CN109897024A - The preparation method and its antitumor application thereof of genistein amino acid ester derivative - Google Patents

Abstract

The present invention relates to the preparation of genistein amino acid ester derivative shown in chemical structure Formulas I and its preparing the application in anti-human colon cancer cell HCT-116 drug:R is selected from Formulas I: the group (a-s) selected from rectangular structure；N is selected from: 3 or 5；

Description

The preparation method and its antitumor application thereof of genistein amino acid ester derivative

Technical field

The present invention relates to the new applications of compound, the specifically preparation method and its work of genistein amino acid ester derivative For the application of anti-tumor drug.

Background technique

Cancer is the second largest cause of death in the world.The Side effect of chemotherapeutics typically results in most of cancers and suffers from Person's stopped treatment.Therefore there is an urgent need to seek safer and more effective chemotherapeutics.It is well known that many anti-tumor drugs are all It is to be isolated from natural plants.Flavonoids is a kind of natural products being widely present in nature, is had extensive raw Object activity, such as anti-inflammatory, anti-oxidant, antitumor etc..Genistein (siskin isoflavonoid) is one kind of flavonoids, by a large amount of Scholar is the study found that it has the effects that antitumor, anti-oxidant, anti-osteoporosis and estrogen-like.However, due to its water solubility Poor, intestinal absorption is few, and bioavilability is low, is easy metabolic inactivation in vivo.In order to improve its pharmacological activity, it is tied Structure modification and transformation, the novel drug candidate for obtaining high-efficiency low-toxicity are of great significance.

Protein is carry for human life activity, and ultimate constituent of the amino acid as protein, participates in body Interior various physiological activities.Amino acid itself has many advantages, such as that structure diversity, functional group be abundant, hypotoxicity.In addition, amino Acid is the substrate of various enteral peptide transporters, can improve the oral availability of drug.For example, valine is oligopeptides transhipment The substrate of albumen (PepT1), therefore valine can enter small enteral in conjunction with PepT1.In recent years, it has been reported that will Drug and amino acid are combined into L-threonine derivatives of high therapeutic index, can be improved the oral availability and anti-tumor activity of drug.For example, figured silk fabrics Ganciclovir (the Valine ester prodrugs of Ganciclovir), oral availability improves about 10 times than Ganciclovir；With amino Acid esters modification 5 FU 5 fluorouracil improves its anti-breast cancer activity.

Therefore, amino acid is introduced into genistein structure by the present invention, is obtained a series of genistein amino-acid esters and is spread out Biology.Preliminary pharmacodynamic experiment shows that derivative shows good antiproliferative activity to colon cancer cell HCT116, and hinders The stagnant cancer cell division period is in the G1 phase.

Summary of the invention

It is an object of that present invention to provide the preparation method of compound shown in chemical structure Formulas I and its preparing anti-human colon cancer Application in HCT-116 drug:

R is selected from Formulas I: selected from any of following a-s building stone；N is selected from: 3 or 5.

A method of above-mentioned chemical structure Formulas I genistein amino acid ester derivative being prepared, it consists essentially of as follows Step:

Genistein is dissolved in n,N-Dimethylformamide (DMF) by step 1, adds KOH, genistein and KOH substance The ratio between amount is 1:1,60 DEG C of reaction 1h.4- bromobutyrate or 6- bromocaproic acid ethyl ester and KI, genistein and bromine are added after 1h The ratio between compound and the amount of substance of KI are as follows: 1:2:0.1 reacts 4~6h.Reaction solution is poured into a small amount of distilled water, is filtered, After obtained filter cake is dissolved with tetrahydrofuran, silica gel is added, is purified by column chromatography, obtains intermediate product 1 or 2:

Compound 1 or 2 is added in about 8mL water step 2, adds 1mol/LKOH solution, compound 1 or 2 and KOH is molten The ratio between amount of substance of liquid are as follows: 1~2mL methanol hydrotropy is added in 1:4, and 60 DEG C of reactions 1~2h, adjusting reaction solution pH are 2-3, are obtained To intermediate product 3 or 4；

Compound 3 or 4 is added to by 1- ethyl-(3- dimethylaminopropyl) carbodiimide by step 3 under condition of ice bath Hydrochloride (EDCl), I-hydroxybenzotriazole (HOBt) composition reaction system in, compound 3 or 4 with EDCl and HOBt object The ratio between amount of matter are as follows: the amino acid methyl ester hydrochloride as shown in a-s structure, acid binding agent N, N '-is added in 1:3:3, stirring at normal temperature 4h Diisopropylethylamine (DIPEA), 4- dimethylamino pyrroles (DMAP), compound 3 or 4 with amino acid methyl ester hydrochloride and The ratio between the amount of DIPEA and DMAP substance are as follows: 1:3:3:2 reacts at room temperature 12h.It obtains shown in the structural formula I as described in right 1 Genistein amino acid ester derivative.

Wherein: R is one kind with a-s building stone；N is 3 or 5.

It is an object of that present invention to provide genistein amino acid ester derivatives shown in Formulas I to be selected from: (4- ((5- hydroxyl -3- (4- hydroxy phenyl) -4- oxo -4H- chromene -7- base) oxygroup) bytyry) tryptophan methyl ester is thin in anti-human colon cancer HCT-116 The value-added activity of born of the same parents.

The invention has the following advantages over the prior art:

The present invention prepares genistein amino acid ester derivative for the first time, and finds (4- ((5- hydroxyl -3- (4- hydroxy phenyl) -4- Oxo -4H- chromene -7- base) oxygroup) bytyry) and tryptophan methyl ester have for human colon cancer cell (HCT-116) it is good anti- Proliferation activity, and block the cancer cell division period in the G1 phase.

Detailed description of the invention

Fig. 1 is the precursor structure of compound prepared by the present invention, and wherein R is selected from any with following a-s building stone Kind；N is selected from: 3 or 5.

Fig. 2 is HCT-116 cell after compound 5j handles 48h, and obvious become occurs for HCT-116 cell cycle distribution Change.

Fig. 3 is cycle result statistics histogram.

Specific embodiment

Following embodiment is intended to illustrate invention rather than limitation of the invention further.

Embodiment 1

4- ((5- hydroxyl -3- (4- hydroxy phenyl) -4- oxo -4H- chromene -7- base) oxygroup) ethyl butyrate (compound 1)

Genistein (2.0g, 7.4mmol) and KOH (0.415g, 7.4mmol) is dissolved in DMF, 60 DEG C of reaction 1h.It adds 4- bromobutyrate (2.11mL, 14.8mmol) and KI (122.8mg, 0.74mmol) react 4~6h.Reaction solution is poured into few It measures in distilled water, suction filtration takes filter cake, and silica gel column chromatography purifying obtains off-white powder.Yield: 81%.¹HNMR(400MHz, DMSO) δ 12.95 (s, 1H), 9.60 (s, 1H), 8.41 (s, 1H), 7.40 (d, J=8.5Hz, 2H), 6.83 (d, J=8.6Hz, 2H), 6.66 (d, J=2.1Hz, 1H), 6.40 (d, J=2.1Hz, 1H), 4.12 (t, J=6.4Hz, 2H), 3.84 (d, J= 5.9Hz, 2H), 3.63 (t, 2H), 2.33 (t, J=7.3Hz, 2H), 2.02-1.92 (s, 3H)

Embodiment 2

4- ((5- hydroxyl -3- (4- hydroxy phenyl) -4- oxo -4H- chromene -7- base) oxygroup) butyric acid (compound 3)

Compound 1 (800.0mg, 2.25mmol) obtained in example 1 is added in about 8mL water to the KOH that 1mol/L is then added 1~2mL methanol hydrotropy is added in solution (4mL), and 60 DEG C of reactions 1~2h, adjusting reaction solution pH are 2-3, obtain off-white powder. Yield: 54%.¹HNMR(400MHz,DMSO)δ16.47(s,1H),12.95(s,1H),9.60 (s,1H),8.41(s,1H), 7.40 (d, J=8.5Hz, 2H), 6.83 (d, J=8.6Hz, 2H), 6.66 (d, J=2.1Hz, 1H), 6.40 (d, J=2.1Hz, 1H), 4.12 (t, J=6.4Hz, 2H), 3.63 (t, 2H), 2.33 (t, J=7.3Hz, 2H)

Embodiment 3

(4- ((5- hydroxyl -3- (4- hydroxy phenyl) -4- oxo -4H- chromene -7- base) oxygroup) bytyry) glycine methyl ester (is changed Close object 5a)

The compound 3 (356.0mg, 1mmol) that example 2 obtains is added to by 1- ethyl-(3- dimethylamino under condition of ice bath Base propyl) carbodiimide hydrochloride (EDCl) (575.0mg, 3mmol), I-hydroxybenzotriazole (HOBt) (405.0 mg, 3mmol) in the reaction system formed, 4h is stirred, glycine methyl ester hydrochloride (377.0mg, 3mmol), acid binding agent N, N '-is added Diisopropylethylamine (DIPEA) (523.0 μ L, 3mmol), 4- dimethylamino pyrroles (DMAP) (244.0mg, 2mmol), room temperature Reaction.With silica gel column chromatography separating purification (ethyl acetate/petroleum ether=3:1；V/V), faint yellow solid is obtained.Yield: 49%.¹HNMR (400MHz, DMSO) δ 12.95 (s, 1H), 9.60 (s, 1H), 8.41 (s, 1H), 8.36 (t, J=6.1Hz, 1H), 7.40 (d, J=8.5Hz, 2H), 6.83 (d, J=8.6Hz, 2H), 6.66 (d, J=2.1Hz, 1H), 6.40 (d, J=2.1Hz, 1H), 4.12 (t, J=6.4Hz, 2H), 3.84 (d, J=5.9Hz, 2H), 3.63 (s, 3H), 2.33 (t, J=7.3Hz, 2H), 2.02–1.92(m,2H).¹³CNMR(101MHz,DMSO)δ180.86, 172.56,170.95,164.96,162.23, 157.97,157.95,154.84,130.62,122.97,121.53,115.55,105.86, 98.85,93.27,68.23, 52.12,41.04,31.56,24.90.

Embodiment 4

(4- ((5- hydroxyl -3- (4- hydroxy phenyl) -4- oxo -4H- chromene -7- base) oxygroup) bytyry) leucine methyl ester (is changed Close object 5o)

The compound 3 (356.0mg, 1mmol) that example 2 obtains is added to by 1- ethyl-(3- dimethylamino under condition of ice bath Base propyl) carbodiimide hydrochloride (EDCl) (575.0mg, 3mmol), I-hydroxybenzotriazole (HOBt) (405.0 mg, 3mmol) in the reaction system formed, 4h is stirred, leucine methyl ester hydrochloride (545.0mg, 3mmol), acid binding agent N, N '-is added Diisopropylethylamine (DIPEA) (523.0 μ L, 3mmol), 4- dimethylamino pyrroles (DMAP) (244.0mg, 2mmol), room temperature Reaction.With silica gel column chromatography separating purification (ethyl acetate/petroleum ether=3:1；V/V), faint yellow solid is obtained.Yield: 64%.¹HNMR (400MHz, DMSO) δ 12.94 (d, J=7.5Hz, 1H), 9.60 (s, 1H), 8.41 (s, 1H), 8.28 (d, J= 7.6Hz, 1H), 7.40 (d, J=8.5Hz, 2H), 6.83 (d, J=8.6Hz, 2H), 6.65 (d, J=2.2Hz, 1H), 6.40 (d, J=2.1Hz, 1H), 4.29 (ddd, J=9.7,7.7,5.3Hz, 1H), 4.09 (h, J=10.2Hz, 2H), 3.61 (d, J =5.7Hz, 3H), 2.41-2.26 (m, 2H), 1.97 (dd, J=13.4,6.7Hz, 2H), 1.58-1.48 (m, 2H), 1.24 (s, 1H), 0.87 (d, J=6.4Hz, 3H), 0.83 (d, J=6.4Hz, 3H)¹³CNMR (101MHz,DMSO)δ180.86, 173.64,172.21,165.00,162.22,157.96,154.84,130.62,122.97, 121.52,115.55, 105.86,98.85,93.25,68.22,52.24,50.68,31.56,24.92,24.72,23.18,21.67.

Embodiment 5

(4- ((5- hydroxyl -3- (4- hydroxy phenyl) -4- oxo -4H- chromene -7- base) oxygroup) bytyry) phenyalanine methyl ester (compound 5k)

The compound 3 (356.0mg, 1mmol) that example 2 obtains is added to by 1- ethyl-(3- dimethylamino under condition of ice bath Base propyl) carbodiimide hydrochloride (EDCl) (575.0mg, 3mmol), I-hydroxybenzotriazole (HOBt) (405.0 mg, 3mmol) in the reaction system formed, 4h is stirred, phenylalanine methyl ester hydrochloride (647.0mg, 3mmol), acid binding agent N is added, N '-diisopropylethylamine (DIPEA) (523.0 μ L, 3mmol), 4- dimethylamino pyrroles (DMAP) (244.0mg, 2mmol), room Temperature reaction.With silica gel column chromatography separating purification (ethyl acetate/petroleum ether=2:1；V/V), faint yellow solid is obtained.Yield: 59%.¹HNMR (400MHz, DMSO) δ 12.96 (s, 1H), 9.61 (s, 1H), 8.44-8.36 (m, 2H), 7.40 (d, J= 8.6Hz, 2H), 7.28-7.19 (m, 5H), 6.83 (d, J=8.6Hz, 2H), 6.60 (d, J=2.2Hz, 1H), 6.37 (d, J= 2.2Hz, 1H), 4.50 (td, J=9.4,5.5Hz, 1H), 3.99 (t, J=6.4Hz, 2H), 3.66-3.57 (m, 3H), 3.04 (dd, J=13.7,5.4Hz, 1H), 2.88 (dd, J=13.7,9.6Hz, 1H), 2.33-2.18 (m, 2H), 1.94-1.82 (m, 2H).¹³CNMR(101MHz,DMSO)δ180.86,172.64,172.07,164.92, 162.21,157.95,154.85, 137.76,130.63,129.49,128.66,126.96,122.97,121.53,115.55,105.85, 98.83,93.25, 68.13,53.95,52.30,37.15,31.55,24.83.

Embodiment 6

(4- ((5- hydroxyl -3- (4- hydroxy phenyl) -4- oxo -4H- chromene -7- base) oxygroup) bytyry) tryptophan methyl ester (is changed Close object 5j)

The compound 3 (356.0mg, 1mmol) that example 2 obtains is added to by 1- ethyl-(3- dimethylamino under condition of ice bath Base propyl) carbodiimide hydrochloride (EDCl) (575.0mg, 3mmol), I-hydroxybenzotriazole (HOBt) (405.0 mg, 3mmol) in the reaction system formed, 4h is stirred, tryptophan methyl ester hydrochloride (764.0mg, 3mmol), acid binding agent N, N '-is added Diisopropylethylamine (DIPEA) (523.0 μ L, 3mmol), 4- dimethylamino pyrroles (DMAP) (244.0mg, 2mmol), room temperature Reaction.With silica gel column chromatography separating purification (ethyl acetate/petroleum ether=4:1；V/V), faint yellow solid is obtained.Yield: 72%.¹HNMR (500MHz, DMSO) δ 12.96 (s, 1H), 10.85 (s, 1H), 9.61 (s, 1H), 8.39 (s, 1H), 8.36 (d, J= 7.5Hz, 1H), 7.51 (d, J=7.9Hz, 1H), 7.40 (d, J=8.5Hz, 2H), 7.35 (d, J=8.1Hz, 1H), 7.17 (d, J=1.9Hz, 1H), 7.07 (t, J=7.4Hz, 1H), 6.99 (t, J=7.4Hz, 1H), 6.85 (d, J=8.6Hz, 2H), 6.59 (d, J=2.0Hz, 1H), 6.38 (d, J=2.1Hz, 1H), 4.56 (dd, J=13.8,7.9Hz, 1H), 4.03 (t, J= 6.4Hz, 2H), 3.60 (s, 3H), 3.12 (ddd, J=23.1,14.6,7.1Hz, 2H), 2.30 (tq, J=14.8,7.3Hz, 2H),1.96–1.88(m,2H).¹³CNMR(126MHz,DMSO)δ180.86, 173.01,172.12,164.94,162.22, 157.96,154.79,136.58,130.62,127.56,124.10,122.98,121.43, 118.87,118.47, 115.57,111.91,110.04,105.87,98.85,93.22,68.23,67.49,53.62,52.24,31.61, 27.57, 25.60,24.86.

Embodiment 7

(4- ((5- hydroxyl -3- (4- hydroxy phenyl) -4- oxo -4H- chromene -7- base) oxygroup) bytyry) methyl-P-tyrosine (is changed Close object 5l)

The compound 3 (356.0mg, 1mmol) that example 2 obtains is added to by 1- ethyl-(3- dimethylamino under condition of ice bath Base propyl) carbodiimide hydrochloride (EDCl) (575.0mg, 3mmol), I-hydroxybenzotriazole (HOBt) (405.0 mg, 3mmol) in the reaction system formed, 4h is stirred, tyrosine methyl ester hydrochloride (695.0mg, 3mmol), acid binding agent N, N '-is added Diisopropylethylamine (DIPEA) (523.0 μ L, 3mmol), 4- dimethylamino pyrroles (DMAP) (244.0mg, 2mmol), room temperature Reaction.With silica gel column chromatography separating purification (ethyl acetate/petroleum ether=2:1；V/V), faint yellow solid is obtained.Yield: 63%.¹HNMR (400MHz, DMSO) δ 12.96 (s, 1H), 9.60 (s, 1H), 9.20 (s, 1H), 8.41 (s, 1H), 8.33 (d, J= 7.7Hz, 1H), 7.40 (d, J=8.6Hz, 2H), 7.00 (d, J=8.4Hz, 2H), 6.83 (d, J=8.6Hz, 2H), 6.66 (s, 1H), 6.64-6.61 (m, 2H), 6.39 (d, J=2.1Hz, 1H), 4.40 (dd, J=14.1,8.5Hz, 1H), 4.04 (t, J=6.0Hz, 2H), 3.59 (s, 3H), 2.88 (d, J=5.5Hz, 1H), 2.79 (d, J=9.2 Hz, 1H), 2.26 (dd, J= 11.6,7.2Hz,2H),1.92–1.87(m,2H).¹³CNMR(101MHz,DMSO)δ 180.86,172.78,172.09, 164.94,162.22,157.96,156.44,154.80,130.62,127.69,122.97,121.55, 115.55, 105.86,98.84,93.22,68.20,60.22,54.38,52.21,36.48,31.59,24.88.

Embodiment 8

6- ((5- hydroxyl -3- (4- hydroxy phenyl) -4- oxo -4H- chromene -7- base) oxygroup) ethyl hexanoate (compound 2)

Genistein (2.0g, 7.4mmol) and KOH (0.415g, 7.4mmol) is dissolved in DMF, 60 DEG C of reaction 1h.It adds 6- bromocaproic acid ethyl ester (2.62mL, 14.8mmol) and KI (122.8mg, 0.74mmol) react 4~6h.Reaction solution is poured into few It measures in distilled water, suction filtration takes filter cake, and silica gel column chromatography purifying obtains faint yellow solid.Yield: 79%.¹HNMR(500MHz, DMSO) δ 12.94 (s, 1H), 9.59 (s, 1H), 8.38 (s, 1H), 7.40 (d, J=8.5Hz, 2H), 6.84 (d, J=8.6Hz, 2H), 6.62 (d, J=2.0Hz, 1H), 6.38 (d, J=2.0Hz, 1H), 4.27 (p, J=7.2Hz, 2H), 4.08 (t, J= 6.4Hz, 2H), 2.15 (t, J=7.3Hz, 2H), 1.77-1.69 (m, 2H), 1.57 (dt, J=14.8,7.3 Hz, 2H), 1.44-1.37 (m, 2H), 1.27 (t, J=7.3Hz, 3H)

Embodiment 9

6- ((5- hydroxyl -3- (4- hydroxy phenyl) -4- oxo -4H- chromene -7- base) oxygroup) caproic acid (compound 4)

Compound 2 (927.0mg, 2.25mmol) obtained in example 8 is added in about 8mL water, is then added 1mol/L's 1~2mL methanol hydrotropy is added in KOH solution (4mL), and 60 DEG C of reactions 1~2h, adjusting reaction solution pH are 2-3, obtain yellow powder. Yield: 46%.Spectrogram information¹HNMR(500MHz,DMSO)δ16.12(s,1H),12.94(s, 1H),9.59(s,1H),8.38 (s, 1H), 7.40 (d, J=8.5Hz, 2H), 6.84 (d, J=8.6Hz, 2H), 6.62 (d, J=2.0 Hz, 1H), 6.38 (d, J =2.0Hz, 1H), 4.08 (t, J=6.4Hz, 2H), 2.15 (t, J=7.3Hz, 2H), 1.77-1.69 (m, 2H), 1.57 (dt, J=14.8,7.3Hz, 2H), 1.44-1.37 (m, 2H)

Embodiment 10

(6- ((5- hydroxyl -3- (4- hydroxy phenyl) -4- oxo -4H- chromene -7- base) oxygroup) caproyl) methyl lactamine (is changed Close object 6b)

The compound 4 (384.0mg, 1mmol) that example 9 obtains is added to by 1- ethyl-(3- dimethylamino under condition of ice bath Propyl) carbodiimide hydrochloride (EDCl) (575.0mg, 3mmol), I-hydroxybenzotriazole (HOBt) (405.0 mg, 3mmol) In the reaction system of composition, 4h is stirred, alanine methyl ester hydrochloride (419.0mg, 3mmol), acid binding agent N, N '-diisopropyl is added Base ethamine (DIPEA) (523.0 μ L, 3mmol), 4- dimethylamino pyrroles (DMAP) (244.0mg, 2mmol), room temperature reaction.With Silica gel chromatograph column separating purification (ethyl acetate/petroleum ether=1:1；V/V), faint yellow solid is obtained.Yield: 51%.¹HNMR (500MHz, DMSO) δ 12.94 (s, 1H), 9.59 (s, 1H), 8.38 (s, 1H), 8.23 (d, J=7.0Hz, 1H), 7.40 (d, J =8.5Hz, 2H), 6.84 (d, J=8.6Hz, 2H), 6.62 (d, J=2.0 Hz, 1H), 6.38 (d, J=2.0Hz, 1H), 4.27 (p, J=7.2Hz, 1H), 4.08 (t, J=6.4Hz, 2H), 3.62 (s, 3H), 2.15 (t, J=7.3Hz, 2H), 1.77-1.69 (m, 2H), 1.57 (dt, J=14.8,7.3Hz, 2H), 1.44-1.37 (m, 2H), 1.27 (d, J=7.3Hz, 3H)¹³CNMR (126MHz,DMSO)δ180.84,173.74,172.51,165.10,162.23, 157.96,154.77,130.61, 122.95,121.54,115.55,105.80,98.79,93.23,68.88,52.21,47.92,35.25, 30.88,28.62, 25.43,25.31,17.43.

Embodiment 11

(6- ((5- hydroxyl -3- (4- hydroxy phenyl) -4- oxo -4H- chromene -7- base) oxygroup) caproyl) valine methyl ester (is changed Close object 6m)

The compound 4 (384.0mg, 1mmol) that example 9 obtains is added to by 1- ethyl-(3- dimethylamino under condition of ice bath Propyl) carbodiimide hydrochloride (EDCl) (575.0mg, 3mmol), I-hydroxybenzotriazole (HOBt) (405.0 mg, 3mmol) In the reaction system of composition, 4h is stirred, valine methyl ester hydrochloride (503.0mg, 3mmol), acid binding agent N, N '-diisopropyl is added Base ethamine (DIPEA) (523.0 μ L, 3mmol), 4- dimethylamino pyrroles (DMAP) (244.0mg, 2mmol), room temperature reaction.With Silica gel chromatograph column separating purification (ethyl acetate/petroleum ether=2:1；V/V), yellow solid is obtained.Yield: 72%.¹HNMR (500MHz, DMSO) δ 12.94 (s, 1H), 9.60 (s, 1H), 8.39 (s, 1H), 8.09 (d, J=8.2Hz, 1H), 7.41- 7.36 (m, 2H), 6.86-6.80 (m, 2H), 6.62 (d, J=2.2Hz, 1H), 6.38 (d, J=2.2Hz, 1H), 4.19 (dd, J =8.1,6.5Hz, 1H), 4.08 (t, J=6.4Hz, 2H), 3.63 (s, 3H), 2.27-2.15 (m, 2H), 2.06-1.98 (m, 1H),1.77–1.69(m,2H),1.61–1.53(m,2H),1.45–1.36 (m,2H),0.90–0.88(m,3H),0.87(d,J =6.8Hz, 3H)¹³CNMR(126MHz,DMSO)δ180.84, 173.00,172.73,165.10,162.22,157.95, 154.78,130.61,122.96,121.54,115.55,105.81,98.79, 93.23,68.88,57.79,52.02, 35.21,30.28,28.60,25.47,19.46,18.77.

Embodiment 12

(6- ((5- hydroxyl -3- (4- hydroxy phenyl) -4- oxo -4H- chromene -7- base) oxygroup) caproyl) phenyalanine methyl ester (compound 6k)

The compound 4 (384.0mg, 1mmol) that example 9 obtains is added to by 1- ethyl-(3- dimethylamino under condition of ice bath Propyl) carbodiimide hydrochloride (EDCl) (575.0mg, 3mmol), I-hydroxybenzotriazole (HOBt) (405.0 mg, 3mmol) In the reaction system of composition, 4h is stirred, addition phenylalanine methyl ester hydrochloride (647.0mg, 3mmol), acid binding agent N, N '-two are different Propylethylamine (DIPEA) (523.0 μ L, 3mmol), 4- dimethylamino pyrroles (DMAP) (244.0mg, 2mmol), room temperature reaction. With silica gel chromatograph column separating purification (ethyl acetate/petroleum ether=3:1；V/V), yellow solid is obtained.Yield: 52%.¹HNMR (500MHz, DMSO) δ 12.96 (s, 1H), 9.61 (s, 1H), 8.39 (s, 1H), 8.28 (d, J=7.9Hz, 1H), 7.40 (d, J =8.6Hz, 2H), 7.28 (d, J=3.8Hz, 1H), 7.27 (d, J=4.0 Hz, 1H), 7.26 (s, 1H), 7.24-7.16 (m, 4H), 6.84 (d, J=8.6Hz, 2H), 6.62 (d, J=2.1Hz, 1H), 6.38 (d, J=2.1Hz, 1H), 4.53-4.46 (m, 1H), 4.03 (t, J=6.4Hz, 2H), 3.61 (s, 3H), 3.04 (dd, J=13.8,5.3Hz, 1H), 2.93-2.85 (m, 1H), 2.10 (t, J=7.2Hz, 2H), 1.72-1.63 (m, 2H), 1.52-1.44 (m, 2H), 1.33-1.23 (m, 2H) .¹³CNMR(126MHz,DMSO)δ180.85,172.70,172.64,165.10, 162.24,157.97,154.78,137.81, 130.62,129.66,129.50,128.64,126.92,122.97,121.55,115.56, 105.81,98.79,93.23, 68.85,53.84,52.25,37.17,35.31,28.57,25.29.

Embodiment 13

(6- ((5- hydroxyl -3- (4- hydroxy phenyl) -4- oxo -4H- chromene -7- base) oxygroup) caproyl) tryptophan (compound 6j)

The compound 4 (384.0mg, 1mmol) that example 9 obtains is added to by 1- ethyl-(3- dimethylamino under condition of ice bath Propyl) carbodiimide hydrochloride (EDCl) (575.0mg, 3mmol), I-hydroxybenzotriazole (HOBt) (405.0 mg, 3mmol) In the reaction system of composition, 4h is stirred, tryptophan methyl ester hydrochloride (764.0mg, 3mmol), acid binding agent N, N '-diisopropyl is added Base ethamine (DIPEA) (523.0 μ L, 3mmol), 4- dimethylamino pyrroles (DMAP) (244.0mg, 2mmol), room temperature reaction.With Silica gel chromatograph column separating purification (ethyl acetate/petroleum ether=3:1；V/V), yellow solid is obtained.Yield: 58%.¹HNMR (500MHz, DMSO) δ 12.96 (s, 1H), 10.86 (s, 1H), 9.60 (s, 1H), 8.38 (s, 1H), 8.24 (d, J=7.6Hz, 1H), 7.50 (t, J=7.8Hz, 1H), 7.40 (d, J=8.5Hz, 2H), 7.34 (d, J=8.1Hz, 1H), 7.16 (d, J= 1.5Hz, 1H), 7.07 (t, J=7.5Hz, 1H), 6.99 (t, J=7.4Hz, 1H), 6.84 (d, J=8.5Hz, 2H), 6.61 (s, 1H), 6.39 (s, 1H), 4.55 (dd, J=13.8,8.0Hz, 1H), 4.02 (t, J=6.3Hz, 2H), 3.59 (d, J= 7.4Hz, 3H), 3.16 (dd, J=14.5,5.5Hz, 1H), 3.04 (dd, J=14.5,8.7 Hz, 1H), 2.17-2.10 (m, 2H), 1.73-1.63 (m, 2H), 1.58-1.48 (m, 2H), 1.33 (dd, J=14.1,7.1 Hz, 2H)¹³CNMR(126MHz, DMSO)δ180.85,173.05,172.68,165.09,162.23,157.96,154.77, 136.57,130.62,127.57, 124.09,122.96,121.56,121.41,118.85,118.48,115.56,111.88,110.07, 105.81,98.80, 93.24,68.84,53.47,52.21,35.33,28.59,27.58,25.34,25.28.

Embodiment 14

External anti-human colon cancer increment active testing.

Cell strain selects HCT-116 (human colon cancer cell), buys in Chinese Academy of Sciences's Shanghai school of life and health sciences.

Method: mtt assay.The HCT-116 cell for taking logarithmic phase to grow, is diluted to 8 × 10⁴A/mL is planted in 96 orifice plates (100 μ L/ Hole).If 5 FU 5 fluorouracil be control group, the untested compound group of DMSO group, blank group and 6 various concentrations, every 100 μ L of hole, Parallel 3 multiple holes.Set 37 DEG C, 5%CO₂Incubator cultivates 48h.20 μ LMTT (3- (4,5- diformazans are added in every hole under the conditions of being protected from light Base thiazole -2- base) -2,5- diphenyltetrazoliumbromide father-in-law's bromide, 5mg/mL) solution, it places into and places 4h in incubator；It is abandoned after 4h Culture solution is removed, 150 μ LDMSO dissolution concussion is added in every hole, surveys 490nmOD value with the full-automatic microplate reader of MK-2, calculates half and inhibit Concentration IC₅₀。

4- ((5- hydroxyl -3- (4- hydroxy phenyl) -4- oxo -4H- chromene -7- base) oxygroup) bytyry) tryptophan first Ester, genistein, 5 FU 5 fluorouracil IC₅₀Calculated result.

1 IC of table₅₀Calculated result

By that can find out above, 4- ((5- hydroxyl -3- (4- hydroxy phenyl) -4- oxo -4H- chromene -7- base) oxygroup) bytyry) Tryptophan methyl ester (compound 5j) anticancer activity is greater than positive control 5 FU 5 fluorouracil and is far longer than genistein parent.

Embodiment 15

Influence of the Flow Cytometry Assay compound to human colon carcinoma (HCT-116) division cycle.

1. method: flow cytometry.Take logarithmic phase cell kind in 6 orifice plates, 100,000 cells of every Kong Yuewu are urinated phonetic with 5- fluorine Pyridine and genistein are positive controls, 3 untested compound concentration are arranged, 2 secondary orifices of each concentration set 37 DEG C, 5%CO₂ Incubator cultivates 48h.After 48h, pastille culture medium is sucked out, cold PBS washs cell surface, and pancreatin digests every hole cell.Centrifugation 3 times Afterwards, 4 DEG C of ethyl alcohol of 1mL70% are added in each sample, are put into 4 DEG C of environment 12h.

2. cell dyeing: after 12h, carrying out the 4th centrifugation, after supernatant carefully is sucked out, 1mL PBS is added, cell is resuspended The 5th centrifugation is then carried out, supernatant is carefully sucked out, 0.5mL dyeing agent solution (buffer+PI dyeing is added in each sample Liquid+RNaseA) cell is resuspended.It is placed under 37 DEG C of environment after being protected from light incubation 30 minutes and carries out flow cytometer detection.

4- ((5- hydroxyl -3- (4- hydroxy phenyl) -4- oxo -4H- chromene -7- base) oxygroup) bytyry) tryptophan first The influence of ester, genistein, 5 FU 5 fluorouracil to cell division cycle.

Cell cycle distribution statistical form after the processing of 2 compound of table

The experimental results showed that compound 4- ((5- hydroxyl -3- (4- hydroxy phenyl) -4- oxo -4H- chromene -7- base) oxygroup) fourth Acyl group) tryptophan methyl ester by retardance HCT-116 cell inhibits cell Proliferation in G1 phase.

Active testing shows that there is genistein amino acid ester derivative good anti-human colon cancer HCT-116 proliferation to live Property, and block the cancer cell division period in the G1 phase.

Claims (3)

1. compound shown in chemical structure Formulas I preparation and its preparing the application in anti-human colon cancer HCT-116 drug:

R is selected from Formulas I: selected from any of following a-s building stone；N is selected from: 3 or 5.

2. application described in claim 1, wherein compound shown in Formulas I is (4- ((5- hydroxyl -3- (4- hydroxy phenyl) -4- oxygen Generation -4H- chromene -7- base) oxygroup) bytyry) tryptophan methyl ester anti-human colon cancer HCT-116 cell Proliferation activity.

3. a kind of method for preparing genistein amino acid ester derivative described in claim 1, it consists essentially of following steps:

Genistein is dissolved in n,N-Dimethylformamide (DMF) by step 1, adds KOH, genistein and KOH substance The ratio between amount is 1:1,60 DEG C of reaction 1h.4- bromobutyrate or 6- bromocaproic acid ethyl ester and KI, genistein and bromine are added after 1h The ratio between compound and the amount of substance of KI are as follows: 1:2:0.1 reacts 4~6h.Reaction solution is poured into a small amount of distilled water, is filtered, institute After obtained filter cake is dissolved with tetrahydrofuran, silica gel is added, is purified by column chromatography, obtains intermediate product 1 or 2:

Compound 1 or 2 is added in about 8mL water step 2, adds 1mol/LKOH solution, compound 1 or 2 and KOH is molten The ratio between amount of substance of liquid are as follows: 1~2mL methanol hydrotropy is added in 1:4, and 60 DEG C of reactions 1~2h, adjusting reaction solution pH are 2-3, are obtained To intermediate product 3 or 4；

Compound 3 or 4 is added to by 1- ethyl-(3- dimethylaminopropyl) carbodiimide by step 3 under condition of ice bath Hydrochloride (EDCl), I-hydroxybenzotriazole (HOBt) composition reaction system in, compound 3 or 4 with EDCl and HOBt object The ratio between amount of matter are as follows: the amino acid methyl ester hydrochloride as shown in a-s structure, acid binding agent N, N '-is added in 1:3:3, stirring at normal temperature 4h Diisopropylethylamine (DIPEA), 4- dimethylamino pyrroles (DMAP), compound 3 or 4 with amino acid methyl ester hydrochloride and The ratio between the amount of DIPEA and DMAP substance are as follows: 1:3:3:2 reacts at room temperature 12h.It obtains shown in the structural formula I as described in right 1 Genistein amino acid ester derivative.

Wherein: R is one kind with a-s building stone；N is 3 or 5.