11(dd,J＝12.0,3.6Hz,2H),3.56(d,J＝2.7Hz,8H),3.53–3.47(m,8H),3.43–3.36(m,6H),3.10(dd,J＝11.7,7.3Hz,2H),3.04(s,2H),2.86(dd,J＝13.6,5.6Hz,4H),2.75–2.68(m,4H),2.59–2.33(m,6H),2.31–2.27(m,2H),2.24–2.05(m,10H),1.97–1.79(m,10H),1.72–1.52(m,12H),1.49–1.30(m,10H),1.18(d,J＝15.9Hz,6H),1.10–1.07(m,6H),1.05–1.03(m,6H).13C NMR(101MHz,CDCl3)δ216.98,216.57,206.60,205.45,173.67,173.61,173.18,173.08,172.62,171.12,164.30,164.29,146.92,146.45,118.75,117.91,77.43,75.70,75.54,74.20,72.56,70.25,70.22,70.15,70.08,70.04,70.01,62.22,61.99,61.96,61.45,60.43,55.76,54.48,53.38,52.12,51.27,46.98,46.96,46.75,45.82,44.43,40.66,39.41,39.36,39.30,39.17,39.03,38.74,38.72,38.27,38.18,36.10,36.05,35.44,35.13,33.81,33.72,33.63,33.33,32.23,31.00,29.87,28.68,28.34,28.22,28.06,27.75,26.58,25.74,21.18,21.12,20.94,20.78,18.69,18.58,18.42,18.22,18.09,17.92,9.42,9.38.HR-ESI-MS(m/z)calcd for C41H62N4O10S,[M+Na]+825.4079,found 825.4078.
Preparation of 3 and 3' isomers of Compound 71% yield Isomer A + B1H NMR(400MHz,CDCl3)δ6.99–6.89(m,2H),6.82–6.66(m,2H),6.54(s,2H),6.09(d,J＝4.1Hz,2H),5.91(s,1H),5.73(s,2H),5.37(d,J＝5.4Hz,2H),4.77(s,1H),4.46–4.43(m,2H),4.27–4.24(m,2H),4.10(dd,J＝12.0,3.6Hz,2H),3.58(d,J＝2.8Hz,14H),3.52–3.48(m,6H),3.39–3.33(m,6H),3.09(q,J＝7.3Hz,4H),3.03(s,2H),2.85(dd,J＝12.7,4.6Hz,4H),2.70(dd,J＝12.8,2.7Hz,2H),2.58–2.32(m,6H),2.30–2.26(m,2H),2.24–2.09(m,10H),1.98–1.76(m,12H),1.72–1.55(m,14H),1.47–1.32(m,10H),1.18(d,J＝15.1Hz,6H),1.08–1.04(m,6H),1.03–1.01(m,6H).13C NMR(101MHz,CDCl3)δ216.98,216.54,206.51,205.35,173.69,173.65,173.08,172.55,171.10,164.27,146.87,146.42,118.69,117.81,77.43,75.63,75.50,74.14,72.47,70.59,70.50,70.47,70.20,70.13,70.10,70.04,62.14,61.95,61.40,60.37,55.81,54.44,53.36,52.08,51.25,46.94,46.71,45.80,44.39,40.65,39.33,39.29,39.25,39.12,39.00,38.23,38.13,36.03,35.41,35.08,33.78,33.70,33.59,33.45,32.20,30.98,29.81,28.68,28.39,28.23,28.01,27.71,26.53,25.78,21.13,21.04,20.90,20.73,18.63,18.51,18.17,18.05.HR-ESI-MS(m/z)calcd for C43H66N4O11S,[M+Na]+869.4341,found 869.4272.
Preparation of Compounds 4 and 4' isomers in 60% yield Isomer A + B1H NMR(400MHz,CDCl3)δ7.10–7.04(m,2H),6.45(s,1H),6.10(s,1H),5.94(s,1H),5.76(s,1H),5.39(d,J＝7.3Hz,1H),5.33(dd,J＝11.7,3.6Hz,1H),4.79(s,1H),4.48(dd,J＝7.5,4.9Hz,2H),4.34–4.24(m,2H),4.10(dd,J＝11.9,3.7Hz,1H),3.50(d,J＝4.1Hz,7H),3.42–3.30(m,7H),3.16–3.11(m,3H),3.06(s,1H),2.92–2.59(m,15H),2.56–2.34(m,10H),2.22–2.18(m,4H),2.12–2.04(m,1H),2.02–1.97(m,1H),1.93–1.72(m,9H),1.69–1.54(m,10H),1.49–1.32(m,9H),1.21(s,3H),1.15(s,3H),1.10–1.06(m,6H),1.04(s,3H),1.02(s,3H),13C NMR(101MHz,CDCl3)δ217.13,216.66,206.48,205.32,174.22,174.14,172.83,172.54,171.93,171.19,164.40,146.88,146.41,118.76,117.91,77.43,75.84,74.13,72.47,69.92,69.88,69.62,62.04,61.47,60.40,55.93,54.49,53.49,52.12,51.32,46.94,46.78,45.87,44.41,40.73,39.51,39.40,39.21,39.11,39.04,38.26,38.18,35.84,33.79,33.66,32.23,31.08,30.77,30.75,30.53,30.50,30.08,29.86,28.93,28.34,28.14,27.98,27.82,26.45,25.74,21.15,20.95,18.65,18.57,18.20,18.08.HR-ESI-MS(m/z)calcd for C38H56N4O9S,[M+Na]+767.3660,found 767.3663.
Preparation of 5 and 5' isomers in 75% yield, Isomer A + B1H NMR(400MHz,CDCl3)δ7.07–6.98(m,4H),6.51(s,2H),6.09(s,2H),5.93(s,1H),5.83(d,J＝11.7Hz,2H),5.38(d,J＝5.9Hz,2H),4.83–4.71(m,1H),4.48–4.45(m,2H),4.29–4.26(m,2H),3.56(d,J＝2.0Hz,8H).3.53–3.48(m,8H),3.39–3.33(m,8H),3.13–3.10(m,4H),3.05(s,2H),2.87–2.83(m,4H),2.70(d,J＝11.8Hz,4H),2.63–2.32(m,12H),2.18(t,J＝7.3Hz,4H),1.92–1.86(m,4H),1.80–1.72(m,4H),1.71–1.56(m,10H),1.44–1.30(m,10H),1.20(s,4H),1.14(s,6H),1.09–1.05(m,6H),1.03(s,6H).13C NMR(101MHz,CDCl3)δ217.15,216.70,206.49,205.26,174.67,173.96,173.91,172.83,172.34,171.78,171.10,164.46,164.43,162.81,146.83,146.35,118.71,117.92,77.43,75.78,74.09,72.44,70.22,70.13,70.08,70.04,70.01,62.00,61.43,61.41,60.42,55.80,54.49,53.45,52.08,51.28,46.91,46.89,46.75,45.83,44.35,40.83,40.64,39.44,39.36,39.28,39.07,39.01,38.65,38.22,38.15,37.97,36.70,35.94,35.91,33.77,33.71,33.63,32.20,31.62,31.05,30.72,30.35,29.99,29.82,28.86,28.30,28.13,27.95,27.78,26.39,25.68,21.17,21.12,21.04,20.92,18.61,18.53,18.40,18.39,18.16,18.05.HR-ESI-MS(m/z)calcd for C40H60N4O10S,[M+Na]+811.3922,found 811.3934.
Preparation of 6 and 6' isomers in 58% yield, Isomer A + B1H NMR(400MHz,CDCl3)δ7.03–6.98(m,4H),6.51(s,2H),6.09(s,2H),5.93(s,1H),5.76(s,2H),5.37(d,J＝7.0Hz,2H),4.46(dd,J＝7.6,4.8Hz,2H),4.27(dd,J＝7.6,4.6Hz,2H),4.09(dd,J＝11.9,3.8Hz,1H),3.58(d,J＝4.2Hz,16H),3.53–3.48(m,8H),3.40–3.32(m,8H),3.12–3.07(m,4H),3.05(s,2H),2.88–2.83(m,2H),2.70(dd,J＝12.7,3.8Hz,2H),2.53–2.37(m,12H),2.17(t,J＝7.4Hz,4H),2.09–2.03(m,2H),1.99–1.86(m,4H),1.83–1.70(m,6H),1.67–1.55(m,10H),1.47–1.29(m,10H),1.14(s,6H),1.09–1.05(m,6H),1.03(s,6H).13C NMR(101MHz,CDCl3)δ217.13,216.66,206.41,205.20,173.83,173.79,172.80,172.19,171.62,171.02,164.35,146.85,146.38,118.67,117.84,77.43,75.77,74.09,72.45,70.46,70.17,70.11,70.09,70.03,70.00,62.00,61.39,60.40,55.81,54.58,54.48,53.45,52.19,52.10,51.29,46.92,46.76,45.84,44.37,43.84,40.85,40.65,39.43,39.34,39.24,39.07,39.00,38.65,38.23,38.16,37.99,36.67,35.96,33.77,33.71,33.63,32.20,31.61,31.04,30.70,30.37,30.32,29.97,29.82,29.16,28.86,28.36,28.19,27.97,27.79,26.41,25.75,25.20,21.19,21.11,21.03,20.92,18.61,18.54,18.40,18.16,18.06,17.90,9.36.HR-ESI-MS(m/z)calcd for C42H64N4O11S,[M+Na]+855.4185,found855.4196.
Example 4: pharmacodynamic experiments of the glaucocalyxin A small molecule probe comprise: cancer cell in vitro inhibitory Activity test
Pharmacodynamic experiments aiming at the growth inhibition effect of HepG2 cells of human liver cancer and Hela cells of human cervical carcinoma.
1. Drugs and reagents: test samples, DMEM, 1640 medium, 10% inactivated bovine serum (FBS), PBS solubles, dimethyl sulfoxide (DMSO), triple solutions (10% SDS + 5% isopropanol +12mM HCl), thiazole blue (MTT), 5-FU (positive control).
2. The instrument comprises the following steps: clean bench, CO2Incubator, multifunctional inverted microscope, centrifuge, and 96-well culture plate with automatic microplate reader.
3. Cell lines: human liver cancer HepG2 tumor cell and human cervical carcinoma Hela cell.
4. Sample preparation: taking 1-6 of the glaucocalyxin A small molecular probe prepared by the method, dissolving a compound by DMSO, ultrasonically dissolving the compound at the concentration of 100mM, and storing the obtained medicinal solution at the temperature of-20 ℃.
5. Experimental methods
Step 1: a drug-MTT assay for adherent cells, further comprising:
the adherent cells comprise human liver cancer HepG2 tumor cells and human cervical carcinoma Hela cells.
Step 1.1: collecting cells in logarithmic growth phase, suspending with complete DMEM medium, and adjusting cell suspension concentration to 3 × 104Perml, inoculate 96 well cell culture plates, 100 mL/well. Standing at 37 deg.C for 5% CO2Culturing in an incubator for 24 hours, removing the supernatant, adding a fresh complete DMEM culture medium, adding 90 mL/hole, adding drug solutions to be tested with different concentrations, 10 mL/hole, and arranging 3 multiple holes per concentration; adding 10mL of DMEM culture medium into the blank hole per hole; background wells were filled with 100 mL/well of medium without cells.
Step 1.2: standing at 37 deg.C for 5% CO2Incubate for 48 hours.
Step 1.3: 100uLMTT solution (0.5mg/mL, incomplete DMEM medium) was added to each well and incubation in the incubator continued for 4 hours.
Step 1.4: terminating the culture after 4 hours, discarding the supernatant, adding 150mL of dimethyl sulfoxide into each well, and placing on a shaking table to shake at low speed for 5min to fully dissolve the crystals.
Step 1.5: the absorbance of each well was measured at 570nm in an enzyme linked immunosorbent assay.
6. The experimental results are as follows: the glaucocalyxin A small molecular probe of the invention has the following growth inhibition effect on human liver cancer HepG2 cells and human cervical carcinoma Hela cells as shown in the following table:
table 1: result of inhibition of small molecular probe of glaucocalyxin A on proliferation of cancer cell strain
7. The experimental results show that: the glaucocalyxin A small molecular probe obtained by the invention shows obvious cell proliferation inhibition activity on human liver cancer HepG2 cells and human cervical carcinoma Hela cell strains.
8. And (4) conclusion: the glaucocalyxin A small molecular probe prepared by the invention has the application prospect of anticancer drugs, and can be applied to the research of glaucocalyxin A 'fishing' target spots and action mechanisms thereof.
The foregoing is illustrative of specific embodiments of the present invention and reference to reagents, equipment, procedures and the like not specifically described herein is to be understood as being modified in light of the common and routine experimentation in the art.
Claims (6)
1. A small molecular probe of a glaucocalyxin A derivative is characterized by having a structure shown in a general formula 1:
n＝1,2,3；m＝2,3。
2. the small molecule probe of glaucocalyxin A derivative of claim 1, wherein: selected from the following compounds:
3. a small molecular probe of a glaucocalyxin A derivative is characterized by having a structure shown in a general formula I':
n＝1,2,3；m＝2,3。
4. the small molecule probe of glaucocalyxin A derivative of claim 3, wherein: selected from the following compounds:
5. a method for preparing the glaucocalyxin a small molecule probe of claim 1 or 3, comprising the steps of:
(1) dissolving glaucocalyxin A and anhydride with a structure shown as a formula 7 in an organic solvent, adding alkali, heating and refluxing under an alkaline condition to perform an esterification reaction to obtain derivative isomers of terminal carboxyl groups of 7 α and 14 β shown as a formula 8;
(2): dissolving the isomer of the end carboxyl derivative of glaucocalyxin A shown in formula 8 and HOBT (1-hydroxybenzotriazole) in an organic solvent, and adding a reporter group biotin-PEG-NH shown in formula 92Adding alkali into the catalyst, and carrying out an amide reaction under an alkaline condition to synthesize a molecular probe general formula I or I' containing an amide group; the catalyst is selected from EDCI (1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride);
in the step (1), the organic solvent is THF or CH2Cl2DMF, DMSO; the acid anhydride is selected from succinic anhydride or glutaric anhydride;
in the step (2), the organic solvent is DMF, THF, CH2Cl2One or more of the above;
in the steps (1) and (2), the alkali is selected from one of triethylamine, pyridine, DMAP, N-methylmorpholine and tetramethylethylenediamine.
6. The use of the glaucocalyxin A molecular probe of claim 1 or 2, wherein the glaucocalyxin A molecular probe is used as a molecular probe of an antitumor drug glaucocalyxin A.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101993359A (en) * | 2009-08-14 | 2011-03-30 | 上海金昊药业开发有限公司 | Fatty acid derivative of glaucocalyxin A, as well as preparation method and applications thereof |
| CN101993370A (en) * | 2009-08-14 | 2011-03-30 | 上海金昊药业开发有限公司 | Glaucocalyxin A acid ester derivative as well as preparation method and application of Glaucocalyxin A acid ester derivative |
| CN103601641A (en) * | 2013-11-01 | 2014-02-26 | 中国人民解放军第二军医大学 | Glaucocalyxin derivatives and application thereof in preparation of antitumor drugs |
-
2020
- 2020-01-16 CN CN202010044590.3A patent/CN111187278A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101993359A (en) * | 2009-08-14 | 2011-03-30 | 上海金昊药业开发有限公司 | Fatty acid derivative of glaucocalyxin A, as well as preparation method and applications thereof |
| CN101993370A (en) * | 2009-08-14 | 2011-03-30 | 上海金昊药业开发有限公司 | Glaucocalyxin A acid ester derivative as well as preparation method and application of Glaucocalyxin A acid ester derivative |
| CN103601641A (en) * | 2013-11-01 | 2014-02-26 | 中国人民解放军第二军医大学 | Glaucocalyxin derivatives and application thereof in preparation of antitumor drugs |
Non-Patent Citations (7)
| Title |
|---|
| MICHAEL MCPHERSON, ET AL.: "Drug receptor identification from multiple tissues using cellular-derived mRNA display libraries", 《CHEMISTRY & BIOLOGY》 * |
| MUH-CHING YEE, ET AL.: "A Cell-permeable, Activity-based Probe for Protein and Lipid Kinases", 《JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| 张煚: "小分子探针在确定活性化合物的生物靶点中的应用", 《药学学报》 * |
| 李显伦: "蓝萼甲素生物素标记物合成", 《中国优秀博硕士学位论文全文数据库(硕士)医药卫生科技辑》 * |
| 沈晓丹: "蓝萼甲素体外细胞毒活性研究", 《中华中医药学刊》 * |
| 程懂懂: "蓝萼甲素分子探针的设计合成及其抗肿瘤活性研究", 《中国优秀博硕士学位论文全文数据库(硕士)工程科技Ⅰ辑》 * |
| 程懂懂等: "原位Cu(I)催化的叠氮-炔环加成反应合成长链双功能分子：蓝萼甲素与生物素的长链轭合分子的制备", 《有机化学》 * |
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